生物检材中氯胺酮及其代谢物的检测

近年来氯胺酮的滥用越来越普遍,建立快速、准确的检测方法越来越重要。氯胺酮在生物体内的代谢物主要有去甲氯胺酮、脱氢去甲氯胺酮等。目前,常用的生物检材有血液、尿液、毛发等。常用的检测方法有气相色谱法、气相色谱-质谱联用法、高效液相色谱法、液相色谱-质谱联用法、高效毛细管电泳法等。本文参考近年来的相关文献对生物检材中氯胺酮及其代谢物的检测方法作一综述,为法医毒物分析等相关领域提供参考。     

Hair and urine analysis: relative distribution of drugs and their metabolites

This work studies the distribution of cocaine and heroin metabolites in hair and urine of living polidrug abusers. Cocaine, benzoylecgonine (BEG), ecgonine methyl ester (EME), morphine, codeine and 6-monoacetylmorphine (6-MAM) were simultaneously extracted and analyzed by GC/MS in SIM mode. The results obtained show a different distribution of heroin and cocaine metabolites in urine and hair. In urine, we generally find BEG and EME for cocaine abuse, and morphine for heroin abuse. In hair, we detect cocaine and MAM as major metabolites for cocaine and heroin abuse, respectively.     

Hair analysis for opiates,cocaine and metabolites. Evaluation of a method by interlaboratory comparison

The aim of this study was to evaluate the performance of a technique for the simultaneous testing of opiates, cocaine and metabolites in hair by interlaboratory comparison. Sixteen forensic and clinical laboratories with different degrees of experience in hair analysis participated voluntarily in the study (no selection criteria were applied). The suggested analytical procedure, the one routinely used in our laboratory, consisted of incubation in HCl 0.1N (45 degrees C, overnight), solid phase extraction (with Bond Elut Certify) cartridges), derivatisation (trimethylsilyl (TMS) derivatives) and GC-MS analysis. Three different mixtures of finely cut (1 mm or less) hair were prepared using drug-users' and drug-free hair: one 'negative' sample (<0.1 ng/mg for morphine, 6-acetylmorphine (6AM), cocaine and benzoylecgonine (BE)), one 'low concentration' sample (between 0.5 and 2 ng/mg) and one 'high concentration' sample (>3 ng/mg). Accuracy and precision (CV% lower than 5.1, 9.9, 5.2, 3.8, 7.3 and 8.3% for morphine, 6AM, codeine, cocaine, BE, and methylecgonine (ME), respectively; range 0.5-5 ng/mg) of the method and homogeneity of the mixtures were evaluated in our laboratory by intraday (CV% lower than 12% for all analytes) and interday analyses (CV% lower than 17% for all analytes except 6AM, 25%). Participants in the study were grouped into: (1) laboratories (n = 6) obtaining the best qualitative and quantitative values, corresponding to those with long experience in hair analysis; (2) laboratories (n = 5) with no reported false positive and/or false negatives; (3) laboratories (n = 5) with one or more reported false positives/false negatives. The results obtained by the labs of the first group were used as reference values. The scatter of data was similar to those obtained in other published studies.     

Hair analysis for fenfluramine and norfenfluramine as biomarkers for N-nitrosofenfluramine ingestion

In this paper, a high performance liquid chromatographic method with fluorescence detection (HPLC-FL) for the determination of fenfluramine (Fen) and norfenfluramine (Norf) in human hair as biomarker metabolites of N-nitrosofenfluramine (N-Fen) is described. Washed and cut hair segments were extracted by ultrasonication for 1h at room temperature in methanol. The extract was evaporated and applied for derivatization with the fluorescent reagent 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). An HPLC-FL analysis was performed using an ODS column with mobile phase composition of acetonitrile and water (65:35, v/v) and monitored at 430 nm (excitation 325 nm). The method was sensitive with detection limits of 36 and 16 pg/mg hair for Fen and Norf, respectively. The linearity was assessed in the range 0.036-144 ng/mg for Fen and 0.016-127 ng/mg for Norf with correlation coefficients larger than 0.999. The method was successfully used for the segmental determination of Fen and Norf in hair samples obtained from hospitalized patients diagnosed with hepatotoxicity and suspected to ingest N-Fen. Both Fen and Norf could be detected in these patients' hair samples in the ranges 43-1389 pg/mg for Fen and 18-680 pg/mg for Norf and the results showed that the patients might ingest N-Fen for a period of not less than 5 months. As well, the method was applied for the determination of Fen and Norf in rats that possess pigmented and non-pigmented hair after an intraperitoneal administration of Fen. Both compounds were determined in black as well as in white hair.     

