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Improvements to the Sample Manipulation Design of a LEAP CTC HTS PAL Autosampler Used for
High-Throughput Qualitative and Quantitative Liquid Chromatography−−Mass Spectrometry Assays
Dieter M. Drexler, Kurt J. Edinger and James J. Mongillo
Journal of Laboratory Automation 2007 12: 152
DOI: 10.1016/j.jala.2007.01.002
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Technical Brief
Improvements to the Sample
Manipulation Design of a LEAP
CTC HTS PAL Autosampler Used
for High-Throughput Qualitative
and Quantitative Liquid
ChromatographyeMass
Spectrometry Assays
Dieter M. Drexler,* Kurt J. Edinger, and James J. Mongillo
Bristol-Myers Squibb Company, Wallingford, CT
Keywords:
LCeMS,
autosampler,
high throughput
o produce high-quality drug candidates and to
support drug discovery decision making, compounds
are subjected to predictive in vitro and in vivo absorption,
distribution, metabolism, excretion, toxicology, drug
metabolism, and pharmacokinetics assays, in which
samples are typically analyzed using automated liquid
chromatographyemass spectrometry (LCeMS).
Depending on sample preparation and clean-up efforts,
matrix components such as salts, nonprecipitated small
molecules, and particulate matter can remain in the
sample, thus having a detrimental effect on the
performance of the autosampler, especially if the sample
manipulation mechanism is syringe-based. This issue is
amplified in high-throughput qualitative and quantitative
LCeMS-based assays due to the large sample load and
associated mechanical ‘‘wear-and-tear’’ of the syringe,
resulting in poor data quality, increased costs, and lost
T
*Correspondence: Dieter M. Drexler, Ph.D, Bristol-Myers Squibb
Company, Pharmaceutical Research Institute, 5 Research Parkway,
Wallingford, CT 06492; Phone: þ1.203.677.6340; Fax:
þ1.203.677.7702; E-mail: dieter.drexler@bms.com
1535-5535/$32.00
Copyright
c
2007 by The Association for Laboratory Automation
doi:10.1016/j.jala.2007.01.002
152 JALA
June 2007
time as a result of sample re-analysis. Described here are
improvements made to the sample manipulation design
and mechanism of a LEAP CTC HTS PAL autosampler.
This setup completely eliminates the contact of samples
and potentially abrasive and corrosive matrix components
with both the syringe plunger and barrel through the use
of a Teflon tubing loop, thus significantly extending the life
of the injection device. In our high-throughput in vitro
assay to assess the metabolic stability of compounds, we
observed a 20-fold increase in the syringe lifetime using
the improved sample manipulation design versus the
standard setup with similar analytical performance such as
reproducibility, accuracy, and precision. ( JALA
2007;12:152–6)
INTRODUCTION
Today’s eﬀorts to synthesize new chemical entities
and to discover new drug candidates occur at an
ever-increasing rate. As a result of the recent shift of
the drug development paradigm to earlier assessment
of a compound’s drug properties and characteristics
such as absorption, distribution, metabolism, excretion, toxicology, drug metabolism, pharmacokinetics,
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Technical Brief
drug safety, and pharmacological selectivity, bioanalytical
groups supporting this endeavor have to deal with a substantial
sample load.1e4 These samples from in vitro and in vivo studies
are analyzed using automated high-throughput analytical processes typically utilizing liquid chromatographyemass spectrometry (LCeMS) techniques to quantitatively measure the
disappearance or in situ appearance of a speciﬁc analyte.5,6
As a prerequisite, the analytical instrumentation (LC pump,
autosampler, LC pre-column, LC analytical column, UV
detector, and mass spectrometer) along with the analytical
methods used needs to be robust, accurate, and reliable to
reproducibly generate high-quality data. The large number
of compounds being screened does not permit extensive sample preparation and clean-up; therefore, these samples may
still contain a substantial amount of matrix components such
as salts, nonprecipitated small molecules, and particulate matter. This can affect not only the LC-UV/MS analysis and data
quality but also the analytical equipment, particularly an autosampler with syringe-based sample manipulation design and
mechanism.
