LC-MS/MS法快速测定头发中可卡因及其代谢物苯甲酰爱康宁

目的建立头发中可卡因(cocaine)及其代谢物苯甲酰爱康宁(benzoylecgonine)的液相色谱-串联质谱(LC-MS/MS)快速测定方法。方法去污处理后的头发中加入氘代内标(可卡因-D_3和苯甲酰爱康宁-D_8),经甲醇超声提取后用Restek Allure PFP丙基柱分离,采用多反应监测模式同时测定可卡因和苯甲酰爱康宁。结果头发中可卡因和苯甲酰爱康宁在0.02~10.00 ng/mg质量分数范围内线性关系良好,检出限均为0.01 ng/mg。结论本研究所建方法样品前处理简单、快速、选择性好,适用于头发中可卡因及代谢物苯甲酰爱康宁的检测。     

可卡因及其代谢物苯甲酰爱康宁胶体金检测试剂盒的研制

目的根据胶体金免疫层析的原理,建立一种可以在现场快速检测可卡因及其代谢物苯甲酰爱康宁的方法。方法将胶体金标记的抗可卡因单克隆抗体均匀浸涂在玻璃纤维膜上,用点膜仪将可卡因的完全抗原以及羊抗鼠多抗在硝酸纤维素膜上划线,分别作为可卡因的检测区（T）和质控区（C）。并且对样品垫用0.2%Tween 20和0.5%PVA封闭处理。样本（尿样、唾液、血样等）中游离的可卡因及其代谢物同包被的完全抗原分别免疫竞争结合胶体金标记的抗可卡因单抗。肉眼观察试剂盒的质控区和检测区上有无紫红色带来判读结果。结果可卡因胶体金检测试剂盒对65种毒品、药物的特异性测试中仅识别可卡因及其代谢物。对人体尿样中的可卡因和苯甲酰爱康宁检测阈值均为300ng/mL。与GC/MS对照试验,其准确性分别为98.9%和98.6%。在常温下能稳定保存2年。结论该可卡因检测试剂盒在5min内可以检测出样本中的可卡因及其代谢物成分。     

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。     

液相色谱-串联质谱法分析生物检材中的河豚毒素

目的建立血液、尿液以及肝中河豚毒素(tetrodotoxin,TTX)的液相色谱-串联质谱分析方法,并进行方法学验证。方法血液、尿液和肝用1%乙酸甲醇溶液去蛋白后,上清液用固相萃取法净化,LC-MS/MS检测。结果血液、尿液和肝中TTX检出限分别为2ng/mL、2ng/mL和4ng/g。血液和尿液在4～100ng/mL、肝在5～100ng/g的范围内线性关系良好,相关系数r≥0.9973;日内精密度和日间精密度均在12.80%以内;回收率大于47.2%。结论所建方法高效、灵敏、准确,可以为河豚毒素中毒的法医学鉴定、临床诊治以及食品安全的监控提供技术保障。     

衍生化-气相色谱法在体内药物分析中的应用

本文对应用衍生化-气相色谱技术检测体液中吗啡、可待因和可卡因及其代谢物苯甲酰爱冈宁的研究及其进展进行了综述。为解决吸毒者血液和尿液等体液中有关毒品及其代谢物的微量分析提供了一种先进、可靠和灵敏的检测技术。     

LC-MS/MS同时分析尿液中15种安眠镇静药物及其代谢物

目的建立尿液中15种常见安眠镇静药物及代谢物的液相色谱-串联质谱分析方法。方法尿液经酶水解、固相萃取后,用C18液相柱分离,以含甲酸铵和甲酸的水、乙腈为流动相梯度洗脱,质谱采用电喷雾电离（ESI）-正负离子模式同时扫描,采用二级质谱多反应监测（MRM）模式检测目标化合物。结果以化合物的保留时间、两对母离子/子离子对定性,尿中常见安眠镇静药物的检测限为0.01～0.5ng/mL（ESI＋）和10ng/mL（ESI-）;相关系数r在0.994以上;日内及日间精密度均在18%以下;绝对回收率在64.80%～116.20%之间。结论方法快速、灵敏、简便、可靠,能同时分析尿液中的15种安眠镇静药物及其代谢物。     

