Pipetted Liquid Sampling
Automated sample
preparation platform
for online LC-MS/MS
bioanalysis
Integrated Sample Processing
Whole Blood
Whole Blood
Centrifugation
Conventional example SCAP procedure
Application of Sample
Elution of Sample Matrix
Elution of Analyte(s)
Concentration of Extract
Conditioning of
Extraction Column
Injection into
HPLC - System
Injection into SCAP
HPLC - System
SCAP PLS system components mounted on a robotic platform
Integrated Sample Processing
Both the healthcare and pharmaceutical industry do a tremendous number of analyses of
substances in biological uids, of which blood
is one of the most common. In the healthcare
industry, complete measurements are done to
diagnose patients to determine the efcacy of
administered drugs or to screen for early
detection of diseases. In the pharmaceutical
industry, measurements are done during the
development process of new drugs (e.g. PK/
TK studies, etc.).
Whole blood is a difcult, complex matrix to
handle. In order to determine and quantify
substances in blood, various intermediate
preparation steps are required (e.g. Blood
Spots, Dialysis, Liquid-Liquid Extraction, Precipitation, (Hemo)Lysis, Membrane Filtration,
Anticoagulation). The major drawback of all
these protocols is the time consuming, error
prone and labour intensive nature of manual
sample pretreatment. The SCAP PLS system
from Prolab GmbH represents a unique approach for biouid analysis and eliminates the
drawbacks of current methods. It delivers a
fully integrated workow from sample collection until the nal result without any manual
sample preparation. A newly designed disposable SCAP PLS system sample cartridge is used
for clean and safe blood collection.
The SCAP PLS cartridge is used for sample
collection directly at the subject. Transferred to
the SCAP PLS system it serves afterwards as a
disposable sample loop prior to the following
automated sample preparation steps.
Comparison of conventional and
SCAP PLS system sample preparation
Advantages
Fully automated sample processing of biouids including whole blood samples
Integrated workow from sample collection
until the nal analysis result
One step sample manipulation, simplies
sample logistics, avoids sample mix-up
Requires a minimum amount of sample for
quantitative results (typically 5 µl)
Minimized infection and contamination risk
due to completely sealed and disposable
sample containers
No re-used syringe or sample loops.
Disposable sample container, eliminates
any carryover
Simplies method development, use of
same generic method for different matrices
Fully automated process saves time and
labour costs versus traditional sample
preparation
Saves additional costs for SPE materials,
solvents and other consumables
Aspirate sampleAttach tip to pipette
Attach and seal with cap
Electronic
pipette
Tip
Insert sample cartridge
into SCAP PLS system rack
Aspirate sample volume
directly from subject
Tip Electronic
pipette
Seal with cap
Aspirate ISTD
Anticoagulant
Esterase inhibitor
Attach tip
to pipette
Insert sample cartridge
into SCAP PLS system rack
Smart Sample Collection
Left: Assembled SCAP PLS system sample cartridge
Right: Numbered SCAP PLS system tip and cap
Sample Collection Process
The SCAP PLS system sample collection process is as easy as any other standard pipetting
task. The SCAP PLS system tip is attached to
an electronic pipette and the desired sample
volume is aspirated. The lower tip end is
sealed with a SCAP PLS system cap. The completely sealed cartridges are then placed into
the 54 position SCAP PLS system rack and are
ready for immediate analysis. Alternatively the
lled sample racks can be stored in a freezer
for later analysis or may be shipped to an
external location.
Sampling of supernatant (e.g. serum, plasma)
or whole blood involves some additional
pipetting steps. First an anticoagulant and if
desired an esterase inhibitor are aspirated.
Additionally the sample may be spiked with
an appropriate internal standard. Finally
the biouid is aspirated and the cartridge is
sealed.
Loaded sample cartridges inserted in the SCAP PLS system rack
SCAP PLS system Standard Sample Collection
SCAP PLS system Biouid Sample Collection
In case of whole blood, samples are directly
collected at the subject (e.g. nger, vein,
heel, ear lobe) and are ready for immediate
analysis without any further manual sample
preparation steps.
Efcient LC-MS/MS Workow
Sample collection
Sample introduction
into flow path
Cartridge transfer
Sonication
Online
extraction
Separation
Cartridge disposal
Cartridges placed into
SCAP PLS system rack
SCAP PLS system robotic gripper tool picks-up sample
cartridge…
…and places it into the SCAP PLS system clamp module… …where it is pierced at both ends and ushed into the
SCAP PLS system Sample Preparation Process
The SCAP PLS system samples directly from
disposable cartridges without prior solid-phase
extraction or any other sample desalting/preprocessing step. After placing the cartridges
into the SCAP PLS system sample rack the
samples may be stored in a freezer, shipped
to another location or are directly analyzed.
