Simultaneous detection of 93 synthetic cannabinoids by liquid chromatography‐tandem mass spectrometry and retrospective application to real forensic samples

The Internet is flooded with steadily changing synthetic cannabinoids in `Spice` products. In routine forensic work, it is difficult to keep the analytical methods for the detection of these analytes up to date. We describe a liquid chromatography‐tandem mass spectrometry method after liquid‐liquid extraction for the detection of 93 synthetic cannabinoids in human serum. The method was validated for selectivity and specificity, matrix effects, and analytical limits (<1 ng/mL for 81 substances) for qualitative analysis. A short quantitative validation regarding linearity and precision data was also conducted. The method was applied to 189 serum samples provided by police authorities. Sixty‐four samples (33.8%) were found positive for at least one synthetic cannabinoid, whereby MDMB‐CHMICA, AB‐CHMINACA, and 5 F‐PB‐22 were the substances most frequently detected. Consumption of these substances and plasma concentrations are linked to symptoms documented by the police. Six case reports are presented. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a simple and sensitive liquid chromatography–tandem mass spectrometry method for quantifying trimetazidine in human plasma

1. A simple and sensitive liquid chromatography–tandem mass spectrometry (LC‐MS‐MS) method for quantifying trimetazidine in human plasma was developed and validated. Sample preparation was based on deproteinating with acetonitrile. 2. Chromatography was performed on a C18 analytical column (5 μm; 150 × 2.1 mm i.d.) and the retention times for trimetazidine and cetirizine (used as the internal standard) were 1.8 and 3.0 min, respectively. The ionization was optimized using an electrospray ionization source and enhanced selectivity was achieved using tandem mass spectrometry. The calibration curve ranged from 0.1 to 200 ng/mL. The inter‐day precision, accuracy and the relative standard deviation (RSD) were all < 15%. The analyte was shown to be stable over the time‐scale of the entire procedure. 3. The robustness of the method was demonstrated by the good reproducibility of the results obtained during the analysis of clinical samples.     

Quantification of six cannabinoids and metabolites in oral fluid by liquid chromatography‐tandem mass spectrometry

Δ⁹‐Tetrahydrocannabinol (THC) is the most commonly analyzed cannabinoid in oral fluid (OF); however, its metabolite 11‐nor‐9‐carboxy‐THC (THCCOOH) offers the advantage of documenting active consumption, as it is not detected in cannabis smoke. Analytical challenges such as low (ng/L) THCCOOH OF concentrations hampered routine OF THCCOOH monitoring. Presence of minor cannabinoids like cannabidiol and cannabinol offer the advantage of identifying recent cannabis intake. Published OF cannabinoids methods have limitations, including few analytes and lengthy derivatization. We developed and validated a sensitive and specific liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for THC, its metabolites, 11‐hydroxy‐THC and THCCOOH quantification, and other natural cannabinoids including tetrahydrocannabivarin (THCV), cannabidiol (CBD), and cannabigerol (CBG) in 1 mL OF collected with the Quantisal device. After solid‐phase extraction, chromatography was performed on a Selectra PFPP column with a 0.15% formic acid in water and acetonitrile gradient with a 0.5 mL/min flow rate. All analytes were monitored in positive mode atmospheric pressure chemical ionization (APCI) with multiple reaction monitoring. Limits of quantification were 15 ng/L THCCOOH and 0.2 µg/L for all other analytes. Linear ranges extended to 3750 ng/L THCCOOH, 100 µg/L THC, and 50 µg/L for all other analytes. Inter‐day analytical recoveries (bias) and imprecision at low, mid, and high quality control (QC) concentrations were 88.7‐107.3% and 2.3‐6.7%, respectively (n = 20). Mean extraction efficiencies and matrix effects evaluated at low and high QC were 75.9–86.1% and 8.4–99.4%, respectively. This method will be highly useful for workplace, criminal justice, drug treatment and driving under the influence of cannabis OF testing. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Simultaneous quantification of 19 nonsteroidal anti-inflammatory drugs in oral fluid by liquid chromatography-high resolution mass spectrometry: Application on ultratrail runner's oral fluid