As part of the high-throughput screening model at our
company, an in vitro assay is used to assess the metabolic
stability of compounds.7 After incubation of the compounds with microsomes, samples are prepared using
protein precipitation8 with a 2:1 (v/v) organic solvent (acetonitrile) to assay media ratio. The amount of the remaining non-metabolized compound in the supernatant is
quantitatively determined by LCeMS analysis. The
LCeMS system employs a LEAP CTC HTS PAL autosampler that uses a syringe to directly manipulate the samples.
It was observed that after a syringe had handled about 500
samples and analyses, the reproducibility, accuracy, and
precision of hourly ‘‘LC-UV/MS system control’’ samples
drifted outside the range of acceptable data. These samples
contain MS-active (verapamil) and UV-active (dimethoxynaphthalene [DMN]) standards, which are analyzed in duplicate to monitor the performance of the analytical setup.
Typical standard deviation values for the UV-active
(DMN) and MS-active (verapamil) standards are in the
range of 4 and 8%, respectively. Upon further investigation
using visual microscopy, it appeared that the syringe experienced abrasion of the polyethylene or Teﬂon plunger tip
and etching on the barrel most likely due to matrix components and/or mechanical ‘‘wear-and-tear.’’ This resulted in
irreproducible sample volumes, which were detrimental to
the bioanalytical assays. Using the hypothesis that the matrix was responsible for the deterioration of the syringe,
a strategy came to mind to prevent the sample from entering the syringe barrel. This report describes improvements
made to the sample manipulation design and mechanism
by aspirating the sample into a Teﬂon tubing loop and thus
eliminating any contact between the sample, its associated
matrix components, and the syringe. This modiﬁed sample
manipulation has resulted in a 20-fold increase in the lifetime of a syringe versus the standard setup with similar analytical performance such as reproducibility, accuracy, and
Figure 1. Syringe needle bending plate to bend the syringe needle 180 around the post at the bottom of the plate.
precision. Approximately 10,000 samples can now be analyzed before the loop and syringe assembly needs to be
replaced. This practical and inexpensive design without
the need for additional hardware such as switching valves
or dual-arm systems has been successfully implemented in
both qualitative and quantitative high-throughput assays
and is routinely being used.7
Figure 2. The original LEAP CTC HTS PAL autosampler syringe
assembly where the sample enters the syringe.
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JALA June 2007 153
Technical Brief
MATERIAL
AND METHODS
Instrumentation and Software
The autosampler used for the LCeMS system was a CTC
HTS PAL (LEAP Technologies, Carrboro, NC). Custom
sample manipulation software macros were programmed using PAL Cycle Composer software (V 1.5.2).
Sample Manipulation Assembly
The threaded needle holder, plunger, and magnetic syringe holders were built in-house using anodized aluminum
based on the dimensions of the commercially available
model. The syringe used was an LC syringe (22 gauge, blunt
needle point, polyethylene plunger tip, #L100.K100) from
Microliter Analytical Supplies (Suwanee, GA). The Teﬂon
tubing (inside diameter 0.025 in., outside diameter 0.06 in.,
#4E02501810) was from Zeus (Orangeburg, SC). The needle
(22 gauge, blunt needle point, #7770-02) was from Hamilton
(Reno, NV). The needle was connected to the Teﬂon tubing
using a VacuTight ﬁtting (#P846) and ferrule (#P840) from
Upchurch Scientiﬁc (Oak Harbor, WA).
The syringe needle was manually bent by 180 using a template made in-house (Fig. 1). The Teﬂon tubing was manually pushed over the needle end and the syringe tip.
HPLC-grade water and HPLC-grade methanol were obtained from EM Science (Darmstadt, Germany). The autosampler wash solvent consisted of water:methanol (80:20,
v/v).
DISCUSSION
The commercially available sample manipulation mechanism
of the autosampler consists of a robotic x, y, z-stage that
moves a removable magnetic holder with the mounted
syringe (Fig. 2) between the stationary sample vials, LC injection port, and wash stations. By vertical movement
(z-direction) of the plunger via a wormgear, solvent is aspirated into, held in, or dispensed from the syringe barrel. This
Figure 3. (a) Modified sample manipulation design where the sample does not enter the syringe. (b) Details of the modified sample
manipulation design. (c) Computer-aided design (CAD) drawing of the modified sample manipulation design (front view). (d) CAD drawing
of the modified sample manipulation design (back view).
