![Role ofBioanalysis 3
2. The active ingredients from natural plants, soil extracts and
microorganisms (fungi, viruses and molds) are isolated, purified and
screened for activity using various pharmacological models. This
approach led to identification of paclitaxel (Taxol®), a drug used to
treat various forms ofcancer.
3. Accidental discovery or serendipity occurs when a drug molecule is
found to work for a different target than the one for which it was
originally synthesized. For example, in the search for novel drugs to
treat cardiac arrhythmias, researchers discovered that imiquimod
(Aldara®) was a novel immunomodulator that boosted the body's
immune system; a new class ofantiviral agents was discovered.
Today, however, the pharmaceutical research process is looking at new and
improved ways to develop drugs, in response to several important scientific
advances that have recently occurred. These advances include the identification
of new and more specific drug targets (as a result of maturation in genomics
and proteomics); successes with tissue growth outside of the living organism;
development of faster, more sensitive and more selective analytical systems
(mass spectrometry); higher throughput (as a result of robotics and laboratory
automation); proliferation in synthesis techniques (combinatorial chemistry);
and advances in computing and information systems (bioinformatics). In
parallel with these scientific advances, business factors have changed with the
consolidation ofdrug companies and the intense pressure to get drugs to market
faster than ever before. The current focus ofdrug discovery research is on rapid
data generation and analysis to identify promising candidates very early in the
development cycle. An optimal lead candidate is selected for further
evaluation.
Combinatorial chemistry techniques allow the synthesis of compounds faster
than ever before, and these greater numbers of compounds are quickly
evaluated for potency and pharmacological activity using high throughput
screening (HTS) techniques. HTS involves performing various microplate
based immunoassays with synthesized compounds or compounds from natural
product isolation. Examples of assay types used in HTS are scintillation
proximity assay (SPA), enzyme linked immunosorbent assay (ELISA),
fluorescent intensity, chemiluminescence, absorbance/colorimetry and bio-
luminescence assays [I]. These HTS tests simulate a specific biological
receptor or target function and a qualitative decision ("hit" or "miss") is
generated [2].](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-10-320.jpg)
![Role ofBioanalysis 5
Toxicology tests in preclinical development examine acute toxicity at
escalating doses and short term toxicity (defined as 2 weeks to 3 months), as
well as the potential of the drug candidate to cause genetic toxicity. Today's
research efforts attempt to utilize as few animals as possible and so more
in vitro tests are conducted. The use of metabonomics for toxicity testing is
making an impact on both drug discovery (to select a lead compound) and
preclinical development (to examine safety biomarkers and mechanisms).
Metabonomics is a technology that explores the potential of combining state of
the art high resolution NMR (Nuclear Magnetic Resonance) spectroscopy with
multivariate statistical techniques. Specifically, this technique involves the
elucidation ofchanges in metabolic patterns associated with drug toxicity based
on the measurement of component profiles in biofluids (i.e., urine). NMR
pattern recognition technology associates target organ toxicity with specific
NMR spectral patterns and identifies novel surrogate markers of toxicity [3].
Also in preclinical development, the pharmacokinetic profile of a drug
candidate is learned. Pharmacokinetics is a specific, detailed analysis which
refers to the kinetics (i.e., time course profile) of drug absorption, distribution
and elimination. The metabolites from the drug are identified in this stage.
Definitive metabolism studies of drug absorption, tissue distribution,
metabolism and elimination are based on the administration of radiolabeled
drug to animals. It is important that the radionuclide is introduced at a position
in the chemical structure that is stable to points of metabolism and conditions
ofacid and base hydrolysis.
Pharmacology testing contains two major aspects-in vivo (animal models) and
in vitro (receptor binding) explorations. Comparisons are made among other
drugs in the particular collection under evaluation, as well as among
established drugs and/or competitive drugs already on the market. More
informative and/or predictive biomarkers are also identified and monitored
from these studies.
Detailed information about the drug candidate is developed at the proper time
in preclinical development, such as the intended route of administration and the
proposed method of manufacturing. In order to supply enough of the drug to
meet the demands of toxicology, metabolism and pharmacology, the medicinal
chemistry and analytical groups work together to determine the source of raw
materials, develop the necessary manufacturing process and establish the purity
of the drug product. The exact synthesis scheme and methodology needed to
produce the drug are recorded in detailed reports. The pharmaceutics research
group develops and evaluates formulations for the drug candidate. These](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-12-320.jpg)
![8 Chapter 1
provided on data supporting the claim of the new drug, its proposed labeling,
its chemistry and results from animal studies. Note that procedures exist that
can expedite the development, evaluation and marketing of new drug therapies
intended to treat patients with life threatening illnesses. Such procedures may
be activated when no satisfactory alternative therapies exist. During Phase I or
Phase II clinical studies, these procedures (also called "Subpart E" for Section
312 of the US Code of Federal Regulations) may be put into action [4].
The company developing the drug must then consider many factors before
further development is undertaken, such as the cost of manufacturing the drug
(which mayor may not involve new equipment purchases or changes in
existing facilities), the estimated time and cost to gain final FDA approval, the
competition the drug may face in the market, its sales potential and projected
sales growth. The return on the company's investment is estimated. It has been
observed in recent years for a major pharmaceutical company that if the return
on investment on a single drug is not 100 million dollars (US) or more, the
company may choose not to develop the drug further; instead, licensing the
drug to a smaller company is one of several options.
1.1.4.4 Phase III
After evidence establishing the effectiveness of the drug candidate has been
obtained in Phase II clinical studies, and the "End of Phase II" meeting with the
FDA has shown a favorable outcome, Phase III studies can begin. These
studies are large scale controlled efficacy studies and the objective is to gain
more data on the effectiveness and safety of the drug in a larger population of
patients. A special population may be used, e.g., those having an additional
disease or organic deficiency such as renal or liver failure. The drug is often
compared with another drug used to treat the same condition. Drug interaction
studies are conducted as well as bioavailability studies in the presence and
absence of food. A Phase III study is a clinical trial in which the patients are
assigned randomly to the experimental group or the control group.
From 1,000 to 5,000 volunteer patients are typically used in a Phase III study;
this aspect of drug development can last from 2 to 3 years. Data obtained are
needed to develop the detailed physician labeling that will be provided with the
new drug. These data also extrapolate the results to the general population and
identify the side effect profile and the frequency of each side effect. Inparallel,
various toxicology, carcinogenicity and metabolic studies are conducted in
animals. The cumulative results from all of these studies are used to establish
statements ofefficacy and safety of the new drug.](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-15-320.jpg)
![Role ofBioanalysis 9
As Phase III progresses, many commercial considerations are put into action.
These matters include pricing, registration, large scale manufacturing and plans
for market launch. The plan for marketing the drug is developed and additional
clinical trials may be started to satisfy new labeling indications or to expand
current indications that define exactly which conditions the drug is intended to
treat. Note that once a drug is approved, physicians are able to prescribe its use
to treat other conditions for which they feel the drug might have a beneficial
effect; this use is known as "off label drug use."
A Treatment IND is a special case in which the FDA may decide to make a
promising new drug available to desperately ill patients as early as possible in
the drug's development [5]. In order for a Treatment IND to be instituted, there
must be significant evidence of drug efficacy, the drug must treat a serious or
life threatening disease (where death may occur in months if no treatment is
received), and/or there is no alternative treatment available for these intended
patients. Treatment INDs, when they occur, are typically made available to
patients during Phase III studies before marketing of the drug begins. Any
patient who receives the drug under a Treatment IND cannot participate in the
definitive Phase III studies.
Another means by which promising and unique experimental agents can be
made available to patients is called "Parallel Track." This policy was developed
in response to the AIDS illness and allows patients with AIDS who cannot
participate in controlled clinical trials to receive the promising investigational
drug [6].
1.1.4.5 New Drug Application (NDA)
The New Drug Application (NDA) is the formal summary of the results of all
animal and human studies, in conjunction with detailed plans for marketing and
manufacturing the drug. Also, information is provided about the drug's
chemistry, analysis, specifications and proposed labeling. The NDA is filed
with the FDA by the drug sponsor who wishes to sell the new pharmaceutical
entity in the United States.
Before final approval may be granted, the FDA conducts a PreApproval
Inspection (PAl) of the manufacturer's facilities because it is very important
that methods used to manufacture the drug and maintain its quality are
sufficient to preserve the drug's identity, strength and purity. This inspection
evaluates the manufacturer's compliance with Good Manufacturing Practices
(GMP), verifies the accuracy of information submitted in the NDA, and](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-16-320.jpg)
![Role ofBioanalysis 11
the wholesalers with packaged drug product so that the drug can be purchased
and used by pharmacies in response to receiving written prescriptions from
physicians.
The product launch announces the new drug to physicians and other medical
professionals. This introduction provides education about the new drug's
characteristics, indications and labeling. Various marketing and advertising
programs are devised and executed.
Phase IV clinical trials are those studies conducted after a product launch to
expand on approved claims, study the drug in a particular patient population, as
well as extend the product line with new formulations. A clinical study after the
drug is sold may be conducted to evaluate a new dosage regimen for a drug,
e.g., fexofenadine (Allegra®) is an antihistamine sold by Aventis (Bridgewater,
NJ USA). The original clinical studies indicated that a dosage of 60 mg, given
every 12 h, was adequate to control symptoms of allergies and rhinitis. Their
product launch was made with this strength and dosage regimen. In response to
competition from a once a day allergy drug, Aventis conducted Phase IV
clinical trials (after the product was on the market) with different dosages and
obtained the necessary data to show that a 180 mg version of Allegra could be
taken once a day and relieve allergy symptoms with similar efficacy as 60 mg
taken twice a day. Aventis then filed the clinical and regulatory documentation,
and obtained approval to market a new dosage form of their drug. Another
example of a post-marketing Phase IV clinical study is the investigation of
whether or not sertraline (Zoloft®), an antidepressant drug, could be taken by
patients with unstable ischemic heart disease. Results suggested that it is a safe
and effective treatment for depression in patients with recent myocardial
infarction or unstable angina [7].
In order to further ensure continued drug product quality, the FDA requires the
submission of Annual Reports for each drug product. Annual Reports include
information pertaining to adverse reaction data and records of production,
quality control and distribution. For some drug products, the FDA requires
affirmative post-marketing monitoring or additional studies to evaluate long
term effects. A drug company also closely monitors all adverse drug
experiences collected after the sale ofa drug and reports them to the FDA.
The FDA has the authority to withdraw a drug from the market at any time in
response to unusual or rare occurrences of life threatening side effects or
toxicity noted in the post-marketing surveillance program. A conclusion that a
drug should no longer be marketed is based on the nature and frequency of the](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-18-320.jpg)
![Role ofBioanalysis 13
targets through proteomics; advances in the fields of combinatorial chemistry,
high throughput screening, and mass spectrometry; and improvements in
laboratory automation and throughput in bioanalysis. The end result of these
process improvements is that compounds can now be synthesized faster than
ever before. These greater numbers of compounds are quickly evaluated for
pharmacological and metabolic activity using high throughput automated
techniques, with the ultimate goal of bringing a drug product to market in a
shorter timeframe.
Some background material is provided next for the reader to gain a better
understanding of four key industry trends: (a) combinatorial chemistry;
(b) advances in automation for combinatorial chemistry, high throughput
screening and bioanalysis; (c) LC-MS/MS analytical detection techniques; and
(d) newer bioanalytical dosing regimens (n-in-l dosing) made possible by the
advances in detection.
1.2.2 Combinatorial Chemistry
A key component of satisfying the high throughput capability and demands of
drug discovery has been the implementation of combinatorial chemistry
techniques to synthesize, purify and confirm the identity of a large number of
compounds displaying wide chemical diversity within a class. In place of
traditional serial compound synthesis, libraries of compounds are created in
96-well plates by interconnecting a set or sets of small reactive molecules,
called building blocks, in many different permutations [8-10]. Today, as many
as 2,000 compounds can be synthesized in a week. Although 96-well plates
serve as the most common format for reaction vessels, 24- and 48-well plates
are also used by medicinal chemists.
These combinatorial chemistry and parallel synthesis strategies are used to
produce a large number of compounds which are then subjected to high
throughput screening to identify biological activity. Automation aids the
chemist in the high throughput synthesis of these compound libraries [11], as
well in the subsequent purification steps required to isolate synthesized
compound from reaction starting materials, reagents and byproducts [12]. The
popular strategic options for the synthesis of combinatorial libraries include
solid-phase, solution-phaseand liquid-phase synthesis.
Solid-phase parallel synthesis uses resins to which the starting material is
attached in order to produce a large number of compounds via split and mix
methods. The solid support matrix used consists of a base polymer, a linker to](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-20-320.jpg)
![14 Chapter 1
join the base polymer to the reactive center, and a functionalized reactive site.
The immobilized reactant is then subjected to a series of chemical reactions to
prepare the desired end product. The use of excess reagents drives reactions to
completion. However, the need for deconvolution approaches to determine the
active components within a pool has limited the utility of solid-phase synthesis.
Since the synthesized compounds are attached to the solid support, this
approach does offer simplified reagent removal via filtration and impurities are
washed away easily during purification. The compound of interest is released
from the polymer support in a final chemical release step.
Solution-phase parallel synthesis techniques are more flexible than solid-phase
techniques and are often used to create focused chemical libraries. Using this
approach, the reactions occur in solution and so are easily monitored by thin
layer chromatography or NMR. The synthesized compound is isolated in one
liquid phase; all non product species are fractionated into an immiscible liquid
phase [13]. A purification step following the reaction is required and common
approaches are liquid-liquid extraction, liquid chromatography, solid-phase
extraction and the use of solid-phase scavengers to remove excess reagents
and/or reaction impurities from crude solutions. These solid-phase scavengers
(functionally modified polymers of polystyrene or bonded silica) are chosen for
their inertness to the reaction products but affinity for reagents and unwanted
byproducts. Scavengers are becoming more popular since they can easily be
adapted to automated purification techniques via filtration [14].
The procedure for use of scavengers follows. Scavenger beads are placed into
the wells ofa flow-through 96-well filtration plate. A reaction block (consisting
of individual wells of a flow-through 96-well plate in which the top and/or
bottom of the wells can be blocked or opened to allow flow and reagent
addition) is placed on top of the filtration plate (loaded with beads), so that
when vacuum is applied the reaction mixture flows out of the reaction block
and through the scavenger bed. A collection plate centered below the filtration
plate isolates the solution.
Liquid-phase parallel synthesis combines the strategic features of solid-phase
synthesis and solution-phase synthesis. This method uses a supporting polymer
(e.g., polyethylene glycol) that is soluble in the reaction media. Selective
precipitation of this polymer can be performed for the purposes ofisolation and
purification. Excess reagents and byproducts are removed by simple filtration
[15].](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-21-320.jpg)
![16 Chapter 1
analysis and screening. The addition of an analytical balance and vortex mixer
on the workstation meet the requirements of automating synthetic chemistry
conditions. A multitasking robotic workstation for synthesis features two
independently controlled robotic arms that dispense reagents and solvents
simultaneously, and can do so in inert environments. Equipment such as this
can be upgraded to perform additional tasks and can interface with some
additional components of a core system for higher throughput and higher
performance.
High throughput screening utilizes robotic-feeding liquid handling
workstations with higher density microplates (384-well and 1536-well formats)
and plate stackers for improved productivity. Automated hit picking is a
hardware and software application that automates the transfer of lead
compounds from their source plates into destination plates for consolidation.
The use of 96- or 384-channel disposable tip pipetting heads allows improved
liquid dispensing capabilities and speeds. The demand for even greater
throughput in screening procedures often requires a larger industrial process
rather than a laboratory workstation approach. Independent workstation
modules can be combined in an assembly line format, consisting ofstorage and
incubation carousels, washers, liquid handlers and plate readers. Modules are
simply added to put in more steps or increase capabilities. This type of system
is capable of running 1,000 96-well plates (96,000 assays) per day and is
compatible with 384-well plates. An ultrahigh throughput example of a fully
integrated automation solution can screen 100,000 compounds in one working
day [16].
Automation for bioanalysis is described in detail in Chapter 5. Briefly
mentioned here, liquid handling workstations with plate grippers have greatly
improved the throughput of sample preparation procedures using 96-well
plates. Automation allows more samples to be processed per unit time and frees
the analyst from most hands-on tasks. Using the microplate format for sample
preparation allows the automation of common procedures including protein
precipitation, liquid-liquid extraction, solid-phase extraction and filtration.
1.2.4 Analytical Instrumentation-LC-MS
1.2.4.1 Introduction
The preferred analytical technique in the bioanalytical research environment is
liquid chromatography-mass spectrometry (LC-MS), used for qualitative and
quantitative drug identification. LC-MS is preferred for its speed, sensitivity](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-23-320.jpg)
![Role ofBioanalysis 17
and specificity. LC is a powerful and universally accepted technique that offers
chromatographic separation of individual analytes within liquid mixtures.
These analytes are subjected to an ionization source and then are introduced
into the mass spectrometer. The mass spectrometer separates or filters these
ions based on their mass-to-charge ratio (m/z) and then sends them on to the
detector [17]. A general scheme of this process is shown in Figure 1.3. The
data generated are used to provide information about the molecular weight,
structure, identity and quantity of specific components within the sample.
1.2.4.2 LC-MS Interface
The LC-MS interface is the most important element of this system. It is the
point at which the liquid from the LC (operated at atmospheric pressure) meets
the mass spectrometer (operated in a vacuum). Advances have occurred over
the years to mate the two techniques [18, 19]. The most common ionization
interface used for bioanalysis is atmospheric pressure ionization (API) which is
a soft ionization process (i.e., provides little fragmentation of a molecular ion).
API is performed as either API-electrospray or atmospheric pressure chemical
ionization (APCI).
1.2.4.2.1 Electrospray Ionization
Electrospray ionization (ESI) generates ions directly from the solution phase
into the gas phase. The ions are produced by applying a strong electric field to
a very fine spray of the analyte in solution. The electric field charges the
surface of the liquid and forms a spray of charged droplets. The charged
droplets are attracted toward a capillary sampling orifice where heated nitrogen
drying gas shrinks the droplets and carries away the uncharged material. As the
droplets shrink, ionized analytes escape the liquid phase through electrostatic
(coulombic) forces and enter the gas phase, where they proceed into the low
pressure region of the ion source and into the mass analyzer [20-22].
IDetector r'--__---'
Figure 1.3. Schematic diagram of the basic components ofa mass spectrometer system.
The mobile phase following separation ofcomponents on a liquid chromatograph flows
into the mass spectrometer for LC-MS analysis.](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-24-320.jpg)
![18 Chapter 1
Analysis by electrospray requires the prior formation of ionized analytes in
solution; for nonionic compounds, ions are prepared by adding acid or base
modifiers to the LC mobile phase solution to promote the electrospray process
[23]. Ionization thus occurs in the liquid phase with ESI.
Pure electrospray is suitable for only capillary LC and capillary
electrophoresis, or conventional LC where the post-column effluent is split
using a zero dead volume T-piece, reducing the flow of liquid entering the
mass spectrometer. In an attempt to extend the range of solvent flow rates
amenable to electrospray, modifications have been made using pneumatic and
thermal assistance [23].
1.2.4.2.2 Atmospheric Pressure Chemical Ionization
APCI is similar to API-ES, but APCI nebulization occurs in a hot vaporization
chamber, where a heated stream of nitrogen gas rapidly evaporates nearly all of
the solvent. The vapor is ionized by a corona discharge needle [24]. The
discharge produces reagent ions from the LC solvent which then ionize the
sample [23]. Ionization thus occurs in the gas phase with APCI.
1.2.4.3 Mass Analyzers
The ionization source and the mass analyzer are linked since the mass analyzer
requires a charged particle in order for separation to occur. The mass analyzer
contains some electric or magnetic field, or combination of the two, which can
manipulate the trajectory of the ion in a vacuum chamber [25]. The
atmospheric pressure ionization interfaces described above are commercially
available with various mass analyzers. The most popular and available mass
analyzer is the quadrupole which will be described here. For information on
product ion scanning, precursor ion scanning and neutralloss/gain, the reader is
referred to the book chapter by Fountain [25].