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a therapeutic class suspected to be used by ultratrail runners. The use of NSAIDs during ultratrails is known to be associated with various adverse effects. To study the prevalence of NSAIDs intake in ultratrail runners, oral fluid (OF) is a relevant matrix as it is noninvasive and easy to collect. The aim of our work was to develop and validate a liquid–liquid extraction followed by a liquid chromatography (LC)–mass spectrometry (MS)/high resolution mass spectrometry (HRMS) method for the simultaneous quantification of 19 NSAIDs in OF. After a comparison of different liquid–liquid extraction methods, a double step liquid–liquid extraction with chloroform was performed on OF collected with Quantisal®, with extraction recoveries higher than 90%. An Accucore AQ column was selected for the chromatographic separation of NSAIDs. The Q Exactive Plus mass spectrometer operated in full scan and ddms2 mode after positive and negative electrospray ionization. Selectivity, carry-over, matrix effect, and linearity were validated for all NSAIDs. Within-day and between-day accuracy and precision were validated for all NSAIDs (<15% for quality control [QC] samples and <20% for lower limit of quantitation [LLOQ]), except within-day accuracy for the LLOQ of mefenamic acid. A stability study was also performed on OF at room temperature and +4°C. The method was applied on OF from runners who participate to Ultra Trail du Mont Blanc®.     

Analysis of 30 synthetic cannabinoids in oral fluid using liquid chromatography‐electrospray ionization tandem mass spectrometry

In recent years, the analysis of synthetic cannabinoids in human specimens has gained enormous importance in the broad field of drug testing. Nevertheless, the considerable structural diversity among synthetic cannabinoids already identified in ‘herbal mixtures’ hampers the development of comprehensive analytical methods. As the identification of the main metabolites of newly appearing substances is very laborious and time‐consuming, the detection of the parent compounds in blood samples is the current approach of choice for drug abstinence testing. Whenever blood sampling is not possible however, the need for alternative matrices arises. In this article, we present a fully validated liquid chromatography‐electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) method for the analysis of 30 synthetic cannabinoids in oral fluid samples collected with the Dräger DCD 5000 collection device. The method proved to be suitable for the quantification of 28 substances. The limits of detection were in the range from 0.015 to 0.9 ng/ml, while the lower limits of quantification ranged from 0.15 to 3.0 ng/ml. The method was successfully applied to 264 authentic samples during routine analysis. A total of 31 samples (12%) was tested positive for at least one of the following synthetic cannabinoids: AM‐694, AM‐2201, JWH‐018, JWH‐019, JWH‐081, JWH‐122, JWH‐203, JWH‐210, JWH‐250, JWH‐307, MAM‐2201, and RCS‐4. Given that stabilization of the collection pads after sampling is warranted, the collection device provides satisfactory sensitivity. Hence, whenever blood sampling is not possible, the Dräger DCD 5000 collection device offers a good tool for the analysis of synthetic cannabinoids in oral fluid in the broad field of drug testing. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous analysis of frequently used licit and illicit psychoactive drugs in breast milk by liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC-MS-MS) method for the quantification of frequently used licit (caffeine, nicotine and cotinine) and illicit drugs (opiates, cocaine, cannabinoids and amphetamines) in breast milk was developed and fully validated. Chromatography was performed on a reverse-phase column using a gradient of 2mM ammonium acetate, pH 6.6, and methyl alcohol as mobile phase at a flow rate of 0.35 mL/min. Separated analytes were quantified by electrospray ionization tandem mass spectrometry in positive ion mode using multiple reaction monitoring. Milk samples were kept at -20 °C until analysis and the compounds under investigation were extracted from the matrix by Bond Elut Certify cartridges. The concentration range covered was LOQ to 1000 ng/mL for all the investigated drugs. Intra- and inter-assay imprecision was less than 20%, analytical recovery ranged between 51.6% and 86.5%, matrix effect between 71.1% and 116.6% and process efficiency between 46.8% and 84.0%. Analytes were stable after three freeze-thaw cycles, after 6 months at -20 °C and after the pasteurization process (differences to the initial concentration always lower than 10%). matrix effect ranged from 77.6% to 116.6%, recovery from 51.6% to 86.5%, and process efficiency from 46.8% to 79.0%. This LC-MS-MS assay was applied to screen samples from the largest Spanish milk bank and samples coming from drug addicted mothers. The developed method provided adequate sensitivity and performance characteristics to prove the presence of only caffeine in a small percentage of samples from milk donating nursing mothers and the presence or absence of most commonly used illicit drugs in breast milk from addicted lactating mothers.     

