Automated Chromatographic Solid-Phase Extraction Using an Autosampler _ American Laboratory

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Automated Chromatographic SolidPhase Extraction
Using an Autosampler
Thursday, September 1, 2016
Print
Mark Hayward
Jonathan Ho
Rick Youngblood
Matthew T. Hardison
Tom Moran
Kim Gamble
Solidphase extraction (SPE) is a preferred tool for isolating target analytes from complex matrices because the
availability of a diverse range of chromatographic sorbents enables targeted approaches based on the specific
chemistry of the analytes and matrices. Also, SPE offers the ability to enrich or preconcentrate analytes in samples.
Enrichment is valuable because it allows one to match the analyte concentrations to the approach used to measure
them. Given these unique capabilities, SPE is considered the “gold standard” in analytical sample preparation.
SPE using a cartridge on a vacuum (or pneumatic pressure) manifold requires proper training, as care must be taken
to maintain appropriate liquid flow in real time. As a result, the number of samples that can be processed is limited to
30 per day per technician. If enrichment is needed, a sample drydown step is required, which reduces throughput.
A parallel approach with tubes, pipet tips, cartridges or 96well plates increases the number of samples, but at a cost.
Parallel liquid flow is highly variable from tube to tube or well to well. Increased variability in parallel flow results in
additional variability in analyte recovery, requiring the use of internal standards and acceptance of lower quality,
specifically, higher LLOQs (lower limits of quantification) and inconsistent assay results. Also, this approach only
addresses sample/solvent flow over the SPE sorbent—not pipetting sample/solvents to each well. Thus, going parallel
only modestly increases sample throughput, but also the probability of human error due to the greater number of
pipetting steps.
To increase SPE throughput, a parallel approach can be coupled with robots to perform the pipetting (robotic SPE,
then LC/MS/MS or GC/MS/MS measurement); however, results are lower in quality (for variable parallel flow reasons
given above) in this twoworkflow process. Another drawback is the additional cost for robots ($200–500k),
programming/integration and technicians with robotic experience. From a results point of view, this seems to mirror the
“speed, cost, quality triangle,” where gains in any one of the three take away from the other two. Breaking through the

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“speed, cost, quality triangle” requires fundamental change in the SPE device to enable both automation and higher
quality using only existing analytical instrumentation instead of high cost robots.
Introduction of precisioncontrolled microSPE using the autosampler
Central to automated, rapid, and higherquality SPE is a patented, singleuse micro cartridge (ITSP, Hartwell, Ga.)15
(Figure 1) containing userdefined, packed chromatographic media. The crimpedon septum and needle guide (upper
80% of cartridge) enable automation by facilitating accurate cartridge transport on a syringe needle. Automated SPE
begins by using the autosampler syringe for cartridge conditioning, sample loading and sample washing over a waste
receptacle. The syringe is used to perform elution over a clean vial or well, and the used cartridge is discarded in a
different waste receptacle. Automation is complete after the syringe mixes the freshly eluted sample and then injects it
into the LC/MS/MS or GC/MS/MS that will be used to measure the sample (see the CTC/PAL autosampler in Figure 2).

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Figure 1 – Internal view of an ITSP SPE singleuse cartridge and how it interfaces with the PAL autosampler
syringe and needle support. This facilitates all SPE cartridge transport and delivery of sample and solvents.

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Figure 2 – PAL RTC autosampler (CTC Analytics, Lake Elmo, Minn.) equipped for online SPELC/MS/MS.
Hoses connect the SPE cartridge tray and syringe wash station to a laboratory solvent waste container.
Used cartridges are typically discarded by the PAL into a box under the wash station and LC valve.
This simple, automated, singleworkflow process integrates SPE directly into the LC/MS/MS or GC/MS/MS software as
an online workflow, and does not require additional skills beyond those needed to operate the LC/MS/MS or
GC/MS/MS. It is simply a different method selection in the MS software already in use with any LC/MS/MS or
GC/MS/MS equipped with a PAL autosampler.
Figure 3 – Execution timeline for SPE in parallel with LC/MS/MS for the