The quadrupole mass analyzer is available as a single quadrupole or is
configured in tandem (called a triple quadrupole) to greatly enhance its
capabilities. A quadrupole mass filter typically consists of four cylindrical
electrodes (rods) to which precise DC and RF voltages can be applied. Note
that the tandem quadrupole mass filters are referred to as Ql and Q3; the
additional Q2 quadrupole has no filtering effect, except to use its RF voltage to
guide ions through the vacuum chamber.](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-25-320.jpg)
![Role ofBioanalysis 19
A mass spectrometer with a single quadrupole is capable of either full scan
acquisition or selected ion monitoring (SIM) detection. In the full scan mode,
the instrument detects signals over a defined mass range during a short period
oftime. All the signals are detected until the full mass range is covered. A mass
spectrum is generated in which ion intensity (abundance) is plotted versus m/z
ratio. Scan mode is used for qualitative analysis when the analyte mass is not
known. Inthe SIM mode, a single stage quadrupole is used as a mass filter and
monitors only a specific m/z ratio, allowing only that m/z ratio to pass through
to the detector. Chemical noise is reduced and a response curve is generated for
a specific ion rather than a mass spectrum. As a result, sensitivity is improved
and this technique is useful for quantitative analysis of a specific analyte.
When an additional quadrupole mass analyzer is configured in tandem and
coupled with a collision cell for gas reactions, several additional analytical
experiments can be conducted: multiple reaction monitoring, product ion
scanning, precursor ion scanning and neutral loss/gain. Tandem mass
spectrometry (MS/MS) achieves unequivocal identification of a drug
substance. In LC-MS/MS, the first analyzer selects a parent ion from the first
quadrupole and filters out all unwanted ions. The selected ion undergoes
collision in another quadrupole via bombardment with gas molecules such as
nitrogen or argon. Fragmentation occurs and the other mass analyzer in the
third quadrupole selects a particular product (daughter) ion. This scenario is
called selected reaction monitoring (SRM) or multiple reaction monitoring
(MRM). Information is provided on the chemical structure using the parent-
daughter ion pair.
The key attractive features of an LC-MS/MS system are its speed, selectivity
for a single MW in the presence of many other constituents and sensitivity
(typically pg/mL concentrations are able to be accurately quantitated).
Additional advantages are good precision and accuracy, and wide dynamic
range. Note that the selectivity of LC-MS/MS reduces the need for complete
chromatographic resolution of individual components, allowing a shorter
analytical run time and higher throughput [26]. Typically the combined
increases in selectivity and sensitivity of LC-MS/MS methods provide a
>10 fold improvement in the limit of quantitation (LOQ) compared with
traditional methods using ultraviolet or fluorescence detection [23]. A typical
LC-MS/MS instrument as used for the analytical quantitation of drugs from
biological matrices is shown in Figure 1.4.
In addition to the single quadrupole (LC-MS) and triple quadrupole (LC-
MS/MS) mass analyzers already discussed, other mass analyzers include:](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-26-320.jpg)
![20 Chapter 1
Figure 1.4. Typical example ofan LC-MS/MS system used for the separation, detection
and quantitation of drugs in biological matrices.
magnetic sector, time-of-flight (TOF) [27, 28], quadrupole orthogonal
acceleration TOF (Q-TOF) [29] and ion trap (IT) mass analyzers [26, 30, 31].
Applications for quantitative bioanalysis are best served by LC-MS (single
quadrupole) and LC-MS/MS (triple quadrupole) for added sensitivity.
However, when gaining information on the metabolic route of a compound is
more important than absolute sensitivity and selectivity for the parent drug, the
use of ion trap and time-of-flight instruments present advantages. IT and TOF
offer greater sensitivity in full scan mode than triple quadrupole MSIMS
detection [32] and multiple analytes can be detected and quantified, as reported
by Cai et af. [33] and Zhang et af. [34]. Another benefit of TOF-MS is its
capability of accurate mass analysis which allows metabolites to be identified
with greater confidence [32].
1.2.4.4 Further Reading
Additional discussion of these mass spectrometry techniques is outside the
scope of this text and the reader is referred to additional resources. Mass
spectrometry is introduced as a tutorial in a book chapter by Fountain [25] and
the fundamentals of electrospray are presented by Gaskel1 [35]. A review by](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-27-320.jpg)
![Role ofBioanalysis 21
Lee and Kerns [26] and a book by Lee [36] detail how LC-MS techniques are
fundamentally established as a valuable tool and used in all phases of drug
development. Kyranos et al. describe applications for LC-MS in drug discovery
[37]. Hoke et at. describe how pharmaceutical research and development have
been transformed by innovations in mass spectrometry based technologies [38].
The impact of mass spectrometry on combinatorial chemistry is described by
Triolo et at. [39] and Sii~muth and Jung [40]. Niessen provides a review of the
principles and applications of LC-MS [41]. Various reviews describe
applications for LC-MS in proteomics [42, 43], analytical toxicology [44], food
analysis [45], forensic and clinical toxicology [46] and forensic sciences [47].
A useful text for students of chemistry and biochemistry who wish to
understand the principles of mass spectrometry while using it as a tool is the
book by Johnstone and Rose [48].
LC-MS/MS techniques for drug separation, detection and quantitation have
become the standard and their use continues to expand in bioanalysis. Several
reviews of the capabilities of high throughput LC-MSIMS for bioanalysis,
including sample preparation schemes, have been published by Brewer and
Henion [49], Plumb et al. [23], Jemal [50], Brockman, Hiller and Cole [51],
and Ackermann et al. [52]. Korfmacher et al. illustrate the important role of
LC-MS/MS API techniques in drug discovery for rapid, quantitative method
development, metabolite identification and multiple drug analysis [53]. Yang
et al. describe the latest LC-MS/MS technologies for drug discovery support
[54] and Rudewicz and Yang discuss the use of LC-MS/MS in a regulated
environment [55]. Law and Temesi share some useful considerations in making
the switch from LC-UV to ESI LC-MS techniques in support of drug discovery
[56]. Rossi et al. describe the use of tandem-in-time MS as a quantitative
bioana1ytica1 tool [57].
Improvements continue to be made in three areas: (1) interfaces from the LC to
the mass spectrometer; (2) multiple sample inlets (e.g., four instead of one [55,
58, 59]); and (3) staggered parallel sample introduction schemes where one
mass spectrometer inlet is shared with two or more LC columns [60-62]. These
advances are described in more detail in Chapter 14, Section 14.4.4. Analysis
times using these novel approaches are very fast, allowing rapid turnaround of
samples to meet the needs of drug development research. Note that LC and
mass spectrometry can be interfaced to NMR to create an LC-NMR-MS
system. Such a configuration has been shown useful for the structural
elucidation of metabolites in urine [63].](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-28-320.jpg)
![22
1.2.5 N-in-l Dosing
Chapter 1
The traditional approach of dosing one animal with one drug and collecting
blood at a series of time points after administration yields valuable information
on a candidate's pharmacokinetics in a living system. However, this procedure
is time consuming and labor intensive, as each individual sample must undergo
analysis in a serial manner. This approach cannot be used to rapidly evaluate
the many hundreds of candidate compounds generated from combinatorial
chemistry, for it would take too much time.
In an effort to improve throughput and reduce cost, n-in-I dosing was devised
in which multiple compounds are dosed in one animal and the selectivity of the
mass spectrometer is used to individually quantitate their concentrations from
the mixture. This approach is also called simultaneous multiple compound
dosing or cassette dosing. The data generated by this approach yield
meaningful pharmacokinetic data in a much shorter time frame, and fewer
animals and fewer numbers of samples are used than in traditional methods.
Examples of the n-in-I approach for rapid pharmacokinetic screening of drug
candidates using LC-MSIMS are published by Olah et al. [64], McLoughlin
et al. [65], Liang et al. [66], Berman et al. [67], Cai et al. [68] and Bayliss and
Frick [69]. A concise review of cassette dosing can be found in the book
chapter by Vora, Rossi and Kindt [70].
The drug screening process is now more manageable using a fewer number of
samples generated from n-in-l dosing techniques. Each compound used for
dosing is quickly evaluated and compared on a relative basis with the other
dosed compounds and the most desirable candidates are selected. A typical
profile ofdrug concentration versus time, observed in one dog after intravenous
dosing with 10 drugs, is shown in Figure 1.5.
Note that the use of pooled plasma from multiple animals dosed with single
unique compounds has also been demonstrated to yield a throughput advantage
in the analysis of bioanalytical samples [71]. Using this approach, 10 animals
may be dosed with one compound each and then all of the I h time point
samples are pooled and subjected to sample preparation, and so on for each
time point. Sample pooling in this manner eliminates the concern from cassette
dosing of potential drug-drug interactions on pharmacokinetics and metabolic
conversion of one compound to another compound already included in the
series. The practice of sample pooling and its associated reduction in workload
is described by Kuo et al. for six proprietary compounds from a class of
antipsychotic agents [72]. Note that some variations of this approach exist,](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-29-320.jpg)
![Role ofBioanalysis 23
1
10
1000
-
:E
S 100
=
e
....
-
e
-
=
~
~
=
<:)
U
=
e
'"
=
Ii:
Dog Intravenous Dose (IO-ill-l)
0.5 mglkg
-+- Cpdl
-X-Cpd2
-:1(- Cpd3
-+-Cpd4
-o-Cpd5
-o·Cpd6
-+-Cpd7
- .... Cpd8
-'-Cpd9
~Cpdl0
24
20
16
12
8
4
0.1 +---+--~I----+---+---+---+-
o
Time (h)
Figure 1.5. Pharmacokinetic profiles of 10 compounds that were dosed intravenously in
one dog. Reprinted with permission from [73]. Copyright 2001 Elsevier Science B.V
such as the cassette accelerated rapid rat screen in which drug candidates are
dosed individually (n=2 rats per compound) in batches of six compounds per
set and then samples are pooled across time points to provide a smaller number
of test samples for analysis [74].
Another method used to reduce the number of animals in the support of drug
discovery is to serially bleed mice (removal of 10-20 ul, blood) and analyze
the small sample volumes using a small capillary LC column prior to MS
analysis [75, 76]. Conventional techniques used to obtain a nine point
pharmacokinetic curve with 4 animals per time point would require the use of
36 mice; using the serial bleeding technique, only 4 mice are used [76]. A
related method to serially bleed an animal is microdialysis, in which a semi-
permeable membrane is surgically implanted in a tissue of a living organism
and the perfusate solution is sampled over time. Thus, little biological fluid is
removed and continuous in vivo sampling is possible. Microdialysis works
especially well for drug transport studies. An overview [77] and some
applications ofmicrodialysis [78-80] provide interesting reading.](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-30-320.jpg)
![Role ofBioanalysis 25
Table 1.2
A list of experiments that are commonly performed to assess the absorption,
distribution, metabolism and elimination (ADME) characteristics of potential lead
compounds in drug discovery
Parameter Examined Typical Experiments
Absorption Caco-2 cells, MDCK cells, PgP transport
in vivo pharmacokinetic profiling
Distribution
Metabolism
Elimination
in vitro protein binding
in vivo tissue distribution studies
Metabolic stability
-Microsomes, sub cellular fractions, hepatocytes
P450 inhibition studies
-Microsomes
P450 induction studies
-Gene chips, multiple dosing
Quantitation ofdrugs and metabolites in biological fluids
drug-drug interactions, clearance, bioavailability and toxicity [81-83].
Instrumentation to accommodate cell maintenance has matured in recent years
to the point where high throughput testing using these in vitro screens is now a
viable approach to investigate the absorption and metabolism of drugs.
Note that in addition to in vivo and in vitro testing, another prediction technique
is called in silica. This approach refers to computer modeling based on
sophisticated software using information on chemical structure, receptors,
enzymes and various other databases of information [84]. Another important
note to mention is that in vitro plasma protein binding measurements (e.g.,
equilibrium dialysis and ultrafiltration) are utilized in drug discovery in a high
throughput manner but are discussed instead in Chapter 6, Section 6.6. These
applications for investigating in vitro absorption and metabolism studies will
now be described in detail: absorption, metabolic stability screening, metabolic
inhibition and induction ofcytochrome P450, and toxicity testing.
1.3.1.2.1 Absorption
It is important to determine whether a drug displays pharmacological activity
when it is administered orally, a desirable route of administration for the
general population. Therefore an estimate of absorption is desired. The](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-32-320.jpg)
![26 Chapter 1
approach used to thoroughly evaluate oral absorption is to assess those
individual factors that contribute to drug passage through the gastrointestinal
membrane, such as solubility in the lumen of the intestine, permeability across
cells, and chemical stability in the stomach and small intestine. Additional
criteria are evaluated, such as lipophilicity of the compound and its hydrogen
bonding potential, and then an overall predictive estimate of absorption
characteristics is made. While this approach is complete, it takes time to
generate all the pieces of data needed.
The in vitro permeability of drugs through Caco-2 cells has been used as a
single predictor of oral absorption in humans. Caco-2 cell monolayers are
derived from human colon adenocarcinoma cells. These cells are grown on
semipermeable membranes and spontaneously differentiate to form confluent
monolayers that mimic intestinal absorption cells. The apical (donor) surface of
the monolayer contains microvilli, as in the intestine. Permeability
measurements are based on the rate of appearance of test compound in a
receiving (basolateral) compartment. Bioanalysis is used to determine the
concentrations of analyte in the basolateral compartment [85, 86]. The cells
also express functional transport proteins and metabolic enzymes.
While the Caco-2 model is fairly predictive, it does have the limitation of
requiring a 21 day culture time with frequent attention required for replenishing
its nutrients. A common concern about the Caco-2 model is that it may not be
truly representative of all absorption pathways in the small intestine. Another
model of absorption, which reduces the tissue culture time to 3 days, is the
MDCK (Madin-Darby Canine Kidney) cell line [87, 88]. This accelerated
permeability model is a feasible alternative to the traditional model that
provides rank ordering ofcompounds with improved turnaround time.
1.3.1.2.2 Metabolic Stability Screening
The metabolic stability of a new chemical entity greatly influences its
pharmacokinetic profile. A drug may have high bioavailability (high absorption
and low first pass metabolism) following oral dosing but extensive and rapid
metabolism can reduce the time it is in the blood as an intact molecule. For
example, ester groups on drugs can be cleaved by esterases in the blood and the
drug is metabolized or biotransformed to a new chemical entity. Note that
metabolites can also be active as well, or even more active than the parent drug.
The cytochrome (CYP) P450 system in the liver is an important enzymatic
pathway for the oxidative metabolism of drugs and is often the primary route
for degradation, regardless of how the drug is administered. It is valuable to](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-33-320.jpg)
![Role ofBioanalysis 27
learn the specific P450 isoenzymes responsible for a drug's metabolism, as the
information can be used to predict the fate of the drug, potential drug-drug
interactions, reactive metabolites and cytotoxic mechanisms.
Many in vitro methodologies for assessing metabolism are used in drug
discovery support. Hepatic microsomes are among the most popular systems in
use. These preparations retain the activity of those enzymes that reside in the
smooth endoplasmic reticulum of cells, such as the cytochrome P450 system,
flavin monooxygenases and glucuronyltransferases. Cultured hepatocytes
retain a broader range of enzymatic activities, including not only the reticular
systems of CYP450 but also cytosolic and mitochondrial enzymes [89]. These
hepatocytes are particularly useful for induction and inhibition studies where
the enzymatic activities in the liver are predicted. Additionally, liver slices are
sometimes used in similar metabolic screens because they retain a wide range
of enzymatic activities, like hepatocytes, but more closely resemble the organ
level of the liver.
Metabolic stability testing ofcompounds is performed in liver microsomes with
a collection of subcellular materials called S9 mix in the presence and absence
of enzymatic cofactors such as NADP+ (Nicotinamide Adenine Dinucleotide
Phosphate). Hepatocytes and liver slices are also used for this stability testing.
Incubations are assembled in a microplate format. At selected time points, the
metabolic reaction is stopped by the addition of cold acetonitrile and then
centrifugation is performed to pellet the proteins at the bottom of the wells. The
supernatants are collected following centrifugation. Substrates are analyzed by
LC-MS/MS interfaced with a 96-well autosampler for high throughput
operation [61, 90-96].
The use of metabolic stability screening to predict clearance is also of interest.
A drug with a high clearance has a high hepatic extraction ratio and so its
maximum oral bioavailability is low. Conversely, a drug with a low clearance
has a low hepatic extraction ratio and its maximum oral bioavailability is high
(assuming complete absorption). Since clearance provides an understanding of
the ability of the body to eliminate a drug, it is often used as a pharmacokinetic
screen in lead selection. The ability of hepatic microsomal stability assessments
to predict in vivo clearance in the rat was retrospectively evaluated for 1163
compounds from 48 research programs at a pharmaceutical research company
[97].](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-34-320.jpg)
![28
1.3.1.2.3 Metabolic Inhibition ofCytochrome P450
Chapter 1
The assessment of a candidate drug's potential to inhibit or induce cytochrome
P450 isoenzymes using in vitro microsomal incubations is one method for
predicting possible in vivo drug-drug interactions. Seven isoenzymes of
cytochrome P450 play dominant roles in drug metabolism: CYPIA2, 2A6,
3A4, 2C9, 2C19, 2D6 and 2E1. One of the strategies adopted to evaluate the
inhibition or induction of CYP450 by a drug is to monitor its effect on the
metabolism of selected compounds known as probe substrates whose specific
metabolic pathway is documented to occur via a single CYP450 enzyme
[98-100]. After incubation and quenching of the reaction, sample preparation
is required to remove proteins and matrix interferences before analysis. Two
reports from the same laboratory detail methods for high throughput CYP
inhibition screening using a cassette dosing strategy [101, 102]. Characteristics
and common properties of inhibitors, inducers and activators of CYP enzymes
are reviewed by Hollenberg [103].
The ADME profile of a drug product can be improved when the effect of CYP
P450 inhibitors and/or inducers is fully known. For example, terfenadine
(Seldane®) was removed from the market (see Section 1.1.5) due to potentially
life threatening drug interactions that could result in abnormalities in the
electrical impulse that stimulates the heart to contract and pump blood. An
active carboxy metabolite of terfenadine, named fexofenadine, was introduced
as Allegra®. Fexofenadine undergoes less metabolism by CYP3A4 isoenzymes
and therefore the effect ofinhibitors or inducers is greatly reduced on CYP3A4
when compared with the effect with terfenadine [104]. Another example of an
improved ADME profile is esomeprazole (Nexium®). Esomeprazole is the (S)-
isomer of omeprazole (Prilosec®) and undergoes less metabolism by
CYP2C19 isoenzymes. A beneficial result is that CYP2C19 polymorphism has
less ofan effect on its pharmacokinetics [104].
1.3.1.3 Toxicity Testing
Toxicity testing is an important component of screening potential lead
compounds [105], as compounds often fail in the development process because
of the harmful effect they may have on cells, organs or organ systems.
Commonly, animals will be administered escalating drug doses and the time
course profile of the drug will be determined using bioanalysis; this technique
is referred to as toxicokinetics. Genotoxicity analysis is another important
toxicity test that is usually performed just prior to phase I clinical trials because](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-35-320.jpg)
![Role ofBioanalysis 29
of the high cost involved; performing this test on all lead compounds would be
prohibitively expensive. An effort has recently begun to move toxicity testing
to an earlier stage in drug development to confirm viable leads more quickly.
Toxicogenomics, the examination of changes in gene expression following
exposure to a toxicant, offers this potential for early detection; it may also
detect human specific toxicants that cause no adverse reaction in rats [106].
Cytotoxicity tests using bioluminescence are proceeding along the lines of high
throughput screening by being miniaturized to 384-well formats [107].
1.3.1.4 Further Reading
A wealth of information is available in published literature concerning the role
of bioanalysis in the support of drug discovery and the lead optimization
process, including descriptions of the various in vivo and in vitro screening
tests. A schematic diagram of this iterative course of action is shown in Figure
1.6. Both general reviews and detailed reports are recommended.
in vivo in vitro
Single Dose
Pharmacokinetics
and
Metabolite
Screening
Metabolic Stability
Clearance
and
Metabolite
Screening
t
I
I
I
_ ...J
L _
continued development
Figure 1.6. Schematic diagram of the lead optimization process and the role of
metabolite screening. Reprinted with permission from [108]. Copyright 2002 John
Wiley & Sons, Ltd.](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-36-320.jpg)
![30 Chapter 1
Venkatesh and Lipper review the role of the development scientist in the
selection and optimization of lead compounds [109]. Kennedy describes the
compound selection and decision making skills necessary to manage the
interface between drug discovery and drug development [110]. Perspectives on
twelve months of lead optimization were provided from a chemistry team
which identified the need for a balance between achieving the needed
throughput and allowing time for appropriate decision making and reflection
[Ill]. The prediction and use of pharmacokinetic properties in early drug
discovery is discussed in several papers [81, 82,112-115] (the report by
Roberts [112] is highly recommended reading). The concept of a bioanalytical
toolbox for performing fast turnaround in drug discovery support is a practical
proposal to describe the choices available to the bioanalyst [116]. The
importance of stereospecific bioanalytical monitoring in drug development is
reviewed by Caldwell [117], with particular emphasis on stereospecific assays
for the individual optical isomers of drugs. Some examples of enantiomeric
separation and quantification in bioanalysis include the analysis of ketoprofen
[118] and fluoxetine [119] in human plasma.