生物检材中海洛因代谢物分析现状

海洛因在体内极易代谢,其主要的代谢产物为单乙酰吗啡、吗啡等。目前,检测海洛因代谢物的常用生物检材有尿液、血液、毛发等;常用分析方法有薄层色谱法、气相色谱法、高效液相色谱法、气相色谱-质谱联用法、液相色谱-质谱联用法、免疫分析法、毛细管电泳法等。本文参考近年来的文献对生物检材中海洛因代谢物的分析方法作一综述,为法医毒物分析等相关领域的研究提供参考。     

The chromatographic analysis of phencyclidine, its metabolites and analogs in biological fluids

Reviews data on analysis of narcotic phencyclidine and its main metabolites and analogs. Pharmacological and toxic characteristics of phencyclidine, conditions of its isolation from biological objects and its detection and measurement by thin layer and gas chromatography and by chromatographic mass-spectrometry are described. Analysis of phencyclidine metabolites is discussed and a scheme of its metabolism is presented. Statistical characteristics of methods of quantitative analysis of phencyclidine in biological objects are presented.     

Hair analysis for diphenhydramine after surreptitious administration to a child

Diphenhydramine is one of the first effective antihistamine agents to have been discovered. The compound is also used for its sedative and antiemetic effects. The first case involving repetitive sedation linked to the use of diphenhydramine as a drug-facilitated crime and subsequent impairment of a 9-year-old female victim is reported. Due to the long delay between the alleged crime and clinical examination, collection of blood or urine was of little value. Hence, the laboratory developed an original approach based on hair testing by LC-MS/MS. A single strand of hair from the victim was sampled about 7 weeks after the last suspected administration and was cut into small segments. After cutting into small pieces, about 20 mg of hair per segment was incubated overnight in a phosphate buffer (pH 8.4). The aqueous phase was extracted with 5 ml of a mixture of methylene chloride/diethyl ether (80/20), in presence of diazepam-d5, used as internal standard (IS). The hair extract was separated on an XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 256.2-152.1 and 167.1 and m/z 289.9-154.0 for diphenhydramine and the IS, respectively. In the hair of the child, diphenhydramine was detected at concentrations in the range 33-39 pg/mg, depending on the segment.     

Diagnosis of chronic alcohol consumption. Hair analysis for ethyl-glucuronide

This paper describes a procedure for the detection and quantification of ethyl-glucuronide (EtG) in hair samples. During method development the efficacy of extraction of EtG from hair was compared in four extraction methods: (a) methanol; (b) methanol:water (1:1); (c) water; and (d) water:trifluoroacetic acid (9:1). In addition, three derivatizing agents were compared as well: N,O-bistrimethylsilyl-trifluoroacetamide (BSTFA): trimethylchlorosilane (TMCS) (99:1), pentafluoropropionic anhydride (PFPA) and heptafluorobutyric anhydride (HFBA). Water was found to be the best extracting solvent and PFPA the best derivatizing agent. Both provided the highest recoveries, with cleaner extracts and more stable derivatives. The final method is as follows: about 100mg of hair are sequentially washed with water and acetone. The decontaminated sample is finely cut with scissors, then the deuterated internal standard (EtG-d5) and 2 mL of water are added. After sonication for 2 h, the sample is maintained at room temperature overnight. Derivatization is performed with PFPA. Derivatives are injected into a GC-MS system in the electronic impact mode. The method shows linearity over the range of concentrations from 0.050 to 5 ng/mg. Detection and quantification limits are 0.025 and 0.050 ng/mg, respectively. Mean recoveries for the three studied concentrations (low, medium and high) are higher than 87%. The coefficients of variation in intra- and inter-assay precision are always lower than 7%. The method is being routinely applied in our lab for the diagnosis of chronic alcohol consumption.     