The cartridges are automatically picked-up by
the autosampler gripper tool and are inserted
into the clamp module. Both ends of the cartridge are pierced to eject the sample into the
analytical ow path. The sample passes
rst through a pulsed ultrasonic module,
where the blood cell components are disintegrated and homogenized. This patented technology avoids any clogging of the uidic path
or the SPE pre-columns. Pre-columns may be
used up to 1000 extractions before exchange.
Subsequently the analyte reaches a restricted
access material column (RAM) to remove
selectively higher molecular components such
as proteins. The trapped lower molecular compounds are eluted again and nally separated
on the analytical LC column.
analytical ow path.
The SCAP PLS system is delivered with a
ready to go control software, which triggers
all necessary auxiliary hardware and valve
switching operations. It can be interfaced to
any lead- ing mass spectrometer and data
acquisition system.
Minimized Sample Volume - Maximized Return
Protein
Analyte
Valve module SCAP PLS system ultrasonic module SCAP PLS system waste box
Clean Sample Introduction SCAP PLS system Valve module
In contrast to current automated HPLC sample
introduction technology the SCAP PLS system
works with an exchangeable sample loop.
Each sample is prepared and processed in a
disposable SCAP PLS system cartridge which
is discarded after sample injection. No glass
syringes, injection ports and time consuming
wash procedures are involved. This ensures
fast and virtually carry-over free operation.
For convenient sample handling, the SCAP PLS
system cartridges are delivered in 54 position
tip boxes. To load the tips a conventional
pipette is used. Prepared cartridges are completely sealed and may be stored or directly
analyzed.
The valve module consists of up to 3 valve
drives and connects the clamp module
containing the disposable sample cartridge to
the analytical separation column. Depending
on the application the drives are equipped
with up to 3pcs. 6-port valves for regular HPLC
or UPLC operation. Only one valve is used if
the sample is injected without prior online
sample preparation. The two valve conguration enables a regular pre-column solid phase
extraction step.
Three valves are recommended if a dual
stage cleaning procedure on a RAM and an
additional SPE pre-column is performed. All
necessary valve switching operations are
controlled by pre-stored timetable events and
may be optimized for the individual analytical
method. Optionally the valve module can be
equipped with high pressure valves for UPLC
operation up to 20’000 psi.
RAM Column
RAM columns allow the direct extraction and
enrichment of hydrophobic, low molecular
analytes from untreated samples such as
haemolysed blood, plasma, serum, milk, fermentation broth, supernatants of cell cultures
as well as food homogenates. It allows fully
automated preparation of the sample prior to
the analytes being separated in the analytical
column. This allows the untreated biouid to
be directly injected without negative effects
on either the column or the results achieved.
Cartridge preparation with electronic pipette
Restricted Access Material principle
Single Valve Option
V1
Waste
Waste
Sample Cartridge
to MS
HPLC Gradient Pump
Analytical Column
Sample Cartridge
V1
V2
Waste
Waste
Waste
SPE or RAM Column
HPLC Gradient Pump
HPLC Loading Pump
to MS
Analytical Column
Sample Cartridge
Waste
Waste
V1
V2
V3
Waste
Waste
RAM SPE Column
SPE Column
Ultra Sonic Module
Dilutor Pump
to MS
HPLC Loading Pump
HPLC Transfer Pump
Analytical Column
HPLC Gradient Pump
The single valve conguration shows how
a sample is loaded directly onto an analytical column without prior sample preparation
steps. Thereby the SCAP PLS system cartridge
acts as a disposable sample loop and is automatically exchanged after each injection. This
setup is limited to a system pressure of 2’500
psi (170 bar)
Dual Valve Option
The dual valve conguration is used if prior
to the separation on an analytical column a
classical SPE cleaning step is required (e.g.
serum, plasma samples). For higher throughput the SPE column is regenerated during the
analytical run. Each sample is introduced into
a disposable SCAP PLS system cartridge which
is automatically exchanged before each run.
No syringes, dilutors, injection ports or time
consuming wash procedures are involved. This
ensures fast, contamination free operation,
with virtually no carry-over. This setup can be
operated either for regular HPLC or fast UPLC
applications up to 20’000 psi.
Column Switching Options
Triple Valve Option
The triple valve conguration enables a dual
stage cleaning step including additional
sample sonication at the SCAP PLS system
ultrasonic module. This setup is recommended
for whole blood samples. First the sample
passes the ultrasonic module where the
blood cell components are disintegrated and
homogenized. The sample reaches the RAM
and SPE column where all unwanted matrix is
discharged. Afterwards the trapped analytes
are desorbed again to the analytical column
with strong organic solvents. If the amount
of organic modier used for backash is too
high, an optional enrichment step may be
included by adding water via a T-piece. While
separation and detection take place, the precolumns are re-equilibrated with the initial
eluent composition to be ready for the next
sample injection.