1.3.2 Prec/inical-ADME and Metabolite Identification
1.3.2.1 ADME Studies
Once a drug candidate has received IND approval, the preclinical development
phase formally begins. With a focus on accelerated timelines, the distinction
between drug discovery and preclinical development is less defined than in past
years. Some activities that were traditionally performed only in the preclinical
phase are now begun in drug discovery with the goal of identifying optimal
lead compounds as early in development as possible.
Once a drug compound reaches the preclinical development phase, its ADME
characteristics are determined in many species of animals as well as in man. At
this point, a defined and validated assay is used over and over for determining
concentrations of drugs from in vitro and in vivo samples. It would be ideal if
one analytical method could be used for every animal species, but in practice a
method needs to be modified slightly and validated for each species. One
method for many species of animals is described in a case study of two
antibiotics [120].
While multiple compounds are encountered in drug discovery, in preclinical
development fewer compounds are in use. Another differentiation between the
two phases of development is that now in the preclinical stage validated](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-37-320.jpg)
![Role ofBioanalysis 31
methods are needed and adherence to Good Laboratory Practices (GLP) is
required.
Preclinical development projects in bioanalysis commonly involve the
determination of drug concentrations in biological fluids after dosing by
multiple routes of administration and in multiple species of animals.
Toxicokinetic studies also continue to be performed which examine the
relationships among dose, pharmacokinetics and toxicity. Tissue distribution
studies are also conducted, frequently with radiolabeled drug. Some common
approaches to assessing tissue distribution are to excise tissues and analyze
extracts by LC-MS/MS, following sample preparation; oxidize excised tissues
and determine the amount of radioactivity contained in each; and perform
autoradiography. This latter technique utilizes thin.whole body slices and the
amount of radioactivity in each organ slice is determined.
1.3.2.2 Metabolite Identification and Characterization
Another important aspect of drug development research is the determination of
the metabolic fate of lead compounds in several animal species. Many new
chemical entities are terminated late in the development stage due to problems
with drug metabolism. In an effort to prevent these late stage failures, it is
useful to know the metabolic fate of a promising lead compound as early as
possible. Typically, metabolites of lead compounds are identified in the
preclinical development phase, although it is becoming more common to
identify major metabolites in the discovery phase. Then, should toxic
metabolites be identified (e.g., some acyl glucuronides [121]), structural
analogs of early drug lead candidates may be designed to block portions of the
molecules that are particularly susceptible to metabolism.
Mass spectrometry coupled with liquid chromatography is an effective
analytical method for metabolite profiling. The integration of data collected
from the ion trap, triple quadrupole and quadrupole/time-of-flight instruments
allows a comprehensive evaluation of biotransformation products. This
approach is routinely used to evaluate metabolites generated from in vitro and
in vivo systems. Once metabolites have been identified and characterized from
definitive ADME studies, they are monitored in subsequent pharmacokinetic
studies; it is common in bioanalysis to monitor concentrations of both parent
drug and one or more metabolites. These parent-metabolite combinations
present a useful assessment of metabolism that can be important to establish
dosing regimens and assess toxicity concerns. Also note that a metabolite can
present an ideal backup candidate to a drug lead, e.g., desloratidine (Clarinex®)](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-38-320.jpg)
![32 Chapter 1
is a metabolite of loratidine (Claritin®) that was developed and FDA approved
for marketing several years after the introduction ofClaritin.
Many LC-MS applications are found in the literature for metabolite
characterization from in vitro [122-124] and in vivo [34, 108] experiments. An
overview of the choices and decisions involved in metabolite identification and
how they can be merged into a systematic approach is described by Clarke
et al. [125]. The mass spectrometric identification of metabolites is reviewed
by Oxford and Monte [126] and strategies for metabolite isolation and
identification are reviewed by Wiltshire [127]. An automated high throughput
approach to metabolite identification is of great practical utility. Lopez et al.
[128] and Kim et al. (129] have demonstrated an automated metabolite
identification approach using an ion trap mass spectrometer. Automated
fraction collection of metabolites for subsequent ion trap MS, MS/MS or NMR
analyses has been described by Dear et al. [130].
1.3.3 Clinical-Pharmacokinetics
A major effort in the clinical phase ofdrug development is the determination of
drug and metabolite concentrations in biological fluids after drug
administration to humans. The pharmacokinetic data obtained are used to
support drug development in assessing the therapeutic index, drug-drug
interactions, design of dosage regimes, etc. Since drugs today are more potent
than in years past and are dosed at lower levels, very sensitive assays are
required to detect the low circulating levels of drugs in biological fluids (often
to pg/mL sensitivity limits). Again, LC-MS/MS is the analysis and detection
technique of choice due to its high sensitivity, selectivity and speed. GLP
assays are also maintained during the clinical phase ofdrug development.
The greatest numbers of samples to be analyzed are found in clinical drug
development, where thousands of samples are often obtained from one clinical
study (conducted at multiple sites). Rapid sample turnaround is sought for'one
or more reasons: important metabolic information from patient samples can be
obtained; the next dose can be determined; or the results used to plan the next
clinical study. In addition to sensitivity, selectivity and ruggedness are also
important to the analytical method. Examples of the increased requirement for
sensitivity as a compound moved from preclinical into the clinical phase of
drug development have been detailed by Dear et al. [131] and Laugher et al.
[132]. In the report by Dear et al., preclinical studies required a lower limit of
quantitation (LLOQ) ofonly 1 ng/mL in rat and dog plasma but the LLOQ
needed for human clinical studies was 50 pg/mL.](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-39-320.jpg)
![Role ofBioanalysis
1.3.4 Therapeutic Drug Monitoring
33
The objective of therapeutic drug monitoring (TDM) is as a guide to provide
optimal drug therapy by maintaining plasma concentrations of a drug within a
desired range. Some examples of drugs for which monitoring is very important
are: digoxin, phenytoin, aminoglycoside antibiotics, theophylline, cyclosporine,
HIV protease inhibitors, and some cardiac agents, antiepileptic drugs and
antidepressant drugs. Should drug concentrations in the sampled biological
fluid exceed the desired range, toxicity may result and/or the desired
therapeutic effect may no longer be achieved. The knowledge of plasma drug
concentrations may explain why a patient does not respond to drug therapy or
why the drug causes an adverse effect. Also, for HIV infections, low drug
concentrations may in some cases lead to drug resistance and treatment failure.
Most drugs can be safely taken without monitoring drug concentrations since
therapeutic endpoints can effectively be evaluated by other means. For
example, blood pressure determinations indicate how well the ~-blocker
atenolol may be working for a patient, and coagulation times accurately
measure the effect of Coumadin®, an anticoagulant drug.
Drugs that are chosen for TDM have the following characteristics in common:
• The range of therapeutic and safe plasma concentrations is narrow
(i.e., a low therapeutic index)
• Toxicity or lack ofeffectiveness of the drug puts the patient at risk
• Patient compliance needs to be monitored
The therapeutic index is defined as the ratio between the maximum and
minimum plasma concentrations of the drug's therapeutic range. A low
therapeutic index (less than 2) means that the dose that commonly yields a sub
therapeutic response is close to the dose that produces some toxicity. Most
drugs have a therapeutic index greater than 2 [133]. Note that the relationship
between drug concentration at the site of action and the pharmacological
response observed in the patient should be considered. TDM using plasma
concentration data can be justified when a correlation has been established.
Assay procedures for a drug in biological fluids are usually performed by a
local or regional clinical laboratory or a national medical laboratory. Standard
methods that are used for detection and quantitation of drugs are immunoassay
techniques and liquid chromatography employing sensitive detection
(fluorescence or mass spectrometry). Once drug concentrations and responses](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-40-320.jpg)
![34 Chapter 1
have been adjusted to achieve the desired therapeutic result, a regular clinical
monitoring program is begun. An informative discussion of the analytical goals
in therapeutic drug monitoring is provided by Bowers [134].
Additional reports provide a further perspective on therapeutic drug
monitoring. The roles for chromatographic analysis in TDM have been
described by Wong [135], Shihabi and McCormick [136] and Binder [137].
Some examples of TDM are reported for flecainide [138], tacrolimus [139],
immunosuppressants [140, 141], an antineoplastic agent (etoposide) [142],
gabapentin [143], oxcarbazepine [144] and mexiletine [145]. TDM in special
populations of patients is discussed by Walson [146]. A case for performing
prospective concentration to clinical response investigations during the early
stages of drug development, rather than the traditional retrospective review, is
proposed by Shaw, Kaplan and Brayman [147].
Acknowledgments
The author is appreciative to Danlin Wu and Mike Lee for their critical review
of the manuscript, helpful discussions and contributions to this chapter. The
line art illustrations were kindly provided by Woody Dells.
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[I] G.R. Nakayama, Curr Opin. Drug Discovery Dev. I (1998) 85-91.
[2] G. Ziokamik, Anal. Chern. (1999) 322A-328A.
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[4] Federal Register USA (1988) October 21.
[5] Federal Register USA (1987) May 22.
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J.T. Bigger, K.R.R. Krishnan, L.T. van Zyl, J.R. Swenson, M.S. Finkel,
C. Landau, P.A. Shapiro, c.J. Pepine, J. Mardekian and W.M. Harrison,
J. Amer. Med. Assn. 288 (2002) 701-709.
[8] D.T. Rossi and MJ. Lovdahl, In: D.T. Rossi and M.W. Sinz, Eds., Mass
Spectrometry in Drug Discovery, Marcel Dekker, New York (2002)
215-244.
[9] R.A. Fecik, K.E. Frank, E.J. Gentry, S.R. Menon, L.A. Mitscher and
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![Role ofBioanalysis 35
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![42 Chapter 2
detection by mass spectrometry (MS) is the preferred analytical technique for
drug analysis. The use of tandem mass spectrometry (MS/MS) that identifies a
specific parent-daughter ion pair allows unequivocal identification of a drug
substance from biological samples. LC-MS/MS is widely used because it offers
unmatched speed, sensitivity and specificity with good precision and wide
dynamic range.
Note that although mass spectrometry allows sensitive and specific detection of
analytes of interest, it benefits tremendously from a well chosen column and
mobile phase to provide adequate chromatographic separation [1]. The
importance of chromatography cannot be overlooked; the column separates the
analytes from the much higher concentrations of endogenous materials present
in samples that can potentially mask an analyte or introduce ion suppression.
Some essential chromatography issues for fast bioanalysis are discussed by
O'Connor [2].
Another very important component in the overall analysis is the choice of
sample preparation technique which influences the cleanliness of the sample
introduced into the chromatographic system. Sample preparation is necessary
because most analytical instruments cannot accept the matrix directly. Three
major goals for sample preparation are to:
1. Remove unwanted matrix components that can cause interferences
upon analysis, improving method specificity
2. Concentrate an analyte to improve its limit ofdetection
3. Exchange the analyte from an environment of aqueous solvent into a
high percentage organic solvent suitable for injection into the
chromatographic system
Some additional goals for the sample preparation step may include removal of
material that could block the tubing of the chromatographic system,
solubilization of analytes to enable injection under the initial chromatographic
conditions and dilution to reduce solvent strength or avoid solvent
incompatibility [3].
The term sample preparation typically encompasses a wide variety of processes
which include aspirating and dispensing liquids, release of drugs from the
sample matrix via hydrolysis or sonication, dilution, filtration, evaporation,
homogenization, mixing and sample delivery. Here, the term will primarily be
used regarding the removal of endogenous compounds from the sample matrix.](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-49-320.jpg)
![Sample Preparation Strategies
Table 2.1
Objectives for bioanalytical sample preparation
43
1. Removal ofunwanted matrix components (primarily protein) that would interfere
with analyte determination
2. Concentration of analyte to meet the detection limits ofthe analytical instrument
3. Exchange of the solvent or solution in which the analyte resides so that it is
compatible with mobile phase for injection into a chromatographic system
4. Removal of selected analyte components if the resolving power of the
chromatographic column is insufficient to separate all the components completely
5. Removal of material that could block the chromatographic tubing or foul the
interface to the detector
6. Dilution to reduce solvent strength or avoid solvent incompatibility
7. Solubilization ofcompounds to enable injection under the initial chromatographic
conditions
8. Stabilization ofanalyte to avoid hydrolytic or enzymatic degradation
Reprinted with permission from [4]. Copyright 1989 Elsevier Science.
The different types of sample matrices encountered in bioanalysis may include
the following: plasma, serum, bile, urine, tissue homogenates, perfusates,
buffer, saliva, seminal fluid, dialysate solution, Caco-2 buffer and hepatocyte
or microsomal incubation solution. Table 2.1 lists the many overall objectives
of sample preparation for drug bioanalysis. Three major objectives from this
list will now be described in more detail: removal of matrix components,
concentration ofanalyte and solvent exchange.
1.1.1 Removal ofMatrix Components
Biological samples cannot usually be injected directly into an analytical system
such as LC-MS/MS because of the multitude of substances present in the
sample matrix that can potentially interfere with the analysis, the
chromatographic column and/or the detector. These materials include proteins,
salts, endogenous macromolecules, small molecules and metabolic byproducts.
If these materials were to be injected, the consequences may include the
following: a rapid deterioration in the separation performance of the
chromatographic column; clogged frits or lines resulting in an increased system
backpressure; impaired selectivity of the sorbent in the column due to
irreversible adsorption of proteins; and detector fouling that may reduce system
performance and require maintenance for cleaning the source. Injection of
matrix substances can also cover up and hide the drug or analyte being
analyzed, making quantitation difficult (and adversely affecting the data).
These materials may also coelute with the analyte of interest, falsely elevating
the data. All samples can benefit from a pretreatment step before analysis in](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-50-320.jpg)
![Sample Preparation Strategies 45
many regards, such as simplicity, time requirements (in terms of speed and
hands-on analyst time), ease of automation, extraction chemistry expertise,
concentration factor and selectivity of the final extract. The particular method
chosen depends on the requirements of the assay as well as the time involved to
run the method. The investment in method development time is also a
consideration. Fortunately, the bioanalytical chemist can choose from a range
of sample preparation methodologies, as listed in Table 2.2.
Examples of two contrasts for sample preparation requirements are drug
discovery and clinical development laboratories. In drug discovery, criteria of
rapid sample turnaround, little time available for method development and
higher limits of quantitation are acceptable. These decisive factors suggest
protein precipitation as a preferred approach. However, in clinical analysis
where drugs are potent and are dosed at low levels, the important criteria of
ultra sensitivity, great selectivity and a rugged method point toward solid-phase
extraction as the technique ofchoice.
Some useful perspectives and reviews of sample preparation approaches for
high throughput LC-MS and LC-MS/MS analyses are described in the
literature [2, 5-8] and in a book chapter by Rossi [9]. Other helpful but more
general discussions of different sample preparation methodologies are found in
various reviews [4, 10-14] and ina book chapter by Kataoka and Lord [3]. A
review by Peng and Chiou [15] is recommended for the reader who would like
to learn more about pharmacokinetics and the overall requirements for
bioanalysis, including sample preparation and analytical method validation.
Table 2.2
Typical choices of sample preparation techniques useful in bioanalysis
• Dilution followed by injection
• Protein precipitation
• Filtration
• Protein removal by equilibrium dialysis or ultrafiltration
• Liquid-liquid extraction
• Solid-supported liquid-liquid extraction
• Solid-phase extraction (off-line)
• Solid-phase extraction (on-line)
• Turbulent flow chromatography
• Restricted access media
• Monolithic columns
• Immunoaffinity extraction
• Combinations ofthe above](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-52-320.jpg)
![46
2.2.2 Dilution Followed by Injection
Chapter 2
Sample dilution is used to reduce the concentration of salts and endogenous
materials in a sample matrix and is commonly applied to urine. Drug
concentrations in urine are usually fairly high and allow this dilution without an
adverse effect on sensitivity; protein concentrations in urine are negligible
under normal physiological conditions. An example of a urine dilution
procedure is reported for indinavir in which 1 mL urine was diluted with
650 j.1L acetonitrile (so that the resulting concentration of organic in the sample
was equal to or less than that of the mobile phase). An aliquot of 6 j.1L was
injected into an LC-MS/MS system [16J. Although coeluting endogenous
species in urine were not seen in the selected ion monitoring mode, their
presence did suppress or enhance the ionization of analytes, leading to
increased variation in MS/MS responses.
Dilution is not typically used for plasma due to the high amounts of protein
present and the greater effect that dilution has on sensitivity. However, dilution
is very attractive for the minimal effort and time involved. One report did
discuss a dilution approach for plasma in a high throughput procedure [17]. In
this report, dog plasma samples were centrifuged, pipetted into wells of a
microplate and then placed on an automated pipettor. A volume of 15 j.1L
plasma was diluted with 485 j.1L of a solution of water/methanol/formic acid
(70:30:0.1, v/v/v) containing internal standard. The samples were sealed, mixed
and 5 j.1L were injected into an LC-MS/MS system. The dilution resulted in a
slightly viscous solution with no observed precipitation. The limit of
quantitation for the dilution assay (2 ng/mL) was 400 times higher than that of
a more selective procedure that also concentrates the analyte (liquid-liquid
extraction; 5 pg/mL LOQ). However, the advantage of the first technique was
that the throughput was 50 times greater. In this case, throughput was a more
important consideration than analyte sensitivity.
2.2.3 Protein Precipitation
Protein precipitation is often used as the initial sample preparation scheme in
the analysis of a new drug substance since it does not require any method
development. A volume of sample matrix (I part) is diluted with a volume of
organic solvent or other precipitating agent (3-4 parts), followed by vortex
mixing and then centrifugation or filtration to isolate or remove the precipitated
protein mass. The supernatant or filtrate is then analyzed directly. Protein
precipitation dilutes the sample. When a concentration step is required, the](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-53-320.jpg)
![Sample Preparation Strategies 47
supernatant can be isolated, evaporated to dryness and then reconstituted before
analysis.
This procedure is popular because it is simple, universal, inexpensive and can
be automated in microplates. However, matrix components are not efficiently
removed and will be contained in the isolated supernatant or filtrate. In MS/MS
detection systems, matrix contaminants have been shown to reduce the
efficiency of the ionization process [1, 16, 18-26]. The observation seen is a
loss in response and this phenomenon is referred to as ionization suppression.
This effect can lead to decreased reproducibility and accuracy for an assay and
failure to reach the desired limit of quantitation.
Protein precipitation techniques can be performed in high throughput systems
using the collection plate format and several reports of these applications are
available [27-31]. Procedures for performing high throughput protein
precipitation are presented in Chapter 6 along with strategies for method
development. The automation of protein precipitation techniques is presented
in Chapter 7.
2.2.4 Filtration
Filtration is important for the removal of material or debris from a sample
matrix so that the chromatographic tubing and column do not become
physically blocked. For example, a precipitated protein mass can be filtered
from solution and the filtrate analyzed directly [28, 32-36]. Filtration is often
used to clarify raw sample matrix before another sample preparation technique
(e.g., turbulent flow chromatography, discussed in Section 2.2.10) as well as
for the filtration ofdebris from the final eluate before injection. Filtration in the
microplate format allows high throughput sample preparation and this approach
is discussed in more detail in Chapter 6, Section 6.4.
2.2.5 Protein Removal
Equilibrium dialysis, ultrafiltration and other membrane based sample
preparation methods are useful for protein removal in bioanalysis [13, 14,
37-40]. Equilibrium dialysis is a classical method to physically separate small
molecular weight analytes from larger molecular weight constituents (e.g.,
protein) in a biological sample matrix. This process occurs by diffusion through
the pores of a selective, semipermeable membrane and is concentration driven.
