Identification of selected psychopharmaceuticals and their metabolites in hair by LC/ESI-CID/MS and LC/MS/MS

Hair samples of patients of psychiatry and hair samples of suicide cases were analysed by liquid-chromatography/ionspray-mass spectrometry (LC/MS) for antidepressants and neuroleptics. Electrospray ionisation (ESI) with in-source collision induced dissociation (ESI/CID) and tandem-mass spectrometry (MS/MS) were used for drug and metabolite identification. Mass spectra library searching was performed using an ESI/CID mass spectra library and a MS/MS spectra library. Furthermore, extracted ion chromatograms were used for the detection of N-desmethyl-metabolites, which were also identified by their fragment-ion spectra. Three examples using these methods are shown: The tricyclic antidepressant maprotiline, the selective serotonin receptor inhibitor (SSRI) citalopram and their desmethylmetabolites as well as the neuroleptic pipamperone were detected and identified in hair extracts. For extraction powdered hair was treated by ultrasonication in methanol and solid-phase extraction was used for sample clean-up prior to LC/MS or MS/MS analysis. These examples demonstrate the power of LC/MS and LC/MS/MS for the detection and identification of drugs in hair extracts using full-scan mode and ESI/CID with library searching or using highly selective LC/MS/MS-analysis with library searching or in multiple reaction monitoring mode.     

GC/MS/Xcalibur技术自动分析疑似固体中管制药物

目的建立疑似含毒检材中国家管制药品的自动筛选方法。方法以我国2007年公布的精神药品和麻醉药品的品种目录为检测目标物,建立质谱用户库;以此为基础,设置气质联用仪自动分析方法对疑拟检材进行常见管制药品的快速筛选。结果建立了库名含多种实用信息、包括230种管制药品的质谱用户库;建立管制药品的质谱自动分析方法,实现＂一针分析出结果＂。结论方法简单、快速、准确可靠,省时省力,能有效提高可疑检材中管制药品筛选分析的速度。     

Electrospray-ionization MS/MS library of drugs as database for method development and drug identification

An ESI MS/MS library of 800 compounds has been developed and a collection of data is now available for Analyst 1.4 and higher. Compounds include forensically important drugs, such as illegal drugs, some deuterated analogues, hypnotics, amphetamines, benzodiazepines, neuroleptics, antidepressants and many others. For setting up the library of product ion spectra, 20-200 ng of the compounds have been injected either by flow injection or via a short LC-column, the precursor ions were chosen from the Q1 scan spectra, and product ion spectra were generated by CID in the collision cell using three different collision energies (20, 35 and 50 eV). Three spectra of each compound have been collected and compound names, CAS numbers, formulas and molecular weights have been added in the database, which has been generated by the Analyst software. The library can be used for compound identification during general unknown screening analysis by combination of Q1 scan techniques and subsequent MS/MS analysis in a second analytical run. Quantitative procedures for multi drug analysis using Multiple Reaction Monitoring can be established by selection of product ions and suitable collision energies from the library. For publication of the spectra, PDF-files have been generated and can be viewed on-line as supplementary data or from the website in alphabetical order: (supplementary data, should be made available via ELSEVIER-WEBSITE or via ).     

Simultaneous determination of THC-COOH and THC-COOH-glucuronide in urine samples by LC/MS/MS