Hair analysis for drug abuse, Part II. Hair analysis for monitoring of methamphetamine abuse by isotope dilution gas chromatography/mass spectrometry

Sectional analysis of methamphetamine abuser's hair was performed by using stable-isotope dilution GC/MS method. Drug concentrations of hair shaft cut into 2-cm sections from the root side were compared with the self-reported drug histories of 11 cases and the results of experiments on monkeys. It was found that in nine of the 11 cases, the relationship between the results of sectional analysis and drug histories coincided, but the sectional analyses of two cases were not consistent with self-reported drug history. The difference in drug concentrations between the regions of scalp hair was also investigated. Our study suggests that hair analysis, especially sectional analysis, may be useful in determining past drug history even though it is not exact.     

Hair analysis: a worthless tool for therapeutic compliance monitoring

Hair analysis has been presented by some authors as a possible tool of investigation for estimating patients' compliance to long-term therapies. This paper summarises the different publications that have been devoted to this topic and highlights the available statistical data presented to support this proposition. Qualitative results of such determinations may be of some interest but due to the enormous interindividual variations of quantitative data, the idea of using hair analysis to ascertain whether a patient has taken his treatment exactly as prescribed, clearly appears to be inapplicable.     

Hair analysis of seven bodybuilders for anabolic steroids,ephedrine, and clenbuterol

Several bodybuilders, all winners of international competitions, were arrested for trafficking of a number of doping agents including anabolic steroids, ephedrine, beta-adrenergics, human chorionic gonadotropin, antidepressants, and diuretics. In accordance with the recent French law against doping, the judge asked to test seven bodybuilders to identify doping practices. Hair and urine specimens were collected for analysis. After decontamination, a 100 mg hair strand was pulverized in a ball mill, hydrolyzed, extracted, and derivatized to be tested by GC/MS for anabolic steroids, beta-adrenergic compounds, ephedrine, and other doping agents. Urine was analyzed for anabolic steroids and metabolites, beta-adrenergic compounds, ephedrine, and human chorionic gonadotropin, in addition to a broad spectrum screening with GC/MS. The following compounds were detected in urine: ephedrine (29 and 36 ng/mL, n = 2), clenbuterol (0.2 to 0.3 ng/mL, n = 3), norandrosterone (4.7 to 100.7 ng/mL, n = 7), norethiocholanolone (0.9 to 161.8 ng/mL, n = 6), stanozolol (1 to 25.8 ng/mL, n = 4), methenolone (2.5 to 29.7 ng/mL, n = 4), testosterone (3 to 59.6 ng/mL, n = 7), epitestosterone (1 to 20.4 ng/mL, n = 7) and ratio testosterone/epitestosterone >6 for four subjects (18.5 to 59.6). The following drugs were detected in hair: ephedrine (0.67 and 10.70 ng/mg, n = 2), salbutamol (15 to 31 pg/mg, n = 3), clenbuterol (15 to 122 pg/mg, n = 6), nandrolone (1 to 7.5 pg/mg, n = 3), stanozolol (2 to 84 pg/mg, n = 4), methenolone (17 and 34 ng/ml, n = 2), testosterone enanthate (0.6 to 18.8 ng/mg, n = 5), and testosterone cypionate (3.3 to 4.8 ng/mg, n = 2). These results document the doping practice and demonstrate repetitive exposure to anabolic compounds and confirm the value of hair analysis as a complement to urinalysis in the control of doping practice.     

