Analytical tech in pre formulation 112070804009

Prepared by: PARTH
Guided by: Dr. Ashok Mahajan

APMC College of Pharmaceutical
education and research,
Himmatnagar

INDEX
I. Introduction
II. Need for a Preformulation study
III. Stages of Preformulation studies
IV. Analytical techniques and instruments for
Preformulation studies
V. Regulatory requirements for Preformulation
VI. Appendix: Physicochemical properties and
analytical testing for drug
VII. References

I .Introduction
 Definition: “Preformulation study is define as the process of
optimizing the delivery of drug through determinations of
physicochemical properties of the new compound that could affect
drug performance and development of an efficacious, stable and
safe dosage form.”

 Preformulation is the study of the chemical and physical properties
of the drug components prior to the compounding process of the
formulation.

 The purpose of the study is to understand the nature and
characteristics of each component and to optimize conditions of the
dosage form manufacture.

Essential information helpful in designing Preformulation evaluation of
new drug:
1. Compound identity % volatiles
2. Structure Observations
3. Formula and molecular weight 7. Analytical methods
4.Therapeutic indication HPLC Assay
Probable human dose TLC Assay
Desired dosage form(s) UV/VIS Spectroscopy
Bioavailability model(s) Synthetic route
Competitive products Probable decay products
5. Potential hazards 8. Key dates
6. Initial bulk lots: Bulk scale up
Lot number Toxicology start date
Crystallization solvent(s) Clinical supplies preparation
IND filing
Particle size range
Phase I testing
Melting point

II.Need for a Preformulation Study
 Scientific and regulatory justifications for acquiring preformulation
data include the following.
1. Establishment of drug specifications intended for toxicologic
evaluation and clinical supply preparations
2. Formulation of clinical supplies and establishment of their
preliminary Specifications
3. Providing scientific data to support dosage form development
and evaluation of product efficacy, quality, stability, and
bioavailability
4. Evaluation of the stability of early developed dosage forms
5. Fulfillment of the requirement of the CMC section of the IND
and subsequent NDA or ANDA

III. Stages of Preformulation Studies
 The preformulation is performed in several stages with different
development cycles, which are discussed in the following.

 Preformulation Report, Part 1: Physicochemical Properties and
Analytical Testing for Drugs

 Preformulation Report, Part 2: Data Supporting the
Development of Dosage Forms

 Preformulation Report, Part 3: Support for Quality Control and
Finished Product Manufacturing

IV.Analytical techniques and instruments for
preformulation studies:
 A preformulation study is performed to gain insight from
physicochemical and biological data into the design and
development of dosage forms.

 Samples are taken in each study and analyzed qualitatively and/or
quantitatively, according to the need.

 Analytical techniques are generally divided into two prevalent
areas in the specific detection and separation sciences.

Analytical Preformulation

Attribute Test
Identity Nuclear magnetic resonance (NMR)
Infra red spectroscopy (IR)
Ultraviolet spectroscopy (UV)
Thin-layer chromatography (TLC)
Differential scanning calorimetry (DSC)
Optical rotation, where applicable

Purity Moisture (water and solvents)
Inorganic elements
Heavy metals
Organic impurities
Differential scanning calorimetry (DSC)
Melting point

Assay and Separation Titration
Ultraviolet spectroscopy (UV)
High-performance liquid chromatography (HPLC)
Thin-layer chromatography (TLC)

A. Specific Detection
 Specific detection is based on specific responses related to the
chemical characteristics of a molecule excited by a certain type of
irradiation.
 In this detection method, measurement of the molecule of interest
may usually be performed without separation from matrix
materials or from other ingredients if appropriate instrumental
adjustments are made.
 Techniques such as Fourier transform IR (FTIR), attenuated total
reflectance (ATR), NIR, Raman spectroscopy are used with
increased regularity.
 The detection of foreign metal contaminants is essential with
inductively coupled plasma spectroscopy (ICP), atomic absorption
(AA), and X-ray fluorescence.