Ultrafiltration separates proteins according to molecular weight and size using
centrifugal force, and is thus based on a pressure differential rather than on a](https://image.slidesharecdn.com/564042-250523094949-10e6d0f3/85/High-Throughput-Bioanalytical-Sample-Preparation-Methods-And-Automation-Strategies-1st-Edition-David-A-Wells-Eds-54-320.jpg)
High Throughput Bioanalytical Sample Preparation Methods And Automation Strategies 1st Edition David A Wells Eds
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- 6. Preface It has been exciting to be involved with the development and implementation of high throughput techniques for sample preparation used for drug analysis in the pharmaceutical industry. My work as an independent consultant and educator in this field has allowed me to work with scientists worldwide having a broad range of expertise. It became evident during my assignments that a single source of information that reviewed the utilization of high throughput sample preparation techniques was not available. This book was written to fulfill this need for my students and colleagues. The text begins with an introductory overview of the role of bioanalysis in pharmaceutical drug development, focused on the particular activities that are performed within each stage of the research process. A fundamental understanding of the strategies for sample preparation is reinforced next, along with essential concepts in extraction chemistry. In order to gain a mastery of knowledge about the available tools needed to perform high throughput sample preparation techniques, several chapters introduce and discuss microplates, accessory products and automation devices. Particular strategies for efficient use of automation within a bioanalytical laboratory are also presented. The subject material then reviews four common sample preparation techniques: protein precipitation, liquid-liquid extraction, solid-phase extraction and various on-line sample preparation approaches. Each technique is discussed with reference to its fundamental principles and strategies for method development and automation. The book concludes with information on recent advances in sample preparation. The important objectives that can be accomplished when the strategies presented in this book are followed include: (a) Improved efficiency in moving discovery compounds to preclinical status with robust analytical methods (b) Return on investment in automation for sample preparation (c) Improved knowledge and expertise of staff It is my sincere desire that the reader finds this book a valuable resource for information on high throughput sample preparation methods for bioanalysis and recommends it to staff and colleagues. v
- 7. vi Preface The need for this book was mentioned previously but this project would never have been completed without inspiration and the support of many individuals. Early in my professional career I was inspired by two outstanding professors who instilled within me the exhilaration of investigative science and the important role of teaching students and nurturing their professional growth. I am indebted to Dr. George A. Digenis at the University of Kentucky, College of Pharmacy, and Dr. Robert E. Lamb at Ohio Northern University, Department of Chemistry for their inspiration and encouragement. As I became an educator in my own career, my inspiration continued from the students with whom I worked and who learned high throughput sample preparation techniques from me. The progress of several of them, in particular, into recognized experts has been rewarding. I have also learned from, and been motivated by, established scientists and colleagues who worked with me to develop leading edge products for solid-phase extraction and implement high throughput drug sample preparation techniques in their laboratories. The production of this book was greatly assisted by the efforts of two individuals in particular. Teresa Wells efficiently managed my large database of 1,800 literature references in sample preparation collected over the years so that I could quickly retrieve published information for use within each chapter. She also tolerated with great patience the long hours and months that were spent assembling the considerable amount of information that went into this book ("Is it done yet?"). I was astonished that an associate, Patt Threinen, volunteered to perform the arduous task of editing this manuscript by carefully reading each chapter. lowe her profound thanks for her understanding, corrections and comments of the many writes and rewrites passed back and forth. Her extensive experience in chemistry, biology, chromatography and sales added an invaluable perspective. This text is more readable because of her indefatigable attention to detail. My colleagues also assisted in this effort by reviewing selected chapters pertinent to their expertise; their discussions and suggestions shaped the subject material into a useful and comprehensive information resource. They are each mentioned within the chapter to which they contributed. In particular, I am grateful for the collaboration with Jing-Tao Wu who coauthored the on-line sample preparation techniques chapter. The staff at Elsevier Science was also very supportive in the production of this book. David A. Wells
- 8. Chapter 1 Role of Bioanalysis in Pharmaceutical Drug Development Abstract Bioanalysis is the quantitative determination of drugs and their metabolites in biological fluids. This technique is used very early in the drug development process to provide support to drug discovery programs on the metabolic fate and pharmacokinetics of chemicals in living cells and in animals. Its use continues throughout the preclinical and clinical drug development phases, into post-marketing support and may sometimes extend into clinical therapeutic drug monitoring. The role of bioanalysis in pharmaceutical drug development is discussed, with focus on the particular activities that are performed within each stage of the development process and on the variety of sample preparation matrices encountered. Recent developments and industry trends for rapid sample throughput and data generation are introduced, together with examples of how these high throughput needs are being met in bioanalysis. 1.1 Overview of the Drug Development Process 1.1.1 Introduction The discovery and development of safe and effective new medicines is a long and complex process. Pharmaceutical companies typically invest 9-15 years of research and hundreds of millions of dollars into this effort; a low rate of success has historically been achieved. The drug development process itself requires the interaction and cooperation of scientists and medical professionals from many diverse disciplines. Some of these disciplines include medicinal chemistry, pharmacology, drug metabolism and pharmacokinetics, toxicology, analytical chemistry, pharmaceutics, statistics, laboratory automation, information technology, and medical and regulatory affairs. A progression of research activities and regulatory filings must operate in parallel, often under severe time constraints. The success of a drug development program depends upon a number of favorable selections, such as targeting a therapeutic area in which an identified drug compound offers outstanding efficacy, identifying the 1
- 9. 2 Drug Discovery L....--> Preclinical Research File IND .... Clinical Development ~> Chapter 1 File NDA ....Regulatory Approval V Combinatorial Chemistry High Throughput Screening Genomics ADME Support in vitro and in vivo Assessments Safety Analysis Metabolism and Pharmacokinetics Pharmacology Phase I Phase II Phase III Manufacturing Sales and Marketing Phase IV Studies Figure 1.1. Schematic diagram of the overall drug development process and the major activities performed within each ofthe four major divisions. optimal chemical structure of the drug molecule that yields the most favorable absorption, distribution, metabolism and elimination profiles, demonstrating safety, satisfying regulatory needs, as well as cost effective manufacturing and extensive sales support in the marketplace.'A schematic diagram of the overall drug development process is outlined in Figure 1.1. Appropriate times for the regulatory filings are indicated. 1.1.2 Drug Discovery Traditionally, drugs have been identified using one ofthree major strategies: 1. A focused and systematic approach is made to synthesize compounds that interact optimally with a target receptor or pharmacological model whose 3-dimensional structure has been elucidated, i.e., rational drug synthesis. An example is the HIV-1 protease enzyme (Human Immunodeficiency Virus) whose function is important for maturation and assembly of infectious viral particles for the disease AIDS (Acquired Immune Deficiency Syndrome). The 3-dimensional structure of the HIV-I protease enzyme is known and antiviral agents have been designed that specifically bind to this key protein and inhibit its function.
- 10. Role ofBioanalysis 3 2. The active ingredients from natural plants, soil extracts and microorganisms (fungi, viruses and molds) are isolated, purified and screened for activity using various pharmacological models. This approach led to identification of paclitaxel (Taxol®), a drug used to treat various forms ofcancer. 3. Accidental discovery or serendipity occurs when a drug molecule is found to work for a different target than the one for which it was originally synthesized. For example, in the search for novel drugs to treat cardiac arrhythmias, researchers discovered that imiquimod (Aldara®) was a novel immunomodulator that boosted the body's immune system; a new class ofantiviral agents was discovered. Today, however, the pharmaceutical research process is looking at new and improved ways to develop drugs, in response to several important scientific advances that have recently occurred. These advances include the identification of new and more specific drug targets (as a result of maturation in genomics and proteomics); successes with tissue growth outside of the living organism; development of faster, more sensitive and more selective analytical systems (mass spectrometry); higher throughput (as a result of robotics and laboratory automation); proliferation in synthesis techniques (combinatorial chemistry); and advances in computing and information systems (bioinformatics). In parallel with these scientific advances, business factors have changed with the consolidation ofdrug companies and the intense pressure to get drugs to market faster than ever before. The current focus ofdrug discovery research is on rapid data generation and analysis to identify promising candidates very early in the development cycle. An optimal lead candidate is selected for further evaluation. Combinatorial chemistry techniques allow the synthesis of compounds faster than ever before, and these greater numbers of compounds are quickly evaluated for potency and pharmacological activity using high throughput screening (HTS) techniques. HTS involves performing various microplate based immunoassays with synthesized compounds or compounds from natural product isolation. Examples of assay types used in HTS are scintillation proximity assay (SPA), enzyme linked immunosorbent assay (ELISA), fluorescent intensity, chemiluminescence, absorbance/colorimetry and bio- luminescence assays [I]. These HTS tests simulate a specific biological receptor or target function and a qualitative decision ("hit" or "miss") is generated [2].
- 11. 4 Chapter 1 Advances in genomics have increased the understanding of certain diseases at the molecular level, i.e., the effect of a gene sequence on a particular illness. The role of the protein encoded by the gene is also studied. Proteins have generally been shown to be good drug targets. The effect ofa drug on a protein, and thus on a specific biochemical pathway, forms the basis for a high throughput screening test. These HTS tests are usually conducted by scientists in pharmacology research groups. Once hits are identified, chemists perform an iterative process to synthesize and screen smaller, more focused libraries for lead optimization in an effort to improve compound activity toward a specific target. Using automated techniques, ultra high throughput can be obtained by the most advanced laboratories and tens of thousands ofcompounds can be screened in one day. In parallel studies, information is learned on a drug molecule's absorption, distribution (including an estimate of protein binding), metabolism and elimination by sampling from dosed laboratory animals (called in vivo testing) and from working cells and/or tissues removed from a living organism (called in vitro testing since the cells are outside a living animal). These important tests are collectively referred to as ADME characteristics (Absorption, Distribution, Metabolism and Elimination). A candidate compound that will potentially meet an important medical need receives an exhaustive review addressing all the key issues concerning its further development. Evaluation of the available data, competitive therapies, expected therapeutic benefit, market opportunities and financial considerations all contribute to the final decision to grant development status to a particular compound. A multifunctional project team is assembled to guide the development efforts into the next phase-preclinical development. By this period in the process, a patent application has been filed to prevent other companies from marketing the same compound and protect the company's investment in the research and development costs. 1.1.3 Preclinical Development The preclinical development process largely consists of a safety analysis (toxicity testing) and continued study into a drug candidate's metabolism and pharmacology. Both in vitro and in vivo tests are conducted; many species of animals will be used because a drug may behave differently in one species than in another. An early assessment of dosing schedules in animal species can be determined, although human dosage regimens are not determined until the subsequent clinical trials in the next development phase (see Section 1.1.4).
- 12. Role ofBioanalysis 5 Toxicology tests in preclinical development examine acute toxicity at escalating doses and short term toxicity (defined as 2 weeks to 3 months), as well as the potential of the drug candidate to cause genetic toxicity. Today's research efforts attempt to utilize as few animals as possible and so more in vitro tests are conducted. The use of metabonomics for toxicity testing is making an impact on both drug discovery (to select a lead compound) and preclinical development (to examine safety biomarkers and mechanisms). Metabonomics is a technology that explores the potential of combining state of the art high resolution NMR (Nuclear Magnetic Resonance) spectroscopy with multivariate statistical techniques. Specifically, this technique involves the elucidation ofchanges in metabolic patterns associated with drug toxicity based on the measurement of component profiles in biofluids (i.e., urine). NMR pattern recognition technology associates target organ toxicity with specific NMR spectral patterns and identifies novel surrogate markers of toxicity [3]. Also in preclinical development, the pharmacokinetic profile of a drug candidate is learned. Pharmacokinetics is a specific, detailed analysis which refers to the kinetics (i.e., time course profile) of drug absorption, distribution and elimination. The metabolites from the drug are identified in this stage. Definitive metabolism studies of drug absorption, tissue distribution, metabolism and elimination are based on the administration of radiolabeled drug to animals. It is important that the radionuclide is introduced at a position in the chemical structure that is stable to points of metabolism and conditions ofacid and base hydrolysis. Pharmacology testing contains two major aspects-in vivo (animal models) and in vitro (receptor binding) explorations. Comparisons are made among other drugs in the particular collection under evaluation, as well as among established drugs and/or competitive drugs already on the market. More informative and/or predictive biomarkers are also identified and monitored from these studies. Detailed information about the drug candidate is developed at the proper time in preclinical development, such as the intended route of administration and the proposed method of manufacturing. In order to supply enough of the drug to meet the demands of toxicology, metabolism and pharmacology, the medicinal chemistry and analytical groups work together to determine the source of raw materials, develop the necessary manufacturing process and establish the purity of the drug product. The exact synthesis scheme and methodology needed to produce the drug are recorded in detailed reports. The pharmaceutics research group develops and evaluates formulations for the drug candidate. These
- 13. 6 Chapter 1 formulations are assessed in vivo by the drug metabolism group. Quality and stability are the goals for this dosage form development effort. In the United States, after the active and inactive ingredients of a formulation containing the candidate compound have been identified and developed, a detailed summary called an Investigational New Drug Application (IND) is prepared. This document contains reports of all the data known to date on a drug candidate's toxicology, metabolism, pharmacology, synthesis, manufacturing and formulation. It also contains the proposed clinical protocol for the first safety study in man. All of the information contained in an IND application is submitted to the United States Food and Drug Administration (FDA). Typically, thousands of pages of documents comprise this IND. The FDA reviews the information submitted and makes a decision whether or not the drug has efficacy and appears safe for study in human volunteers. The IND becomes effective if the FDA does not disapprove the application within 30 days. The drug sponsor is then approved to begin clinical studies in humans. When questions arise, the FDA responds to the IND application with a series of inquiries to be answered and a dialogue begins between the drug sponsor and the FDA. 1.1.4 Clinical Development 1.1.4.1 Introduction Clinical trials are used to judge the safety and efficacy of new drug therapies in humans. Drug development is comprised of four clinical phases: Phase I, II, III and IV (Table 1.1). Each phase constitutes an important juncture, or decision point, in the drug's development cycle. A drug can be terminated at any phase for any valid reason. Should the drug continue its development, the return on investment is expected to be high so that the company developing the drug can realize a substantial and often sustained profit for a period of time while the drug is still covered under patent. 1.1.4.2 Phase I Phase I safety studies constitute the "first time in man." The objective is to establish a safe dosage range that is tolerated by the human body. These studies involve a small number of healthy male volunteers (usually 20-80) and may last a few months; females are not used at this stage because of the unknown effects of any new drug on a developing fetus. Biological samples are taken
- 14. Role ofBioanalysis 7 Table 1.1 Objectives of the four phases in clinical drug development and typical numbers of volunteers or patientsinvolved PhaseI PhaseII Establishsafe Demonstrate dosingrangeand efficacy, identify assesspharmaco- sideeffectsand kinetics; also called assesspharmaco- Firsttime in man kinetics (FTIM) PhaseIII Gaindata on safety and effectiveness in a largerpopulation of patients; assess pharmacokinetics PhaseIV Expandon approvedclaimsor demonstrate new claims;examine specialdrug-drug interactions; assess pharmacokinetics 20-80 male volunteers 200-800 patients 1,000-5,000 patients A fewthousand to severalthousand patients from these volunteers to assess the drug's pharmacokinetic characteristics. During a Phase I study, information about a drug's safety and pharmacokinetics is obtained so that well controlled studies in Phase II can be developed. Note that Institutional Review Boards (IRE) are in place at hospitals and research institutions across the country to make sure that the rights and welfare of people participating in clinical trials are maintained. IREs ensure that participants in clinical studies are fully informed and give their written permis- sion before the studies begin. IREs are monitored by the FDA. 1.1.4.3 Phase II Phase II studies are designed to demonstrate efficacy, i.e., evidence that the drug is effective in humans to treat the intended disease or condition. A Phase II controlled clinical study can take from several months to two years and uses from 200 to 800 volunteer patients. These studies are closely monitored for side effects as well as efficacy. Animal studies may continue in parallel to determine the drug's safety. A meeting is held between the drug sponsor and the FDA at the end of Phase II studies. Results to date are reviewed and discussion about the plan for Phase III studies is held. Additional data that may be needed to support the drug's development are outlined at this time and all information requirements are clarified. A month prior to this meeting, the drug sponsor submits the protocols for the Phase III studies to the FDA for its review. Additional information is
- 15. 8 Chapter 1 provided on data supporting the claim of the new drug, its proposed labeling, its chemistry and results from animal studies. Note that procedures exist that can expedite the development, evaluation and marketing of new drug therapies intended to treat patients with life threatening illnesses. Such procedures may be activated when no satisfactory alternative therapies exist. During Phase I or Phase II clinical studies, these procedures (also called "Subpart E" for Section 312 of the US Code of Federal Regulations) may be put into action [4]. The company developing the drug must then consider many factors before further development is undertaken, such as the cost of manufacturing the drug (which mayor may not involve new equipment purchases or changes in existing facilities), the estimated time and cost to gain final FDA approval, the competition the drug may face in the market, its sales potential and projected sales growth. The return on the company's investment is estimated. It has been observed in recent years for a major pharmaceutical company that if the return on investment on a single drug is not 100 million dollars (US) or more, the company may choose not to develop the drug further; instead, licensing the drug to a smaller company is one of several options. 1.1.4.4 Phase III After evidence establishing the effectiveness of the drug candidate has been obtained in Phase II clinical studies, and the "End of Phase II" meeting with the FDA has shown a favorable outcome, Phase III studies can begin. These studies are large scale controlled efficacy studies and the objective is to gain more data on the effectiveness and safety of the drug in a larger population of patients. A special population may be used, e.g., those having an additional disease or organic deficiency such as renal or liver failure. The drug is often compared with another drug used to treat the same condition. Drug interaction studies are conducted as well as bioavailability studies in the presence and absence of food. A Phase III study is a clinical trial in which the patients are assigned randomly to the experimental group or the control group. From 1,000 to 5,000 volunteer patients are typically used in a Phase III study; this aspect of drug development can last from 2 to 3 years. Data obtained are needed to develop the detailed physician labeling that will be provided with the new drug. These data also extrapolate the results to the general population and identify the side effect profile and the frequency of each side effect. Inparallel, various toxicology, carcinogenicity and metabolic studies are conducted in animals. The cumulative results from all of these studies are used to establish statements ofefficacy and safety of the new drug.