人体中可卡因代谢物：尿液中四种未报道过的可卡因代谢物的鉴定

在体内,可卡因由于酶的作用和化学过程迅速转变为代谢物,因此通常尿中仅能提出很少可卡因。尿中两种主要代谢物为苯甲酰牙子碱及牙子碱甲基酯。Amber 最近报道可卡因在尿中14～17%以苯甲酰牙子碱形式被提出,12～21%以牙子碱甲基酯形式被提出。另外报道的体内尿中可卡因代谢物包括:降可卡因、牙子碱、苯甲酰降牙子碱、脱水芽子碱甲基酯、苯甲酰环上羟基化的可卡因、羟基、甲氧基取代的苯甲酰芽子碱。本文报道一种扩展的GC/MS 方法,用于分析药物试验中收集的1例尿样,该     

SPE-HPLC/MS/MS检验体液中赛拉嗪及DMA

目的建立了一种高效液相色谱串联三重四极杆质谱同时测定体液中赛拉嗪及2,6-二甲基苯胺的分析方法。方法样品经HLB固相萃取柱提取净化,Waters Atlantis d C18色谱柱分离,正电离条件下进行选择监视扫描模式检测。结果方法的回收率为70.5%~79.8%,RSD为8.2%~10.5%。赛拉嗪及2,6-二甲基苯胺在血液和尿液中的检出限分别为0.4 ng/mL和0.3 ng/mL,定量限分别为1.2 ng/mL和1.0 ng/mL。结论本方法灵敏度高、特异性好、重现性好,适用于赛拉嗪中毒的血液和尿液检测。     

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。     

LC-MS/MS法检测人血液中的利多卡因及其代谢物

目的建立人血液样品中利多卡因及其代谢物单乙基甘氨酰二甲苯胺(MEGX)和甘氨酰二甲苯胺(GX的液相色谱-串联质谱(LC-MS/MS)分析方法。方法人血液样品经乙腈沉淀蛋白后,Zorbax RRHD Plus C18柱(2.1mm×50mm,1.8μm)分离,以0.1%甲酸和乙腈为流动相进行洗脱。质谱采用电喷雾正离子化(ESI+),多反应监测扫描模式。结果血液中利多卡因、MEGX和GX在相应范围内线性良好,相关系数(r 0.9950)。在血液中检出限分别为0.1 ng/mL、0.5 ng/mL和0.2 ng/mL,方法回收率为92.7%～103.9%,准确度为92.9%～113.1%,日内、日间精密度的相对标准偏差均不超过13.0%。在1例肌肉注射利多卡因死者心血中利多卡因、MEGX和GX的质量浓度分别为1.520μg/mL、0.019μg/mL和0.266μg/mL。结论本方法灵敏度高、选择性好,适用于血液中利多卡因及其代谢物的检测,可为利多卡因临床治疗过程中的药物浓度监测和法医学鉴定提供技术支撑。     

A Direct Aqueous Derivatization GSMS Method for Determining Benzoylecgonine Concentrations in Human Urine

A sensitive and reliable method for extraction and quantification of benzoylecgonine (BZE) and cocaine (COC) in urine is presented. Propyl‐chloroformate was used as derivatizing agent, and it was directly added to the urine sample: the propyl derivative and COC were then recovered by liquid–liquid extraction procedure. Gas chromatography–mass spectrometry was used to detect the analytes in selected ion monitoring mode. The method proved to be precise for BZE and COC both in term of intraday and interday analysis, with a coefficient of variation (CV) <6%. Limits of detection (LOD) were 2.7 ng/mL for BZE and 1.4 ng/mL for COC. The calibration curve showed a linear relationship for BZE and COC (r² >0.999 and >0.997, respectively) within the range investigated. The method, applied to thirty authentic samples, showed to be very simple, fast, and reliable, so it can be easily applied in routine analysis for the quantification of BZE and COC in urine samples.     

