A validated GC–MS method for the determination of Δ9-tetrahydrocannabinol and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in bile samples

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    A validated GCMS method for the determinationof D9-tetrahydrocannabinol and 11-nor-D9-tetrahydrocannabinol-9-carboxylic acid in bile samples

    Ioannis Papoutsis Panagiota Nikolaou Artemisia Dona Constantinos Pistos

    Maria Stefanidou Chara Spiliopoulou Sotirios Athanaselis

    Received: 21 October 2011 / Accepted: 9 November 2011 / Published online: 3 December 2011

    Japanese Association of Forensic Toxicology and Springer 2011

    Abstract We present a validated gas chromatography-

    mass spectrometry method for the determination of D9-tet-rahydrocannabinol and 11-nor-D9-tetrahydrocannabinol-9-carboxylic acid in bile samples. The method includes protein

    precipitation with acetonitrile after enzymatic hydrolysis,

    and solid-phase extraction followed by silylation using

    N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimeth-

    ylchlorosilane. The limit of detection was 0.30 ng/ml and the

    limit of quantitation was 1.00 ng/ml. The calibration curves

    were linear within the dynamic range of 1.00500 ng/ml

    (R2 C 0.993) and the absolute recovery for both analytes

    was higher than 87.5%. Accuracy and precision were less

    than 8.8% and 8.2%, respectively. The developed method

    was applied for the analysis of bile samples obtained from 21

    forensic cases.

    Keywords D9-Tetrahydrocannabinol 11-Nor-D9-tetrahydrocannabinol-9-carboxylic acid Bile GCMS


    Cannabis, the most commonly used illicit drug throughout

    the world [1], is often implicated in forensic cases like deaths

    of drug addicts, driving under the influence of drugs, acci-

    dents, and others. The increasing use of cannabis threatens

    road safety because it is known to be a causative factor in

    some traffic accidents [26]. D9-Tetrahydrocannabinol

    (THC) is the main psychoactive constituent of the plant

    Cannabis sativa [7, 8] and it has a complex pharmacokinetic

    profile due to its high lipid solubility, protein binding and

    large distribution volume [3, 9]. THC is extensively metab-

    olized primarily to 11-hydroxy-D9-tetrahydrocannabinol(THC-OH), which is further metabolized to the carbox-

    ylic acid metabolite 11-nor-D9-tetrahydrocannabinol-9-car-boxylic acid (THCCOOH) [1013]. The concentrations of

    THC and its metabolites in biological samples correlate

    with recent drug use and impaired human performance or

    behavior; quantification of THC in blood is absolutely nec-

    essary in cases of accidents where victims or other involved

    persons are affected by cannabis [2, 11, 14]. Generally,

    plasma or whole blood concentrations of cannabinoids are

    required for the evaluation of driving ability under the

    influence of cannabis, for monitoring drug addicts under

    rehabilitation, and for the investigation of forensic cases

    [8, 12, 15]. In forensic cases, analysis of alternative matrices,

    such as bile, may be required especially when urine or blood

    samples are not available or cannabinoid blood concentra-

    tion data do not provide adequate answers to questions

    posed by the investigation [12]. The bile concentrations of

    drugs, including cannabinoids, are generally severalfold

    higher than the respective blood concentrations, so determi-

    nation of cannabinoids in bile remains an important post-

    mortem tool for documenting chronic cannabinoid use [16].

    Many analytical methods for determining cannabinoids

    in different biological matrices have been developed.