A fast method using liquid-liquid extraction and HPLC/tandem-mass spectrometry (LC/MS/MS) was developed for the simultaneous detection of 11-Nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid beta-glucuronide (THC-COOH-glucuronide) and 11-Nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. This highly specific method, which combines chromatographic separation and MS/MS analysis, can be used for the confirmation of positive immunoassay results even without hydrolysis of the sample or derivatisation of extracts. Liquid-liquid extraction was optimised: with ethylacetate/diethylether (1:1, v/v) THC-COOH-glucuronide and THC-COOH could be extracted in one step. Molecular ions of the glucuronide (MH(+), m/z 521) and THC-COOH (MH(+), m/z 345) were generated using a PE/SCIEX turboionspray source in positive ionisation mode; specific fragmentation was performed in the collision cell of an API 365 triple-quadrupole mass spectrometer and yielded major fragments at m/z 345 (for THC-COOH-glucuronide) and m/z 327 as well as m/z 299 for both cannabinoids. Chromatographic separation was performed using a reversed-phase C8 column and gradient elution with 0.1% formic acid/1 mM ammonium formate and acetonitrile/0.1% formic acid. Retention times were 22.2 min for the glucuronide and 26.8 min for THC-COOH. After enzymatic hydrolysis of urine samples with beta-glucuronidase/arylsulfatase (37 degrees C, 5 h), THC-COOH-glucuronide was no longer detectable by LC/MS/MS in urine samples. However, the THC-COOH concentration was increased. For quantitation of THC-COOH, THC-COOH-D(3) was added to the urine samples as internal standard prior to analysis. From the difference of THC-COOH in the native urine and urine after enzymatic hydrolysis, molar concentration ratios of THC-COOH-glucuronide/THC-COOH in urine samples of cannabis users were determined and found to be between 1.3 and 4.5.     

Detection of the synthetic drug 4-fluoroamphetamine (4-FA) in serum and urine

4-Fluoroamphetamine (4-FA) was detected in the blood and urine of two individuals suspected for driving under the influence (DUI). The test for amphetamines in urine subjected to immunoassay screening using the CEDIA DAU assay proved positive. Further investigations revealed a 4-FA cross-reactivity of about 6% in the CEDIA amphetamine assay. 4-FA was qualitatively detected in a general unknown screening for drugs using GC/MS in full scan mode. No other drugs or fluorinated phenethylamines were detected. A validated GC/MS method was established in SIM mode for serum analysis of 4-FA with a limit of detection (LOD) of 1 ng/mL and a lower limit of quantification (LLOQ) of 5 ng/mL. Intra-assay precision was approx. 4% and inter-assay precision approx. 8%. Applying this method, the 4-FA serum concentrations of the two subjects were determined to be 350 ng/mL and 475 ng/mL, respectively. Given the pharmacological data of amphetamine, 4-FA psychoactive effects are to be expected at these serum levels. Both subjects exhibited sympathomimetic effects and psychostimulant-like impairment accordingly.     

Drug-facilitated sexual assault using tetrahydrozoline

Drug-facilitated sexual assault (DFSA) has been defined as the use of a chemical agent to facilitate a sexual assault. We report two cases of the use of tetrahydrozoline for DFSA. We believe this is the first report with urinary quantification of tetrahydrozoline levels postassault. Blood and urine were obtained c. 20 h postexposure in two cases of reported DFSA. Tetrahydrozoline was not detected in blood but was identified in urine in both victims. After initial identification in the urine using the 2010 update to the AAFS mass spectrometry database library, tetrahydrozoline was quantified at 114 and 150 ng/mL, respectively, using GC/MS. Two unique clinical features reported in these cases were intermittent periods of consciousness and postexposure vomiting. Use of GC/MS was successful in identifying tetrahydrozoline in the 100 ng/mL range up to 20 h postexposure. For victims with late presentation, urine may be a better sample for evaluation for tetrahydrozoline.     

Time of drug elimination in chronic drug abusers. Case study of 52 patients in a "low-step" detoxification ward

The elimination time of illicit drugs and their metabolites is of both clinical and forensic interest. In order to determine the elimination time for various drugs and their metabolites we recruited 52 volunteers in a protected, low-step detoxification program. Blood samples were taken from each volunteer for the first 7 days, daily, urine sample for the first 3 weeks, daily. Urine was analyzed using a fluorescence-polarization immunoassay (FPIA) and gas chromatography/mass spectrometry (GC/MS), serum using GC/MS. The elimination times of the drugs and/or their metabolites in urine and serum as well as the tolerance intervals/confidence intervals were determined. Due to the sometimes extremely high initial concentrations and low cut-off values, a few of the volunteers had markedly longer elimination times than those described in the literature. The cut-off values were as follows: barbiturates II (200ng/ml), cannabinoids (20ng/ml), cocaine metabolites (300ng/ml), opiates (200ng/ml). GC/MS detected the following maximum elimination times: total morphine in urine up to 270.3h, total morphine and free morphine in serum up to 121.3h, monoacetylmorphine in urine up to 34.5h, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in urine up to 433.5h, THC-COOH in serum up to 74.3h, total codeine in urine up to 123h, free codeine in urine up to 97.5h, total codeine in serum up to 29h, free codeine in serum up to 6.3h, total dihydrocodeine (DHC) in urine up to 314.8h, free DHC in urine up to 273.3h, total and free DHC in serum up to 50.1h. Cocaine and its metabolites were largely undetectable in the present study.     