1.UV spectroscopy
 UV absorptions are mainly electronic in nature and are associated with
resonating structures in the molecule.
 The UV quantitative determination, generally performed in solution, is based
on the Beer–Lambert law.
 In a Preformulation study, Certain UV techniques are worthy of discussion:
 Solubility,
 Dissolution rate,
 Molecular weight
 pKa
 Assay (potency)
 Mixtures:
 - resolving compound products
 Stability studies : hydrolysis, oxidation ,when degradation products
have a different absorption maximum from the parent compound

 ionization of benzoic acid affect its aqueous solubility, is also manifested in the
ultraviolet absorption spectrum.
 Figure shows the UV spectra obtained for a 5 mg/mL solution of benzoic acid in
methanol (i.e., the protonated form) and for the same concentration of substance
dissolved in 0.1N sodium hydroxide solution (i.e., the ionized form).
 The spectrum of the protonated form is dominated by the peak at 228nm (molar
absorptivity =11,900 L/mole), while the analogous peak of the ionized form is
slightly blue-shifted to 225nm and is significantly less intense (molar absorptivity
¼8640 L/mole)

2.IR Spectroscopy
 Used for fingerprint identification of a drug molecule and the proof
of its structure.
 IR absorption bands are characteristic of the functional group of a
molecule as well as the structure configuration.
 The wavelength of the IR spectrum is 750–2500 μm.
 The sampling preparation techniques for IR determination are
solution, drug dispersion in a KBr pellet, Nujol mulls, and
direct determination by microscopic ATR preparation.
 An example of modern IR equipment is FTIR, which gives
better quality determination.

Analytical tech in pre formulation 112070804009

3.Raman Spectroscopy
 When a particle is irradiated at a certain frequency, radiation
scattered by the molecule contains photons of the same frequency
as the incident radiation and may contain photons (weak signal)
with a changed or shifted frequency.
 A nondestructive tool and requires little or no sample preparation.
 A sample may be analyzed in solid or powder form or in an
aqueous solution and placed in glass containers such as an NMR
tube, GC vial, test tube, light-path cell, or glass bottle.
 Aside from structure elucidation and functional group analysis,
FT-Raman may be used for quantitative determination of
polymorphs in a Preformulation study.

4.NIR Spectroscopy
 NIR is making significant progress through recent advances in
pharmaceutical analysis.
 The advantage of this technique is the rapidity of analytical
determinations without sample preparation and the use of
solvent.
 The NIR spectrum is primarily related to the overtone
variation. Hence, the absorption bands are generally weaker
than those in the IR.
 The wavelength of the NIR spectrum is defined as 2500–3000
μm.
 The detection method is nondestructive.
 Therefore, it is suitable for use in on-line monitoring and meets
100% inspection requirements in quality control practice.

5.X-Ray Diffraction
 obtains information on substance structure at the atomic
level.
 This technique allows measurement of both crystalline and
noncrystalline materials.
 The analysis is nondestructive in nature and handles
samples in the form of powders, solids, and liquids.
 The X-ray diffraction of a single crystal is employed for the
determination of the absolute chemical structure.
 Quantitative ratios of two polymorphs and their
percentage of crystallinity may also be determined.

 the XRPD pattern of benzoic acid is shown in Figure
 One may define this particular crystal form by the angles of the five
most intense scattering peaks, namely 8.15, 10.21, 16.24, 17.20, and
21.67 degrees 2q. Through use of the Bragg equation ,nzλ= 2d sin θ,
calculated d-spacings for the five most intense scattering peaks,
namely 10.840, 8.657, 5.453, 5.151, and 4.098 A ° , actually constitute a
better definitionof this particular crystal form.

6. NMR Spectroscopy
 NMR involves the absorption of electromagnetic radiation in the
radiofrequency of a longer wavelength spectrum.
 When a sample is placed with atomic nuclei of hydrogen (1H,
protons), fluorine (15F), or phosphorous (31P) in a magnetic field,
absorption of energy will occur.
 The nuclei shift from the preferred orientation with lowest energy
to a less preferred, high-energy orientation at a particular
frequency.
 Thus a plot of frequency versus intensity of radiation results in the
NMR spectrum of a material.
 Spectra of NMR can be obtained in liquids or in solids.
 NM R spectra gives information about structure and atomic
environment of molecule,

 It consist of resonance bands associated with the carbon
atoms in the aromatic nucleus and the carbon atom of the
carboxylic acid group.

7. Mass Spectroscopy
 Mass spectra is the result of detection of charged particles or
ions separated according to their mass to charge (m/e) ratio
after ionization and acceleration through magnetic field.

 Mass spectra gives information about molecular weight of
substance and what its degraded or metabolic products will be.

 MALDI like techniques are employed for high molecular
weight substances like certain proteins.