Comparison of extraction methods for methamphetamine and its metabolites in tissue

Five extraction methods were examined for analysis of methamphetamine and its major metabolites in tissue samples. The extraction methods studied were an acetone extraction method, an ethanol extraction method, an ammonium sulfate method, dialysis, and a direct solvent extraction. Acetone, ethanol, and dialysis methods showed no interference from endogenous components using thin-layer chromatography and gas chromatography, and gave satisfactory recovery of methamphetamine, amphetamine, and p-hydroxymethamphetamine when added to rabbit liver. These methods, however, proved time-consuming. The ammonium sulfate method and direct solvent extraction method were simple and more rapid, but recovery of the polar metabolite was poor.     

Postmortem redistribution of morphine and its metabolites

The postmortem redistribution of morphine, morphine-3-glucuronide, morphine-6-glucuronide and total morphine was assessed in 40 heroin-related deaths. In blood taken from subclavian, heart, and femoral regions, concentrations of morphine and its metabolites were similar. While there was a trend for higher concentrations in heart blood, when compared with femoral or subclavian blood, this was not significant. There was also no significant difference in concentrations between admission and autopsy blood in which the postmortem interval was on average 59 h. From our observations, significant postmortem redistribution of morphine and its metabolites seems unlikely.     

Hair analysis for driving licence in cocaine and heroin users. An epidemiological study

Diagnosis of drug exposure is strongly supported by analysis of hair samples. In the province of Brescia, Italy, for regranting driving license to drug addicts or occasional abusers, a control programme was adopted including analysis of illicit drugs in two hair segments (0-3 and 3-6 cm) and in urine. From January 1998 to April 1999, upon request of the Local Medical Commission, 697 hair samples were tested in our laboratory. One hundred and eighty subjects resulted positive in hair for one or two of the controlled drug classes (73.3% for cocaine, 10% for opiates, 16.7% for both). Positive subjects were classified by residence, age, sex and license category. Seventy-two subjects were called back after 6-12 months and submitted to a second hair and urine analysis: in 34 cases the result of the first analysis was confirmed (19 negatives, 15 positives for one or both drug classes). Another 37 cases tested positive at the first control and negative at the second, suggesting the hypothesis that a strict control may have a significant deterrent function. The high percentage of negative results at the second control may be explained by the prevalence of cocaine users in the examined population. Our results allow us to conclude that the strict application of control rules lead to a decrease of social risk behaviours.     

Hair analysis of opiates in mothers and newborns for evaluating opiate exposure during pregnancy

The increasing interest in toxicological hair analysis as a marker of human exposure to xenobiotics such as illicit substances or therapeutic drugs, has been made feasible by the extension of mass spectrometry, a highly sensitive method of detection. A newborn exposed to drugs in utero can suffer from a varying degree of withdrawal syndrome, a few days after birth. If of opiate origin, the withdrawal syndrome can be treated with morphine, among other therapeutics, but it is not easy to diagnose because of atypical symptoms presented by neonates and especially when maternal drug addiction has not been revealed. To assess and measure toxicological factors linked with the appearance and the severity of this syndrome, maternal and neonatal matrices such as urine, meconium and hair were collected during a protocol approved by the ethical committee. Opiates in particular were measured with GC-MS and potential combined dependences (cannabis, cocaine, amphetamine, LSD and benzodiazepines) and/or substitutive therapeutics (methadone or buprenorphine) were also assessed in 17 mother/neonate couples. Gestational opiate exposure profiles were drawn up and linked with the observed withdrawal syndromes. A withdrawal syndrome seems to appear more frequently after foetal exposure to an association of opiates/substitutive molecules (8 out of 10 withdrawal syndromes observed in this study), although the impact of cocaine and benzodiazepines must also be taken into account. The results obtained in neonatal hair make it possible to affirm foetal drug exposure and are in accordance, for the majority, with the appearance of a neonatal withdrawal syndrome (NWS). Neonatal hair analysis could contribute to assess in utero exposure to opiates, particularly when results in urine and meconium are negative or when these matrices are not available.     