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analysis of 71 drugs in urine. In this process, overnight measurement of
two 96well plates of samples per LC/MS/MS is routine and results are
ready for review when the lab opens in the morning.
In the singleworkflow procedure, SPE and LC/MS/MS (or GC/MS/MS) are performed serially, but in parallel with each
other (see Figure 3). As shown, cycle time is frequently a function of the LC/MS/MS (or GC/MS/MS) measurement and
not the SPE. With this form of parallelism, the only cost in time is SPE of the first sample. By combining the processes it
is considerably faster than the twoworkflow process and requires nothing more than a good initial choice in
autosampler in order to achieve complete automation.
The top of the ITSP microSPE cartridge enables precise automation, while the bottom of the cartridge performs
chromatographic SPE, cleaning and enriching the sample to produce highquality results and maintaining the
LC/MS/MS or GC/MS/MS to ensure longterm, robust operation. As shown in Figure 1, the syringe needle is placed at
the frit containing the packed chromatographic sorbent to transfer sample, without significant dispersion, directly to the
packed sorbent. Flow of the sample and solvents is positive liquid pressure, syringe pumpdriven (flow adjustable at a
resolution of ±10 nL/sec) and is precise and accurate. The extracolumn volume of the cartridge (Figure 1, below
sorbent) is just 16 μL.
Elution volumes of 50–100 μL facilitate control of the analyte concentrations delivered for measurement. As a result,
sample volume can be adjusted to match the capability (sensitivity) of the measurement instrumentation and the
necessary cutoffs (LLOQs, S/N ≥20) for proper interpretation of test results. This is achieved without the customary dry
down step used with vacuum or pneumatically driven flow forms of SPE, which tend to require ≥5× larger elution
volumes.
This concept is best illustrated with common, inuse assays. For broadpanel drug measurement in urine samples
using a midrange LC/MS/MS, 225 μL of sample is loaded onto the SPE cartridge and is then eluted with 75 μL. This
3× analyte enrichment achieves the necessary 1ng/mL cutoffs for lowdose drugs and produces LC peaks for all
drugs—sufficiently intense for automatic integration across the concentration ranges observed in real samples. For
measurement of the same broadpanel drug assay in oral fluid samples, 1 mL of sample is loaded onto the SPE
cartridge and is then eluted with 75 μL. This 13× enrichment provides the 0.2ng/mL cutoffs for lowdose drugs. For
measurement of priority pollutants in drinking water, 10 mL of water sample is loaded onto the SPE cartridge, followed
by 50 μL elution to achieve a 200× analyte enrichment and LLOQs at or near singledigit partspertrillion levels. In all
of these examples, the analytes are ready to measure immediately after SPE without performing drydown of the
eluent.
The ITSP microSPE cartridge provides accurate syringe pump flow control over the packed sorbent bed, allowing
SPE separations to be performed at their van Deemter optimum velocity6
(see Figure 4). This finding was
unanticipated, because with singleuse SPE devices it is expected that lower flow7
yields higher recovery. The data
clearly shows that accurate flow control and optimization are equally important for SPE and LC. With method
optimization experiments performed in the same ways as LC, SPE recoveries are systematically high and precise (not
common with other forms of SPE, particularly for ion exchange and chelation mechanisms). The accuracy and
variance in the final test results depend most on the LC/MS/MS or GC/MS/MS measurement (results CV = 3–5%)
rather than the recovery of SPE sample preparation performed by the CTC PAL autosampler (Figure 2).

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Figure 4 – van Deemter curves for reversedphase (C18 endcapped) SPE using two different particle
diameters (10 and 50 μm). The image also shows the source of variable recovery with vacuum or
pneumatically driven flow, because as flow drifts away from the optimum, recovery suffers. Δ%Recovery
squared (relative variance, σ2, in the amount of sample recovered) is the dimension plotted on the yaxis; the
numbers indicated on the yaxis are absolute %recovery (not adjusted based on IS) as an aid to the reader.
This is analogous to the %RSD approach to determining plate height described by Neue,6 but is used here
only for flow optimization.
Additional capabilities
Automated method development/optimization experiments can be carried out for multiple sorbents and solvents as a
series of 5–6 run lists (Design of Experiments [DOE]), where each list provides optima fed into the next list. The sum of
these lists (including measurement/elimination of breakthrough of all condition, load and wash steps; flow
optimization; and a sample loading study) can be measured in three days to result in a highly optimized SPE method.
ITSP SPE achieves precise chromatographic separations, preconcentration of sample (without drydown), robust
operation and total automation.
PALbased SPE does not limit one to a single dimension of SPE. For lipid profiling, uncharged oils can be isolated
from fatty acids and phospholipids using anionexchange SPE. The phospholipids can then be isolated from the fatty
acids using chelation in a second SPE step (easily achieved with a PAL autosampler). Finally, each of the three
isolated samples (oils, fatty acids and phospholipids) can be directed to the LC/MS/MS or GC/MS/MS measurement
approach best suited for that lipid class. Similarly, for proteomics, reversedphase SPE can be used for desalting,

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followed by a second chelation SPE to isolate peptides from phosphopeptides for separate LC/MS/MS measurement
of the large numbers of peptides. In these examples of 2D SPE, the PAL is often operated as a standalone sample
preparation device; multiple instrument types and/or methods can process the different compound classes for the
same samples in parallel. This is also useful in applications in which the analytical measurement time significantly
exceeds the SPE time. For example, in the GC/MS/MS measurement of large pesticide panels in food, one standalone
PAL autosampler can use SPE to clean up enough QuEChERS extracts for continuous, aroundtheclock
measurement by five GC/MS/MS systems.8
Conclusion
A novel microSPE device offers highly effective automation using only existing analytical instrumentation while
simultaneously improving SPE performance.
References
1. Gamble, K. U.S. Patent 6969615; 11/1/2005.
2. Gamble, K. and Martin, W. EU Patent 1174701; 09/11/2007.
3. Gamble, K. CDN Patent 2316648; 7/4/2004.
4. Gamble, K. and Martin, W. U.S. Patent 7001774; 2/21/2006.
5. Gamble, K., Fitzgerald, R. U.S. Patent 7798021, 9/21/2010.
6. Neue, U.D. HPLC Columns: Theory, Technology, and Practice; Wiley VCH: Weinheim, 1997, p 13.
7. Jordan, L. Automating a solid phase extraction method. LC·GC 1993, 11, 634–8.
8. Lehotay, S.J.; Han, L. et al. Automated minicolumn solidphase extraction cleanup for highthroughput analysis of chemical
contaminants in foods by lowpressure gas chromatography—tandem mass spectrometry. Chromatographia 2016, 79, 1113–30.
Mark Hayward, Rick Youngblood and Kim Gamble are with ITSP Solutions Inc. (ITSP), 10 South Carolina St., Hartwell,
Ga. 30643, U.S.A.; tel.: 7063958300; email: mark.hayward@itspsolutions.com; www.itspsolutions.com. Jonathan Ho
and Tom Moran are with Shimadzu Scientific Instruments, Somerset, N.J., U.S.A. Matthew T. Hardison is with
Assurance Scientific Laboratories, Bessemer, Ala., U.S.A.
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