Detection and quantification of psychotropic drug etaqualone in human hair using GC–MS/MS

In this study, sensitive analytical procedure for detection and quantification of etaqualone in human hair samples using gas chromatography tandem mass spectrometry (GC–MS/MS) was newly established, and applied it to authentic human samples obtained from an abuser. In this method, the hair samples were treated with hydrochloric acid and then extracted with ethyl ether. The ether layer was dried in a warm water bath, and the residue was reconstituted in ethyl acetate, followed by GC–MS/MS analysis. Multiple reaction monitoring (MRM) mode was used for data collection, and quantitative analysis was performed using internal standard method. Good linear relationship within the concentration range of 1–100 pg/mg were obtained in calibrators for the hair samples showing its correlation coefficient value was 0.9993. The lower limit of quantitation in this study was 1 pg/mg and the recovery rate examined ranged from 100.4% to 108.5%. The intra-day precision and accuracy were less than 5.0% and 5.8%, respectively. The inter-day precision and accuracy were lower than 6.4% and 4.6%, respectively. Using this established method, etaqualone could be detected in the hair sample obtained from a suspected user to be level of 65.2 pg/mg. It should be expected that the method established in this study would contribute to rapid detection and identification of psychotropic drug etaqualone among multiple fields including forensic investigation, clinical application and of course public health matters.     

液相色谱串联质谱法检测马尿中32种蛋白同化激素

目的:建立马尿中32种蛋白同化激素的液相色谱串联质谱(LC-MS/MS)检测方法。方法:利用Agilent 6410A LC-MS/MS仪器;色谱柱采用Agilent Zorbax XDB-C18柱(2.1×100 mm,3.5μm);流动相采用0.1%甲酸水溶液(A)和乙腈(B)进行梯度洗脱;离子检测方式为多反应离子检测(MRM);正离子检测。结果:建立了马尿中32种蛋白同化激素的检测方法,药物浓度线性方程的相关系数均大于0.992,日内和日间精密度基本小于20%,提取回收率在84%以上且基质效应多在80%~120%之间,LOD和LOQ满足相关马术检测机构的要求。结论:本方法操作简单,经方法学验证符合相关标准,能满足马术项目的兴奋剂检查要求,可以应用于常规的兴奋剂检测。     

GC-PCI-MS/MS and LC-ESI-MS/MS databases for the detection of 104 psychotropic compounds (synthetic cannabinoids,synthetic cathinones,phenethylamine derivatives)

Designer psychotropic compounds continue to be a major problem in Japan and all around the world. Electron impact mass spectrometry (GC-EI-MS) and liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) data on these compounds have been widely reported. In this report, we present a detection method that has been rarely utilized to analyze these types of compounds, gas chromatography with positive chemical ionization and tandem mass spectrometry (GC-PCI-MS/MS). We report on the development of GC-PCI-MS/MS and LC-ESI-MS/MS databases of 104 psychotropic compounds, including 32 cannabinoid derivatives, 29 cathinone derivatives, 34 phenethylamine derivatives, and several other designer compounds. Using this database, we were able to detect 5 psychotropic compounds in an actual forensic autopsy case. If GC-PCI-MS/MS is used together with the more established methods of GC-EI-MS and LC-ESI-MS/MS, we believe the forensic toxicology community could be better prepared to deal with the challenges of these ever-changing compounds.     

Qualitative analysis of synthetic opioids,synthetic hallucinogens and LSD in urine using LC-MS/MS