154 JALA
June 2007
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Technical Brief
solvent, whether wash solution or sample containing matrix
components, can affect and potentially damage the barrel
and plunger tip, resulting in non-reproducible sample
volumes.
Our strategy was to implement a design that completely
eliminated the contact of the sample with the syringe. Furthermore, we wanted to maintain the concept of a removable
magnetic syringe holder by simply replacing it with our design, and thus being able to switch between standard and
modiﬁed versions within seconds.
The improved sample manipulation mechanism consists
of a standard syringe that is connected to the needle via a Teflon tubing loop. The length and consequently the volume of
the tubing can be manually adjusted so that the sample does
not enter the syringe barrel. This assembly is mounted on
a modiﬁed removable magnetic holder (Fig. 3aed) and will
be referred to as the ‘‘unit.’’ To accommodate the tubing,
the syringe is offset from the center, which is addressed by
the modiﬁed plunger holder. An example of the functionality
is illustrated by the following procedure using a custom software macro. This particular protocol is routinely used in
both qualitative and quantitative high-throughput in vitro
LCeMS assays such as the assessment of the metabolic stability of compounds.7 In this case, the modiﬁed design
resulted in a 20-fold increase in the lifetime of a syringe compared to the commercially available model with similar analytical performance such as reproducibility, accuracy, and
precision as determined via statistical data obtained by
hourly ‘‘LC-UV/MS system control’’ standards. Notably,
the volume of sample and wash solvents manipulated by
means of Teﬂon tubing and software macro as well as any
protocol action is customizable, which enables the presented
design to be applied to many different assays (Table 1).
CONCLUSIONS
An improved sample manipulation design and mechanism
for a LEAP CTC HTS PAL autosampler has been developed. The setup eliminates any contact of the sample, including potentially destructive matrix components, with the
syringe barrel and plunger tip, thus signiﬁcantly increasing
the lifetime of the syringe with analytical performance such
as reproducibility, accuracy, and precision similar to that
of the original syringe assembly. The modiﬁed unit is routinely used in high-throughput qualitative and quantitative
LCeMS assays with excellent data reproducibility, precision,
and accuracy.
Table 1. Example of a custom software macro used with the modified sample manipulation design
Step
#
0
1
2
3
4
5
6
7
8
9
10
11
Action
Unit is at home position
Start sample sequence via software
Unit moves needle into wash station
Syringe plunger ascends to aspirate 100 mL wash solvent
Syringe plunger descends to dispense 100 mL wash solvent
Syringe plunger ascends to aspirate 55 mL wash solvent
Unit moves to home position
Syringe plunger further ascends to aspirate 10 mL air
Unit moves needle into sample vial
Syringe plunger further ascends to aspirate 20 mL of the
sample
Unit moves needle into LC injection port
Syringe plunger descends to dispense 17 mL sample into
sample loop
12
13
14
2-s wait time
Injection port actuates
Syringe plunger descends completely
15
16
17
18
19
Unit moves needle into wash station
Syringe plunger ascends to aspirate 100 mL wash solvent
Syringe plunger descends to dispense 100 mL wash solvent
Unit moves to home position
Injection port actuates
Comments
Plunger is descended completely in the syringe barrel
Lubricates plunger and washes sample manipulation unit
Maintains prime of the syringe
Air plug separates sample from wash solvent
The injector port has a 5 mL sample loop; 17 mL will overfill the sample loop
three times, which improves accuracy and reproducibility in bioanalytical
assays; LC eluent bypasses sample loop (¼ offline)
LC eluent flows through sample loop (¼ online) and LCeMS analysis starts
Remaining wash solvent in the syringe barrel washes injection port with the
flow going to waste
Washes sample manipulation unit
LC eluent bypasses sample loop (¼ offline)
The subsequent sample is prepared as described above starting with step 3.
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JALA June 2007 155
Technical Brief
ACKNOWLEDGMENT
5. Ackermann, B. L.; Berna, M. J.; Murphy, A. T. Recent advances in use of
The authors would like to thank Ms. Bethanne Warrack for critical proof
LC/MS/MS for quantitative high-throughput bioanalytical support of
drug discovery. Curr. Top. Med. Chem. 2002, 2, 53e66.
reading of the manuscript.
6. Jemal, M. High-throughput quantitative bioanalysis by LC/MS/MS.
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