8. Metal analysis
a) Atomic absorption spectroscopy
b) ICP Spectroscopy
c) X-Ray fluorescence
 Pharmaceutical compounds such as ferrous sulfate, ferrous
gluconate, zinc undecylenate, and magnesium stearate (a
commonly used excipient)
 Sodium, potassium, zinc detection for certain preparations like
protamine zinc insulin etc.
 presence of metal in pharmaceuticals, even in trace amounts, is a
form of contaminant. For example, metallic ions may act as a
catalyst in oxidation that may be detected in drug products.

B. Separation Sciences
1. Thin-Layer Chromatography
 impurity profiling in drug development
 Involves most convenient, least inexpensive and portable
equipment
 microscopic technique (it uses a very small sample) is
simple and has a short development time
 general detection technique is to spray a sample with a
detecting agent, which reacts chemically with the
ingredient to be detected or visual observation under
short- or long-wave UV light is also employed.
 The disadvantages of TLC include reproducibility,
detection inconsistency, person-to-person variations,
documentation, and electronic data reduction.

 HPTLC:
 to overcome some drawbacks of TLC, especially in
quantitative determination, A high-performance instrument
has been developed with
 a fully automated sample applicator,
 a solvent-developing and -evaporating chamber,
 a precision-made dryer,
 a color developing agent
 sprayer,
 a light control chamber for visual or photographic
observation, and a reflective spectrophometric detector
 Ingenious methods of quantitative determination are available
that use a flame ionization detector (FID)

2. High-Pressure Liquid Chromatography
 HPLC is used extensively in the laboratory for quantization of
drugs and related components. Identification of a drug
component can simultaneously be determined by retention
times in the chromatogram.
 reliable analytical tool for Preformulation study because of
the high-resolution capacity, accuracy, and reproducibility of
the equipment
 Its primary function includes
 search for and detection of impurities in drug substances,
 stability evaluation of dosage forms in terms of detection
and quantization of degradation products.
 UV detector coupling with Micro-bore HPLC equipment is the
most important analytical instrument for Preformulation

3. Capillary Electrophoresis:
 A separate technique employing narrow-bore tubes (10–200
μm i.d.) for high-efficiency resolution of both large and small
molecules
 In free solution capillary electrophoresis (CE), the separation
and migration of the molecules through the capillary are
based on electrophoretic migration (based on net charge)
and electrosomotic flow (the bulk flow of electrolyte buffer)
 Other mechanisms for separation depend on molecular size,
isoelectric focusing, and hydrophobicity
 Modification of CE is micellar electrokinetic
chromatography (MEKC), widely used for the separation of
nonpolar compounds

4. Gas Chromatography:
 GC is used for speedy separation or for high-resolution
separation of volatile or thermal labile substances. GC has
good sensitivity, with detection limits of 1 ppb to 100 ppm.
 With the advances in HPLC, GC is utilized less often. It is still
used for the analysis of retained solvents, such as the USP test
for volatile organic solvents.
5. Ion Chromatography:
 Ion chromatography is a modified version of HPLC with a
capacity for precise and highly sensitive detection of inorganic
ions in a complex matrix.
 IC has instrumental configurations similar to those of HPLC,
but the stationary phase is an ion-exchange column, and the
detector can be either an electrochemical detector or a
colorimeter with a mixer to carry out color formation by
chemical reaction with the detected ion

6. Supercritical Fluid Chromatography:

 SFC uses highly compressed gas above its critical
temperature and pressure instead of an organic solvent
as the solvent phase
 Gases such as carbon dioxide, nitrous oxide, and
ammonia are commonly used
 The SFC detecting systems are those commonly used in
GC, that is, FID.
 Major advantage is allowance in the analysis for thermal
unstable compounds

C .Thermal Analytical methods
1) Differential Scanning Calorimetry
2) Hot Stage Microscopy
3) Thermal Gravimetric Analysis
4) Solution Calorimetry
 DSC is a precise method of measuring the endothermic and
exothermic behaviors of sample materials.
 TGA measures the weight change (gains and losses) as a
function of temperature or time is recorded which provides
information about the material’s thermal stability and
compositional analysis (e.g., moisture content of the
materials).

 The gas evolved during the heating process may be
detected with FTIR or MS to provide additional
information.
 TGA may be used to determine moisture content related to
weight loss in isothermal or nonisothermal stability
studies.
 Preformulation study, differentiation of polymorph from
hydrate or identification of monohydrate from among
other hydrates by DSC alone may not be possible.