Differentiation of side chain isomers of ring-substituted amphetamines using gas chromatography/infrared/mass spectrometry (GC/IR/MS).

Common analytical methods used for identifying samples obtained from clandestine laboratories were evaluated for their ability to differentiate between possible amphetamine isomers and homologs. A series of ring-substituted (4-methyl, 4-methoxy, and 3,4-methylenedioxy) amphetamine and N-methylphenethylamine isomers was analyzed using color tests, thin-layer chromatography, gas chromatography/mass spectrometry (GC/MS) and GC/infrared (GC/IR). The N-acetyl derivatives of the isomers were analyzed using GC/IR/MS. GC/IR/MS readily differentiated the 4-methylphenylalkylamine isomers. MS and IR spectra were also obtained for each pair of the 4-methoxyphenylalkylamine isomers and the 3,4-methylenedioxyphenylalkylamine isomers, but differentiation via GC/IR/MS was difficult. The N-acetyl derivatives of each pair of isomers could be readily differentiated using GC/IR/MS. Good library researchable spectra for N-acetylamphetamine could be obtained for IR identification with 10 ng (on-column) and MS identification with 2 ng. The spectrometrically independent IR and MS data obtained for the N-acetyl derivatives indicated that the combination of GC/IR/MS can add a significant level of confidence in the analysis of ring-substituted arylalkylamines.     

Detection and assay of cis- and trans-isomers of 4-methylaminorex in urine, plasma and tissue samples

The 4-methylaminorex (4-MAX) is an amphetamine-related psychostimulant drug that has appeared on the clandestine market with a street name of "U4Euh". This compound exists as four stereoisomers, trans-4R,5R, trans-4S,5S, cis-4R,5S and cis-4S,5R, of which the cis forms have been classified as Schedule I substances in the US. The increasing variety of designer drugs has highlighted the importance of detection, identification, and quantitative measurement of these drugs, including 4-MAX, in biological samples. In the present study, the isomers of 4-MAX were detected in urine of rats treated with the drugs by some but not all of the on-site immunoassays tested, mainly as amphetamine or methamphetamine. To facilitate identification of 4-MAX by laboratories specialized in drug analysis, the electron-ionization mass spectrum and TLC data for underivatized 4-MAX using a routine laboratory drug-screening procedure is provided. In addition, a GC/MS method is described for the quantitative determination of cis- and trans-4-MAX as tert-butyldimethylsilyl-derivatives in plasma, urine and tissue.     

Synthesis of 2,3- and 3,4-methylenedioxyphenylalkylamines and their regioisomeric differentiation by mass spectral analysis using GC-MS-MS

3,4-methylenedioxyamphetamine (MDA) derivatives are increasingly abused central nervous system stimulants with neurotoxic properties. In recent years a number of controlled substance analogs (designer drugs) with high structural variety reached the illegal market making their identification an arduous task. The underivatized compounds give very similar or even virtually identical electron impact mass spectra containing mainly intense C(n)H(2n+2)N(+) immonium ions. Using tandem mass spectrometry (MS-MS) the additional structural information contained in the collision induced dissoziation (CID) mass spectra of molecular ions using electron impact (EI) and especially chemical ionization (CI) allowed an unequivocal differentiation of 18 studied regioisomeric 1-(methylenedioxyphenyl)-2-propanamines and 1-(methylenedioxyphenyl)-2-butanamines.Further synthetic methods are presented for 1-(3,4-methylenedioxyphenyl)-N-propyl-2-butanamine, N-isopropyl-1-(3,4-methylenedioxyphenyl)-2-butanamine and four 1-(2, 3-methylenedioxyphenyl)-2-butanamines. N-alkylated 1-(3, 4-methylenedioxyphenyl)-2-butanamine compounds (e.g. MBDB) are also known to be abused psychoactive agents (entactogenes) without the sympatomimetic effects of the 3,4-methylenedioxyamphetamines.     