    Immunoassays are generally used for screening of urine

    samples for cannabis use, but the chromatographic confir-

    mation of positive results is mandatory [13]. For this

    purpose, gas chromatography (GC) [7, 11, 14, 15, 1719]

    or liquid chromatography (LC) [2, 4, 5, 8, 12, 13, 2025]

    methods using mainly mass spectrometry (MS) and MS/

    MS detectors have been used for the determination of THC

    I. Papoutsis P. Nikolaou (&) A. Dona C. Pistos M. Stefanidou C. Spiliopoulou S. AthanaselisDepartment of Forensic Medicine and Toxicology,

    School of Medicine, National and Kapodistrian University

    of Athens, 75 Mikras Asias, 115 27 Athens, Greece

    e-mail: pan_nik@hotmail.com


    Forensic Toxicol (2012) 30:5158

    DOI 10.1007/s11419-011-0126-1

  • and its metabolites in blood [2, 4, 5, 8, 11, 12, 14, 17, 19,

    20, 22, 24, 25] or plasma [7, 13, 15, 18, 21], urine [11, 13,

    1921], oral fluid [20], and tissues [18, 23, 26]. To our

    knowledge, there is no validated method published con-

    cerning the determination of THC and its carboxylic acid

    metabolite THCCOOH in bile samples that could be used

    for the study of the distribution of cannabinoids and the

    establishment of cannabis use. The aim of this study was

    the development, optimization, and validation of a GCMS

    method for the determination of THC and THCCOOH

    concentrations in bile. The developed method was suc-

    cessfully applied in the analysis of bile samples from dead

    drug addicts during the investigation of the respective

    forensic cases.

    Materials and methods

    Chemicals and reagents

    Methanolic standard stock solutions of THC (1.0 mg/ml),

    THCCOOH (0.1 mg/ml), THC-d3 (0.1 mg/ml) and THC-

    COOH-d3 (1.0 mg/ml) were purchased from LGC Pro-

    mochem (Molsheim, France) and all standards were

    [99.9% pure. Bond Elut LRC Certify II Solid PhaseExtraction (SPE) columns were obtained from Varian

    (Houten, Netherlands). N,O-bis(trimethylsilyl)trifluoroace-

    tamide (BSTFA) with 1% trimethylchlorosilane (TMCS),

    pentafluoropropionic anhydride (PFPA) 99%, 2,2,3,3,3-

    pentafluoro-1-propanol (PFPOH) 97%, methyl iodide

    (CH3I) 99%, and b-glucuronidase (from Helix pomatia,400,000 units/g) were purchased from Sigma-Aldrich

    (Steinheim, Germany). The stock enzyme was diluted with

    1.0 M acetate buffer (pH 5.0) to prepare the working

    b-glucuronidase solution (100,000 units/ml). All solvents(methanol, hexane, ethyl acetate, acetone, acetonitrile,

    isooctane, acetic acid) were HPLC grade and were pur-

    chased from Merck (Darmstadt, Germany). Drug-free bile

    samples, verified as negative for drugs by GCMS, were

    pooled and used for the preparation of spiked calibrators

    and quality control (QC) samples.

    Preparation of standard solutions

    Combined stock solutions of THC and THCCOOH were

    diluted with methanol to prepare seven calibrator working

    solutions (0.02, 0.10, 0.20, 0.50, 1.50, 4.00, and 10.0 lg/ml).Bile samples for calibration curves were prepared by

    spiking pooled drug-free bile (950 ll) with 50 ll of theabove-mentioned working solutions, yielding concentra-

    tions for each analyte of 1.00, 5.00, 10.0, 25.0, 75.0, 200,

    and 500 ng/ml. QC working solutions were prepared at

    concentrations of 0.06, 2.00, and 8.00 lg/ml. Blank bilesamples (950 ll) were fortified with 50 ll of the appro-priate QC working solutions to give low (3.00 ng/ml),

    medium (100 ng/ml), and high (400 ng/ml) concentration

    QC samples for each analyte. A working internal standard

    solution containing THC-d3 and THCCOOH-d3 at con-

    centrations of 0.50 lg/ml was prepared by mixing theappropriate volumes of the corresponding stock solutions

    and then by diluting with methanol. Fresh working solu-

    tions were prepared on each day of analysis.

    Sample preparation

    A volume of 50 ll of the mixed working internal standardsolution (0.50 lg/ml) was added to the calibrator, QC, andreal bile samples (1.0 ml). All samples were vortex-mixed

    for 15 s. As a result, all calibrators, QC, and real samples

    contained 25.0 ng/ml of THC-d3 and THCCOOH-d3.

