Pipetted Liquid Sampling

Automated samplepreparation platform for online LC-MS/MS bioanalysis

Integrated Sample Processing

Integrated Sample Processing Advantages

Fully automated sample processing of bio-fluids including whole blood samples

Integrated workflow from sample collectionuntil the final analysis result

One step sample manipulation, simplifiessample logistics, avoids sample mix-up

Requires a minimum amount of sample for quantitative results (typically 5 µl)

Minimized infection and contamination riskdue to completely sealed and disposablesample containers

No re-used syringe or sample loops. Disposable sample container, eliminates any carryover

Simplifies 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

SCAP PLS system components mounted on a robotic platform

Both the healthcare and pharmaceutical indus-try do a tremendous number of analyses ofsubstances in biological fluids, of which bloodis one of the most common. In the healthcareindustry, complete measurements are done todiagnose patients to determine the efficacy ofadministered 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 difficult, complex matrix to handle. In order to determine and quantifysubstances in blood, various intermediatepreparation steps are required (e.g. BloodSpots, Dialysis, Liquid-Liquid Extraction, Precipi-tation, (Hemo)Lysis, Membrane Filtration,Anticoagulation). The major drawback of allthese protocols is the time consuming, errorprone and labour intensive nature of manualsample pretreatment. The SCAP PLS system from Prolab GmbH represents a unique ap-proach for biofluid analysis and eliminates the drawbacks of current methods. It delivers a fully integrated workflow from sample collec-tion until the final result without any manual sample preparation. A newly designed dispos-able 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

Whole Blood Whole Blood

Centrifugation

Conventional example SCAP procedure

Application of Sample

Elution of Sample Matrix

Elution of Analyte(s)

Concentration of Extract

Conditioning ofExtraction Column

Injection into HPLC - System

Injection into SCAP HPLC - System

Aspirate sampleAttach tip to pipette Attach and seal with cap

Electronicpipette

Tip

Insert sample cartridge into SCAP PLS system rack

Aspirate sample volume directly from subject

Tip Electronicpipette

Seal with capAspirate ISTD Anticoagulant Esterase inhibitor

Attach tip to pipette

Insert sample cartridge into SCAP PLS system rack

Smart Sample Collection

Sample Collection Process SCAP PLS system Standard Sample Collection

SCAP PLS system Biofluid Sample Collection

The SCAP PLS system sample collection pro-cess 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 com-pletely sealed cartridges are then placed into the 54 position SCAP PLS system rack and are ready for immediate analysis. Alternatively the filled 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 biofluid is aspirated and the cartridge is sealed.

In case of whole blood, samples are directlycollected at the subject (e.g. finger, vein,heel, ear lobe) and are ready for immediateanalysis without any further manual samplepreparation steps.

Left: Assembled SCAP PLS system sample cartridgeRight: Numbered SCAP PLS system tip and cap

Loaded sample cartridges inserted in the SCAP PLS system rack

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.

Efficient LC-MS/MS Workflow

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/pre-processing 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 car-tridge are pierced to eject the sample into the analytical flow path. The sample passes

first through a pulsed ultrasonic module, where the blood cell components are disinte-grated and homogenized. This patented tech-nology avoids any clogging of the fluidic 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 com-pounds are eluted again and finally separated on the analytical LC column.

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 flushed into the analytical flow path.

Sample collection Sample introduction into flow path

Cartridge transfer Sonication Online extraction

Separation Cartridge disposalCartridges placed into SCAP PLS system rack

Minimized Sample Volume - Maximized Return

Clean Sample Introduction SCAP PLS system Valve module 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, fer-mentation 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 biofluid to be directly injected without negative effects on either the column or the results achieved.

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 configura-tion 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.

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 com-pletely sealed and may be stored or directly analyzed.

Valve module SCAP PLS system ultrasonic module SCAP PLS system waste box

Cartridge preparation with electronic pipette Restricted Access Material principle

Protein

Analyte

Column Switching Options

Single Valve OptionThe single valve configuration shows how a sample is loaded directly onto an analyti-cal column without prior sample preparation steps. Thereby the SCAP PLS system cartridge acts as a disposable sample loop and is auto-matically exchanged after each injection. This setup is limited to a system pressure of 2’500 psi (170 bar)

Dual Valve Option The dual valve configuration 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 through- put 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.