Screening and quantification of antipsychotic drugs in human brain tissue by liquid chromatography-tandem mass spectrometry: application to postmortem diagnostics of forensic interest

A quantitative LC-MS/MS method has been developed for the simultaneous determination of 17 antipsychotic drugs in human postmortem brain tissue. Sample preparation was performed using Hybrid Solid Phase Extraction-Precipitation technology for the removal of endogenous protein and phospholipid interferences. The chromatographic separation was performed for 16 min on a C8 column, which used a gradient elution of formate ammonium and acetonitrile, and a flow rate gradient. Triple quadrupole mass spectrometry was employed to generate tandem mass spectrometric (MS/MS) data of the target analytes to select the ion m/z signals. Quantitation of the analytes was performed by operating in the dynamic multiple reaction monitoring (dMRM) mode using an electrospray ionization interface. Calibration curves prepared in the spiked brain tissue were linear in the range 20-8000 ng/g (r(2)>0.993) for all drugs (except olanzapine). Within- and between-day coefficients of variation were lower than 25% for all drugs at the LOQ. The LOQ in the matrix ranged between 2 ng/g and 80 ng/g. The method was successfully applied to the unequivocal identification and accurate quantification of antipsychotic drugs in human postmortem brain tissues: therefore, this method can be used in forensic investigations.     

Pancuronium bromide (Pavulon) isolation and identification in aged autopsy tissues and fluids

The isolation and detection of pancuronium bromide was developed for aged autopsy samples to identify and confirm this compound in questioned tissue samples. A novel protocol was optimized for the isolation of the target drug in highly decomposed tissues. Solid-phase extraction (SPE) cartridges containing styrene-divinylbenzene were investigated. This polymer retained quaternary drugs and facilitated sequential elution upon washing with commonly available solvents. The semi-purified SPE samples were prescreened by pyrolysis GC-MS. A candidate specimen was then confirmed by microbore high-performance liquid chromatography/electrospray-ionization/mass spectrometry (microHPLC-ESI-MS/MS) with a triple-quadrupole mass spectrometer. The developed procedures provided a qualitative or semiquantitative (at best) basis for the investigation of difficult cases involving overdoses of polar drugs.     

液相色谱-串联质谱法测定血液和尿液中秋水仙碱

目的建立检测血液和尿液中秋水仙碱的液相色谱-串联质谱法。方法0.5mL血液或尿液以丁丙诺啡为内标,经pH9.2硼酸盐缓冲溶液碱化后,用乙酸乙酯进行提取,在ZORBAX SB-C18液相柱(150mm×2.1mm×5μm)上以V(甲醇)∶V(20mmol/L乙酸铵和0.1%甲酸缓冲溶液)=80∶20为流动相,流速为0.2mL/min,采用电喷雾正离子模式离子化、多反应监测模式检测秋水仙碱,内标法定量。结果血液、尿液中秋水仙碱与内标丁丙诺啡色谱分离良好,秋水仙碱在0.1~50 ng/mL内均具有良好的线性,相关系数>0.9990,最低检出限为0.05ng/mL,方法回收率为94%~116%,日内与日间精密度(RSD)均小于8.5%。结论所建LC-MS-MS方法灵敏度高、操作简便、快速、准确,适用于血液及尿液等生物检材中痕量秋水仙碱成分的检测。     

Analysis of illicit dietary supplements sold in the Italian market: Identification of a sildenafil thioderivative as adulterant using UPLC–TOF/MS and GC/MS