ABSTRACT

Synthetic opioids and synthetic hallucinogens continue to expand in the illicit drugs market where they are designed and synthesized to avoid detection in routine drug tests. It is therefore crucial for laboratories to develop analytical methods for the detection of these drugs in urine. A method for the simultaneous analysis of synthetic opioids (including fentanyl, furanyl fentanyl, U-47700 and AH-7921), phenylalkylamine hallucinogens (25C-NBOMe, 25B-NBOMe, 25I-NBOMe, 25H-NBOMe, 25C-NBOH, 25B-NBOH, 25I-NBOH and 25H-NBOH) along with LSD, 2-oxo-3-OH-LSD (Oxo-LSD), psilocin and bufotenine was hence developed. Briefly, urine samples (0.5 mL) underwent enzymatic hydrolysis and matrix clean-up using supported-liquid extraction (SLE) prior to analysis on LC-MS/MS. The SLE extraction recoveries ranged from 62.8% to 97.4% and limits of detection between 0.1–3 ng/ml were achieved. The matrix effects results ranged from 70.6% to 124.9% and there was no carryover up to 100 ng/ml for psilocin and bufotenine and 30 ng/ml for the rest of the analytes. Extracted urine samples were stable for up to three days for Oxo-LSD and seven days for the rest when stored at 4°C. The method was validated to be accurate and precise for each analyte at their individual cut-off points. It was also successfully applied on suspected drug abusers’ urine samples.     

Analysis of fentanyl and sufentanil in hair by GC/MS/MS

Fentanyl and sufentanil are potent narcotic analgesics used only in hospitals as anaesthetic agents. The dependence potential of fentanyl is known. As they are given in doses at the microgram level and their elimination half-life is in the order of a few hours, detection in body fluids is possible only for a short time after administration. Radioimmunological methods are the only ones capable of detecting fentanyl in hair, as normal GC/MS methods for hair analysis are not sensitive enough to detect the drugs after doses in the order of micrograms. We therefore chose GC/MS/MS to analyse fentanyl and sufentanil in two cases where the drugs were given under controlled conditions over several days. The concentration was in the order of less than 100 pg/mg hair.     

Detection of ethyl glucuronide in dried human blood using LC-MS/MS

Ethyl glucuronide, as a direct metabolite of ethanol degradation, has proven useful as a long-term marker in many forensic applications. The inability to determine ethyl glucuronide in dried blood left a missing link in many investigations. Here, we describe a new method based on mass spectrometry in a Pauli-type ion trap in order to determine this substance in dried blood samples.     

Postmortem distribution and redistribution of synthetic cathinones

Purpose

Synthetic cathinones are powerful psychostimulants that have been associated with fatal intoxications. Because of changes that take place following death, postmortem toxicology results require careful interpretation. The purpose of this study was to evaluate the distribution of synthetic cathinones in postmortem specimens in a series of 50 cathinone-positive fatalities.

Methods

Liquid chromatography–quadrupole time-of-flight-mass spectrometry was used to quantitatively identify cathinones in central blood (n = 51), peripheral blood (n = 31), urine (n = 33), liver (n = 22), vitreous humor (n = 1) and stomach contents (n = 1). The distribution of cathinones and the potential for postmortem redistribution was assessed.

Results

Among the 50 cases investigated, a total of nine synthetic cathinones (α-PVP, ethylone, methylone, butylone, MDPV, methedrone, pentylone, 4-MEC, and MDPBP) were identified in 139 specimens. The number of specimens per case ranged from one to six. In cases that included central blood or liver, together with a peripheral blood source, the central/peripheral (C/P) or liver/peripheral (L/P) ratio was calculated to estimate the potential for postmortem redistribution (n = 21 C/P; n = 11 L/P). Methylone and ethylone appeared to exhibit the greatest potential for postmortem redistribution, producing C/P ratios of 4.0 (1.5–6.1) and 2.9 (0.5–9.2), respectively. In contrast, the C/P ratio for α-PVP was 1.1 (0.5–1.9). Differences in C/P ratios between methylone and α-PVP were statistically significant (α = 0.05).

Conclusions

Although synthetic cathinones may exhibit low to moderate postmortem redistribution, significant variability exists due to site- and time-dependent factors. This, in combination with their overall instability, necessitates careful interpretation of postmortem toxicology results.

