LC/MS Application Note
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1 LC/MS Application Note Automated Sample Preparation for Therapeutic Drug Monitoring by LC-MS
2 Automated Sample Preparation for Therapeutic Drug Monitoring by LC-MS Christian Berchtold 1, Irene Wegner 1,Timm Hettich 1, Reto Bolliger 2, Guenter Boehm 2, Goetz Schlotterbeck 1 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 1200 Agilent MS 6460 TripleQ-MS Tested 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. Sample 4 C Vortex mixer Centrifuge Injection valves Fig. 1: Configuration of the PAL RTC used to perform the experiments described here. Solvents Sample/reagent room temperature Column oven 2 IngeniousNews 04/2017
3 μl blood μl water μl solvent 1. Vortex 2. Centrifuge Inject 2 μl supernatant Fig.2: Typical workflow for the sample preparation of whole blood samples for therapeutic drug monitoring. Direct sampling from primary tubes with high process safety 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): Acetonitril 0.1 mol/l ZnSO 4 in Methanol Methanol 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 3000g for 10 min. Full vial, clean needle Empty vial, clean needle, no sample Blocked needle (clogging) Full vial, sample foaming 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. LCMS-P 80 Tool Transfer conditions Vial full, needle clean # samples #correct detections Rate % Needle clogged % Vial empty, needle clean Vial full, sample foaming % % 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. 2 sensor transitions 1 2 Sensor Correct Transfer No sample Clot Foam IngeniousNews 04/2017 3
4 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.
5 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 diagnostics. Spiked and blank (in red) whole blood samples were analyzed. Carbamazepine Carbamazepine 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 min Column: Waters Coretecs C18 2.1x100 mm 2.7 μm MS Parameter: Mode: positive MRM: 237 >194 Blood Volume: 10 μl / Injection volume: 2 μl [1] Linder et al. Bioanalysis (2015) 1(16) Diclophenac Diclophenac 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 min Column: Zorbax SB-C8 4.6x50 mm 1.8 μm MS Parameter: Mode: positive MRM: 296 >249 Blood Volume: 10 μl / Injection volume: 2 μl [2] Kearney et al. BMJ. (2006) 332(7553): Hydroxy vitamin D3 25-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 min Column: Waters Coretecs C18 2.1x100 mm 2.7 μm MS Parameter: Mode: positive MRM: 401>383 Blood Volume: 10 μl / Injection volume: 20 μl [3] Shah et al. Nutrition Journal (2011) 10:4 10 μg/ml Carbamazepine 233 ng/ml Diclophenac 80 ng/ml 25-hydroxy vitamin D3 LOD 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.
6 Legal Statements Imprint CTC Analytics AG reserves the right to make improvements and/or changes to the product(s) described in this document at any time without prior notice. CTC Analytics AG makes no warranty of any kind pertaining to this product, including but not limited to implied warranties of merchantability and suitability for a particular purpose. Under no circumstances shall CTC Analytics AG be held liable for any coincidental damage or damages arising as a consequence of or from the use of this document CTC Analytics AG. All rights reserved. Neither this publication nor any part hereof may be copied, photocopied, reproduced, translated, distributed or reduced to electronic medium or machine readable form without the prior written permission from CTC Analytics AG, except as permitted under copyright laws. CTC Analytics AG acknowledges all trade names and trademarks used as the property of their respective owners. PAL is a registered trademark of CTC Analytics AG Switzerland Date of print: CTC Analytics AG Industriestrasse 20 CH-4222 Zwingen Switzerland T Contact: info@ctc.ch Visit our homepage for more information.
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