HR-MS/MS as a way to avoid false positive quantification of 27 psychoactive compounds in venous and VAMS-collected blood.

HR-MS/MS as a way to avoid false positive quantification of 27 psychoactive compounds in venous and VAMS-collected blood.

Julia Mironenka (1); Anna Lenartowicz (1); Adrian Soboń (1,2); Rafał Szewczyk (1,2); Katarzyna Krupczyńska-Stopa (1,2); Maciej Stopa (1,2); Andrzej Kwaśnica (3)
1 – LabExperts sp. z o. o., Gdańsk, Poland; 2 – Bioanalytic sp. z o. o., Gdańsk, Poland; 3 – Lab4Tox sp. z o. o., Wrocław, Poland

Abstract

Introduction

Compound presence confirmation is one of the key aspects of targeted and quantitative analysis in forensics applications. High resolution tandem mass spectrometry (HR-MS/MS) may become a major tool for this kind of analysis because of its mass accuracy resulting in unparallel specificity, all combined with broad linearity and scanning speed achieved in the newest hardware on the market. In this work we show a modern HR-MS/MS method for a sensitive detection of 27 analytes with high confidence in blood and blood collected on a volumetric absorptive microsampling (VAMS) probe. Analyzed compounds include: Tramadol, Morphine, Fentanyl, Methadone, Codeine, Alprazolam, Diazepam, Flunitrazepam, Lorazepam, Oxazepam, Zolpiclone, Clonazepam, Nordiazepam, Zolpidem, Hydroxyzine, Amphetamine, Methamphetamine, MDA, MDMA, MDEA, THC, Cocaine, 6-acetylmorphine, 7-aminoflunitrazepam, 7-aminoclonazepam, Benzoylecgonine, THC-COOH.

Methods:

Briefly, sample preparation for the quantitation of selected compounds from blood and blood collected by VAMS included: spiking of internal standards, protein precipitation and extraction with acetonitrile:methanol (1:1) with 0.1% formic acid, evaporation under nitrogen stream and reconstitution in LC mobile phase. A quantitative LC-MS/MS method for 27 psychoactive compounds and 5 internal standards was developed and validated. Compounds were analyzed using reversed-phase chromatography on ExionAC LC coupled with ZenoTOF 7600 (SCIEX) operating in positive electrospray ionization scheduled MRMhr mode acquiring full MS/MS spectra for each compound. Analyte presence confirmation was based on 5 criteria out of which the most important was MS/MS spectrum, the mass defect of precursor and quantifier ion. Data were processed in SCIEX OS software.

Preliminary Data:

We decided to transfer the method from triple quad instrument to HR-MS/MS because of difficult data interpretation at low concentrations of the analytes in real-life samples where MRM ratio and matrix interferences leads to data misinterpretation. Developed processing method use multi-level confirmation and combined scoring based on HR data where result is affected by 5 criteria that have different decision weights: MS/MS library confirmation (50%), precursor ion mass defect (20%), quantifier ion mass defect (20%), precursor ion isotope pattern (5%) and retention time (5%). Each criterium contributes in the combined score calculation and has its own range of 3-level acceptance in ppm, % or seconds, respectively. The cut-off point of combined score was estimated at 60% after series of analysis and allows flawless reporting of compounds from DRUID list. To achieve full potential of scoring library has to be updated with MS/MS spectra obtained with optimized for each analyte collision energy and collected in spiked at different levels matrix samples.

The method was validated separately for blood and blood collected on VAMS and fulfilled the following criteria for each analyte: linearity (R ≥ 0.995), working range (LLOQ – 50 ng/ml), reproducibility (%CV ≤ 15%, accuracy 80 – 120% – based on two different QC-concentration levels) and recovery (%CV ≤ 15%). For majority of analytes included in the method LLOQ was in the range 0.01- 0.05 ng/ml (ex. Lorazepam, Diazepam, Flunitrazepam, Kodeine, Morphine, MDA, Oxazepam) for blood and 0.05-0.1 ng/ml for VAMS sampler. For analytes such as Tramadol, Fentanyl, Methadone and Cocaine the LLOQ ranged between 0.001- 0.005 ng/ml for blood and 0.01-0.05 for VAMS.

Novel aspect:

Sensitive, specific and false positive resistant HR-MS/MS quantitation of 27 psychoactive compounds in blood and blood collected by VAMS.

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