Simultaneous determination of phytoestrogens and key metabolites in breast cancer patients’ urine by liquid chromatography–tandem mass spectrometry

A novel, selective and sensitive liquid chromatography–tandem mass spectrometry method has been developed and validated for the simultaneous determination of phytoestrogens and their key metabolites in human urine in this study. This method includes internal standard (IS) screening, analytical sample preparation procedure establishment, and linear range investigation. The analytical sample was extracted by liquid–liquid extraction from urine sample. The phytoestrogens and related key metabolites were separated with Agilent Zorbax Eclipse XDB-C18 chromatographic column using methanol and water as mobile phase. The Quattro premier MICROMASS mass spectrometer in negative ion selected reaction monitoring (SRM) mode using electrospray ionization was applied to detect the phytoestrogens and key metabolites. To validate the developed liquid chromatography–tandem mass spectrometry method, the intra- and inter-day precisions, specificity, sensitivity, reproducibility, and sample detective concentration range were evaluated. This is the first reported phytoestrogens analysis and validation study that demonstrates the feasibility of using liquid chromatography–electrospray ionization mass spectrometry to simultaneously analyze ten analytes including both phytoestrogens and their key metabolites in urine samples collected for epidemiological studies in human.     

New oxymesterone metabolites in human by gas chromatography‐tandem mass spectrometry and their application for doping control

Oxymesterone (17α‐methyl‐4, 17β‐dihydroxy‐androst‐4‐ene‐3‐one) is one of the anabolic androgenic steroids (AAS) banned by the World Anti‐Doping Agency (WADA). The biotransformation of oxymesterone is performed in vitro by human heptocytes and human urinary metabolic profiles are investigated after single dose of 20 mg to two adult males as well. Cell cultures and urine samples were hydrolyzed by β‐glucuronidase, extracted, and reacted with N‐Methyl‐N‐trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide (NH₄I), and dithioerythritol. After derivatization, a gas chromatography triple quadruple tandem mass spectrometry (GC‐MS/MS) using full scan and MS/MS modes was applied. The total ion chromatographs of the blank and the positive samples are compared, and 7 new metabolites were found. In addition to the well‐known 17‐epioxymesterone, oxymesterone is metabolized by 4‐ene‐reduction, 3‐keto‐reduction, 11β‐hydroxylation, and 16ξ‐hydroxylation. Based on the behavior of the MS/MS results of product ion and precursor ion modes, a GC‐MS/MS method has been developed monitoring these metabolites. The structures of metabolite 2 and 4 are tentatively identified as 17α‐methyl‐3β, 17β‐dihydroxy‐5α‐androstane‐4‐one and 17α‐methyl‐3α, 4ξ, 17β‐trihydroxy‐5α‐androstane, respectively. Detection of oxymesterone using new metabolites M2 and M4 can extend the detection window up to 4 days since the parent steroid was not detectable. Copyright © 2015 John Wiley & Sons, Ltd.     

