Accepted Manuscript Title: Simultaneous determination of 9 -tetrahydrocannabinol, cannabidiol, cannabinol and 11-nor-9 -tetrahydrocannabinol-9-carboxylic acid in hair using liquid chromatography–tandem mass spectrometry Author: S. Dulaurent J.M. Gaulier L. Imbert A. Morla G. Lachˆatre PII: DOI: Reference:

S0379-0738(14)00019-X http://dx.doi.org/doi:10.1016/j.forsciint.2014.01.004 FSI 7479

To appear in:

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Received date: Revised date: Accepted date:

28-8-2013 20-12-2013 5-1-2014

Please cite this article as: S. Dulaurent, J.M. Gaulier, L. Imbert, A. Morla, G. Lachˆatre, Simultaneous determination of Delta9 -tetrahydrocannabinol, cannabidiol, cannabinol and 11-nor-Delta9 -tetrahydrocannabinol-9-carboxylic acid in hair using liquid chromatographyndashtandem mass spectrometry., Forensic Science International (2014), http://dx.doi.org/10.1016/j.forsciint.2014.01.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title Page (with authors and addresses)

Simultaneous determination of Δ9-tetrahydrocannabinol, cannabidiol, cannabinol and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in hair using liquid chromatography– tandem mass spectrometry.

Dulaurent S., 1Gaulier J.M., 1,2Imbert L., 3Morla A. and 1,2Lachâtre G.

CHU Dupuytren, Department of Pharmacology and Toxicology, Unit of biological and

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University of Limoges, 87032 Limoges, France

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AB Sciex France, 91940 Les Ulis, France

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*Corresponding author: Sylvain DULAURENT

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forensic toxicology, 87042 Limoges, France

CHU Dupuytren

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Department of Pharmacology and Toxicology

87042 LIMOGES Cedex, France Phone: + 33 555 056 140

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Fax: +33 555 056 162

(e-mail : [email protected])

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*Manuscript (without author details)

ABSTRACT For several years, hair analyses have become a powerful tool to investigate past exposure towards xenobiotics. In the case of illicit drugs and more precisely of cannabis exposure, four compounds are usually investigated: Δ9-tetrahydrocannabinol (THC), the main active

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compound of cannabis, one of its metabolites [11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)] and two cannabinoids (cannabinol and cannabidiol). Up until now, the

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hair determination of the carboxylic metabolite of THC, which has been described as the only

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marker allowing distinguishing consumption and passive exposure, has been performed using a gas chromatography–tandem mass spectrometry method. The aim of this study was to

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develop a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantitative determination of the four markers. The sample preparation was

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based on an alkaline hydrolysis of hair samples followed by a liquid-liquid extraction of compounds in acidic conditions using a hexane/ethyl acetate mixture. The method was

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validated and the results were satisfactory: intra- and inter-assay accuracies below 9% and relative standard deviation below 15% for the four compounds. Moreover, the limit of

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quantification for THC-COOH, the most challenging compound, was validated at 0.2 pg/mg. This concentration is in accordance with the recommendations made by a scientific society which specialises in hair testing. It makes it possible to distinguish the kind of exposure to

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cannabis.

KEY WORDS: Liquid chromatography coupled with tandem mass spectrometry, hair, cannabis exposure diagnosis.

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Introduction

In France, as in other countries, cannabis is the most widely used illicit drug. In forensic as well as in clinical contexts, Δ9-tetrahydrocannabinol (THC), the main active compound of

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cannabis, and two of its metabolites [11-hydroxy- Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)] are regularly investigated

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in blood [1]. With the purpose of determining whether a person is a regular cannabis

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consumer or not (i.e. in the case of restitution of a driving licence), these substances can be assayed in hair with two different approaches: 1), THC, cannabidiol (CBD), cannabinol

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(CBN) determination in hair, and 2) THC-COOH determination in hair. Nevertheless, it is well known that THC-COOH determination in hair allows unequivocally distinguishing

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consumption to passive exposure [2].

Numerous methods have been proposed for the determination of cannabinoids and

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metabolites in hair: (a) THC-COOH [3-8], (b) THC, CBD, CBN, [8-10], and (c) THC-COOH and THC, CBD, CBN [11,12]. Almost all of them are gas chromatography methods, requiring

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a derivatization step for the analysis of THC-COOH [8,11,12]. This analytical step is time consuming, often costly and responsible for the deterioration of the injection liner together with the deterioration of the column of the chromatographic system. For the diagnosis of an

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active consumption, the guidelines proposed by the Society of Hair Testing (SoHT) normally require the use of a gas chromatography coupled with a tandem mass spectrometry system (GC-MS/MS) [13]. Indeed, the recommended THC-COOH cut-off in hair (0.0002 ng/mg) is not reachable with a single mass spectrometer, except with the use of a sophisticated approach using a two-dimensional GC-GC-MS system [14]. Moreover, analytical applications requiring a GC-MS/MS are rare in toxicological laboratories: the determination of THC-COOH in hair is one of them. In the present article, we describe an alternative method for the determination

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of cannabinoids in hair using a liquid chromatography coupled with a tandem mass spectrometry system (LC-MS/MS), an analytical tool with a wide field of applications in toxicological laboratories, in contrast with a GC-MS/MS system. Another advantage of such an analytical method is the simultaneous determination in the same hair lock, with one sample

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preparation and one injection, of THC-COOH, THC, CBD and CBN at concentrations

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compatible with a reliable interpretation in all clinical or forensic contexts.

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Material and methods

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Chemicals and reagents

THC, CBD, CBN, THC-COOH, THC-COOH-D3 and THC-D3 were supplied by LGC

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Promochem (Molsheim, France). Methanol was purchased from Carlo Erba Reactifs (Val de Reuil, France). Ethyl acetate (Suprasolv), hexane (Suprasolv), pentan-1-ol (Normapur), hydroxide

(Normapur),

acetic

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sodium

acid

(Normapur),

toluene

(Chromanorm),

dichloromethane (Chromanorm) and chlorhydric acid (Normapur) were supplied by VWR (Fontenay-sous-bois, France). Pure water was obtained using a Millipore Direct Q purification

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system (Saint Quentin en Yvelines, France).

Assay procedure First of all, hair strands (samples to be analyzed and blank hair samples) were decontaminated with two two minute water washes followed by two one minute dichloromethane washes (the last dichloromethane wash was kept to be tested for cannabinoids). After drying, they were cut into small pieces (

Simultaneous determination of Δ9-tetrahydrocannabinol, cannabidiol, cannabinol and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in hair using liquid chromatography-tandem mass spectrometry.

For several years, hair analyses have become a powerful tool to investigate past exposure towards xenobiotics. In the case of illicit drugs and more p...
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