 The DSC thermogram of benzoic acid is shown and is seen to consist
 entirely of an endothermic transition associated with the melting
phase transition of the compound.
 No thermal events were observed at the lower temperatures
indicative of the existence of a solvatomorphic crystal form.
 Under the conditions of measurement, the melting endothermic
transition is characterized by an onset temperature of 121.9C, a peak
maximum of 123.7C, and an enthalpy of fusion equal to 138.9 J/g

V. Regulatory requirements for Preformulation
A. Regulatory Compliance:
 FDA initiatives and other government regulations influence
pharmaceutical manufacturing operations, including
Preformulation studies and quality control systems.
1) Current Good Manufacturing Practices:
 The cGMP is an FDA mandatory quality program designed to
ensure that pharmaceutical products are consistently produced
and controlled according to the quality standards appropriate
to their intended use.
2) Good Laboratory Practice:
 GLP covers research activities like raw data,
documentation, standard operating procedures (SOP),
protocols, final reports, and specimens (with some
exceptions) must be retained.

3) International Conference on Harmonization:
 The ICH is intended to avoid duplication efforts for product
registration and manufacturing in world trade from the United
States, the European Union, and Japan to harmonize regulatory
criteria and procedures.
 Ultimately, there will be one set of global requirements. Area of
interest are
 Stability testing
 Quality specification (including impurities)
 Validation of manufacturing procedures and analytical
methods
 CMC sections for product registration
 Toxicity testing
 Clinical testing of biotechnology-derived products

B. Quality Control for a Preformulation Study
 Personnel Qualification and Training
 Analytical Method Validation
 Written analytical procedure
 Instrument calibration
 Validation parameters: accuracy, precision, linearity,
sensitivity
 System suitability criteria: the minimum acceptable
performance criteria before each analysis
 Documentation and Standard Operating Procedures

APPENDIX 1: PHYSICOCHEMICAL PROPERTIES AND
ANALYTICAL TESTING FOR DRUG SUBSTANCE
Chemical Structure
Empirical Formula Molecular Weight
 Lot # of Drug Used: Reported By:
 Assay: Position:
 Reference: Notebook # Date of Report Issued:
1. Chemical Properties 2. Identification of Drug Substance
Chemical structure UV
Molecular weight IR
Empirical formula NMR
Elemental analysis (C, H, N, O, Cl, etc.) Mass spectroscopy
TLC Rf and HPLC retention time
Melting point

3. Titration Methods 6. Synthetic Impurities
Nonaqueous titration with curve  Starting materials a: name and
Other titration methods structure
4. Chromatographic Techniques  Pivotal impurities b: name and
and Method Description structure
TLC  Degradant from synthesis c: name and
HPLC structure
GPC  Other minor impurities d: name and
Others structure
5. Proposed Assay Methods for  Description of Method of Detection
Drug Substance 7. HPLC Data
Titration Impurities Retention Time
 UV 1.
 HPLC 2.
 GC 3.
 Others Typical chromatogram attached
Description 8. Optical Rotation
Typical spectrograph attached (Figure )
Comments

9. Solubility 11. Partition Coefficient
 Solubility in water and organic solvents: Value:
mg/ml System:
 Aqueous Solubility as a Function of 12. Loss on Drying
Temperature Drying temperature:
Temperature ( C) Solubility :mg/ml Time period:
Aqueous Solubility at Various Buffered pH Condition: _ in oven, _ in vacuo
Values LOD in percent:
pH Solubility (mg/ml)
Buffer system
Solubility with Surfactants
Surfactant/Concentration Solubility (mg/ml)
10. Dissociation Constant, pKa
pKa value:
Method of determination:

VII . References
1. Satinder Ahuja, Stephen Scypinski, Handbook of Modern
Pharmaceutical Analysis, pp173-233.
2. Leon Lachman, Herbet A.Lieberman, A theory and practice of
Industrial Pharmacy, special Indian edition -2009,pp 171-196
3. M.E.Aulton, Pharmaceutics The science of Dosage Form
Design, Second edition, pp113-138
4. Gilbert S. Banker, Christopher T. Rhodes, Modern
Pharmaceutics, Fourth edition, Marcel dekker,Inc.
5. Moji Christianah Adeyeye , Harry G. Brittain,
Preformulation in solid dosage form development, Informa
healhcare Inc.-2008,pp1-15,115-145.

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