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LC/MS Application Note

Automated Sample Preparation for Therapeutic Drug Monitoring by LC-MS

2 IngeniousNews 04/2017

Solvents Sample/reagent storage@ room temperature

Injection valves

Column oven

CentrifugeVortex mixerSample storage @ 4°C

Automated Sample Preparation for Therapeutic Drug Monitoring by LC-MSChristian Berchtold1, Irene Wegner1,Timm Hettich1, Reto Bolliger2, Guenter Boehm2, Goetz Schlotterbeck1

1 University of Applied Sciences and Arts Northwestern Switzerland FHNW, Institute for Chemistry & Bioanalytics , Gründenstrasse 40, 4132 Muttenz

2 CTC Analytics AG; Industriestrasse 20, 4222 Zwingen, Switzerland

Keywords: Whole blood, primary container, sample preparation, protein precipitation, centrifugation, LC-MS, therapeutic drug monitoring TDM

Introduction

Automated sample preparation reduces the costs per sample and avoids sample handling errors. This is especially important in therapeutic drug monitoring or diagnostics based on blood samples. The use of robots is well established in these fields since a long time. Usually expensive and highly specialized pipetting robots are used for a high number of samples. However, most of these systems are not designed with a direct interface for LC-MS applications. In this article the parameters necessary to automatically prepare whole blood samples for online LC-MS applications in the field of diagnostics and TDM have been investigated. The PAL RTC (instrument view below) can perform additional sample prep steps, e.g. standard and reagent addition, centrifugation, liquid / liquid extraction (LLE). A typical workflow is shown in fig.1.

Materials and Methods

PAL RTC autosampler with a 1 mL syringe, LC MS P80 injection tool, vortex mixer, centrifuge (fig. 1)

Agilent LC 1200Agilent MS 6460 TripleQ-MSTested laboratory blood was supplied by blood donation in

primary containers (Vacuette™, Vacutainer™, etc.).All chemicals were purchased from Sigma-Aldrich.Hematocrit levels were prepared by mixing defined ratios of

plasma and erythrocytes.

Fig. 1: Configuration of the PAL RTC used to perform the experiments described here.

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Direct sampling from primary tubes with high process safety

The LCMS-P 80 Tool for the PAL RTC features a special needle that allows direct sampling from primary tubes. With adapters it can transport primary tubes to and from modules on the system, e.g. for vortexing. Different adapters allow the use of different formats of primary tubes.The optical sensor positioned on the sample loop (orange triangles in the sketch above) of the tool monitores the aspiration of sample, air gap or bubbles. Different sampling situations (injection with air gaps, clogging, bubbles) generate distinct patterns of the sensor signal triggering defined actions of the system, e.g. repeat of washing step, or move to next sample.

How reliable does the sensor detect different error conditions?

A series of tests was performed to assess if the LCMS-P80 injection tool on the PAL RTC can discriminate between a correct transfer of liquid (blood/reagent) and a number of error conditions, listed below (Tab. 1):

- Full vial, clean needle - Empty vial, clean needle, no sample - Blocked needle (clogging) - Full vial, sample foaming

Transferconditions

# samples #correct detections

Rate

Vial full, needle clean

80 80 100 %

Needle clogged 160 160 100 %

Vial empty,needle clean

200 200 100 %

Vial full,sample foaming

160 160 100 %

Tab. 1: Number of correct detections with respect to the transfer conditions.

Fig. 4: The different detection scenarios of the optical sensor on the sample loop of the LCMS-P 80 tool.

1

22 sensortransitions

LCMS-P 80 Tool

Sensor

Correct Transfer No sample Clot Foam

Fig. 3: Amongst the precipitation agents tested (acetonitrile, methanol and 0.1 M ZnSO4) acetonitrile worked best in terms of pellet formation. Shown are pellets from the precipitation of 50µL of blood and centrifugation @ 3000g for 10 min.

+ 250 μLwater

50 - 200 μLblood

+ 750 μLsolvent

1. Vortex2. Centrifuge

Inject 2 μLsupernatant

Fig.2: Typical workflow for the sample preparation of whole blood samples for therapeutic drug monitoring.