     

Detection and quantification of lorazepam in human hair by GC-MS/NCI in a case of traffic accident

A traffic accident caused by a man who declared that he was driving under influence of drugs (Temesta), led our laboratory to develop a procedure for the detection and the quantification of lorazepam in human hair. The method involves decontamination of hair with dichloromethane, incubation in Soerensen buffer (pH 7.6) in the presence of lorazepam-d₄, liquid-liquid extraction with diethylether-chloroform (80:20, v/v) at pH 8.4, derivatization by silylation and detection by GC-MS/NCl. The increasing concentrations of lorazepam from the end to the roots of a 16-cm-long hair strand (i.e. 31 pg/mg, 40 pg/mg and 49 pg/mg) proved that the driver had taken the drug over a long period of time.     

Detection of colchicine by means of LC-MS/MS after mistaking meadow saffron for bear's garlic

The accidental ingestion of plant material containing colchicines is a rare cause of lethal poisoning. The death of a married couple who mistook colchicines for Bear's garlic (Allium ursinum) and prepared the latter as a salad is the tragic topic of this article. After discussing the specificity of the histological findings, a chemicotoxicological method using liquid chromatographic mass spectrometric (LC-MS/MS) is presented. Toxicological analyses using LC-MS/MS revealed colchicine concentrations between 36.6 ng/mL and 98.3 ng/mL in the heart blood and between 22.7 ng/mL and 78.4 ng/mL in the femoral blood of the victims.     

用液相色谱-串联质谱联用技术检测4种刺激剂类禁用物质

目的:使用液相色谱-串联质谱联用技术,为世界反兴奋剂机构(WADA)在检测窗口中拟增加的4种刺激剂甲磺美嗪(amezinium methylsulfate,I)、吗拉宗(morazone,II)、贝美格(3-ethyl-3-methylglu-tarimide,III)和阿拉明(metaraminol,IV)建立了常规检测方法,并对方法进行考察和验证。方法:用5%的醋酸铅水溶液将样品中蛋白质等大分子沉淀后,离心10分钟,取上清液上机检测。质谱采用电喷雾电离源(ESI),在正离子模式下,通过多反应监测(MRM)模式对样品进行分析。结果:经过定性和定量分析,得到上述四种刺激剂的检测限(LOD)分别为0.1、0.5、5和0.5 ng/m L;定量限(LOQ)分别为0.3,1.5,15和1.5 ng/m L;线性范围(r2>0.99)分别在0.3~5000、1.5~3000、15~3000和1.5~4500 ng/m L之间。每种刺激剂在低、中和高3个浓度范围的基质效应均在98%~120%之间;准确度误差在9%以内;日内精密度的标准偏差低于7.7%,日间精密度的标准偏差不超过10%。结论:本研究建立的检测方法具有样品前处理简单、灵敏度高、准确度好和基质干扰较小等特点,满足兴奋剂检测实验室的要求,并已经应用于常规检测中。     

Detection and quantification of 12 anabolic steroids and analogs in human whole blood and 20 in hair using LC-HRMS/MS: application to real cases

We developed and validated a method to detect and quantify 12 anabolic steroids in blood (androstenedione, dihydrotestosterone, boldenone, epitestosterone, mesterolone, methandienone, nandrolone, stanozolol, norandrostenedione, tamoxifene, testosterone, trenbolone) and eight more in hair samples (nandrolone phenylpropionate, nandrolone decanoate, testosterone propionate, testosterone benzoate, testosterone cypionate, testosterone decanoate, testosterone phenylpropionate, testosterone undecanoate) using liquid chromatography coupled to high-resolution mass spectrometry. This method used a benchtop Orbitrap mass spectrometer operating with an APCI probe under positive ionization mode. Analysis was realized in full scan experiment with a nominal resolving power of 140,000. After addition of the internal standard (testosterone-D3) and incubation in phosphate buffer pH = 5 for hair, 200 μL of blood and 30 mg of hair samples were extracted with heptane. LOQ and LOD were determined at 5 and 1 ng mL⁻¹ in whole blood and 10 to 100 pg mg⁻¹ and 2 to 20 pg mg⁻¹ in hair according to the compounds, respectively. The method was linear in the 5–1000 ng mL⁻¹ range in whole blood and between 10 or 100 pg mg⁻¹ and 1000 pg mg⁻¹ in hair with correlation coefficients >0.99, and intra- and inter-day accuracy and precision were <14.8% for all compounds except for some esters in hairs (<19.9%) probably due to an important matrix effect for these compounds. This sensitive and specific method to detect anabolic steroids has been successfully applied to two real cases, for which various anabolic steroids in whole blood, urine, and hair were identified and quantified.