毛发中合成类固醇兴奋剂分析的进展

合成类固醇类药物是使用频率最高的一类运动兴奋剂。本文对毛发中合成类固醇类兴奋剂分析研究的现状进行了综述,着重介绍了分析的方法学研究和该领域国内外的最新研究动态,并对毛发检材应用于兴奋剂检测作了展望。     

Hair analysis to document non-fatal pesticide intoxication cases

We reported two non-fatal cases of intoxication with pesticides namely alachlor and carbofuran. Hair stand samples were collected from two men approximately 1 year after alachlor intoxication for case 1, and 14 days after the last exposure for case 2. Hair analysis was performed using a liquid chromatography-tandem mass spectrometry method. In case 1, alachlor was detected in the 5 analysed hair segments (concentrations between 12 and 136 pg/mg) and its metabolites were not detected. In case 2, carbofuran and its main metabolite (3-hydroxycarbofuran) were detected in the hair strand (global analysis) at the concentrations of 207 and 164 pg/mg, respectively. However, additional data are required in order to interpret such results.     

丙泊酚及其代谢物在大鼠体内的动态分布

目的研究丙泊酚及其代谢物(4-羟基丙泊酚和葡萄糖醛酸丙泊酚)在大鼠体内的动态分布规律,为丙泊酚麻醉相关的死亡案件法医学鉴定提供实验依据。方法 72只SD大鼠随机分为12组,禁食12h,经尾静脉注射12.6mg/kg丙泊酚,给药后不同时间点处死,取心、肝、肺、肾、脑、肌肉、睾丸、静脉血,检材分为两份,一份经环己烷及乙腈液液萃取法提取后,用气相色谱-串联质谱仪检测丙泊酚含量,并用高效液相色谱-串联质谱仪检测PG含量,另一份检材经乙腈液液提取后经MSTFA衍生化,用气相色谱-串联质谱仪检测4-羟基丙泊酚含量。结果丙泊酚经大鼠尾静脉进入体内,迅速向各组织器官内分布。由数据可看出丙泊酚在体内各组织消除迅速,并于30min后趋于平稳。丙泊酚在大鼠肝、肾、脑中分布较多,PG在大鼠中主要分布在肝脏,4-羟基丙泊酚在大鼠肝脏、肾脏、肺脏分布较多。结论经尾静脉注射丙泊酚后大鼠体内丙泊酚及其代谢物的动态分布规律可以为丙泊酚麻醉相关的死亡案件的法医学鉴定提供实验依据。     

Detection of cocaine and its metabolites in breast milk

A method was developed for measuring cocaine and its metabolites, benzoylecgonine, ecgonine methyl ester, norcocaine, ecgonine ethyl ester, cocaethylene, and m-hydroxybenzoylecgonine, in breast milk by gas chromatography/mass spectrometry. Limits of detection for this method ranged from 2.5 to 10 ng/mL, and limits of quantitation ranged from 5 to 50 ng/mL. For each of the compounds measured by this method, linear response was demonstrated to 750 ng/mL. Breast milk was collected from 11 mothers who admitted to drug use during pregnancy and ten drug-free volunteers serving as control subjects. Cocaine was detected in six of the specimens obtained from drug-exposed subjects, and in none of the drug-free control subjects. In breast milk specimens where cocaine and one or more of its metabolites were detected, the concentration of parent compound was greater than any of the metabolites. The highest cocaine concentration found was over 12 microg/mL. Breast-fed infants of cocaine abusing mothers may be exposed to significant amounts of drug orally.     

氯胺酮滥用的研究进展

氯胺酮为非竞争性NMDA受体阻断剂,作为一种分离性麻醉药应用于临床,其作用机制复杂,由于其具有一定的致幻作用而被滥用于各种娱乐场所,是目前药物滥用中的一个新问题。本文在阐述氯胺酮的药理学及毒理学特性的基础上,复习了氯胺酮与NMDA受体、多巴胺受体等神经生物学相关受体的相互作用,综述氯胺酮滥用的现状、滥用的可能生理机制及其滥用后的检测方法等方面的研究进展。