    Subsequently, 1 ml of 0.1 M acetate buffer (pH 7.0) and

    200 ll of working b-glucuronidase solution were added,the tubes were capped, vortex-mixed for 15 s, and stored at

    37C for 16 h. Following the enzymatic hydrolysis, pro-teins were precipitated with 3.0 ml of acetonitrile, added

    dropwise while vortex mixing. Then the samples were

    centrifuged at 2000 rpm for 5 min, the supernatant was

    decanted into a clean glass tube, and the solvent was

    evaporated under a gentle stream of N2 at 40C toapproximately 0.5 ml. The pH of all samples was adjusted

    to 7 with the addition of 5.0 ml of a mixture of 0.1 M

    acetate buffer (pH 7.0) with methanol (95:5, v/v, mixture

    A). Bond Elut LRC Certify II SPE columns were condi-

    tioned with 2 ml of methanol and 2 ml of mixture A prior

    to sample loading. The samples were applied to the col-

    umns at a flow rate of approximately 1.0 ml/min. The

    columns were washed subsequently with 2 ml of mixture A

    and 100 ll of acetone, and they were dried under highvacuum (C10 mmHg) for 3 min. THC was eluted twice

    with 2.0 ml of a freshly prepared mixture of hexane:ethyl

    acetate (90:10, v/v). The THC eluates were collected in

    clean tubes and evaporated to dryness under a gentle

    stream of N2 at 40C. Then the columns were washed againwith 3.0 ml of methanol 50% and 100 ll of ethyl acetateand they were dried under high vacuum (C10 mmHg) for

    3 min. THCCOOH was eluted twice with 2.0 ml of a

    freshly prepared mixture of hexane:ethyl acetate:acetic

    acid (90:10:1, v/v/v). The THCCOOH eluates were also

    collected in clean tubes and evaporated to dryness under a

    gentle stream of N2 at 40C. All the residues were deriv-atized by adding 30 ll of acetonitrile and 30 ll of BSTFAwith 1% TMCS, vortex mixing, and heating at 70C for30 min. After cooling the tubes, the samples were injected

    (1.0 ll) into the GCMS system.

    52 Forensic Toxicol (2012) 30:5158


  • GCMS conditions and apparatus

    Chromatographic analysis was performed on an Agilent

    6890N/5975 GCMS system, equipped with an HP-5MS

    column (30 m 9 0.25 mm i.d., 0.25 lm film thickness).Helium was used as carrier gas at a flow rate of 1.0 ml/min.

    Injections were carried out in the splitless mode using an

    Agilent 7683B autosampler system. The initial column

    temperature of 150C with 1-min hold was increased at arate of 30C/min to the final column temperature of 300Cand held for 11 min. The injector, ion source, and interface

    temperatures were maintained at 280, 230, and 280C,respectively. The above GC conditions were chosen after

    optimization of the developed GCMS method. The mass

    spectrometer was operated in electron-impact ionization

    (EI, 70 eV) and selective ion monitoring (SIM) modes for

    this assay. The mass fragments used for the qualitative

    analysis of analytes were m/z 371, 386, and 303 for THC

    (374 for THC-d3), and 371, 473, and 488 for THCCOOH

    (374 for THCCOOH-d3). For quantification, mass fragment

    m/z 371 was used for THC and THCCOOH, and m/z 374

    was used for the deuterated analogs.

    The pH meter used was a 691 digital model (Metrohm,

    Switzerland) with a glass electrode. An evaporator using

    nitrogen (Reacti-Vap Pierce, Model 18780, Rochford, IL,

    USA), a furnace (J.P. Selecta, Spain), and a cooled cen-

    trifuge (Sigma 4K10, Germany) were also used.

    Results and discussion

    Method development and optimization

    We developed and optimized a sensitive, selective, and

    specific GCMS method for the determination of THC and

    THCCOOH in bile samples. The developed method

    includes enzymatic hydrolysis using b-glucuronidase priorto protein precipitation with acetonitrile and SPE using

    Bond Elut LRC Certify II columns followed by silylation

    using BSTFA with 1% TMCS in acetonitrile environment.