- 16. Role ofBioanalysis 9 As Phase III progresses, many commercial considerations are put into action. These matters include pricing, registration, large scale manufacturing and plans for market launch. The plan for marketing the drug is developed and additional clinical trials may be started to satisfy new labeling indications or to expand current indications that define exactly which conditions the drug is intended to treat. Note that once a drug is approved, physicians are able to prescribe its use to treat other conditions for which they feel the drug might have a beneficial effect; this use is known as "off label drug use." A Treatment IND is a special case in which the FDA may decide to make a promising new drug available to desperately ill patients as early as possible in the drug's development [5]. In order for a Treatment IND to be instituted, there must be significant evidence of drug efficacy, the drug must treat a serious or life threatening disease (where death may occur in months if no treatment is received), and/or there is no alternative treatment available for these intended patients. Treatment INDs, when they occur, are typically made available to patients during Phase III studies before marketing of the drug begins. Any patient who receives the drug under a Treatment IND cannot participate in the definitive Phase III studies. Another means by which promising and unique experimental agents can be made available to patients is called "Parallel Track." This policy was developed in response to the AIDS illness and allows patients with AIDS who cannot participate in controlled clinical trials to receive the promising investigational drug [6]. 1.1.4.5 New Drug Application (NDA) The New Drug Application (NDA) is the formal summary of the results of all animal and human studies, in conjunction with detailed plans for marketing and manufacturing the drug. Also, information is provided about the drug's chemistry, analysis, specifications and proposed labeling. The NDA is filed with the FDA by the drug sponsor who wishes to sell the new pharmaceutical entity in the United States. Before final approval may be granted, the FDA conducts a PreApproval Inspection (PAl) of the manufacturer's facilities because it is very important that methods used to manufacture the drug and maintain its quality are sufficient to preserve the drug's identity, strength and purity. This inspection evaluates the manufacturer's compliance with Good Manufacturing Practices (GMP), verifies the accuracy of information submitted in the NDA, and
- 17. 10 Chapter 1 evaluates manufacturing controls for the preapproval batches of drug formulation that were specified in the NDA. A collection of samples may be taken for analysis by other laboratories to confirm drug purity, strength, etc. Once the FDA receives the NDA, it undergoes a completeness review to ensure that sufficient information has been submitted to justify the filing. If deficiencies in the required information exist, then a "refuse to file" letter may be issued to the drug sponsor. This completeness review must be finished within 60 days of filing the NDA. When a drug application is considered complete by the FDA, there is no formal time requirement in which that NDA must be acted upon. The speed of review typically depends on how unique the drug is and on the workload of the agency at the time. Typically such a review can take 2-3 years, although "fast track" status for a novel drug can allow for a shorter time for complete review. After the NDA has been thoroughly evaluated, communication takes place with the drug sponsor about medical and scientific issues that may arise. The FDA will tell the applicant when more data is needed, when conclusions made in reports are not justified by the data, and when changes need to be made in the application. At the end of this review period, one of the following actions may occur: (1) The NDA may be "not approvable" and deficiencies in the application are clearly noted; (2) the NDA may be "approvable" after minor deficiencies are corrected, after labeling changes are made, and/or after studies are conducted that will investigate particular clinical issues; or (3) the NDA may be approved with no corrective action or delay necessary. When a director within the FDA having the sufficient authority signs an approved letter, the drug product can be legally marketed on that day in the United States. Typically, however, the precise approval date is not expected in advance and sufficient time is needed by the drug company to prepare manufacturing for the product launch. 1.1.5 Manufacturing andSale Plans for a drug's manufacturing are under way in parallel with efforts to complete studies needed for the NDA. Large quantities of product need to be synthesized, the formulation must be made consistently, and product packaging must be finalized. Also, quality control tests must be put into place to ensure reliable and consistent manufacturing of finished product as well as confirm drug stability in the finished dosage form. Should impurities or degradants be discovered, immediate efforts are made to identify the source of the impurity or degradation and eliminate it from the finished product. Manufacturing supplies
- 18. Role ofBioanalysis 11 the wholesalers with packaged drug product so that the drug can be purchased and used by pharmacies in response to receiving written prescriptions from physicians. The product launch announces the new drug to physicians and other medical professionals. This introduction provides education about the new drug's characteristics, indications and labeling. Various marketing and advertising programs are devised and executed. Phase IV clinical trials are those studies conducted after a product launch to expand on approved claims, study the drug in a particular patient population, as well as extend the product line with new formulations. A clinical study after the drug is sold may be conducted to evaluate a new dosage regimen for a drug, e.g., fexofenadine (Allegra®) is an antihistamine sold by Aventis (Bridgewater, NJ USA). The original clinical studies indicated that a dosage of 60 mg, given every 12 h, was adequate to control symptoms of allergies and rhinitis. Their product launch was made with this strength and dosage regimen. In response to competition from a once a day allergy drug, Aventis conducted Phase IV clinical trials (after the product was on the market) with different dosages and obtained the necessary data to show that a 180 mg version of Allegra could be taken once a day and relieve allergy symptoms with similar efficacy as 60 mg taken twice a day. Aventis then filed the clinical and regulatory documentation, and obtained approval to market a new dosage form of their drug. Another example of a post-marketing Phase IV clinical study is the investigation of whether or not sertraline (Zoloft®), an antidepressant drug, could be taken by patients with unstable ischemic heart disease. Results suggested that it is a safe and effective treatment for depression in patients with recent myocardial infarction or unstable angina [7]. In order to further ensure continued drug product quality, the FDA requires the submission of Annual Reports for each drug product. Annual Reports include information pertaining to adverse reaction data and records of production, quality control and distribution. For some drug products, the FDA requires affirmative post-marketing monitoring or additional studies to evaluate long term effects. A drug company also closely monitors all adverse drug experiences collected after the sale ofa drug and reports them to the FDA. The FDA has the authority to withdraw a drug from the market at any time in response to unusual or rare occurrences of life threatening side effects or toxicity noted in the post-marketing surveillance program. A conclusion that a drug should no longer be marketed is based on the nature and frequency of the
- 19. 12 Chapter 1 adverse effects and how the drug compares with other treatments. Some drugs that were withdrawn from the market between 1997 and 2000 include the following: Rezulin® (troglitazone), Propulsid® (cisapride), Raxar® (grepafloxacin) and Trovan® (trovafloxacin), Duract® (bromfenac), Redux® (desfenfluramine), Posicoreo (mibefradil), Seldane® (terfenadine), Hismanal® (astemizole), Pondimin® (fenfluramine) and Lotronex® (alosetron). These drugs were all removed for one of the following reasons: liver toxicity, cardiac arrhythmias, drug interactions or heart damage (cardiac valve disease); the exception was alosetron which caused ischemic colitis. In rare cases, a drug may be returned to the market after withdrawal but only when very strict and limiting measures for its continued use are put into place (e.g., Propulsid). Propulsid is still available under a special investigational use designation, which means that the drug is available to people with severely debilitating conditions for which the benefits of taking the drug clearly outweigh the risks. Certain eligibility criteria must be met by each patient and additional physician office visits and paperwork are required. These limitations are put into place to assure that Propulsid will only be given to those people whose particular medical condition warrants its use. A natural thought when a drug is taken off the market is, "How did the drug make it through clinical trials successfully?" Most often, the withdrawal occurs because of adverse effects that were not seen before marketing the drug. A rare side effect that may occur in 0.01% of the population may not be scientifically validated until the statistical population of patients taking the drug is large enough. Other times, hints ofthe problem may be noted through a retrospective review of data from clinical studies, but not the serious events that eventually lead to the withdrawal. Sometimes, there simply may not be any indication at all. Also, a serious side effect may only be noted when an approved drug is used in a different manner than the clinical studies were designed to investigate. Many complex factors go into the drug approval process; ultimately, the decision for a new drug approval is a balance of risks versus benefits. 1.2 Industry Trends 1.1.1 Introduction Advances in many different disciplines have occurred to change the way drug discovery is performed today compared with even five years ago. These advances include sequencing of the human genome; identification of more drug
- 20. Role ofBioanalysis 13 targets through proteomics; advances in the fields of combinatorial chemistry, high throughput screening, and mass spectrometry; and improvements in laboratory automation and throughput in bioanalysis. The end result of these process improvements is that compounds can now be synthesized faster than ever before. These greater numbers of compounds are quickly evaluated for pharmacological and metabolic activity using high throughput automated techniques, with the ultimate goal of bringing a drug product to market in a shorter timeframe. Some background material is provided next for the reader to gain a better understanding of four key industry trends: (a) combinatorial chemistry; (b) advances in automation for combinatorial chemistry, high throughput screening and bioanalysis; (c) LC-MS/MS analytical detection techniques; and (d) newer bioanalytical dosing regimens (n-in-l dosing) made possible by the advances in detection. 1.2.2 Combinatorial Chemistry A key component of satisfying the high throughput capability and demands of drug discovery has been the implementation of combinatorial chemistry techniques to synthesize, purify and confirm the identity of a large number of compounds displaying wide chemical diversity within a class. In place of traditional serial compound synthesis, libraries of compounds are created in 96-well plates by interconnecting a set or sets of small reactive molecules, called building blocks, in many different permutations [8-10]. Today, as many as 2,000 compounds can be synthesized in a week. Although 96-well plates serve as the most common format for reaction vessels, 24- and 48-well plates are also used by medicinal chemists. These combinatorial chemistry and parallel synthesis strategies are used to produce a large number of compounds which are then subjected to high throughput screening to identify biological activity. Automation aids the chemist in the high throughput synthesis of these compound libraries [11], as well in the subsequent purification steps required to isolate synthesized compound from reaction starting materials, reagents and byproducts [12]. The popular strategic options for the synthesis of combinatorial libraries include solid-phase, solution-phaseand liquid-phase synthesis. Solid-phase parallel synthesis uses resins to which the starting material is attached in order to produce a large number of compounds via split and mix methods. The solid support matrix used consists of a base polymer, a linker to
- 21. 14 Chapter 1 join the base polymer to the reactive center, and a functionalized reactive site. The immobilized reactant is then subjected to a series of chemical reactions to prepare the desired end product. The use of excess reagents drives reactions to completion. However, the need for deconvolution approaches to determine the active components within a pool has limited the utility of solid-phase synthesis. Since the synthesized compounds are attached to the solid support, this approach does offer simplified reagent removal via filtration and impurities are washed away easily during purification. The compound of interest is released from the polymer support in a final chemical release step. Solution-phase parallel synthesis techniques are more flexible than solid-phase techniques and are often used to create focused chemical libraries. Using this approach, the reactions occur in solution and so are easily monitored by thin layer chromatography or NMR. The synthesized compound is isolated in one liquid phase; all non product species are fractionated into an immiscible liquid phase [13]. A purification step following the reaction is required and common approaches are liquid-liquid extraction, liquid chromatography, solid-phase extraction and the use of solid-phase scavengers to remove excess reagents and/or reaction impurities from crude solutions. These solid-phase scavengers (functionally modified polymers of polystyrene or bonded silica) are chosen for their inertness to the reaction products but affinity for reagents and unwanted byproducts. Scavengers are becoming more popular since they can easily be adapted to automated purification techniques via filtration [14]. The procedure for use of scavengers follows. Scavenger beads are placed into the wells ofa flow-through 96-well filtration plate. A reaction block (consisting of individual wells of a flow-through 96-well plate in which the top and/or bottom of the wells can be blocked or opened to allow flow and reagent addition) is placed on top of the filtration plate (loaded with beads), so that when vacuum is applied the reaction mixture flows out of the reaction block and through the scavenger bed. A collection plate centered below the filtration plate isolates the solution. Liquid-phase parallel synthesis combines the strategic features of solid-phase synthesis and solution-phase synthesis. This method uses a supporting polymer (e.g., polyethylene glycol) that is soluble in the reaction media. Selective precipitation of this polymer can be performed for the purposes ofisolation and purification. Excess reagents and byproducts are removed by simple filtration [15].
- 22. Role ofBioanalysis 1.2.3 Automation 15 Automation is playing an important role in allowing researchers to meet the high throughput demands in today's research environment. A combination of robotics, liquid handling workstations and/or improved formats such as microplate sample preparation have been introduced to allow high speed analyses in combinatorial chemistry, high throughput screening and bioanalysis. An example of a typical liquid handling workstation is shown in Figure 1.2. In combinatorial chemistry, automated workstations are available that are specifically configured for either the organic synthesis step or for the subsequent purification step. Benchtop synthesizers can perform up to 20 reactions in flasks with hands-on control. All synthesis functions (mix, heat, cool, wash, empty, cleave) have been incorporated into a single module that fits on the benchtop. A multifunctional workstation assists with the following functions: reagent preparation, reaction mapping, off-line reaction incubation, liquid-liquid extraction, compound dissolution, and compound aliquoting for Figure 1.2. Typical example of a liquid handling workstation used to automate various sample preparation processes in drug development, the Genesis RSP. Photo reprinted with permission from Tecan.
- 23. 16 Chapter 1 analysis and screening. The addition of an analytical balance and vortex mixer on the workstation meet the requirements of automating synthetic chemistry conditions. A multitasking robotic workstation for synthesis features two independently controlled robotic arms that dispense reagents and solvents simultaneously, and can do so in inert environments. Equipment such as this can be upgraded to perform additional tasks and can interface with some additional components of a core system for higher throughput and higher performance. High throughput screening utilizes robotic-feeding liquid handling workstations with higher density microplates (384-well and 1536-well formats) and plate stackers for improved productivity. Automated hit picking is a hardware and software application that automates the transfer of lead compounds from their source plates into destination plates for consolidation. The use of 96- or 384-channel disposable tip pipetting heads allows improved liquid dispensing capabilities and speeds. The demand for even greater throughput in screening procedures often requires a larger industrial process rather than a laboratory workstation approach. Independent workstation modules can be combined in an assembly line format, consisting ofstorage and incubation carousels, washers, liquid handlers and plate readers. Modules are simply added to put in more steps or increase capabilities. This type of system is capable of running 1,000 96-well plates (96,000 assays) per day and is compatible with 384-well plates. An ultrahigh throughput example of a fully integrated automation solution can screen 100,000 compounds in one working day [16]. Automation for bioanalysis is described in detail in Chapter 5. Briefly mentioned here, liquid handling workstations with plate grippers have greatly improved the throughput of sample preparation procedures using 96-well plates. Automation allows more samples to be processed per unit time and frees the analyst from most hands-on tasks. Using the microplate format for sample preparation allows the automation of common procedures including protein precipitation, liquid-liquid extraction, solid-phase extraction and filtration. 1.2.4 Analytical Instrumentation-LC-MS 1.2.4.1 Introduction The preferred analytical technique in the bioanalytical research environment is liquid chromatography-mass spectrometry (LC-MS), used for qualitative and quantitative drug identification. LC-MS is preferred for its speed, sensitivity
- 24. Role ofBioanalysis 17 and specificity. LC is a powerful and universally accepted technique that offers chromatographic separation of individual analytes within liquid mixtures. These analytes are subjected to an ionization source and then are introduced into the mass spectrometer. The mass spectrometer separates or filters these ions based on their mass-to-charge ratio (m/z) and then sends them on to the detector [17]. A general scheme of this process is shown in Figure 1.3. The data generated are used to provide information about the molecular weight, structure, identity and quantity of specific components within the sample. 1.2.4.2 LC-MS Interface The LC-MS interface is the most important element of this system. It is the point at which the liquid from the LC (operated at atmospheric pressure) meets the mass spectrometer (operated in a vacuum). Advances have occurred over the years to mate the two techniques [18, 19]. The most common ionization interface used for bioanalysis is atmospheric pressure ionization (API) which is a soft ionization process (i.e., provides little fragmentation of a molecular ion). API is performed as either API-electrospray or atmospheric pressure chemical ionization (APCI). 1.2.4.2.1 Electrospray Ionization Electrospray ionization (ESI) generates ions directly from the solution phase into the gas phase. The ions are produced by applying a strong electric field to a very fine spray of the analyte in solution. The electric field charges the surface of the liquid and forms a spray of charged droplets. The charged droplets are attracted toward a capillary sampling orifice where heated nitrogen drying gas shrinks the droplets and carries away the uncharged material. As the droplets shrink, ionized analytes escape the liquid phase through electrostatic (coulombic) forces and enter the gas phase, where they proceed into the low pressure region of the ion source and into the mass analyzer [20-22]. IDetector r'--__---' Figure 1.3. Schematic diagram of the basic components ofa mass spectrometer system. The mobile phase following separation ofcomponents on a liquid chromatograph flows into the mass spectrometer for LC-MS analysis.
- 25. 18 Chapter 1 Analysis by electrospray requires the prior formation of ionized analytes in solution; for nonionic compounds, ions are prepared by adding acid or base modifiers to the LC mobile phase solution to promote the electrospray process [23]. Ionization thus occurs in the liquid phase with ESI. Pure electrospray is suitable for only capillary LC and capillary electrophoresis, or conventional LC where the post-column effluent is split using a zero dead volume T-piece, reducing the flow of liquid entering the mass spectrometer. In an attempt to extend the range of solvent flow rates amenable to electrospray, modifications have been made using pneumatic and thermal assistance [23]. 1.2.4.2.2 Atmospheric Pressure Chemical Ionization APCI is similar to API-ES, but APCI nebulization occurs in a hot vaporization chamber, where a heated stream of nitrogen gas rapidly evaporates nearly all of the solvent. The vapor is ionized by a corona discharge needle [24]. The discharge produces reagent ions from the LC solvent which then ionize the sample [23]. Ionization thus occurs in the gas phase with APCI. 1.2.4.3 Mass Analyzers The ionization source and the mass analyzer are linked since the mass analyzer requires a charged particle in order for separation to occur. The mass analyzer contains some electric or magnetic field, or combination of the two, which can manipulate the trajectory of the ion in a vacuum chamber [25]. The atmospheric pressure ionization interfaces described above are commercially available with various mass analyzers. The most popular and available mass analyzer is the quadrupole which will be described here. For information on product ion scanning, precursor ion scanning and neutralloss/gain, the reader is referred to the book chapter by Fountain [25]. The quadrupole mass analyzer is available as a single quadrupole or is configured in tandem (called a triple quadrupole) to greatly enhance its capabilities. A quadrupole mass filter typically consists of four cylindrical electrodes (rods) to which precise DC and RF voltages can be applied. Note that the tandem quadrupole mass filters are referred to as Ql and Q3; the additional Q2 quadrupole has no filtering effect, except to use its RF voltage to guide ions through the vacuum chamber.
- 26. Role ofBioanalysis 19 A mass spectrometer with a single quadrupole is capable of either full scan acquisition or selected ion monitoring (SIM) detection. In the full scan mode, the instrument detects signals over a defined mass range during a short period oftime. All the signals are detected until the full mass range is covered. A mass spectrum is generated in which ion intensity (abundance) is plotted versus m/z ratio. Scan mode is used for qualitative analysis when the analyte mass is not known. Inthe SIM mode, a single stage quadrupole is used as a mass filter and monitors only a specific m/z ratio, allowing only that m/z ratio to pass through to the detector. Chemical noise is reduced and a response curve is generated for a specific ion rather than a mass spectrum. As a result, sensitivity is improved and this technique is useful for quantitative analysis of a specific analyte. When an additional quadrupole mass analyzer is configured in tandem and coupled with a collision cell for gas reactions, several additional analytical experiments can be conducted: multiple reaction monitoring, product ion scanning, precursor ion scanning and neutral loss/gain. Tandem mass spectrometry (MS/MS) achieves unequivocal identification of a drug substance. In LC-MS/MS, the first analyzer selects a parent ion from the first quadrupole and filters out all unwanted ions. The selected ion undergoes collision in another quadrupole via bombardment with gas molecules such as nitrogen or argon. Fragmentation occurs and the other mass analyzer in the third quadrupole selects a particular product (daughter) ion. This scenario is called selected reaction monitoring (SRM) or multiple reaction monitoring (MRM). Information is provided on the chemical structure using the parent- daughter ion pair. The key attractive features of an LC-MS/MS system are its speed, selectivity for a single MW in the presence of many other constituents and sensitivity (typically pg/mL concentrations are able to be accurately quantitated). Additional advantages are good precision and accuracy, and wide dynamic range. Note that the selectivity of LC-MS/MS reduces the need for complete chromatographic resolution of individual components, allowing a shorter analytical run time and higher throughput [26]. Typically the combined increases in selectivity and sensitivity of LC-MS/MS methods provide a >10 fold improvement in the limit of quantitation (LOQ) compared with traditional methods using ultraviolet or fluorescence detection [23]. A typical LC-MS/MS instrument as used for the analytical quantitation of drugs from biological matrices is shown in Figure 1.4. In addition to the single quadrupole (LC-MS) and triple quadrupole (LC- MS/MS) mass analyzers already discussed, other mass analyzers include:
- 27. 20 Chapter 1 Figure 1.4. Typical example ofan LC-MS/MS system used for the separation, detection and quantitation of drugs in biological matrices. magnetic sector, time-of-flight (TOF) [27, 28], quadrupole orthogonal acceleration TOF (Q-TOF) [29] and ion trap (IT) mass analyzers [26, 30, 31]. Applications for quantitative bioanalysis are best served by LC-MS (single quadrupole) and LC-MS/MS (triple quadrupole) for added sensitivity. However, when gaining information on the metabolic route of a compound is more important than absolute sensitivity and selectivity for the parent drug, the use of ion trap and time-of-flight instruments present advantages. IT and TOF offer greater sensitivity in full scan mode than triple quadrupole MSIMS detection [32] and multiple analytes can be detected and quantified, as reported by Cai et af. [33] and Zhang et af. [34]. Another benefit of TOF-MS is its capability of accurate mass analysis which allows metabolites to be identified with greater confidence [32]. 1.2.4.4 Further Reading Additional discussion of these mass spectrometry techniques is outside the scope of this text and the reader is referred to additional resources. Mass spectrometry is introduced as a tutorial in a book chapter by Fountain [25] and the fundamentals of electrospray are presented by Gaskel1 [35]. A review by
- 28. Role ofBioanalysis 21 Lee and Kerns [26] and a book by Lee [36] detail how LC-MS techniques are fundamentally established as a valuable tool and used in all phases of drug development. Kyranos et al. describe applications for LC-MS in drug discovery [37]. Hoke et at. describe how pharmaceutical research and development have been transformed by innovations in mass spectrometry based technologies [38]. The impact of mass spectrometry on combinatorial chemistry is described by Triolo et at. [39] and Sii~muth and Jung [40]. Niessen provides a review of the principles and applications of LC-MS [41]. Various reviews describe applications for LC-MS in proteomics [42, 43], analytical toxicology [44], food analysis [45], forensic and clinical toxicology [46] and forensic sciences [47]. A useful text for students of chemistry and biochemistry who wish to understand the principles of mass spectrometry while using it as a tool is the book by Johnstone and Rose [48]. LC-MS/MS techniques for drug separation, detection and quantitation have become the standard and their use continues to expand in bioanalysis. Several reviews of the capabilities of high throughput LC-MSIMS for bioanalysis, including sample preparation schemes, have been published by Brewer and Henion [49], Plumb et al. [23], Jemal [50], Brockman, Hiller and Cole [51], and Ackermann et al. [52]. Korfmacher et al. illustrate the important role of LC-MS/MS API techniques in drug discovery for rapid, quantitative method development, metabolite identification and multiple drug analysis [53]. Yang et al. describe the latest LC-MS/MS technologies for drug discovery support [54] and Rudewicz and Yang discuss the use of LC-MS/MS in a regulated environment [55]. Law and Temesi share some useful considerations in making the switch from LC-UV to ESI LC-MS techniques in support of drug discovery [56]. Rossi et al. describe the use of tandem-in-time MS as a quantitative bioana1ytica1 tool [57]. Improvements continue to be made in three areas: (1) interfaces from the LC to the mass spectrometer; (2) multiple sample inlets (e.g., four instead of one [55, 58, 59]); and (3) staggered parallel sample introduction schemes where one mass spectrometer inlet is shared with two or more LC columns [60-62]. These advances are described in more detail in Chapter 14, Section 14.4.4. Analysis times using these novel approaches are very fast, allowing rapid turnaround of samples to meet the needs of drug development research. Note that LC and mass spectrometry can be interfaced to NMR to create an LC-NMR-MS system. Such a configuration has been shown useful for the structural elucidation of metabolites in urine [63].