Triple Valve OptionThe triple valve configuration 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 modifier used for backflash is too high, an optional enrichment step may be included by adding water via a T-piece. While separation and detection take place, the pre-columns are re-equilibrated with the initial eluent composition to be ready for the next sample injection.

V1

Waste

Waste

Sample Cartridge

to MS

HPLC Gradient Pump

Analytical Column

Sample CartridgeV1

V2

Waste

Waste

WasteSPE

or R

AM C

olum

n

HPLC Gradient Pump

HPLC Loading Pump

to MSAnalytical Column

Sample Cartridge

Waste

Waste

V1

V2

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Waste

RAM

SPE

Col

umn

SPE

Colu

mn

Ultra Sonic Module

Dilutor Pump

to MS

HPLC Loading Pump

HPLC Transfer Pump

Analytical Column

HPLC Gradient Pump

Example Application

Method8 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 aspi-rated into the sample cartridge together with 1 µL of internal standard solution (containing the ISTD’s for all 4 compounds).

The mass spectrometer was operated in posi-tive electrospray ionization mode with dwell times of 50 msec for each MRM transition and an HPLC flow 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.

DataValidation runs containing 2 calibration curves and 6 replicates of the 3 levels of quality con-trol were analyzed. Typical calibration curves for Bosentan and one of the metabolites are presented beneath. The lower limit of quanti-fication for all 4 compounds and a picture for a typical chromatogram are shown (traces for ISTD’s are not shown).

DescriptionA new method for the detection of Bosentan and three metabolites is presented. Bosen-tan 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 4000TM operating in positive electrospray ionization mode.

Calibration curve for Bosentan (r = 0.9979)

Calibration curve for one Bosentan metabolite (r = 0.9977)

LLOQ for Bosentan and metabolites

Typical chromatogram

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, min0.0

2.0e44.0e46.0e48.0e41.0e51.2e51.4e51.6e51.8e52.0e52.2e52.4e52.6e52.8e53.0e53.2e53.4e53.6e53.8e54.0e54.2e54.4e54.6e54.8e55.0e55.2e5

Inte

nsity

, cps

2.74

Time, min

2.74Max. 1875.0 cps.

0

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nsity

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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.

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Anal

yte

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/ IS

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0 40 80 120 160 200 240 280 320 360 400 440 480 500

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yte

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Analyte Conc. / IS Conc.

Bosentan

Basic SCAP PLS system consisting of: SCAP PLS system Clamp moduleSCAP PLS system GripperValve Drive module (excl. valve drives and valves)SCAP PLS system Ultrasonic moduleSCAP PLS system Waste BoxSCAP PLS system Starter Kit

SCAP PLS system AccessoriesSCAP PLS system RackSet of 6pcs. SCAP PLS system RacksElectronic Pipette

SCAP PLS system ConsumablesSCAP PLS system Tip Box, 54 Tips and Caps, numberedSCAP PLS system Tip Refill, 110 Tips and Caps not numbered, bulk

PAL Configuration: 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-axisPAL CycCompCD Cycle Composer SoftwarePAL 1VlvDrv* PAL 1-Valve Drive ModulePAL 2VlvDrv* PAL 2-Valve Drive ModulePAL 3VlvDrv* PAL 3-Valve Drive ModuleC2V-1006D-CTC-K 6-port* VICI Cheminert Injection Valve 0.25mm bore, 5000psiC72VX-1696D-CTC-K* 6-port VICI Cheminert Injection Valve, 0.25mm bore, 15’000psiPAL StkCooler12MT Peltier cooled Stack for 6pcs. SCAP RacksPAL MTHolder Trayholder for 1pc. SCAP Rack

*Choose one depending on your application

Prolab GmbH acknowledges all tradenames and trademarks used as the property oftheir respective owners.

SCAP system® is a registered trademark of Prolab, Patent Pending

Kägenstrasse 17CH-4153 ReinachSwitzerland

Tel: +41 61 712 01 28Fax: +41 61 712 01 48Web: www.prolab.che-mail: info@prolab.ch

System Configuration

SCAP PLS system tips and caps

SCAP PLS system clamp module

Electronic pipette

SCAP PLS system Ultrasonic module

SCAP PLS system tip box numbered

SCAP PLS system gripper

Valve module

SCAP PLS system rack

SCAP PLS system waste box