MICROLAB® ROBOTICS

Automated Plasma Protein Precipitation and

Filtration for LC/MS Sample Preparation with
the MICROLAB® STAR
Plasma samples are commonly analyzed
by LC/MS for the presence of drug
metabolites during pre-clinical and
clinical trials. Sample preparation for these analyses
requires precipitating the plasma protein from solution,
which is traditionally done by centrifugation. However,
this method is time consuming and does not
reliably remove all particulate matter from the cleared
plasma. 96 well format vacuum filtration is an efficient
alternative to centrifugation for plasma sample
preparation. Vacuum filtration significantly decreases
MICROLAB STAR
sample preparation time while producing cleaner filtrate
for HPLC/HPLC-MS analysis.

Hamilton Company, in conjunction with Varian, Inc.,

offers a fully-automated plasma protein precipitation
sample preparation protocol using the MICROLAB® STAR
and the Varian Captiva™ system.
Starting with plasma samples in tubes, this system
produces cleared plasma in 96 deep well microplates
ready for analysis in 21 minutes. The MICROLAB
STAR pipetting workstation offers 4, 8, 12 or 16
independent channels and Monitored Air Displacement
(MAD) pipetting technology, which ensures consistent
pipetting of viscous liquids, such as precipitated protein
samples. In addition, the liquid handling capabilities of
the STAR are highly adaptable and can accurately pipette
a range of liquids, including methanol or acetonitrile
precipitation solvents as well as the viscous precipi-
tated plasma samples. For this application, the STAR is
equipped with the MICROLAB AVS vacuum system, the
iSWAP internal gripper and the Autoload positive identi-
fication system.
The Varian Captiva 96 well 0.45µm filter plate is designed
for improved filtration, minimizing clogged wells. The
Varian filter plate uses a 7µm depth filter over the 0.45µm
filter, trapping precipitated protein throughout the 7µm
filter. In addition, the Captiva filter plate is made of
polypropylene, providing a filter with low surface activity
and low non-specific binding capacity.
R
Here we present results of plasma samples prepared
with the STAR and the Varian Captiva filter system. The
prepared samples were tested for post-filtration volume,
clarity, protein concentration and quantitative amount of
the analyte diazepam by HPLC analysis.
Equipment and Materials
Equipment
• MICROLAB STAR, 8 channels with the iSWAP plate
handler and MICROLAB AVS vacuum system
Chemicals
• Acetonitrile (Burdick & Jackson, Muskegon, MI)
• Methanol (Sigma, St. Louis, MO)
• Diazepam (Sigma, St. Louis, MO)
• Defined Bovine Calf Serum
(HyClone, Logan, UT. P/N: SH30073)
• Bovine Serum Albumin (BSA) (Sigma, St. Louis, MO)
• BCA Protein Assay Reagent Kit
(Pierce Biotechnology, Rockford, IL)
Labware
• Varian Captiva 0.45µl Plasma Protein Precipitation
96-well Filter Plate
• Hamilton Company 96 deep well collection plate
(P/N: 6471-01)
• MICROLAB STAR CO-RE disposable tips, high volume
(1000 µl), unfiltered (P/N: 235904)
• Costar round bottom 96-well plate
• Cell culture tube 12 x 100 mm
Protocol
Deck Layout
The required labware is loaded on STAR labware carriers,
including three tube carriers, one landscape microplate carrier,
one tip carrier and one reagent carrier. The labware carriers
are either manually loaded or automatically loaded with the
Autoload option, if barcode positive identification is required.
The MICROLAB AVS is integrated into the deck of the STAR. Any
plate transfers in and out of the AVS vacuum system during the
process are performed by the iSWAP internal plate-handler.
Methanol Method
• Position the AVS plate carrier over the collection plate in the
AVS elution chamber.
• Aliquot 250µl methanol into the deep well microplate.
• Using fresh tips, pipette 50µl serum sample into the deep
well plate with the methanol and mix three times.
• With the same tips, aspirate the 300µl sample/methanol
solution from deep well microplate and dispense into the
Captiva filter plate.
• Repeat the above two steps to complete the Captiva
filter plate.
• Apply vacuum at 10 inches Hg for 2 minutes to collect the
precipitate-free samples in the collection plate.
• Move the Captiva plate to the conditioning side of the AVS
to allow access to the collected samples.
• Cleared plasma samples were observed for lack of visible
precipitates, measured for volume and protein concentration
and submitted for HPLC analysis.
Acetonitrile Method
• Position the AVS plate carrier over the collection plate in the
AVS elution chamber.
• Aliquot 250µl acetonitrile into the deep well microplate.
• Using fresh tips, aspirate 50µl serum sample.
• With sample still in the tip, aspirate 250µl acetonitrile from
the deep well microplate.
• Dispense all 300µl solution into the Captiva filter plate.
• Repeat above three steps to complete the Captiva filter plate.
• Apply vacuum at 10 inches Hg for 2 minutes to the filter
plate and collect the precipitate-free samples in the
collection plate.
• Move the Captiva plate to the conditioning side of the AVS to
allow access to the collected samples.
• Cleared plasma samples were observed for lack of visible
precipitates and the volumes were measured. Protein
concentration was then measured and the samples were
submitted for HPLC analysis.
Protein Analysis
Protein concentration in cleared serum was determined
using the BCA Protein Assay Reagent Kit (Pierce
Biotechnology, Rockford, IL). Protein concentrations were
determined and reported with reference to BSA standards. The
BSA standard curve was determined using 0.125, 0.25, 0.5 and
1.0 mg/ml BSA solutions.
HPLC Analysis
HPLC was preformed using the Hamilton HxSil C18 4.6 x 150mm
5.0µl column (P/N 79868) (Hamilton Company, Reno, NV), with
acetonitrile:water (60:40) flowing isocratically for the methanol
method. The samples were detected at 254 nm.
Results
Sample Clarity and Volume
All samples from both precipitation methods were visually
observed to be clear of residual particulates from the filtration
process. The collected filtrate volumes were measured and
averaged (Table 1).
Method Volume Relative Average (µl)
Range (µl) Standard Deviation
Methanol 255.3-265.6 4.88% 259.7
Acetonitrile 255.9-270.0 5.44% 263.6
Table 1: Recovered volumes. The recovered volumes were
measured to determine the consistency of sample processing.
Protein Concentration
The protein concentrations of the cleared samples were deter-
mined to assess the efficiency of the STAR in facilitating complete
protein precipitation. The protein concentration of the plasma
prior to precipitation was11.7mg/ml. The protein concentration
intheclearedplasmafrom the methanol method was found to be
0.07mg/ml. The protein concentration in the cleared plasma from
the acetonitrile method was 0.06mg/ml. This is less than 0.6% of Throughput and Capacity
the protein concentration of the unprocessed samples.
The plasma precipitation and filtration method with methanol
for 96 samples was completed in 21 minutes. The acetonitrile
method was completed in 22 minutes. This is in contrast to
the manual centrifugation method, which requires 60 minutes
Sample Absorption Concentration for 96 samples. In addition, the STAR can automate up to four
96 well plates or 384 samples without user intervention for
Cleared plasma either method. Four plates with the methanol method can be
(methanol method) 0.124 0.07 mg/ml completed in 68 minutes, while the acetonitrile method requires
Cleared plasma 72 minutes (Table 3).
(acentonitrile method) 0.114 0.06 mg/ml