     

液相色谱串联质谱法分析马尿中司坦唑醇代谢产物

目的:分析司坦唑醇在马尿中的主要代谢产物,并描述其主要代谢产物的浓度随时间变化曲线,为马尿中检测司坦唑醇提供参考。方法:收集服药前和单次口服司坦唑醇片剂后的马尿样本,经酶解和液液萃取后进行液相色谱串联质谱(LC-MS/MS)检测,分析司坦唑醇在马尿中的代谢产物。结果:对服用司坦唑醇后的马尿进行分析,检出了药物原型和5种代谢产物;建立了司坦唑醇的4种代谢产物的浓度随时间变化曲线。结论:本实验对服用司坦唑醇马尿的代谢产物进行分析,但由于药物浓度低和杂质问题的影响,使得代谢产物的分析不够完善;4种代谢产物浓度随时间变化曲线对于马尿中检测司坦唑醇的代谢物的选择、检测窗口期具有一定的参考价值。     

LC-MS／MS同时测定人血浆中对乙酰氨基酚和右氯苯那敏浓度

目的高效液相色谱·串联质谱联用法（LC—MS／MS）同时测定人血浆中对乙酰氨基酚和右氯苯那敏浓度。方法样品用甲醇进行沉淀蛋白后进样,Zorbax Eclipse TC—C18色谱柱,以甲醇旬．1％甲酸的1mmol·L^-1甲酸铵溶液,采用梯度洗脱进行分离,3200QTRAP型质谱仪的多重反应监测（MRM）扫描方式进行检测。结果对乙酰氨基酚线性范围为：0．05-20μg·mL^-1,定量下限为0．05μg·mL^-1,准确度与精密度结果显示对乙酰氨基酚日间、日内RSD均≤5．50％,相对偏差一8．08％～5．73％,方法提取回收率92．0％～99．0％,稳定性测定值与添加值的相对偏差均〈15％;右氯苯那敏线性范围为：0．05～20ng·mL^-1,定量下限为0．05ng·mL^-1,准确度与精密度结果显示右氯苯那敏日间、日内RSD均≤13．71％,相对偏差-1．29％～1．71％,方法提取回收率89．8％～101．4％,稳定性测定值与添加值的相对偏差均〈15％。结论本研究所建立的方法快速、灵敏、专属性强、重复性高,可用于同时测定人血浆中对乙酰氨基酚和右氯苯那敏浓度。     

采用LC-MS/MS检测人尿中睾内脂方法研究

目的:建立人尿中睾内脂的LC-MS/MS检测方法。方法:采用Agilent Zorbax XDB-C18柱(2.1×50mm,3.5μm),流动相采用pH3.5甲酸铵缓冲液(A)和乙腈(B)进行梯度洗脱,离子检测方式为多反应离子检测(MRM);离子极性为正离子,定量的离子对为m/z301→121,定性的离子对为m/z301→225。结果:测定人尿中内源性物质不干扰样品和内标,样品分析时间为7分钟,睾内脂线性范围为0.1~50μg/ml,提取回收率大约为60%,日内和日间的RSD均小于10%。结论:本方法经考察符合国际反兴奋剂机构(WADA)的要求,可以应用于常规的兴奋剂检测工作。     

