= Emerging. More than 5 years before clinical availability.
= Expected to be clinically available in 1 to 4 years.
= Clinically available now.
MSACL 2018 EU : Tiphaine

MSACL 2018 EU Abstract

Topic: Small Molecules

Quantification and Library Identification of 248 Compounds Commonly Detected in Systematic Toxicology Analysis Using an Automated Extraction Coupled to LC-MS/MS

Robin Tiphaine (Presenter)
CHU of Limoges

Presenter Bio: Tiphaine Robin obtained a State Doctorate of Pharmacy in 2017 (University of Limoges, France). She is currently PhD student in analytical chemistry. The main objective of her thesis is to develop innovative LC-MS screening procedures for applications in clinical and forensic toxicology. These works are under the supervision of both the Department of Pharmacology and Toxicology of the Limoges University Hospital (Pr Franck Saint-Marcoux and Dr Souleiman El Balkhi) and Shimadzu Corporation (Stephane Moreau, Shimadzu France, Marne-la-Vallée, France; Alban Huteau, Shimadzu France, Marne-la-Vallée, France). The Mass Spectrometry Business Unit (Overseas) of Shimadzu Corporation, with Neil Loftus and Alan Barnes, is also greatly involved in these developments.

Authors: Tiphaine Robin1; Alan Barnes2; Neil Loftus2; Sylvain Dulaurent1, Pierre Marquet1; Souleiman El Balkhi1, Franck Saint-Marcoux1;
1 - CHU de Limoges, Limoges, France; 2 -– Shimadzu Corporation, Manchester, UK

Short Abstract

We report a fully automated extraction method carried out by a programable liquid handler directly coupled to an LC-MS/MS system for the identification and quantification of 248 compounds of interest. The acquisition was performed in positive and negative ionization mode with up to 15 MRM transitions per compound (MRM Spectrum mode). The method was validated according to the requirements of ISO 15189. A robustness study performed in 16 compounds demonstrated that samples could be quantified using a calibration curve dating up to one month.

Long Abstract

Introduction

Forensic and toxicological drug screening has a number of challenges with importance for rapid sample analysis and reporting with the need for quantitative results. Extraction procedures typically used for LC-MS/MS analysis often require manuals steps in sample preparation. To eliminate these manual steps and decrease sample extraction time, a fully automated extraction method was developed. To identify and quantify over 240 compounds in a single run, an MRM spectrum mode method was developed. The acquisition was performed in positive and negative mode with up to 15 MRM transitions per compound, each with optimized collision energy. Ion intensities from each transition were used to construct an MRM spectrum that could be used to search against registered library spectra.

Methods

The method was developed to screen whole blood, plasma and urine for a panel of 248 compounds including anticoagulants, antidepressants, antiepileptics, antipsychotics, barbiturates, beta-blockers, benzodiazepines, diuretics and drugs of abuse (cannabinoids, cocaine, amphetamines, opioids). For each compound, the calibration curve was designed by taking into account its therapeutic range. Extraction procedure was fully automated on a programmable liquid handler (CLAM-2000, Shimadzu Corporation) and the extract was directly injected and analysed on a LC-MS/MS system (LCMS 8060, Shimadzu Corporation) with no human intervention. The method was validated according to the requirements of ISO 15189 for the 248 compounds. A robustness study was performed to evaluate how long acceptable quantitative accuracy could be provided by using historic calibration curve data files for toxicological applications. For this, freshly prepared quality control samples (containing 16 compounds out of 248) were quantified using historic calibration standard data files from up to 4 weeks old.

Results

Extracts were automatically prepared in 8 min using 50µL of sample to which 100µL of acetonitrile and 10µL of deuterated internal standard compounds were added (CLAM-2000, Shimadzu Corporation). After mixing, samples were filtered into LC vials and 3µL injected for analysis. Chromatographic separation was performed in 18 min by reversed phase UHPLC (Nexera LC, Shimadzu Corporation using a Restek Biphenyl 2.7µm 2.1x100mm column). About 26 min were needed to obtain the first results, then the system produced a result every 18 min (extraction and separation being performed in parallel). The MRM transitions were measured throughout the entire analysis period using MRM peak intensity to create an MRM-spectrum . MRM transitions were set with 1msec pause time and from 1 to 15 msec dwell time. All the compounds were successfully validated for our predefined accuracy and CV acceptance criteria, depending on the matrix. Investigation in to the system stability and robustness by repeat quality control analysis demonstrated that samples could be quantified with an acceptable uncertainty using a calibration curve dating up to one month.

Conclusions & Discussion

A fully automated LC-MS/MS method was validated to identify and quantify a panel of about 250 compounds. MRM spectrum added confidence in compound identification reducing both false positive and false negative results and MRM transition enabled quantification of the identified compound.

This assay has now been implemented for routine toxicological quantitative screening patient samples. Methods for drug screening typically operate on a >24h sample to result cycle time requiring first preliminary analysis to identify target compounds and secondary analysis for quantitative analysis. In this work sample preparation and analysis was completed in under 30min, a time rapid enough to provide treatment choice, for example, between acetylcysteine and naloxone. Whilst patients may exhibit symptoms consistent with opioid overdose, drug combinations also need to be considered necessitating a screening workflow to broaden the panel of compounds analysed.


References & Acknowledgements:


Financial Disclosure

DescriptionY/NSource
GrantsyesShimadzu corporation
Salaryno
Board Memberno
Stockno
Expensesno

IP Royalty: no

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

no