- 29. 22 1.2.5 N-in-l Dosing Chapter 1 The traditional approach of dosing one animal with one drug and collecting blood at a series of time points after administration yields valuable information on a candidate's pharmacokinetics in a living system. However, this procedure is time consuming and labor intensive, as each individual sample must undergo analysis in a serial manner. This approach cannot be used to rapidly evaluate the many hundreds of candidate compounds generated from combinatorial chemistry, for it would take too much time. In an effort to improve throughput and reduce cost, n-in-I dosing was devised in which multiple compounds are dosed in one animal and the selectivity of the mass spectrometer is used to individually quantitate their concentrations from the mixture. This approach is also called simultaneous multiple compound dosing or cassette dosing. The data generated by this approach yield meaningful pharmacokinetic data in a much shorter time frame, and fewer animals and fewer numbers of samples are used than in traditional methods. Examples of the n-in-I approach for rapid pharmacokinetic screening of drug candidates using LC-MSIMS are published by Olah et al. [64], McLoughlin et al. [65], Liang et al. [66], Berman et al. [67], Cai et al. [68] and Bayliss and Frick [69]. A concise review of cassette dosing can be found in the book chapter by Vora, Rossi and Kindt [70]. The drug screening process is now more manageable using a fewer number of samples generated from n-in-l dosing techniques. Each compound used for dosing is quickly evaluated and compared on a relative basis with the other dosed compounds and the most desirable candidates are selected. A typical profile ofdrug concentration versus time, observed in one dog after intravenous dosing with 10 drugs, is shown in Figure 1.5. Note that the use of pooled plasma from multiple animals dosed with single unique compounds has also been demonstrated to yield a throughput advantage in the analysis of bioanalytical samples [71]. Using this approach, 10 animals may be dosed with one compound each and then all of the I h time point samples are pooled and subjected to sample preparation, and so on for each time point. Sample pooling in this manner eliminates the concern from cassette dosing of potential drug-drug interactions on pharmacokinetics and metabolic conversion of one compound to another compound already included in the series. The practice of sample pooling and its associated reduction in workload is described by Kuo et al. for six proprietary compounds from a class of antipsychotic agents [72]. Note that some variations of this approach exist,
- 30. Role ofBioanalysis 23 1 10 1000 - :E S 100 = e .... - e - = ~ ~ = <:) U = e '" = Ii: Dog Intravenous Dose (IO-ill-l) 0.5 mglkg -+- Cpdl -X-Cpd2 -:1(- Cpd3 -+-Cpd4 -o-Cpd5 -o·Cpd6 -+-Cpd7 - .... Cpd8 -'-Cpd9 ~Cpdl0 24 20 16 12 8 4 0.1 +---+--~I----+---+---+---+- o Time (h) Figure 1.5. Pharmacokinetic profiles of 10 compounds that were dosed intravenously in one dog. Reprinted with permission from [73]. Copyright 2001 Elsevier Science B.V such as the cassette accelerated rapid rat screen in which drug candidates are dosed individually (n=2 rats per compound) in batches of six compounds per set and then samples are pooled across time points to provide a smaller number of test samples for analysis [74]. Another method used to reduce the number of animals in the support of drug discovery is to serially bleed mice (removal of 10-20 ul, blood) and analyze the small sample volumes using a small capillary LC column prior to MS analysis [75, 76]. Conventional techniques used to obtain a nine point pharmacokinetic curve with 4 animals per time point would require the use of 36 mice; using the serial bleeding technique, only 4 mice are used [76]. A related method to serially bleed an animal is microdialysis, in which a semi- permeable membrane is surgically implanted in a tissue of a living organism and the perfusate solution is sampled over time. Thus, little biological fluid is removed and continuous in vivo sampling is possible. Microdialysis works especially well for drug transport studies. An overview [77] and some applications ofmicrodialysis [78-80] provide interesting reading.
- 31. 24 1.3 Specific Roles for Bioanalysis in Drug Development 1.3.1 Drug Discovery-Lead Optimization 1.3.1.1 Screening in vivo for Pharmacokinetic Properties Chapter 1 Combinatorial chemistry and high throughput screening techniques synthesize and identify a large number of compounds that may be potential leads for continued development. Chemists synthesize and screen chemical analogs of the hits to further improve and refine a drug's activity. However, the chemist alone cannot identify the best analog for continued study because it is often the case that good leads in vitro are not good leads in vivo due to problems with absorption, metabolism or toxicity. Screening these leads is an important task in a process known as lead optimization. Bioanalysis is the quantitative determination of drugs and their metabolites in biological fluids. Bioanalytical scientists play an integral role in the lead optimization process by performing studies to gain information on a molecule's absorption, distribution (including an estimate of protein binding), metabolism and elimination. These important ADME tests determine the likelihood of a drug candidate continuing into the preclinical phase of drug development. A summary of the experiments commonly performed to assess ADME characteristics is listed in Table 1.2. Each sample of biological fluid from an in vitro or in vivo study is subjected to bioanalysis to determine the concentration of drug at specific time points. Bioanalysis includes the acts of sample preparation and analysis by LC-MS/MS methods. This bioanalytical technique is utilized throughout the development lifetime of all new drugs. Analysis of the drug concentration versus time data yields important pharmacokinetic information that is used in the decision making process of whether or not a new molecule should be a candidate for further development. 1.3.1.2 Screening in vitro for Pharmacokinetic Properties The use of animals to assess ADME characteristics is a costly and time consuming process. While animals are used, especially for toxicokinetic studies to assess drug toxicity, current trends are to use in vitro screens which have matured in recent years and been shown to be fairly predictive. These in vitro methodologies use enzymes, tissues and cell cultures to allow researchers to screen for drug characteristics such as cell absorption, metabolic stability,
- 32. Role ofBioanalysis 25 Table 1.2 A list of experiments that are commonly performed to assess the absorption, distribution, metabolism and elimination (ADME) characteristics of potential lead compounds in drug discovery Parameter Examined Typical Experiments Absorption Caco-2 cells, MDCK cells, PgP transport in vivo pharmacokinetic profiling Distribution Metabolism Elimination in vitro protein binding in vivo tissue distribution studies Metabolic stability -Microsomes, sub cellular fractions, hepatocytes P450 inhibition studies -Microsomes P450 induction studies -Gene chips, multiple dosing Quantitation ofdrugs and metabolites in biological fluids drug-drug interactions, clearance, bioavailability and toxicity [81-83]. Instrumentation to accommodate cell maintenance has matured in recent years to the point where high throughput testing using these in vitro screens is now a viable approach to investigate the absorption and metabolism of drugs. Note that in addition to in vivo and in vitro testing, another prediction technique is called in silica. This approach refers to computer modeling based on sophisticated software using information on chemical structure, receptors, enzymes and various other databases of information [84]. Another important note to mention is that in vitro plasma protein binding measurements (e.g., equilibrium dialysis and ultrafiltration) are utilized in drug discovery in a high throughput manner but are discussed instead in Chapter 6, Section 6.6. These applications for investigating in vitro absorption and metabolism studies will now be described in detail: absorption, metabolic stability screening, metabolic inhibition and induction ofcytochrome P450, and toxicity testing. 1.3.1.2.1 Absorption It is important to determine whether a drug displays pharmacological activity when it is administered orally, a desirable route of administration for the general population. Therefore an estimate of absorption is desired. The
- 33. 26 Chapter 1 approach used to thoroughly evaluate oral absorption is to assess those individual factors that contribute to drug passage through the gastrointestinal membrane, such as solubility in the lumen of the intestine, permeability across cells, and chemical stability in the stomach and small intestine. Additional criteria are evaluated, such as lipophilicity of the compound and its hydrogen bonding potential, and then an overall predictive estimate of absorption characteristics is made. While this approach is complete, it takes time to generate all the pieces of data needed. The in vitro permeability of drugs through Caco-2 cells has been used as a single predictor of oral absorption in humans. Caco-2 cell monolayers are derived from human colon adenocarcinoma cells. These cells are grown on semipermeable membranes and spontaneously differentiate to form confluent monolayers that mimic intestinal absorption cells. The apical (donor) surface of the monolayer contains microvilli, as in the intestine. Permeability measurements are based on the rate of appearance of test compound in a receiving (basolateral) compartment. Bioanalysis is used to determine the concentrations of analyte in the basolateral compartment [85, 86]. The cells also express functional transport proteins and metabolic enzymes. While the Caco-2 model is fairly predictive, it does have the limitation of requiring a 21 day culture time with frequent attention required for replenishing its nutrients. A common concern about the Caco-2 model is that it may not be truly representative of all absorption pathways in the small intestine. Another model of absorption, which reduces the tissue culture time to 3 days, is the MDCK (Madin-Darby Canine Kidney) cell line [87, 88]. This accelerated permeability model is a feasible alternative to the traditional model that provides rank ordering ofcompounds with improved turnaround time. 1.3.1.2.2 Metabolic Stability Screening The metabolic stability of a new chemical entity greatly influences its pharmacokinetic profile. A drug may have high bioavailability (high absorption and low first pass metabolism) following oral dosing but extensive and rapid metabolism can reduce the time it is in the blood as an intact molecule. For example, ester groups on drugs can be cleaved by esterases in the blood and the drug is metabolized or biotransformed to a new chemical entity. Note that metabolites can also be active as well, or even more active than the parent drug. The cytochrome (CYP) P450 system in the liver is an important enzymatic pathway for the oxidative metabolism of drugs and is often the primary route for degradation, regardless of how the drug is administered. It is valuable to
- 34. Role ofBioanalysis 27 learn the specific P450 isoenzymes responsible for a drug's metabolism, as the information can be used to predict the fate of the drug, potential drug-drug interactions, reactive metabolites and cytotoxic mechanisms. Many in vitro methodologies for assessing metabolism are used in drug discovery support. Hepatic microsomes are among the most popular systems in use. These preparations retain the activity of those enzymes that reside in the smooth endoplasmic reticulum of cells, such as the cytochrome P450 system, flavin monooxygenases and glucuronyltransferases. Cultured hepatocytes retain a broader range of enzymatic activities, including not only the reticular systems of CYP450 but also cytosolic and mitochondrial enzymes [89]. These hepatocytes are particularly useful for induction and inhibition studies where the enzymatic activities in the liver are predicted. Additionally, liver slices are sometimes used in similar metabolic screens because they retain a wide range of enzymatic activities, like hepatocytes, but more closely resemble the organ level of the liver. Metabolic stability testing ofcompounds is performed in liver microsomes with a collection of subcellular materials called S9 mix in the presence and absence of enzymatic cofactors such as NADP+ (Nicotinamide Adenine Dinucleotide Phosphate). Hepatocytes and liver slices are also used for this stability testing. Incubations are assembled in a microplate format. At selected time points, the metabolic reaction is stopped by the addition of cold acetonitrile and then centrifugation is performed to pellet the proteins at the bottom of the wells. The supernatants are collected following centrifugation. Substrates are analyzed by LC-MS/MS interfaced with a 96-well autosampler for high throughput operation [61, 90-96]. The use of metabolic stability screening to predict clearance is also of interest. A drug with a high clearance has a high hepatic extraction ratio and so its maximum oral bioavailability is low. Conversely, a drug with a low clearance has a low hepatic extraction ratio and its maximum oral bioavailability is high (assuming complete absorption). Since clearance provides an understanding of the ability of the body to eliminate a drug, it is often used as a pharmacokinetic screen in lead selection. The ability of hepatic microsomal stability assessments to predict in vivo clearance in the rat was retrospectively evaluated for 1163 compounds from 48 research programs at a pharmaceutical research company [97].
- 35. 28 1.3.1.2.3 Metabolic Inhibition ofCytochrome P450 Chapter 1 The assessment of a candidate drug's potential to inhibit or induce cytochrome P450 isoenzymes using in vitro microsomal incubations is one method for predicting possible in vivo drug-drug interactions. Seven isoenzymes of cytochrome P450 play dominant roles in drug metabolism: CYPIA2, 2A6, 3A4, 2C9, 2C19, 2D6 and 2E1. One of the strategies adopted to evaluate the inhibition or induction of CYP450 by a drug is to monitor its effect on the metabolism of selected compounds known as probe substrates whose specific metabolic pathway is documented to occur via a single CYP450 enzyme [98-100]. After incubation and quenching of the reaction, sample preparation is required to remove proteins and matrix interferences before analysis. Two reports from the same laboratory detail methods for high throughput CYP inhibition screening using a cassette dosing strategy [101, 102]. Characteristics and common properties of inhibitors, inducers and activators of CYP enzymes are reviewed by Hollenberg [103]. The ADME profile of a drug product can be improved when the effect of CYP P450 inhibitors and/or inducers is fully known. For example, terfenadine (Seldane®) was removed from the market (see Section 1.1.5) due to potentially life threatening drug interactions that could result in abnormalities in the electrical impulse that stimulates the heart to contract and pump blood. An active carboxy metabolite of terfenadine, named fexofenadine, was introduced as Allegra®. Fexofenadine undergoes less metabolism by CYP3A4 isoenzymes and therefore the effect ofinhibitors or inducers is greatly reduced on CYP3A4 when compared with the effect with terfenadine [104]. Another example of an improved ADME profile is esomeprazole (Nexium®). Esomeprazole is the (S)- isomer of omeprazole (Prilosec®) and undergoes less metabolism by CYP2C19 isoenzymes. A beneficial result is that CYP2C19 polymorphism has less ofan effect on its pharmacokinetics [104]. 1.3.1.3 Toxicity Testing Toxicity testing is an important component of screening potential lead compounds [105], as compounds often fail in the development process because of the harmful effect they may have on cells, organs or organ systems. Commonly, animals will be administered escalating drug doses and the time course profile of the drug will be determined using bioanalysis; this technique is referred to as toxicokinetics. Genotoxicity analysis is another important toxicity test that is usually performed just prior to phase I clinical trials because
- 36. Role ofBioanalysis 29 of the high cost involved; performing this test on all lead compounds would be prohibitively expensive. An effort has recently begun to move toxicity testing to an earlier stage in drug development to confirm viable leads more quickly. Toxicogenomics, the examination of changes in gene expression following exposure to a toxicant, offers this potential for early detection; it may also detect human specific toxicants that cause no adverse reaction in rats [106]. Cytotoxicity tests using bioluminescence are proceeding along the lines of high throughput screening by being miniaturized to 384-well formats [107]. 1.3.1.4 Further Reading A wealth of information is available in published literature concerning the role of bioanalysis in the support of drug discovery and the lead optimization process, including descriptions of the various in vivo and in vitro screening tests. A schematic diagram of this iterative course of action is shown in Figure 1.6. Both general reviews and detailed reports are recommended. in vivo in vitro Single Dose Pharmacokinetics and Metabolite Screening Metabolic Stability Clearance and Metabolite Screening t I I I _ ...J L _ continued development Figure 1.6. Schematic diagram of the lead optimization process and the role of metabolite screening. Reprinted with permission from [108]. Copyright 2002 John Wiley & Sons, Ltd.
- 37. 30 Chapter 1 Venkatesh and Lipper review the role of the development scientist in the selection and optimization of lead compounds [109]. Kennedy describes the compound selection and decision making skills necessary to manage the interface between drug discovery and drug development [110]. Perspectives on twelve months of lead optimization were provided from a chemistry team which identified the need for a balance between achieving the needed throughput and allowing time for appropriate decision making and reflection [Ill]. The prediction and use of pharmacokinetic properties in early drug discovery is discussed in several papers [81, 82,112-115] (the report by Roberts [112] is highly recommended reading). The concept of a bioanalytical toolbox for performing fast turnaround in drug discovery support is a practical proposal to describe the choices available to the bioanalyst [116]. The importance of stereospecific bioanalytical monitoring in drug development is reviewed by Caldwell [117], with particular emphasis on stereospecific assays for the individual optical isomers of drugs. Some examples of enantiomeric separation and quantification in bioanalysis include the analysis of ketoprofen [118] and fluoxetine [119] in human plasma. 1.3.2 Prec/inical-ADME and Metabolite Identification 1.3.2.1 ADME Studies Once a drug candidate has received IND approval, the preclinical development phase formally begins. With a focus on accelerated timelines, the distinction between drug discovery and preclinical development is less defined than in past years. Some activities that were traditionally performed only in the preclinical phase are now begun in drug discovery with the goal of identifying optimal lead compounds as early in development as possible. Once a drug compound reaches the preclinical development phase, its ADME characteristics are determined in many species of animals as well as in man. At this point, a defined and validated assay is used over and over for determining concentrations of drugs from in vitro and in vivo samples. It would be ideal if one analytical method could be used for every animal species, but in practice a method needs to be modified slightly and validated for each species. One method for many species of animals is described in a case study of two antibiotics [120]. While multiple compounds are encountered in drug discovery, in preclinical development fewer compounds are in use. Another differentiation between the two phases of development is that now in the preclinical stage validated
- 38. Role ofBioanalysis 31 methods are needed and adherence to Good Laboratory Practices (GLP) is required. Preclinical development projects in bioanalysis commonly involve the determination of drug concentrations in biological fluids after dosing by multiple routes of administration and in multiple species of animals. Toxicokinetic studies also continue to be performed which examine the relationships among dose, pharmacokinetics and toxicity. Tissue distribution studies are also conducted, frequently with radiolabeled drug. Some common approaches to assessing tissue distribution are to excise tissues and analyze extracts by LC-MS/MS, following sample preparation; oxidize excised tissues and determine the amount of radioactivity contained in each; and perform autoradiography. This latter technique utilizes thin.whole body slices and the amount of radioactivity in each organ slice is determined. 1.3.2.2 Metabolite Identification and Characterization Another important aspect of drug development research is the determination of the metabolic fate of lead compounds in several animal species. Many new chemical entities are terminated late in the development stage due to problems with drug metabolism. In an effort to prevent these late stage failures, it is useful to know the metabolic fate of a promising lead compound as early as possible. Typically, metabolites of lead compounds are identified in the preclinical development phase, although it is becoming more common to identify major metabolites in the discovery phase. Then, should toxic metabolites be identified (e.g., some acyl glucuronides [121]), structural analogs of early drug lead candidates may be designed to block portions of the molecules that are particularly susceptible to metabolism. Mass spectrometry coupled with liquid chromatography is an effective analytical method for metabolite profiling. The integration of data collected from the ion trap, triple quadrupole and quadrupole/time-of-flight instruments allows a comprehensive evaluation of biotransformation products. This approach is routinely used to evaluate metabolites generated from in vitro and in vivo systems. Once metabolites have been identified and characterized from definitive ADME studies, they are monitored in subsequent pharmacokinetic studies; it is common in bioanalysis to monitor concentrations of both parent drug and one or more metabolites. These parent-metabolite combinations present a useful assessment of metabolism that can be important to establish dosing regimens and assess toxicity concerns. Also note that a metabolite can present an ideal backup candidate to a drug lead, e.g., desloratidine (Clarinex®)
- 39. 32 Chapter 1 is a metabolite of loratidine (Claritin®) that was developed and FDA approved for marketing several years after the introduction ofClaritin. Many LC-MS applications are found in the literature for metabolite characterization from in vitro [122-124] and in vivo [34, 108] experiments. An overview of the choices and decisions involved in metabolite identification and how they can be merged into a systematic approach is described by Clarke et al. [125]. The mass spectrometric identification of metabolites is reviewed by Oxford and Monte [126] and strategies for metabolite isolation and identification are reviewed by Wiltshire [127]. An automated high throughput approach to metabolite identification is of great practical utility. Lopez et al. [128] and Kim et al. (129] have demonstrated an automated metabolite identification approach using an ion trap mass spectrometer. Automated fraction collection of metabolites for subsequent ion trap MS, MS/MS or NMR analyses has been described by Dear et al. [130]. 1.3.3 Clinical-Pharmacokinetics A major effort in the clinical phase ofdrug development is the determination of drug and metabolite concentrations in biological fluids after drug administration to humans. The pharmacokinetic data obtained are used to support drug development in assessing the therapeutic index, drug-drug interactions, design of dosage regimes, etc. Since drugs today are more potent than in years past and are dosed at lower levels, very sensitive assays are required to detect the low circulating levels of drugs in biological fluids (often to pg/mL sensitivity limits). Again, LC-MS/MS is the analysis and detection technique of choice due to its high sensitivity, selectivity and speed. GLP assays are also maintained during the clinical phase ofdrug development. The greatest numbers of samples to be analyzed are found in clinical drug development, where thousands of samples are often obtained from one clinical study (conducted at multiple sites). Rapid sample turnaround is sought for'one or more reasons: important metabolic information from patient samples can be obtained; the next dose can be determined; or the results used to plan the next clinical study. In addition to sensitivity, selectivity and ruggedness are also important to the analytical method. Examples of the increased requirement for sensitivity as a compound moved from preclinical into the clinical phase of drug development have been detailed by Dear et al. [131] and Laugher et al. [132]. In the report by Dear et al., preclinical studies required a lower limit of quantitation (LLOQ) ofonly 1 ng/mL in rat and dog plasma but the LLOQ needed for human clinical studies was 50 pg/mL.