LC-MS/MS法研究左旋去甲基苯环壬酯在比格犬体内的生物利用度

目的评价左旋去甲基苯环壬酯在比格犬体内生物利用度。方法 LC-MS/MS法测定比格犬口服和静脉给药后体内的血药浓度,计算药代动力学参数和绝对生物利用度。结果比格犬口服给药2 mg/kg后5 min即可测得血中药物含量,口服给药后1 h左旋去甲基苯环壬酯在比格犬体内血药浓度达峰,给药后36 h血中药物浓度均已降至LLOQ以下。比格犬口服和静脉注射2 mg/kg左旋去甲基苯环壬酯的t1/2分别为（5.36±0.82）h和（6.73±2.00）h,Cmax分别为（141.88±51.03）和（591.84±144.61）μg/L;AUC（0-36 h）分别为（962.42±220.96）和（1572.06±365.80）μg.L-1.h;用AUC（0-36 h）计算比格犬口服单剂量左旋去甲基苯环壬酯的绝对生物利用度为（61.22±10.78）%。结论左旋去甲基苯环壬酯口服给药吸收迅速,绝对生物利用度较高,具有良好的开发前景。     

LC-MS/MS法研究左旋去甲基苯环壬酯在比格犬体内的生物利用度

目的评价左旋去甲基苯环壬酯在比格犬体内生物利用度。方法 LC-MS/MS法测定比格犬口服和静脉给药后体内的血药浓度,计算药代动力学参数和绝对生物利用度。结果比格犬口服给药2 mg/kg后5 min即可测得血中药物含量,口服给药后1 h左旋去甲基苯环壬酯在比格犬体内血药浓度达峰,给药后36 h血中药物浓度均已降至LLOQ以下。比格犬口服和静脉注射2 mg/kg左旋去甲基苯环壬酯的t1/2分别为(5.36±0.82)h和(6.73±2.00)h,Cmax分别为(141.88±51.03)和(591.84±144.61)μg/L;AUC(0-36 h)分别为(962.42±220.96)和(1572.06±365.80)μg.L-1.h;用AUC(0-36 h)计算比格犬口服单剂量左旋去甲基苯环壬酯的绝对生物利用度为(61.22±10.78)%。结论左旋去甲基苯环壬酯口服给药吸收迅速,绝对生物利用度较高,具有良好的开发前景。     

人尿中6种利尿剂的液相色谱-串联质谱联用检测方法研究

目的:使用液相色谱-串联质谱联用(LC-MS/MS)技术,为氯拉扎尼(chlorazanil,I)、氯索隆(cicletanine,II)、西氯他宁(chlorxolone,III)、苄氢氯噻嗪(benzylhydrochlorothiazide,IV)、芬喹唑(fenquizone,V)和美替克仑(meticrane,VI)等6种利尿剂建立在人尿中的检测方法并进行方法验证。方法:将尿样用同等体积的5%乙酸铅水溶液沉淀蛋白质并离心后,使用电喷雾电离源的正、负离子切换模式,以多反应监测(MRM)模式进行定性和定量分析。结果:上述利尿剂I-VI的最低检测限(LOD)分别为1、0.1、0.01、1、2和10 ng/m L;线性范围(R2>0.99)分别为3~300 ng/m L、0.3~500 ng/m L、0.03~4000 ng/m L、3~1500 ng/m L、6~600 ng/m L和30~1600 ng/m L;6种药物在低、中和高三个浓度水平的日内变异系数均小于8%,日间变异系数均小于12%,检测的准确度在80%至120%之间;基质效应均在83%~115%范围内。结论:新建方法具有前处理简单、分析速度快、灵敏度高和重现性好等特点,已应用于我们实验室的兴奋剂常规检测中。     

Determination of dihydrocodeine in hair of opiate addicts by GC/MS

Summary After the examination of more than 300 hair samples of suspected heroin abusers, a large number of which proved positive, we can say that high concentrations of dihydrocodeine can be determined either in addition to, or in the place of, morphine and also frequently in combination with codeine. The opiates were extracted after dissolving the hair samples in NaOH and hydrolysis with HCI. The quantitative determination of dihydrocodeine was achieved by derivatisation with HFBA using GC/MS at m/u = 497. Dihydrocodeine is used in antitussive drugs. After the examination of individual hair samples, it was obvious that some heroin consumers had switched to dihydrocodeine. This may lead to the conclusion that dihydrocodeine itself is used either as an intoxicating drug or to reduce withdrawal symptoms. The increasing number of positive samples should be noted by the legal authorities.