Profiling cocaine residues and pyrolytic products in wastewater by mixed‐mode liquid chromatography–tandem mass spectrometry

This work provides a new analytical method for the determination of cocaine, its metabolites benzoylecgonine and cocaethylene, the pyrolytic products anhydroecgonine and anhydroecgonine methyl ester, and the pharmaceutical levamisole in wastewater. Samples were solid‐phase extracted and extracts analyzed by liquid chromatography–tandem mass spectrometry using, for the first time in the illicit drug field, a stationary phase that combines reversed‐phase and weak cation‐exchange functionalities. The overall method performance was satisfactory, with limits of detection below 1 ng/L, relative standard deviations below 21%, and percentages of recovery between 93% and 121%. Analysis of 24‐hour composite raw wastewater samples collected in Santiago de Compostela (Spain) and Brasilia (Brazil) highlighted benzoylecgonine as the compound showing the highest population‐normalized mass loads (300–1000 mg/day/1000 inhabitants). In Brasilia, cocaine and levamisole loads underwent an upsurge on Sunday, indicating a high consumption, and likely a direct disposal, of cocaine powder on this day. Conversely, the pyrolytic product resulting from the smoke of crack, anhydroecgonine methyl ester, and its metabolite anhydroecgonine were relatively stable over the four days, agreeing with a non‐recreational‐associated use of crack.     

Significant enhancement of 11‐Hydroxy‐THC detection by formation of picolinic acid esters and application of liquid chromatography/multi stage mass spectrometry (LC‐MS3): Application to hair and oral fluid analysis

Formation of picolinic acid esters of hydroxylated drugs or their biotransformation products is a promising tool to improve their mass spectrometric ionization efficiency, alter their fragmentation behaviour and enhance sensitivity and specificity of their detection. The procedure was optimized and tested for the detection of cannabinoids, which proved to be most challenging when dealing with alternative specimens, for example hair and oral fluid. In particular, the detection of the THC metabolites hydroxyl‐THC and carboxy‐THC requires ultimate sensitivity because of their poor incorporation into hair or saliva. Both biotransformation products are widely accepted as incorporation markers to distinguish drug consumption from passive contamination. The derivatization procedure was carried out by adding a mixture of picolinic acid, 4‐(dimethylamino)pyridine and 2‐methyl‐6‐nitrobenzoic anhydride in tetrahydrofuran/triethylamine to the dry extraction residues. Resulting derivatives were found to be very stable and could be reconstituted in aqueous or organic buffers and subsequently analyzed by liquid chromatography‐mass spectrometry (LC‐MS). Owing to the complex consecutive fragmentation patterns, the application of multistage MS3 proved to be extremely useful for a sensitive identification of doubly picolinated hydroxy‐THC in complex matrices. The detection limits – estimated by comparison of corresponding signal‐to‐noise ratios – increased by a factor of 100 following picolination. All other species examined, like cannabinol, THC, cannabidiol, and carboxy‐THC, could also be derivatized exhibiting only moderate sensitivity improvements. The assay was systematically tested using hair samples and exemplarily applied to oral fluid. Concentrations of OH‐THC identified in THC‐positive hair samples ranged from 0.02 to 0.29pg/mg. Copyright © 2014 John Wiley & Sons, Ltd.     

Separation of positional isomers of nine 2‐phenethylamine‐derived designer drugs by liquid chromatography–tandem mass spectrometry

The synthesis of positional isomers of designer drugs is a common way of bypassing legal restrictions. For forensic case work, and especially for the legal assessment of cases, there is a need for screening methods capable of the unequivocal identification of positional isomers. The presented liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) method facilitates separation of positional isomers of 9 2‐phenethylamine‐derived designer drugs in different matrices including seized materials, hair, serum, and urine specimens. Chromatographic separation was achieved on a biphenyl phase using gradient elution with a total runtime of 26 minutes. The limit of detection was 25 pg/mg for hair samples and ranged from 0.1 ng/mL to 0.5 ng/mL for serum and from 0.2 ng/mL to 1.2 ng/mL for urine samples. The method proved to be selective and sensitive and showed good chromatographic resolution (R ≥ 1.2). The method was successfully applied to routine case samples.     