Identification of pharmaceutical active ingredients sildenafil and tadalafil and the characterization of a dimethylated thio-derivative of sildenafil, called thioaildenafil or thiodimethylsildenafil, in illicit dietary supplements were described.A multi-residual ultra-performance liquid chromatography–time of flight mass spectrometry (UPLC–TOF/MS) method was developed to screen for the presence of the phosphodiesterase-5 (PDE-5) inhibitors sildenafil, tadalafil, and vardenafil and their analogues thioaildenafil and thiohomosildenafil in powders and pharmaceutical dosage forms. The study was developed in connection with an operation supervised by the Italian Medicines Agency (A.I.F.A.), aimed to monitor dietary supplements in the Italian market. In two of the eleven specimens under investigation, high-resolution mass spectrometry (HR-MS) allowed the identification of the PDE-5 inhibitors sildenafil and tadalafil, while another specimen proved to contain a unapproved dimethylated thioderivative of sildenafil, thioaildenafil or thiodimethylsildenafil, identified for the first time in Italy as adulterant in food supplements.     

Application of spectral libraries for characterization of oxidizers in post-blast residues by electrospray mass spectrometry

The goal of this paper is to demonstrate that electrospray mass spectrometry when used with spectral libraries becomes a useful method for rapid identification of inorganic oxidizers commonly present in commercial and improvised explosives. Electrospray ionization mass spectra (ESI-MS) of oxidizers reveal a series of characteristic cluster ions. Such a set of cluster ions can be regarded as a "fingerprint" of a particular oxidizer. With the aid of a spectral library, tentative identification of inorganic oxidizer becomes automated and an easy-to-implement process. The oxidizer identity may be further confirmed by using tandem mass spectrometry (MS/MS). Because it is not necessary to separate the components before analysis, results become available in real time. Little sample consumption and the ease of sample preparation should also be noted. The methodology can be readily employed on various ESI-MS systems that are already in use in analytical laboratories. This is another important benefit as the ESI-MS instrumentation is becoming increasingly common in forensic laboratories.     

Simultaneous identification/determination system for phentolamine and sildenafil as adulterants in soft drinks advertising roborant nutrition

An easily available, simultaneous identification/determination procedure for phentolamine (PHE) and sildenafil (SIL) in adulterated dietary supplements was established by using a combination of three different analytical methods; thin-layer chromatography (TLC), liquid chromatography–mass spectrometry (LC/MS) and a high-performance liquid chromatography (HPLC)/photo-diode-array. The sample solution for TLC was applied to silica gel 60 F₂₅₄ plates with chloroform/ammonia solution (28)/methanol (70:5:3, lower layer) and chloroform/diethylamine/methanol (15:3:2) as the developing solvent. Spots were located under UV radiation at 254 nm. Mass spectra of PHE and SIL by LC/MS were investigated with electrospray ionization (ESI) interface, under both positive and negative ion mode. The HPLC analysis was performed on a column of Wakosil 5C18 (4.6 mm×150 mm, 5 μm) with water/methanol/acetonitrile/triethylamine (580:250:170:1) adjusted with phosphoric acid to pH 3.0 as the mobile phase, and the effluent was monitored with a photo-diode-array detector. Quantitative HPLC analysis of PHE and SIL were detected at 280 nm. When this procedure was applied to commercial soft drinks, PHE and SIL were identified and determined at a concentration of 17 mg PHE and 44 mg SIL per bottle, respectively. The procedure described here is available for the screening of PHE and SIL in adulterated supplements.     

GC‐MS and GC‐MS/MS in PCI Mode Determination of Mescaline in Peyote Tea and in Biological Matrices

Peyote, a cactus containing the hallucinogen mescaline, is used to induce altered states of consciousness in religious ceremonies or for recreational purpose. This study reports a case of an underage boy suspected of mescaline abuse. For this purpose, we analyzed a dark green liquid sample found in the bedroom of the boy whose urine and hair samples were collected shortly after the drink was found. A method by gas chromatography‐mass spectrometry tandem mass spectrometry (GC‐MS/MS) in positive chemical ionization mode was developed and validated in terms of linearity, specificity, accuracy, and sensitivity for mescaline determination at the low concentrations present in hair. GC‐MS analysis of the liquid identified mescaline, while urine was negative; GC‐MS/MS segmental hair analysis identified mescaline in the proximal segment (root to 2 cm), while the distal segments were negative. Although peyote was uncommonly encountered, its use was confirmed by segmental hair analysis that can provide long‐term information about drugs use.     