Max. 5.2e5 cps.
Hydroxy Desmethyl Bosentan,
retention time 1.94 minutes
Desmethyl Bosentan,
retention time
2.51 minutes
Bosentan,
retention time
2.74 minutes
Hydroxy Bosentan, 537.59,
retention time 2.08 minutes
Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
4.2e5
4.4e5
4.6e5
4.8e5
5.0e5
5.2e5
Intensity, cps
2.74
Time, min
2.74
Max. 1875.0 cps.
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
Intensity, cps
Hydroxy Desmethyl Bosentan,
retention time 1.94 minutes
Desmethyl Bosentan,
retention time
2.51 minutes
Bosentan,
retention time
2.74 minutes
Hydroxy Bosentan, 537.59,
retention time 2.08 minutes
Analyte Conc. / IS Conc.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.3
Analyte Area / IS Area
0 400 800 1200 1600 2000 2400 2800 3200 3600 4000
0.00
04080 120 160 200 240 280 320 360 400 440 480 500
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
Analyte Area / IS Area
Analyte Conc. / IS Conc.
Description
A new method for the detection of Bosentan
and three metabolites is presented. Bosentan and its three metabolites were spiked in
whole blood and analyzed by the SCAP PLS
system. Mass spectrometric detection was
performed on an MDS Sciex API 4000
operating in positive electrospray ionization
mode.
Method
8 levels of calibration and 3 levels of quality
control samples were spiked in whole blood
with a linear range from 1.00 ng/mL to 4096
ng/mL for Bosentan and 2.00 to 512 ng/mL
for the three metabolites.
For measurement of calibration and quality
control samples, 5 µL of each level were aspirated into the sample cartridge together with
1 µL of internal standard solution (containing
the ISTD’s for all 4 compounds).
Example Application
TM
Bosentan
The mass spectrometer was operated in positive electrospray ionization mode with dwell
times of 50 msec for each MRM transition
and an HPLC ow rate of 300 µL/min on the
analytical column. The whole blood samples
were cleaned on a Merck RAM column,
preconcentrated on a C18 trapping column and
separated on a C6-Phenyl HPLC column.
Data
Validation runs containing 2 calibration curves
and 6 replicates of the 3 levels of quality control were analyzed. Typical calibration curves
for Bosentan and one of the metabolites are
presented beneath. The lower limit of quantication for all 4 compounds and a picture for
a typical chromatogram are shown (traces for
ISTD’s are not shown).
Calibration curve for Bosentan (r = 0.9979)
Calibration curve for one Bosentan metabolite
(r = 0.9977)
LLOQ for Bosentan and metabolites
Typical chromatogram
Basic SCAP PLS system consisting of:
SCAP PLS system Clamp module
SCAP PLS system Gripper
Valve Drive module
(excl. valve drives and valves)
SCAP PLS system Ultrasonic module
SCAP PLS system Waste Box
SCAP PLS system Starter Kit
SCAP PLS system Accessories
SCAP PLS system Rack
Set of 6pcs. SCAP PLS system Racks
Electronic Pipette
SCAP PLS system Consumables
SCAP PLS system Tip Box, 54 Tips and Caps,
numbered
SCAP PLS system Tip Rell, 110 Tips and Caps
not numbered, bulk
System Conguration
Electronic pipette
SCAP PLS system Ultrasonic module
SCAP PLS system gripper
Valve module
SCAP PLS system tips and caps
SCAP PLS system waste box
SCAP PLS system tip box numbered
SCAP PLS system rack
SCAP PLS system clamp module
PAL Conguration:
The following CTC Analytics PAL components are required to operate a SCAP System
(Available either through CTC Analytics or Prolab GmbH)
PAL HTS9 or PAL HTS9-xt PAL80 instrument 80cm X-axis
PAL CycCompCD Cycle Composer Software
PAL 1VlvDrv* PAL 1-Valve Drive Module
PAL 2VlvDrv* PAL 2-Valve Drive Module
PAL 3VlvDrv* PAL 3-Valve Drive Module
C2V-1006D-CTC-K 6-port* VICI Cheminert Injection Valve 0.25mm bore, 5000psi
C72VX-1696D-CTC-K* 6-port VICI Cheminert Injection Valve, 0.25mm bore, 15’000psi
PAL StkCooler12MT Peltier cooled Stack for 6pcs. SCAP Racks
PAL MTHolder Trayholder for 1pc. SCAP Rack
*Choose one depending on your application
SCAP system® is a registered trademark of
Prolab, Patent Pending
Kägenstrasse 17
CH-4153 Reinach
Switzerland
Tel: +41 61 712 01 28
Fax: +41 61 712 01 48
Web: www.prolab.ch
e-mail: info@prolab.ch
Prolab GmbH acknowledges all tradenames
and trademarks used as the property of
their respective owners.
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