Sensitive and rapid determination of pyrethroids in human blood by gas chromatography–tandem mass spectrometry with ultrasound‐assisted dispersive liquid‐liquid microextraction

In this study, a sensitive and fast procedure of ultrasonic‐assisted dispersive liquid‐liquid microextraction (UADLLME) coupled with gas chromatography–tandem mass spectrometry (GC–MS/MS) for the determination of major pyrethroid pesticides (permethrin, tetramethrin, bifenthrin, fenvalerate, flucythrinate, fluvalinate, fenpropathrin, deltamethrin, and cyhalothrin) in blood samples was developed. Response surface methodology (RSM) combined with Box‐Behnken design (BBD) and ANOVA function was used to optimize key factors affecting the extraction efficiency of UADLLME procedure. Target compounds were analyzed by GC‐MS/MS. Under the optimal conditions, good linearity (R²>0.99) was achieved for all the analytes in the concentration range of 0.5 to 100 μg L^‐1. The recoveries for spiked samples at 3 concentration levels were between 70.2 and 91.8%, with relative standard deviations (RSD) lower than 10%. Very low limits of detection (LODs) and limits of quantification (LOQs) ranging from 0.01 to 0.1 μg L^‐1 and from 0.03 to 0.3 μg L^‐1 were achieved. This method was successfully applied to the determination of low concentration of pyrethroids in blood samples from real forensic cases. High sensitivity, fast determination, simplicity in operation, small sample volume, and low usage of organic solvents are the advantages of this method. This methodology is of important value for sensitive and quick determination of residue pesticides and metabolites, study of residue pesticides behavior in human body, as well as application in real forensic cases.     

Development of an ultra‐high performance liquid chromatography–tandem mass spectrometry method for the determination of anabolic steroids currently available on the black market in the Czech Republic and Slovakia

Over the past decade, the use of anabolic androgenic steroids (AAS) by recreational bodybuilders and regular gym members has been increasing exponentially. However, there is a lack of knowledge on AAS products sold in the Czech Republic and Slovakia. The aim of this study was to assess the quantity of active substances in AAS products obtained from AAS users in the Czech Republic and Slovakia. In addition, the study also examines the current trends in counterfeit AAS products used by recreational bodybuilders and regular gym members. For this purpose, the authors developed and validated a universal ultra‐high performance liquid chromatography with tandem mass spectrometry method to determine the most popular doping substances in different pharmaceutical formulations (oil‐based injectables, tablets, and capsules). This method was successfully utilized for the analysis of 358 AAS products. Our results showed that 58.9% AAS products analyzed contained the declared active substances at the declared concentrations, 15.9% contained no active substance, 16.8% were under‐concentrated, 4.5% contained active substances different from those declared on the label, and 3.6% products were over‐concentrated. Alarmingly, the results demonstrated that over 40% of the AAS analyzed failed to meet label claims and therefore may pose serious health risks to consumers. This study also highlighted that the availability of AAS should be more rigorously restricted and their quality closely monitored in order to protect AAS users. In conclusion, the authors have developed a precise, accurate, sensitive, selective, and robust method for the routine screening of products containing anabolic androgenic steroids.     

