MSACL 2016 EU Abstract

Automated Sample Preparation of Whole Blood for Therapeutic Drug Monitoring and Diagnostics by LC-MS Using a Commercial Autosampler

Thomas Preiswerk (Presenter)
CTC Analytics AG

Authorship: Christian Berchtold 1 , Irene Wegner 1 ,Timm Hettich 1 , Reto Bolliger 2 , Guenter Boehm 2 , Goetz Schlotterbeck 1, Thomas Preiswerk 2
1 University of Applied Sciences and Arts Northwestern Switzerland FHNW, Institute for Chemistry and Bioanalytics , Gründenstrasse 40, 4132 Muttenz, 2 CTC Analytics AG; Industriestrasse 20, CH-4222 Zwingen, Switzerland

Short Abstract

In this poster the parameters necessary to automatically prepare whole blood samples for on-line LC-MS applications in the field of diagnostics and therapeutic drug monitoring have been investigated. A strategy and the most important parameters are shown for the optimization of a PAL RTC autosampler for the preparation of whole blood.

Long Abstract

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 project the parameters necessary to automatically prepare whole blood samples for on-line LC-MS applications in the field of diagnostics and TDM have been investigated. A strategy and the most important parameters are shown for the optimization of a PAL RTC autosampler for the preparation of whole blood.

Methods

An integrated system consisting of a PAL RTC autosampler equipped with a 1 mL syringe, a LC-MS injection tool, a vortexer and a centrifuge was used. Tested laboratory blood was supplied by blood donation and applied into 1.5 ml standard LC-vials with magnetic caps for vial transport. Methanol, acetonitrile, 0.1 mol/L ZnSO4 solutions in methanol and a mix of isopropanol/water have been tested to achieve protein precipitation. Blood volumes in the range of 50 to 250 µL were investigated at centrifugation times of 1 to 20 minutes (3000 g). Amino acids, acyl-carnitins and steroids as endogenous analytes were chosen as model substances for diagnostics. Diclofenac, carbamazepine and immunosuppressiveswere used as examples for therapeutic drug monitoring applications.

Preliminary Data or Plenary Speakers Abstract

A strategy for the method development is shown.

On-line sample preparation of whole blood within standard vials consists of the important tasks, protein precipitation with organic solvent, vortexing, centrifugation, and taking aliquots of the supernatant for injection.

The direct sample preparation in 1.5 mL vials has been shown to work for blood volumes between 50 and 200 µL. Solvents for precipitation such as methanol, acetonitrile and 0.1M ZnSO4 methanol solution are well suited to prepare these volumes in blood. Mixtures using isopropanol and water caused insufficiently cleared solutions for further analysis. The experiments show that for sufficient protein precipitation a percentage of at least 75% organic solvent should be used.

For most metabolites a duration of vortexing of 90 seconds (at 1400 rpm) appears sufficient. Exceptions are highly protein bound drugs such as immunosuppressants which need prolonged durations (up to 20 min).

A centrifugation time of more than 2 minutes is necessary at 3000 g especially for methanol extraction, since methanol causes less dense pellets of higher volume compared to acetonitrile. Therefore the needle penetration depth of the sampler needs to be adjusted depending on the solvent and amount of blood sample. The needle penetration should not exceed 25 mm, for an amount of 50 µL blood sample, due to the pellet height. At about 22 mm up to 200 µL aliquots might be used for further analysis. However, since the solution after crashing consists of a high portion of organic solvent it should be considered to dilute further with water, to use online SPE (e.g. immunosuppressant) or to inject only low volumes in order to maintain the chromatographic peak shape. For the analytes used in this study, injection volumes of < 50 µL or the use of online SPE were sufficient.

Novel Aspect

Systematic investigation of parameters necessary to perform direct sample preparation of whole blood using a commercial autosampler with centrifuge.


References & Acknowledgements:

[1] Linder et al. Bioanalysis (2015) 4 (16), 2013-2039

[2] Seger et al. Nature Protocols (2009) 4 (4), 526-534

[3] Fingerhut et al. Rapid Communications in Mass Spectrometry (2014) 28 (8), 965-973


Financial Disclosure

DescriptionY/NSource
Grantsno
SalaryyesCTC Analytics AG
Board Memberno
Stockno
Expensesno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

yes