    In most GCMS methods, a derivatization step is

    required to increase stability and improve the chromato-

    graphic performance of the cannabinoids during analysis.

    This part of the sample preparation is not needed in LC

    methods [2, 4, 5, 8, 12, 13, 2025], and the cannabinoids

    may be analyzed directly. However, LC-MS-MS instru-

    mentation is not widely available for routine analysis in

    forensic laboratories worldwide, and it remains a more

    expensive analytical technique when compared with

    GCMS. Furthermore, in some previously published

    methods [14, 15, 21] the compound identification criteria

    were not fulfilled as only one characteristic transition ion

    was monitored [21], or in some GCMSMS [14] and

    GCMS methods using chemical ionization [15], two

    qualifier ions required in addition to the primary ion were

    not provided for identification.

    During the development and optimization of the GCMS

    method, a derivatization procedure was preferred and dif-

    ferent reagents (BSTFA with 1% TMCS, CH3I, PFPA with

    PFPOH) were tested. Derivatization of both analytes was

    achieved using all the reagents tested. Silylation resulted in

    increased sensitivity, so the conditions of this derivatization

    reaction (e.g., solvent environment, temperature, time) were

    optimized. Conditions of acetonitrile environment, tem-

    perature of 70C, and time duration of 30 min were optimalfor silylation of the cannabinoids. Furthermore, different

    chromatographic conditions were tested and the optimal

    values were selected according to the peak areas of both

    silylated analytes and their resolution.

    Optimization of the extraction procedure was also per-

    formed. The previously published methods for the determi-

    nation of THC and THCCOOH in biological fluids use either

    SPE [2, 5, 7, 8, 11, 13, 15, 17, 20, 21, 24, 25] or liquidliquid

    extraction (LLE) [12, 14, 22, 23]. When the LLE technique

    was tested using different organic solvents (hexane, ethyl

    acetate, and isooctane) or a mixture of hexane:ethyl acetate

    (60:40, 70:30, 80:20, and 90:10, v/v) at different pH values

    (4.0, 5.0, 6.0, and 7.0), there were many interferences from

    endogenous compounds, so the matrix effect influenced the

    derivatization of both analytes and reduced the recovery of

    the method. In our study, SPE was chosen for sample prep-

    aration, because it ensures a rapid, reproducible, and simple

    process that gives clean extracts suitable for a derivatization

    procedure. Bond Elut LRC Certify II SPE columns were

    selected in this study due to their chemical properties

    (nonpolar and strong anion exchange) and for their suit-

    ability for the analysis of acidic drugs. When these columns

    were tested, a low matrix effect was observed and the

    recovery results were high for both analytes ([85%). Dif-ferent ratios of the eluting solvent mixture of hexane:ethyl

    acetate for THC (70:30, 80:20, 90:10, and 95:5, v/v) and

    hexane:ethyl acetate:acetic acid for THCCOOH (70:30:1,

    80:20:1, 90:10:1, and 95:5:1, v/v/v) were evaluated. It was

    found that the eluting mixtures of hexane:ethyl acetate

    (90:10, v/v) and hexane:ethyl acetate:acetic acid (90:10:1,

    v/v/v) gave very good cleanup of the bile samples, and they

    gave high and reproducible extraction efficiency values for

    THC and THCCOOH, respectively.

    Method validation

    The combination of protein precipitation and SPE, prior to

    silylation, proved to be useful for the determination

    of THC and THCCOOH concentrations in bile. The

    developed method was validated in terms of selectivity,

    specificity, linearity, limit of detection (LOD), limit of

    Forensic Toxicol (2012) 30:5158 53


  • quantification (LOQ), recovery, precision, accuracy, and

    robustness according to international guidelines regarding

    bioanalytical method validation [27, 28].

    The selectivity of the method was adequate with minimal

    matrix effects for all blank bile samples (n = 6); no inter-

    ference from endogenous compounds was observed at the

    retention times of the analytes. A representative SIM chro-

    matogram of a blank bile samp...


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