Simultaneous determination of opiates,cocaine and major metabolites of cocaine in human hair by gas chromotography/mass spectrometry (GC/MS)

A procedure is presented for the simultaneous identification and quantification of morphine (MOR), codeine (COD), ethylmorphine (EM), 6-monoacetylmorphine (6-MAM), cocaine (COC), benzoylecgonine (BZE), ecgonine methylester (EME) and cocaethylene (CE), contained in the hair of opiates and cocaine addicts. The method involves decontamination in dichloromethane, pulverization in a ball mill, heat-acid hydrolysis, addition of deuterated internal standards, liquid-liquid extraction and gas chromatography/mass spectrometry (GC/MS) after silylation. The limit of detection (LOD) was ~0.1–0.8 ng/mg for each drug, using a 30-mg hair sample. The method is reproductible, with a coefficient of variation (CV) of ~8–17%. Cocaine and 6-monoacetylmorphine were the major compounds detected in cases of cocaine (14 cases) and heroin (68 cases) intake. Concentrations were in the range 0.4–78.4 ng/mg (COC), 0.0–36.3 ng/mg (BZE), 0.0–1.6 ng/mg (EME), 0.0–2.1 ng/mg (CE), 0.0–84.3 ng/mg (6-MAM), 0.2–27.1 ng/mg (MOR) and 0.1–19.6 ng/mg (COD). An application in forensic sciences, involving multi-sectional analysis, is given.     

Simultaneous analyses of cocaine, cocaethylene, and their possible metabolic and pyrolytic products

A method was developed for simultaneously analyzing cocaine (COC), benzoylecgonine (BZE), norbenzoylecgonine (BNE), norcocaine (NCOC), ecgonine (ECG), ecgonine methyl ester (EME), m-hydroxybenzoylecgonine (HBZE), anhydroecgonine methyl ester (AEME), cocaethylene (CE), norcocaethylene (NCE), and ecgonine ethyl ester (EEE) in blood, urine, and muscle. Available deuterated analogs of these analytes were used as internal standards. Proteins from blood and muscle homogenate were precipitated with cold acetonitrile. After the removal of acetonitrile by evaporation, the supernatants and urine were subjected to solid-phase extraction. The eluted analytes were converted to their hydrochloride salts and derivatized with pentafluoropropionic anhydride and 2,2,3,3,3-pentafluoro-1-propanol. The derivatized products were analyzed by a gas chromatograph (GC)/mass spectrometer by selected ion monitoring. The limit of detection (LOD) for COC, BZE, NCOC, EME, CE, NCE, and EEE was 2ng/ml, while the LODs for BNE, ECG, HBZE, and AEME were 25, 640, 50, and 13 ng/ml, respectively. This method was successfully applied in analyzing 13 case samples from aviation accident pilot fatalities and motor vehicle operators. AEME concentrations found in the 13 samples were consistent with those produced solely by the GC inlet pyrolysis of COC controls in blood. Anhydroecgonine cannot be used as a marker for the abuse of COC by smoking because it is also pyrolytically produced from COC metabolites on the GC inlet. The developed method can be effectively adopted for analyzing COC and related compounds in urine, blood, and muscle by a single extraction with increased sensitivity through formation of hydrochloride salts and using a one-step derivatization.     