超分子溶剂萃取-气相色谱-串联质谱法检测尿液中苯二氮䓬类药物

目的建立一种尿液中9种苯二氮?类药物的超分子溶剂样品气相色谱-串联质谱(gas chromatography-tandem mass spectrometry,GC-MS/MS)分析方法。方法含9种苯二氮?类药物对照品的尿液样品用四氢呋喃和1-己醇组成的超分子溶剂进行液液萃取,取溶剂层氮吹至干,残余物用甲醇复溶后进行GC-MS/MS分析,数据采集方式为多反应监测模式,采用内标法定量。结果尿液中地西泮、咪达唑仑、氟硝西泮和氯氮平质量浓度在1~100ng/mL,劳拉西泮和阿普唑仑质量浓度在5~100ng/mL,硝西泮和氯硝西泮质量浓度在2~100ng/mL,艾司唑仑在质量浓度0.2~100ng/mL范围内具有良好的线性关系,相关系数为0.9991~0.9999,定量下限为0.2~5ng/mL,提取回收率为81.12%~99.52%,日内精密度[相对标准偏差(relative standard deviation,RSD)]和准确度(偏倚)分别小于9.86%、9.51%;日间精密度(RSD)和准确度(偏倚)分别小于8.74%、9.98%。室温和-20℃条件下,尿液中9种药物在15d内具有良好的稳定性。8名志愿者单摄口服阿普唑仑片后,在8~72h内尿液中阿普唑仑的质量浓度为6.54~88.28ng/mL。结论本研究建立的尿液中9种苯二氮?类药物的超分子溶剂萃取-GC-MS/MS分析方法,简便、快速、准确、灵敏,可为临床治疗及司法鉴定中苯二氮?类药物中毒监测提供技术支持。     

Positive and negative ion mass spectrometry of benzophenones, the acid-hydrolysis products of benzodiazepines

Positive electron impact (EI), positive chemical ionization (CI), and negative CI mass spectra of 14 benzophenones are presented. In the positive EI mode, intense molecular peaks appeared for most compounds; some other peaks due to splitting at both sides of the carbonyl group also appeared. In the positive CI mode, [M + 1]+ quasi-molecular ions together with [M + C2H5]+ peaks were observed for all compounds; some fragment peaks were common to those in the positive EI mode. In the negative CI mode, the spectra were much simpler than those in the positive EI or CI mode. In the 1 Torr negative CI mode, some spectra showed only single molecular anions; in the 0.01 Torr negative CI mode, halogen or nitro peaks appeared in addition to the molecular anions. An extraction procedure for benzophenones from human urine and plasma after heating in strong acid, and their separation by gas chromatography (GC) are also presented to serve for their actual identification by GC/mass spectrometry.     

Determination of butane in respiratory gases by means of GC/MS and GC/MS-MS

Butane is inhaled in order to achieve a pleasurable state of intoxication. An overdose can lead to death. In two deaths from our own investigation material the circumstances were suspicious for the inhalation of liquid gas, and the presence of butane should be demonstrated in the respiratory gases. For detection, a method of ion trap gas chromatography/mass spectrometry (GC/MS) and tandem-mass spectrometry (GC/MS-MS) was developed, whereby the gas samples from the lung tissue were directly injected into the GC. The GC/MS tests revealed the presence of butane. Moreover, it was found that during the MS-MS tests reaction products appeared which had formed in the ion trap. Systematic investigations of these reaction products showed that these appeared regularly and could be used as additional backup for the proof of butane. Thus phenomena in the ion trap were used which would not have been expected to occur in normal mass spectrometry or tandem-MS and had not been described in the forensic literature so far. The detected amount of butane could be quantified by means of serial dilutions with nitrogen and room air. The described method shows that small molecules or gases can be demonstrated with the ion trap mass spectrometer.