Enantiomeric separation and quantification of R/S‐amphetamine in serum using semi‐automated liquid‐liquid extraction and ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry

The amphetamine molecule contains a chiral center and its enantiomers exhibit differences in pharmacological effects, with the S‐enantiomer mediating most of the central nervous system stimulating activity. The majority of prescribed amphetamine consists of the pure S‐enantiomer, but therapeutic formulations containing the R‐enantiomer in various proportions are also available. Illegal amphetamine remains available mainly as a racemic mixture of the R‐ and S‐enantiomers. To distinguish between legal and illegal consumption of amphetamine a method for enantiomeric separation and quantification of R/S‐amphetamine in serum was developed and validated using ultra‐high performance supercritical fluid chromatography‐tandem mass spectrometry (UHPSFC‐MS/MS). Sample preparation prior to UHPSFC‐MS/MS analysis was performed by a semi‐automated liquid–liquid extraction method. The UHPSFC‐MS/MS method used a Chiralpak AD‐3 column with a mobile phase consisting of CO₂ and 0.1% ammonium hydroxide in 2‐propanol/methanol (50/50, v/v). The injection volume was 2 μL and run time was 4 minutes. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions (m/z 136.1 > 119.0 and m/z 136.1 > 91.0). The calibration range was 12.5–1,000 nM for each analyte. The between‐assay relative standard deviations were in the range of 1.3–3.0%. Recovery was 73% and matrix effects ranged from 95 to 100% when corrected with internal standard. After development and validation, the method has been successfully implemented in our laboratory for both separation and quantification of R/S‐amphetamine and has proved to be a reliable and useful tool for distinguishing intake of R‐ and S‐amphetamine in authentic patient samples.     

Liposomes as potential masking agents in sport doping. Part 1: analysis of phospholipids and sphingomyelins in drugs and biological fluids by aqueous normal‐phase liquid chromatography‐tandem mass spectrometry

In the present work, aqueous normal‐phase liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS), in different acquisition modes, was employed for the direct analysis and profiling of nine phospholipid classes (phosphatidic acids, phosphatidylserines, phosphatidylethanolamines, lysophosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols, phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins) in biological and pharmaceutical matrices. After chromatographic separation by a diol column, detection and elucidation of phospholipid and sphingomyelin classes and molecular species were performed by different scan acquisition modes. For screening analysis, molecular ions [M + H]⁺ were detected in positive precursor ion scan of m /z 184 for the classes of phosphatidylcholines, lyso‐phosphatidylcholines and sphingomyelins; while phosphatidylethanolamines and lyso‐phosphatidylethanolamines were detected monitoring neutral loss scan of 141 Da; and phosphatidylserines detected using neutral loss scan of 184 Da. Molecular ions [M‐H]^‐ were instead acquired in negative precursor ion scan of m /z 153 for the classes of phosphatidic acids and phosphatidylglycerols; and of m /z 241 for the phosphatidylinositols. For the identification of the single molecular species, product ion scan mass spectra of the [M + HCOO]^‐ ions for phosphatidylcholines and [M + H]⁺ ions for the other phospholipids considered were determined for each class and compared with the fragmentation pattern of model phospholipid reference standard. By this approach, nearly 100 phospholipids and sphingomyelins were detected and identified. The optimized method was then used to characterize the phospholipid and sphingomyelin profiles in human plasma and urine samples and in two phospholipid‐based pharmaceutical formulations, proving that it also allows to discriminate compounds of endogenous origin from those resulting from the intake of pharmaceutical products containing phospholipidic liposomes. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of 21 drugs in oral fluid using fully automated supported liquid extraction and UHPLC‐MS/MS