- 40. Role ofBioanalysis 1.3.4 Therapeutic Drug Monitoring 33 The objective of therapeutic drug monitoring (TDM) is as a guide to provide optimal drug therapy by maintaining plasma concentrations of a drug within a desired range. Some examples of drugs for which monitoring is very important are: digoxin, phenytoin, aminoglycoside antibiotics, theophylline, cyclosporine, HIV protease inhibitors, and some cardiac agents, antiepileptic drugs and antidepressant drugs. Should drug concentrations in the sampled biological fluid exceed the desired range, toxicity may result and/or the desired therapeutic effect may no longer be achieved. The knowledge of plasma drug concentrations may explain why a patient does not respond to drug therapy or why the drug causes an adverse effect. Also, for HIV infections, low drug concentrations may in some cases lead to drug resistance and treatment failure. Most drugs can be safely taken without monitoring drug concentrations since therapeutic endpoints can effectively be evaluated by other means. For example, blood pressure determinations indicate how well the ~-blocker atenolol may be working for a patient, and coagulation times accurately measure the effect of Coumadin®, an anticoagulant drug. Drugs that are chosen for TDM have the following characteristics in common: • The range of therapeutic and safe plasma concentrations is narrow (i.e., a low therapeutic index) • Toxicity or lack ofeffectiveness of the drug puts the patient at risk • Patient compliance needs to be monitored The therapeutic index is defined as the ratio between the maximum and minimum plasma concentrations of the drug's therapeutic range. A low therapeutic index (less than 2) means that the dose that commonly yields a sub therapeutic response is close to the dose that produces some toxicity. Most drugs have a therapeutic index greater than 2 [133]. Note that the relationship between drug concentration at the site of action and the pharmacological response observed in the patient should be considered. TDM using plasma concentration data can be justified when a correlation has been established. Assay procedures for a drug in biological fluids are usually performed by a local or regional clinical laboratory or a national medical laboratory. Standard methods that are used for detection and quantitation of drugs are immunoassay techniques and liquid chromatography employing sensitive detection (fluorescence or mass spectrometry). Once drug concentrations and responses
- 41. 34 Chapter 1 have been adjusted to achieve the desired therapeutic result, a regular clinical monitoring program is begun. An informative discussion of the analytical goals in therapeutic drug monitoring is provided by Bowers [134]. Additional reports provide a further perspective on therapeutic drug monitoring. The roles for chromatographic analysis in TDM have been described by Wong [135], Shihabi and McCormick [136] and Binder [137]. Some examples of TDM are reported for flecainide [138], tacrolimus [139], immunosuppressants [140, 141], an antineoplastic agent (etoposide) [142], gabapentin [143], oxcarbazepine [144] and mexiletine [145]. TDM in special populations of patients is discussed by Walson [146]. A case for performing prospective concentration to clinical response investigations during the early stages of drug development, rather than the traditional retrospective review, is proposed by Shaw, Kaplan and Brayman [147]. Acknowledgments The author is appreciative to Danlin Wu and Mike Lee for their critical review of the manuscript, helpful discussions and contributions to this chapter. The line art illustrations were kindly provided by Woody Dells. References [I] G.R. Nakayama, Curr Opin. Drug Discovery Dev. I (1998) 85-91. [2] G. Ziokamik, Anal. Chern. (1999) 322A-328A. [3] J.K. Nicholson, J. Connelly, J.C. Lindon and E. Holmes, Nature Reviews I (2002) 153-161. [4] Federal Register USA (1988) October 21. [5] Federal Register USA (1987) May 22. [6] Federal Register USA (1990) May 21. [7] A.H. Glassman, C.M. O'Connor, R.M. Califf, K. Swedberg, P. Schwartz, J.T. Bigger, K.R.R. Krishnan, L.T. van Zyl, J.R. Swenson, M.S. Finkel, C. Landau, P.A. Shapiro, c.J. Pepine, J. Mardekian and W.M. Harrison, J. Amer. Med. Assn. 288 (2002) 701-709. [8] D.T. Rossi and MJ. Lovdahl, In: D.T. Rossi and M.W. Sinz, Eds., Mass Spectrometry in Drug Discovery, Marcel Dekker, New York (2002) 215-244. [9] R.A. Fecik, K.E. Frank, E.J. Gentry, S.R. Menon, L.A. Mitscher and H. Telikepalli, Med. Res. Rev. 18 (1998) 149-185.
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- 48. Chapter 2 Fundamental Strategies for Bioanalytical Sample Preparation Abstract It can be a challenge to develop bioanalytical methods that selectively separate drugs and metabolites from endogenous materials in the sample matrix. Fortunately, many different sample preparation techniques are available in order to meet the desired objectives for an assay. Successful implementation of these procedures relies on having a fundamental understanding of the strategies for sample preparation and the chemistry of the extraction process. This chapter first discusses the objectives for bioanalytical sample preparation. The many different techniques available to the bioanalyst are then introduced as a preview to the full content available in subsequent chapters of this book. Finally, some essential concepts related to extraction chemistry in sample preparation are presented: the influence of sample pH on ionization, the effects of anticoagulants and storage conditions on clot formation, and procedures for determining the matrix effect and extraction efficiency for a sample preparation method. 2.1 Importance of Sample Preparation 2.1.1 Introduction Sample preparation is a technique used to clean up a sample before analysis and/or to concentrate a sample to improve its detection. When samples are biological fluids such as plasma, serum or urine, this technique is described as bioanalytical sample preparation. The determination of drug concentrations in biological fluids yields the data used to understand the time course of drug action, or pharmacokinetics, in animals and man and is an essential component of the drug discovery and development process. A reliable analytical method is achieved with the successful combination of efficient sample preparation, adequate chromatographic separation and a sensitive detection technique. Liquid chromatography (LC) coupled with 41
- 49. 42 Chapter 2 detection by mass spectrometry (MS) is the preferred analytical technique for drug analysis. The use of tandem mass spectrometry (MS/MS) that identifies a specific parent-daughter ion pair allows unequivocal identification of a drug substance from biological samples. LC-MS/MS is widely used because it offers unmatched speed, sensitivity and specificity with good precision and wide dynamic range. Note that although mass spectrometry allows sensitive and specific detection of analytes of interest, it benefits tremendously from a well chosen column and mobile phase to provide adequate chromatographic separation [1]. The importance of chromatography cannot be overlooked; the column separates the analytes from the much higher concentrations of endogenous materials present in samples that can potentially mask an analyte or introduce ion suppression. Some essential chromatography issues for fast bioanalysis are discussed by O'Connor [2]. Another very important component in the overall analysis is the choice of sample preparation technique which influences the cleanliness of the sample introduced into the chromatographic system. Sample preparation is necessary because most analytical instruments cannot accept the matrix directly. Three major goals for sample preparation are to: 1. Remove unwanted matrix components that can cause interferences upon analysis, improving method specificity 2. Concentrate an analyte to improve its limit ofdetection 3. Exchange the analyte from an environment of aqueous solvent into a high percentage organic solvent suitable for injection into the chromatographic system Some additional goals for the sample preparation step may include removal of material that could block the tubing of the chromatographic system, solubilization of analytes to enable injection under the initial chromatographic conditions and dilution to reduce solvent strength or avoid solvent incompatibility [3]. The term sample preparation typically encompasses a wide variety of processes which include aspirating and dispensing liquids, release of drugs from the sample matrix via hydrolysis or sonication, dilution, filtration, evaporation, homogenization, mixing and sample delivery. Here, the term will primarily be used regarding the removal of endogenous compounds from the sample matrix.
- 50. Sample Preparation Strategies Table 2.1 Objectives for bioanalytical sample preparation 43 1. Removal ofunwanted matrix components (primarily protein) that would interfere with analyte determination 2. Concentration of analyte to meet the detection limits ofthe analytical instrument 3. Exchange of the solvent or solution in which the analyte resides so that it is compatible with mobile phase for injection into a chromatographic system 4. Removal of selected analyte components if the resolving power of the chromatographic column is insufficient to separate all the components completely 5. Removal of material that could block the chromatographic tubing or foul the interface to the detector 6. Dilution to reduce solvent strength or avoid solvent incompatibility 7. Solubilization ofcompounds to enable injection under the initial chromatographic conditions 8. Stabilization ofanalyte to avoid hydrolytic or enzymatic degradation Reprinted with permission from [4]. Copyright 1989 Elsevier Science. The different types of sample matrices encountered in bioanalysis may include the following: plasma, serum, bile, urine, tissue homogenates, perfusates, buffer, saliva, seminal fluid, dialysate solution, Caco-2 buffer and hepatocyte or microsomal incubation solution. Table 2.1 lists the many overall objectives of sample preparation for drug bioanalysis. Three major objectives from this list will now be described in more detail: removal of matrix components, concentration ofanalyte and solvent exchange. 1.1.1 Removal ofMatrix Components Biological samples cannot usually be injected directly into an analytical system such as LC-MS/MS because of the multitude of substances present in the sample matrix that can potentially interfere with the analysis, the chromatographic column and/or the detector. These materials include proteins, salts, endogenous macromolecules, small molecules and metabolic byproducts. If these materials were to be injected, the consequences may include the following: a rapid deterioration in the separation performance of the chromatographic column; clogged frits or lines resulting in an increased system backpressure; impaired selectivity of the sorbent in the column due to irreversible adsorption of proteins; and detector fouling that may reduce system performance and require maintenance for cleaning the source. Injection of matrix substances can also cover up and hide the drug or analyte being analyzed, making quantitation difficult (and adversely affecting the data). These materials may also coelute with the analyte of interest, falsely elevating the data. All samples can benefit from a pretreatment step before analysis in
- 51. 44 Chapter 2 order to remove interfering components and attain a selective technique for the desired analytes. 2.1.3 Concentration ofAnalyte When blood is collected from an animal or human test subject, plasma or serum is commonly isolated by centrifugation. When an anticoagulant (e.g., heparin, EDTA or sodium citrate) is added to the blood immediately upon collection, plasma is obtained following centrifugation; when blood is first allowed to clot at room temperature and then centrifuged, serum is obtained. A drug of unknown concentration is contained in the isolated plasma (or serum) sample. If one milliliter (mL) of plasma is subjected to sample preparation (interferences are removed), and the drug concentration is determined in a final solvent volume of 1 mL, then no concentration has taken place. However, if this plasma were prepared for analysis and also concentrated, by having a final solvent volume of 0.1 mL instead of 1 mL, analysis of that same aliquot volume will improve the detection limit by a factor often (1.0/0.1=10). It is the goal of many, but not all, sample preparation techniques to concentrate the analyte before analysis so that the limit of detection and quantitation can be improved. A common method to concentrate analytes is to evaporate a given solvent volume to dryness and then reconstitute in a smaller volume of mobile phase compatible solvent. 2.1.4 Solvent Exchange Pure aqueous (water based) samples cannot usually be injected into an analytical instrument because of matrix components and incompatibility with the mobile phase used in the chromatographic system (solubility and peak shape concerns). Instead, the analyte is exchanged from a 100% aqueous solution into a percentage of aqueous in organic solvent, such as methanol or acetonitrile, or into a 100% organic solvent. The final solution containing the analyte is now compatible with the mobile phase of the liquid chromatography system used for separation and detection. Solvent exchange occurs using various sample preparation techniques as described in this chapter. 2.2 General Techniques for Sample Preparation 2.2.1 Introduction Many different sample preparation techniques are available for choosing a method to perform bioanalytical sample preparation. These techniques vary in
- 52. Sample Preparation Strategies 45 many regards, such as simplicity, time requirements (in terms of speed and hands-on analyst time), ease of automation, extraction chemistry expertise, concentration factor and selectivity of the final extract. The particular method chosen depends on the requirements of the assay as well as the time involved to run the method. The investment in method development time is also a consideration. Fortunately, the bioanalytical chemist can choose from a range of sample preparation methodologies, as listed in Table 2.2. Examples of two contrasts for sample preparation requirements are drug discovery and clinical development laboratories. In drug discovery, criteria of rapid sample turnaround, little time available for method development and higher limits of quantitation are acceptable. These decisive factors suggest protein precipitation as a preferred approach. However, in clinical analysis where drugs are potent and are dosed at low levels, the important criteria of ultra sensitivity, great selectivity and a rugged method point toward solid-phase extraction as the technique ofchoice. Some useful perspectives and reviews of sample preparation approaches for high throughput LC-MS and LC-MS/MS analyses are described in the literature [2, 5-8] and in a book chapter by Rossi [9]. Other helpful but more general discussions of different sample preparation methodologies are found in various reviews [4, 10-14] and ina book chapter by Kataoka and Lord [3]. A review by Peng and Chiou [15] is recommended for the reader who would like to learn more about pharmacokinetics and the overall requirements for bioanalysis, including sample preparation and analytical method validation. Table 2.2 Typical choices of sample preparation techniques useful in bioanalysis • Dilution followed by injection • Protein precipitation • Filtration • Protein removal by equilibrium dialysis or ultrafiltration • Liquid-liquid extraction • Solid-supported liquid-liquid extraction • Solid-phase extraction (off-line) • Solid-phase extraction (on-line) • Turbulent flow chromatography • Restricted access media • Monolithic columns • Immunoaffinity extraction • Combinations ofthe above
- 53. 46 2.2.2 Dilution Followed by Injection Chapter 2 Sample dilution is used to reduce the concentration of salts and endogenous materials in a sample matrix and is commonly applied to urine. Drug concentrations in urine are usually fairly high and allow this dilution without an adverse effect on sensitivity; protein concentrations in urine are negligible under normal physiological conditions. An example of a urine dilution procedure is reported for indinavir in which 1 mL urine was diluted with 650 j.1L acetonitrile (so that the resulting concentration of organic in the sample was equal to or less than that of the mobile phase). An aliquot of 6 j.1L was injected into an LC-MS/MS system [16J. Although coeluting endogenous species in urine were not seen in the selected ion monitoring mode, their presence did suppress or enhance the ionization of analytes, leading to increased variation in MS/MS responses. Dilution is not typically used for plasma due to the high amounts of protein present and the greater effect that dilution has on sensitivity. However, dilution is very attractive for the minimal effort and time involved. One report did discuss a dilution approach for plasma in a high throughput procedure [17]. In this report, dog plasma samples were centrifuged, pipetted into wells of a microplate and then placed on an automated pipettor. A volume of 15 j.1L plasma was diluted with 485 j.1L of a solution of water/methanol/formic acid (70:30:0.1, v/v/v) containing internal standard. The samples were sealed, mixed and 5 j.1L were injected into an LC-MS/MS system. The dilution resulted in a slightly viscous solution with no observed precipitation. The limit of quantitation for the dilution assay (2 ng/mL) was 400 times higher than that of a more selective procedure that also concentrates the analyte (liquid-liquid extraction; 5 pg/mL LOQ). However, the advantage of the first technique was that the throughput was 50 times greater. In this case, throughput was a more important consideration than analyte sensitivity. 2.2.3 Protein Precipitation Protein precipitation is often used as the initial sample preparation scheme in the analysis of a new drug substance since it does not require any method development. A volume of sample matrix (I part) is diluted with a volume of organic solvent or other precipitating agent (3-4 parts), followed by vortex mixing and then centrifugation or filtration to isolate or remove the precipitated protein mass. The supernatant or filtrate is then analyzed directly. Protein precipitation dilutes the sample. When a concentration step is required, the
- 54. Sample Preparation Strategies 47 supernatant can be isolated, evaporated to dryness and then reconstituted before analysis. This procedure is popular because it is simple, universal, inexpensive and can be automated in microplates. However, matrix components are not efficiently removed and will be contained in the isolated supernatant or filtrate. In MS/MS detection systems, matrix contaminants have been shown to reduce the efficiency of the ionization process [1, 16, 18-26]. The observation seen is a loss in response and this phenomenon is referred to as ionization suppression. This effect can lead to decreased reproducibility and accuracy for an assay and failure to reach the desired limit of quantitation. Protein precipitation techniques can be performed in high throughput systems using the collection plate format and several reports of these applications are available [27-31]. Procedures for performing high throughput protein precipitation are presented in Chapter 6 along with strategies for method development. The automation of protein precipitation techniques is presented in Chapter 7. 2.2.4 Filtration Filtration is important for the removal of material or debris from a sample matrix so that the chromatographic tubing and column do not become physically blocked. For example, a precipitated protein mass can be filtered from solution and the filtrate analyzed directly [28, 32-36]. Filtration is often used to clarify raw sample matrix before another sample preparation technique (e.g., turbulent flow chromatography, discussed in Section 2.2.10) as well as for the filtration ofdebris from the final eluate before injection. Filtration in the microplate format allows high throughput sample preparation and this approach is discussed in more detail in Chapter 6, Section 6.4. 2.2.5 Protein Removal Equilibrium dialysis, ultrafiltration and other membrane based sample preparation methods are useful for protein removal in bioanalysis [13, 14, 37-40]. Equilibrium dialysis is a classical method to physically separate small molecular weight analytes from larger molecular weight constituents (e.g., protein) in a biological sample matrix. This process occurs by diffusion through the pores of a selective, semipermeable membrane and is concentration driven. Ultrafiltration separates proteins according to molecular weight and size using centrifugal force, and is thus based on a pressure differential rather than on a
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- 56. LP18022. C-18 (90) Sweet revenge. © 28Jan58; LP18029. C-19 (91) One man army. © 7Jan58; LP18026. C-20 (92) The general's lady. © 14Jan58; LP18027. C-21 (93) The imitation Jesse James. © 4Feb58; LP18030. C-22 (94) Wyatt rides shotgun. © 18Feb58; LP18032. C-23 (95) The Kansas Lily. © 11Feb58; LP18031. C-24 (96) Wyatt fights. © 25Feb58; LP18033. C-25 (97) Ballard and truth. © 4Mar58; LP18034. C-27 (99) The schoolteacher. © 11Mar58; LP18035. C-28 (100) Big brother Virgil. © 25Mar58; LP18036. C-29 (101) It had to happen. © 1Apr58; LP18037. C-30 (102) The county seat war.