Table 2: Residual protein concentration. The protein

concentration of the cleared plasma samples was tested
to ensure sufficient protein was removed from the plasma.
Method 96 samples 384 samples
99.4% total protein was removed from the samples. (one plate) (four plates)
MICROLAB STAR with Methanol 21 minutes 68 minutes
MICROLAB STAR with Acetonitrile 22 minutes 72 minutes
Sample Recovery Manual (Centrifugation) 60 minutes 240 minutes
HPLC analysis was performed to quantitate the recovery of Table 3: Throughput and capacity. The 96 well format
diazepam. Figure 1a and b show the ration of analyte area counts to plasma protein precipitation method greatly decreases sample
processing time over the manual centrifugation method. In
standard area counts, averaged, across the columns and compared
addition, up to four plates can be processed without user
across the rows A-H. The average ratio for the methanol method intervention with the MICROLAB STAR.
was 97.8% with a relative standard deviation of 1.18%. The
average ratio for the acetonitrile method was 96.6% with a relative
standard deviation of 2.12%.
Discussion
The MICROLAB STAR with the Varian Captiva™ filter system
Figure 1a automatically performs plasma sample transfer, protein
Diazepam Recovery with Methanol Method
precipitation and vacuum filtration for 96 samples on a single
instrument in 21 minutes. With the integrated MICROLAB AVS
100.00% vacuum system and the iSWAP internal gripper, up to four plates
80.00% can be completed without user intervention, eliminating the
60.00%
manual handling required by other systems. Conducting plasma
40.00%
20.00%
protein precipitation by vacuum filtration on the MICROLAB STAR
0.00% with Varian Captiva plates yields filtrates of consistent volume,
A B C D E F G H
free of residual particulates, ready for HPLC/HPLC-MS analysis.
Rows

MICROLAB STAR Plasma Protein Precipitation Benefits

Figure 1b • Up to four plates completed in 68 minutes
Diazepam Recovery with Acetonitrile Method
without intervention

100.00% • Integrated AVS complete vacuum system

80.00%

60.00%
• iSWAP internal gripper for moving collection and filter plates
40.00%
• Flexible liquid handling capabilities for successful pipetting of
20.00%
both volatile solvents and viscous fluids
0.00%
A B C D E F G H
Rows

Figure 1a and b: The ratio of the average recovered

diazepam and the standard, compared across rows A-H.
Samples were prepared with the methanol precipitation
method on the MICROLAB STAR (a). Samples were prepared
with the acetonitrile method on the MICROLAB STAR (b).
http://www.hamiltoncompany.com/robotics Lit. No. RR-0306-03 © Hamilton Company 7/03 3k Printed in U.S.A.

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