Collection of oral fluid (OF) is easy and non‐invasive compared to the collection of urine and blood, and interest in OF for drug screening and diagnostic purposes is increasing. A high‐throughput ultra‐high‐performance liquid chromatography‐tandem mass spectrometry method for determination of 21 drugs in OF using fully automated 96‐well plate supported liquid extraction for sample preparation is presented. The method contains a selection of classic drugs of abuse, including amphetamines, cocaine, cannabis, opioids, and benzodiazepines. The method was fully validated for 200 μL OF/buffer mix using an Intercept OF sampling kit; validation included linearity, sensitivity, precision, accuracy, extraction recovery, matrix effects, stability, and carry‐over. Inter‐assay precision (RSD) and accuracy (relative error) were <15% and 13 to 5%, respectively, for all compounds at concentrations equal to or higher than the lower limit of quantification. Extraction recoveries were between 58 and 76% (RSD < 8%), except for tetrahydrocannabinol and three 7‐amino benzodiazepine metabolites with recoveries between 23 and 33% (RSD between 51 and 52 % and 11 and 25%, respectively). Ion enhancement or ion suppression effects were observed for a few compounds; however, to a large degree they were compensated for by the internal standards used. Deuterium‐labelled and ¹³C‐labelled internal standards were used for 8 and 11 of the compounds, respectively. In a comparison between Intercept and Quantisal OF kits, better recoveries and fewer matrix effects were observed for some compounds using Quantisal. The method is sensitive and robust for its purposes and has been used successfully since February 2015 for analysis of Intercept OF samples from 2600 cases in a 12‐month period. Copyright © 2016 John Wiley & Sons, Ltd.     

UPLC-MS/MS法测定小鼠血浆及脑组织中马钱子碱浓度

目的建立小鼠血浆及脑组织中马钱子碱浓度的UPLC-MS/MS测定方法 ,用于小鼠马钱子碱药动学研究。方法色谱柱为Acquity UPLC BEHC18柱（2.1mm×50mm,1.7μm）。流动相为水（含0.1%甲酸）-甲醇（75：25,v/v）,流速0.25mL.min-1,柱温40℃。吗氯贝胺为内标,采用ESI＋模式,多重反应选择离子检测,马钱子碱m/z395→324,吗氯贝胺m/z269→182。样品处理采用液液萃取。结果马钱子碱在小鼠血浆中线性范围为5.016～5016ng.mL-1,脑组织中线性范围为3.009～725.4ng.g-1,日内精密度（RSD）均〈8.5%,日间精密度（RSD）均〈11.2%,方法回收率范围血浆中为91.7%～112.9%,脑组织中为95.6%～107.2%。结论本方法简单、灵敏、准确,适用于小鼠体内马钱子碱浓度测定及药动力学研究。     

A simple and reliable procedure for the determination of psychoactive drugs in oral fluid by gas chromatography-mass spectrometry

A simple and reliable gas chromatography-mass spectrometry method for identifying and quantifying psychoactive drugs in oral fluid is described. Substances under investigation were: psychostimulant drugs (amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxiamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, phentermine), cocaine and metabolites (benzoylecgonine, cocaethylene, and ecgonine methyl esther), cannabinoids (delta-9-tetrahydrocannabinol, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol, 11-hydroxy-delta-9-tetrahydrocannabinol, cannabinol and cannabidiol), opiates (6-monoacetylmorphine, morphine and codeine), hypnotics (flurazepam, flunitrazepam, dipotassium chlorazepate, alprazolam, diazepam and oxazepam), antidepressant drugs (amitryptiline, paroxetine and sertraline), antipsychotic drugs (haloperidol, chlorpromazine and fluphenazine) chlormethiazole, loratidine, hydroxyzine, diphenhydramine, valproic acid and gabapentin. After the addition of deuterated analogues of morphine, 3,4-methylenedioxymethamphetamine, (+/-)-11-nor-9-carboxy-delta-9-tetrahydrocannabinol and clonazepam as internal standards, all the compounds were simultaneously extracted from oral fluid by solid-phase extraction procedure. Acid compounds were eluted with acetone while basic and neutral compounds with dichloromethane:isopropanol:ammonium (80:20:2, v/v/v). Chromatography was performed on a methylsilicone capillary column and analytes, derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide, were determined in the selected-ion-monitoring (SIM) mode. Mean recovery ranged between 44.5 and 97.7 % and quantification limit between 0.9 and 44.2 ng/ml oral fluid for the different analytes. The developed analytical methodology was applied to investigate the presence of psychoactive drugs in oral fluid from injured individuals attending the emergency room (MACIUS project).