- 57. © 8Apr58; LP18038. C-31 (103) Doc Holliday rewrites history. © 6May58; LP18042. C-32 (104) The underdog. © 22Apr58; LP18040. C-33 (105) My husband. © 10Jun58; LP18047. C-34 (106) The frame-up. © 3Jun58; LP18046. C-35 (107) Dig a grave for Ben Thompson. © 20May58; LP18044. C-36 (108) One. © 15Apr58; LP18039. C-37 (109) Two. © 29Apr58; LP18041. C-38 (110) Three. © 13May58; LP18043. C-39 (111) Four. © 27May58; LP18045. D-1 (112) The hole-up. © 16Sep58; LP18048. D-2 (113) Peace-maker. © 23Sep58; LP18049. D-3 (114) The bounty killer. © 30Sep58; LP18050. D-4 (115) Caught by a whisker. © 7Oct58; LP18051.
- 58. D-5 (116) The mysterious cowhand. © 14Oct58; LP18052. D-6 (117) The Gatling gun. © 21Oct58; LP18053. D-7 (118) Cattle thieves. © 28Oct58; LP18220. D-8 (119) Remittance man. © 4Nov58; LP18054. D-9 (120) King of the frontier. © 11Nov58; LP18055. D-10 (121) Truth about gun fighting. © 18Nov58; LP18221. D-11 (122) Frontier woman. © 25Nov58; LP18056. D-12 (123) Santa Fe war. © 2Dec58; LP18057. D-13 (124); Plague carrier. © 9Dec58; LP18058. D-14 (125) Kill the editor. © 16Dec58; LP18059. D-15 (126) Little brother. © 23Dec58; LP18060. D-16 (127) Reformation of Doc Holliday. © 30Dec58; LP18061.
- 59. D-17 (128) A good man. © 6Jan59; LP18300. D-18 (129) Death for a stolen horse. © 13Jan59; LP18062. D-19 (130) Last stand at Smoky Hill. © 20Jan59; LP18063. D-20 (131) Earp ain't wearin' guns. © 3Feb59; LP18065. D-21 (132) The muleskinner. © 27Jan59; LP18064. D-22 (133) Bat jumps the reservation. © 10Feb59; LP18066. D-23 (134) Truth about Rawhide Geraghty. © 17Feb59; LP18067. D-24 (135) She almost married Wyatt. © 24Feb59; LP18068. D-25 (136) Horse race. © 3Mar59; LP18069. D-26 (137) Juvenile delinquent—1878. © 10Mar59; LP18070. D-27 (138) One murder—fifty suspects. © 17Mar59; LP18071. D-28 (139) How to be a sheriff. © 24Mar59; LP18072.
- 60. D-29 (140) The Judas goat. © 31Mar59; LP18073. D-30 (141) The cyclone. © 7Apr59; LP18074. D-31 (142) Doc Fabrique's greatest case. © 14Apr59; LP18075. D-32 (143) The actress. © 21Apr59; LP18076. D-33 (144) Love and Shotgun Gibbs. © 28Apr59; LP18077. D-34 (145) Dodge is civilized. © 5May59; LP18078. D-35 (146) Little gray home in the west. © 12May59; LP18079. D-36 (147) Kelley was Irish. © 19May59; LP18080. D-37 (148) Arizona comes to Dodge. © 26May59; LP18081. D-38 (149) Dodge City—hail and farewell. © 1Sep59; LP18082. D-39 (150) The trail to Tombstone. © 8Sep59; LP18083. THE LIFE AND TEACHING OF JESUS: LESSONS 1-13, PARTS 1 & 2. American University
- 61. & Council of Churches, National Capital Area. 13 hr., sd., b&w, 16 mm. © American University & Council of Churches, National Capital Area; 17Sep60; MP11105. THE LIFE AND TEACHINGS OF SOCRATES. See PLATO'S APOLOGY, THE LIFE AND TEACHINGS OF SOCRATES. THE LIFE AND TIMES OF CLEOPATRA. See CLEOPATRA. LIFE AT THE TOP. Romulus Films. Released by Royal Films International. 117 min., sd., b&w, 35 mm. Based on the novel by John Braine. © Romulus Films, Ltd.; 1Dec65; LP33181. LIFE BETWEEN TIDES. Encyclopaedia Britannica Films. 11 min., sd., color, 16 mm. (Basic life science for the upper elementary grades, unit 5: Plant and animal relationships) Eastman color. © Encyclopaedia Britannica Films, Inc.; 30Aug63; MP13538. LIFE CAN BE MISERABLE. See MISTER MAGOO. LIFE CYCLE OF CERATOCYSTIS ULMI, THE DUTCH ELM DISEASE FUNGUS. Iowa State University of Science & Technology. 3 min., si., color, 16 mm.
- 62. © Iowa State University a.a.d.o. Iowa State University of Science & Technology; 17Dec68; MP19375. LIFE CYCLE OF THE ANT. Film Associates of California. 4 min., si., color, 8 mm. (Life science series, film no. LS/INV/2) © Film Associates of California: 28Nov66; MP16699. LIFE CYCLE OF THE DRAGONFLY. Film Associates of California. 4 min., si., color, 8 mm. (Life science series, film no. LS/INV/4) © Film Associates of California; 28Nov66; MP16701. LIFE CYCLE OF THE FLY. McGraw-Hill Book Co. 13 min., sd., b&w, 16 mm. © McGraw-Hill Book Co., Inc.; 30Dec60; MP11284. LIFE CYCLE OF THE MONARCH, A STORY OF METAMORPHOSIS. Ken Middleham Productions. 11 min., sd., color, 16 mm. Photographer: Ken Middleham. © Ken Middleham Productions; 1Jan66; MP15757. LIFE ENDEATH. Robert J. Kaplan. 7 min., sd., color, 16 mm. © Robert J. Kaplan; 24Jul67; MU7870. A LIFE FOR A LIFE. See DR. KILDARE.
- 63. LIFE FROM THE SUN. Visual Education Films. 15 min., sd., color, 16 mm. © Visual Education Films, Inc.; 8Nov60; MP10907. LIFE FUNCTIONS. See CELL BIOLOGY: LIFE FUNCTIONS. LIFE IN A CUBIC FOOT OF SOIL. Coronet Instructional Films. 11 min., sd., b&w, 16 mm. © Coronet Instructional Films, a division of Esquire, Inc.; 27May63; MP13657. LIFE IN A MEDIEVAL TOWN. Coronet Instructional Films. 16 min., sd., b&w, 16 mm. © Coronet Instructional Films, a division of Esquire, Inc.; 1Feb65; MP15076. LIFE IN A VACANT LOT. Encyclopaedia Britannica Films. 10 min., sd., color, 16 mm. (Basic life science) © Encyclopaedia Britannica Films, Inc.; 4Jul66; MP16291. LIFE IN ANCIENT ROME. Encyclopaedia Britannica Films. 14 min., sd., color, 16 mm. Eastman color. © Encyclopaedia Britannica Films, Inc.; 9Jul64; MP14298. LIFE IN ANCIENT ROME: THE FAMILY. Coronet Instructional Films. 11 min.,
- 64. sd., b&w, 16 mm. © Coronet Instructional Films, a division of Esquire, Inc.; 6Oct59; MP9868. LIFE IN GRASSLANDS (ARGENTINE PAMPAS) Coronet Instructional Films. 11 min., sd., b&w, 16 mm. © Coronet Instructional Films, a division of Esquire, Inc.; 3Apr61; MP11540. LIFE IN HOT RAIN FORESTS (AMAZON BASIN) Coronet Instructional Films. 14 min., sd., b&w, 16 mm. © Coronet Instructional Films, a division of Esquire, Inc.; 10Apr61; MP11543. LIFE IN MEDITERRANEAN LANDS. Coronet Instructional Films. 11 min., sd., b&w, 16 mm. 2d ed. © Coronet Instructional Films, a division of Esquire, Inc.; 2Jan63; MP13573. LIFE IN PARCHED LANDS. Time. 29 min., sd., color, 16 mm. (The world we live in) Derived from The desert a volume in the Life nature library. Produced by Time-Life Broadcast with National Educational Television. © Time, Inc.; 12Dec68; MP19176. LIFE IN THE BALANCE. See THE DETECTIVES. 2816. LIFE IN THE DANCE HALL: F-U-N. See
- 65. DR. KILDARE. LIFE IN THE HIGH ANDES. Coronet Instructional Films. 11 min., sd., b&w, 16 mm. © Coronet Instructional Films, a division of Esquire, Inc.; 27Apr61; MP11544. LIFE IN THE JAMESTOWN COLONY. Coronet Instructional Films. 40 min., si., color, 8 mm. (Living in early America series) NM: revision & additions. © Coronet Instructional Films, division of Esquire, Inc.; 17Feb67; MP17854. LIFE IN THE OASIS: NORTH AFRICA. Coronet Instructional Films. 11 min., sd., b&w, 16 mm. © Coronet Instructional Films, a division of Esquire, Inc.; 2Apr62; MP12594. LIFE IN THE OCEAN. Film Associates of California. 16 min., sd., color, 16 mm. Eastman color. © Film Associates of California; 22Jul55; MP13859. LIFE IN THE THIRTIES. See PROJECT 20. LIFE IN YOUR HANDS. Smith Kline & French Laboratories. 11 min., sd., b&w, 16 mm. © Smith Kline & French Laboratories; 2Dec61; MP11938.
- 66. LIFE INSURANCE—WHAT IT MEANS AND HOW IT WORKS. Visualscope. 13 min., sd., color, 16 mm. Appl. author: Stanford Sobel. © Visualscope, Inc.; 13Dec60; MP11776. LIFE, LIBERTY AND THE PURSUIT OF GEORGE APPLEBY. See THE RED SKELTON HOUR. THE LIFE MACHINE. See DR. KILDARE. THE LIFE OF A DRAGONFLY. Film Associates of California. 10 min., sd., color, 16 mm. Eastman color. © Film Associates of California; 25Feb64; MP14041. THE LIFE OF CHRIST IN FOCUS. George Mihovich & City Film Center of N.Y. 33 min., sd., color, 16 mm. Eastman color. © George Mihovich; 26May64; LP29257. LIFE OF RILEY. National Broadcasting Co. Approx. 25 min. each, sd., b&w, 16 mm. © National Broadcasting Co., Inc. After you're gone. © 29Nov57; LP25651. All American brain. © 1Feb57; LP26756. Aloha, Riley, goodbye. © 21Dec56;
- 67. LP26749. Anchors away. © 31Oct57; LP25644. Annie's radio romance. © 18Mar58; LP25663. The auction. © 6Aug54; LP26709. Babs & Junior try home economics. © 1Oct54; LP26718. Babs and the Latin. © 7Oct57; LP25643. Babs comes home. © 12Apr57; LP26765. Babs' dream house. © 22Feb57; LP26759. Babs gets engaged. © 27Aug54; LP26712. Babs moves out. © 4May56; LP25203. Babs' new job. © 18Dec54 (in notice: 1955); LP25190. Babs' used car. © 23Nov53; LP26670. Babs' wedding. © 30Dec55; LP25189. Baby Chester's first words. © 21Nov57; LP25650. The barracks bag. © 24Mar58; LP25665. Benefit for Egbert. © 16Nov56; LP26743.
- 68. The big sacrifice. © 2Sep55; LP25169. Blessed event. © 9Nov56; LP26742. The blockade. © 3Sep54; LP26714. Bowling beauties. © 15Dec57; LP25654. A bride for Otto. © 18Jun54; LP26702. Brotherhood of B.P.L.A. © 19Feb54; LP26685. Brotherly love. © 10Dec54; LP26728. Buttering up a millionaire. © 7Sep56; LP26734. Candid camera. © 18Jan57; LP26754. The car pool. © 26Nov54; LP26726. Change in command. © 28Dec56; LP26751. Chicken ranch. © 28May54; LP26699. The circus comes to town. © 2Nov53; LP26664. Come back little Junior. © 24Dec54; LP26730. The contestant. © 10Feb56; LP25206. Darling, I am growing old.
- 69. © 23Sep55; LP25172. A day at the beach. © 14Dec56; LP26747. Deep in the heart of. © 11Jan57; LP26753. Destination Brooklyn. © 10Sep54; LP26716. Destination Del Mar Vista. © 21Sep56; LP26736. The diet. © 23Jul54; LP26707. Do it yourself. © 30Sep55; LP25173. The dog watch. © 24Sep54; LP26717. Double, double date. © 7Dec56; LP26746. Down for the count. © 7Sep57; LP25639. The duck hunting trip. © 8Oct54; LP26719. Dudley comes to town. © 15Apr58; LP25667. Dudley, the burglar. © 14Oct55; LP25175. Expectantly yours. © 6Apr56; LP25199.
- 70. The famous Chester Riley, Jr. (The famous Chester A. Riley) © 4Nov55; LP25178. Father-in-law vs. father-in-law. © 19Apr57; LP26766. The first quarrel. © 31Aug56; LP26733. Foreign intrigue. © 25Jan57; LP26755. Framed. © 7Nov57; LP25646. Friends are where you find them. © 5Oct56; LP26738. From rags to riches. © 20Apr56; LP25201. Getting Riley's goat. © 8Feb57; LP26757. Ghost town. © 28Oct55; LP25177. Gillis' childhood friend. © 6Sep57; LP25638. Going steady. © 8Nov57; LP25647. Gossip. © 29Aug57; LP25636. The gruesome twosome. © 3Oct57; LP25641. A guest from England. © 19Nov57; LP25649.
- 71. The gymnasium. © 13Aug54; LP26710. Happy birthday, Little Chester. © 6Oct57; LP25642. Head of the family. © 16Dec55; LP25185. Help for Honeybee. © 29Apr58; LP25669. Here comes Constance. © 7May58; LP25670. The high cost of Riley. © 5Apr57; LP26764. His brother-in-law's keeper. © 24Feb56; LP25193. Homeless Otto. © 10May57; LP26769. Honeybee's mother. © 23Nov56; LP26744. House divided. © 22Mar57; LP26762. House for sale. © 27Apr56; LP25202. Job open. © 3Dec54; LP26727. Junior gets a car. © 13Apr56; LP25200. Junior quits school. © 20Dec55; LP25186. Junior, the chief magistrate.
- 72. © 15Oct54; LP26720. Junior wins soapbox derby. © 27Feb53; LP26642. Junior's double date. © 31Dec53; LP26683. Junior's future. © 8Mar57; LP26761. Junior's secret. © 27Aug54; LP26713. Junior's vacation. © 23Oct53; LP26662. Juvenile delinquent. © 4Jan57; LP26752. The letter. © 20Dec57; LP25656. Letter of introduction. © 26Aug55; LP25168. Light-fingered Babs. © 9Jul54; LP26705. Little Awful Annie. © 18Dec57; LP25655. Live modern. © 22Apr58; LP25668. Look, Peg, I'm dancin'. © 11Jun54; LP26701. Love comes to Waldo Binny. © 16Sep55; LP25171. A man's pride. © 26Oct56; LP26740.
- 73. The marines have landed. © 9Sep55; LP25170. Meet the neighbor. © 25Jun54; LP26703. Middle age blues. © 25Nov55; LP25181. Mrs. Aircraft Industries. © 1Dec57; LP25652. Movie struck. © 10Dec57; LP25653. Music hath charms. © 22Dec57; LP25657. The new den. © 11Sep57; LP25640. The new job. © 14Sep56; LP26735. Night shift. © 2Jul54; LP26704. Nobody down here likes me. © 14Nov57; LP25648. The oily birds. © 30Dec57; LP25659. The Otis Yonder story. © 24Mar58; LP25664. Out to pasture. © 12Feb54; LP26684. Partnership. © 2Mar56; LP25194. Pay the penalty. © 21Oct55; LP25176. Peg's birthday present. © 18Sep53; LP26657.
- 74. The price of fame. © 8Apr58; LP25666. Puppy love. © 19Nov54; LP26725. Repeat performance. © 18Nov55; LP25180. Return to Blue View. © 29Mar57; LP26763. Riley and the beaux art ball. © 5Mar54; LP26687. Riley and the boss' niece. © 10Dec53; LP26675. Riley and the cop. © 16Nov53; LP26668. Riley and the foreman's gift. © 30Apr54; LP26692. Riley and the suggestion contest. © 14May54; LP26695. Riley and the widow. © 23Dec55; LP25187. Riley balances the budget. © 18Dec53; LP26677. Riley brightens the corner. © 20Dec53; LP26678. Riley buys a statue. © 22Oct54; LP26721.
- 75. Riley buys a wrestler. © 31Dec54; LP26731. Riley cultivates Babs. © 1May53; LP26649. Riley engages an escort. © 17Apr53; LP26647. Riley faces fatherhood. © 11Sep53; LP26656. Riley faces Mother's Day. © 3Dec53; LP26673. Riley gets engaged. © 24Apr53; LP26648. Riley hears bells. © 9Oct53; LP26660. Riley hires a nurse. © 2Nov56; LP26741. Riley holds the bag. © 30Nov53; LP26672. Riley in a rut. © 9Nov53; LP26666. Riley in Brooklyn. © 24Dec53; LP26680. Riley in the wild blue yonder. © 3Sep54; LP26715. Riley invades the fight game. © 13Jan53; LP26640.
- 76. Riley meets a rival. © 29May53; LP26652. Riley meets the press. © 21May54; LP26697. Riley outwits Cupid. © 22May53; LP26651. Riley proves his manhood. © 2Jan53; LP26639. Riley, surprise witness. © 26Feb54; LP26686. Riley takes a roomer. © 7May54; LP26693. Riley takes out insurance. © 9Apr54; LP26690. Riley takes up art. © 27Dec53; LP26681. Riley teaches Junior boxing. © 11Dec53; LP26676. Riley, the animal lover. © 4Sep53; LP26655. Riley, the executive type. © 10Apr53; LP26646. Riley, the friendly neighbor. © 5Nov54; LP26723.
- 77. Riley, the heir. © 2Apr54; LP26689. Riley, the newsboy. © 23Apr54; LP26691. Riley, the tycoon. © 11Nov55; LP25179. Riley, the typical worker. © 5Jun53; LP26653. Riley, the worrier. © 27Nov53; LP26671. Riley trades his house. © 11May56; LP25204. Riley unites the family. © 7May54; LP26694. Riley versus numerology. © 29Oct54; LP26722. Riley wins a trip. © 14Dec56; LP26748. Riley's allergy. © 9Mar56; LP25195. Riley's anniversary. © 20Nov53; LP26669. Riley's burning ambition. © 30Oct53; LP26663. Riley's bursted bubble. © 10Mar58; LP25661. Riley's business venture. © 13Mar53; LP26644.
- 78. Riley's club for service wives. © 23Mar56; LP25197. Riley's family reunion. © 6Mar53; LP26643. Riley's good deed. © 26Mar54; LP26688. Riley's haunted house. © 6Nov53; LP26665. Riley's lonely night. © 19Oct56; LP26739. Riley's lost weekend. © 20Feb53; LP26641. Riley's love letters. © 23Dec53; LP26679. Riley's old flame. © 2Oct53; LP26659. Riley's raffle. © 27Dec55; LP25188. Riley's second honeymoon. © 4Dec53; LP26674. Riley's separation. © 13Nov53; LP26667. The Rileys step out. © 27Mar53; LP26645. Riley's stomach ache. © 28Aug53; LP26654.
- 79. Riley's surprise package. © 25Sep53; LP26658. Riley's surprise party. © 16Oct53; LP26661. Riley's Uncle Baxter. © 8May53; LP26650. Riley's ups and downs. © 29Aug57; LP25637. Riley's uranium mine. © 28Dec53; LP26682. Riley's wild oats. © 12Nov54; LP26724. School board critic. © 21May54; LP26698. Shower for Babs. © 9Dec55; LP25183. Singing cowboy. © 4Jun54; LP26700. Sister Cissy returns. © 14May54; LP26696. The song writer. © 17Feb56; LP25192. Stage door Riley. © 16Mar56; LP25196. The stray dog. © 26Apr57; LP26767. Strolling through the park. © 21Dec56; LP26750. The stupid Cupids. © 20Aug54; LP26711.
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