Acetonitril 0.1 mol/L ZnSO4

in MethanolMethanol

Comparison of different cleaning procedures

Whole blood consists of water, salt, proteins, lipids and intact erythrocytes, which tend to coagulate on surfaces or in contact with organic solvents. Effective cleaning procedures to avoid precipitation in the system as well as the deposition of biofilms are vital. The Scanning Electron Microscope pictures below show the middle section of 3 sample loops of LCMS-P80 tools after 500 injections of whole blood samples and cleaned with 3 different procedures between each sample.

Simple procedure:1 x 100 μL water, 0.1% formic acid.Visible deposition of materials.

Standard procedure:2 x 100 μL water, 0.1% formic acid/ 2 x 100 μL methanol 0.1% formic acid/ 2 x 100 μLwater, 0.1% formic acid.No visible deposition of materials.

Advanced procecedure:100 μL Decon™ Contrad™ 70 followed by standard procedure.Slight deposition of materials.

Examples for Therapeutic Drug Monitoring

The LCMS-P 80 tool was used to prepare samples for several typical drugs for TDM as well as compounds relevant in diag-nostics. Spiked and blank (in red) whole blood samples were analyzed.

CarbamazepineCarbamazepine is a representative of an anti-epileptic drug and very important to monitor in blood to set a proper and individual dosage. [1]Method: Eluent A: water 0.1% Formic acid / Eluent B: methanol 0.1% formic acid // Flow: 0.45 ml/min // Runtime: 6 minColumn: Waters Coretecs C18 2.1x100 mm 2.7 μmMS Parameter: Mode: positive MRM: 237 >194Blood Volume: 10 μL / Injection volume: 2 μL[1] Linder et al. Bioanalysis (2015) 1(16) 2013-2039

10 μg/mL Carbamazepine

DiclophenacDiclophenac is a nonsteroidal anti-inflammatory drug and a representative compound for drug monitoring acording its moleculare properties [2] Method: Eluent A: water 2 mM NaF / Eluent B: acetonitril 2 mM NaF // Flow: 0.5 ml/min // Runtime: 7 minColumn: Zorbax SB-C8 4.6x50 mm 1.8 μmMS Parameter: Mode: positive MRM: 296 >249Blood Volume: 10 μL / Injection volume: 2 μL[2] Kearney et al. BMJ. (2006) 332(7553):1302-8

233 ng/mL Diclophenac25-Hydroxy vitamin D325-hydroxy vitamin D3 is an important marker for the storage depot of vitamin D [3]Method: Eluent A: water 0.1% Formic acid / Eluent B: methanol 0.1% formic acid // Flow: 0.45 ml/min // Runtime: 6 minColumn: Waters Coretecs C18 2.1x100 mm 2.7 μmMS Parameter: Mode: positive MRM: 401>383Blood Volume: 10 μL / Injection volume: 20 μL[3] Shah et al. Nutrition Journal (2011) 10:4

80 ng/mL 25-hydroxy vitamin D3LOD of the method approx. 2 ng/mL

Conclusions

- The new LCMS-P 80 tool is capable of pipetting whole blood with normal hematocrit levels (30-50%) directly from primary tubes with good accuracy and precision, applying one average correction factor. For high hematocrit levels a different correction factor should be applied.- Wash procedures were established to minimize carryover of analytes. A procedure to minimize the deposition of blood on system surfaces was shown to be successful.- The system consisting of a PAL RTC autosampler, connected to an Agilent LC-MS system can perform sample prep and LC-MS analysis for whole blood samples from primary tubes. The performance of the system was tested with several typical compounds in therapeutically relevant concentrations.- The sensor of the LCMS-P80 tool reliably detects error conditions and greatly increases process safety.

Imprint

Date of print: 11.2017

CTC Analytics AGIndustriestrasse 20CH-4222 ZwingenSwitzerland T +41 61 765 81 00Contact: info@ctc.ch

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