Screening for cocaine metabolite fails to detect an intoxication

Testing for the presence of cocaine (COC) is common in postmortem and clinical laboratories. COC use may be detected by screening urine specimens for COC metabolite. In the forensic arena, screening positive results are confirmed by a more specific and sensitive technique, such as gas chromatography-mass spectrometry. This article reports the case of an individual who died of COC intoxication but whose immunoassay screen (EMIT) for COC metabolite was negative. Gas chromatography-mass spectrometry analysis of the urine detected benzoylecgonine (BE) at a concentration of 75 ng/mL and COC at 55 ng/mL. These concentrations explain the negative screening result since the cutoff concentration of the assay was 300 ng/mL for BE. The reported cross reactivity with COC was 25,000 ng/mL. However, heart blood concentrations of COC and BE were 18,330 and 8640 ng/mL, respectively. The results from this case provide evidence that an EMIT test alone may fail to detect COC use. Individuals utilizing results of drug screening by immunoassay must be aware of the limitations of this testing methodology.     

Application of discriminant analysis to differentiate between incorporation of cocaine and its congeners into hair and contamination

The requirement to differentiate between incorporation and external contamination of drugs into hair is undisputed, in particular when dealing with compounds which are administered by sniffing or inhalation (e.g. cocaine). With the aim of making this discrimination, hair samples from cocaine (COC) users (group IN) and seized cocaine samples (group OUT) were compared regarding the parameters benzoylecgonine (BZE), ecgonine methyl ester (EME), ecgonine (ECG), anhydroecgonine methyl ester (AEME), cocaethylene (CE) and norcocaine (NCOC). Since most of these compounds may be minor by-products of COC or be formed by biotransformation or chemical degradation, the stability of each substance was carefully examined. COC was found to be converted into significant amounts of BZE, EME and ECG even under mild extraction conditions, while traces of NCOC proved to be a ubiquitous by-product of COC. Cocaine positive hairs and seized cocaine samples (diluted to relevant concentrations) were equally preprocessed and analyzed by LC-MS-MS. Out of the metabolites listed above, NCOC, CE and AEME (each normalised to COC) were significantly increased in the incorporation group (i.e. hair samples from cocaine users). Based on this approach, a statistical discriminant analysis enabled us to make a prediction (and estimation of uncertainty) for each cocaine positive hair sample as to its likelihood of belonging to the group of cocaine users or of being contaminated.     

Validation of an ion-trap gas chromatographic-mass spectrometric method for the determination of cocaine and metabolites and cocaethylene in post mortem whole blood

The coingestion of cocaine (COC) and ethanol is a very frequent occurrence and is known to increase the risk of morbidity and mortality. The formation occurs of a transesterification product, the cocaethylene (CE), which is even more toxic than cocaine. In order to study the role of ethanol as an agent of interaction in lethal cocaine intoxication, and to establish its influence in post mortem cocaine concentrations, an ion-trap gas chromatographic-mass spectrometric method (GC-MS) was validated to quantify simultaneously the agent and its biotransformation products, benzoylecgonine (BE), ecgoninemethylester (EME) and the 'biomarker' of the interaction, the CE present in whole blood. Deuterated internal standards were added to 2 ml of post mortem whole blood and extracted in Bond Elut Certify columns. The residues were evaporated and derivatized with N-methyl-N-t-butyldimethylsilyltrifluoroacetamide (MTBSTFA). Detection was performed by electron impact ionization. The monitored ions were m/z 82/85 for EME-tert-butyldimethylsilyl (TBDMS)/EME-d3-TBDMS; m/z 182/185 for COC/COC-d3; m/z 196/199 for CE/CE-d3 and m/z 282/285 for BE-TBDMS/BE-d3-TBDMS. The limits of detection and quantification were found to be 25 ng and 50 ng ml(-1), respectively, for COC and CE, and 50 and 100 ng ml(-1) for BE and EME. Accuracy was different for each of the compounds, varying from 65 to 98%. The dynamic range of the assay was 50-2000 ng ml(-1).     

Detection and quantification of low levels of benzoylecgonine in equine urine

Cocaine (COC) is a highly addictive plant alkaloid expressing strong psychostimulatory effect. It has no medical use in equine veterinary practice. The contamination of the environment with cocaine such as its presence on the US paper currency has been reported few times. There are anecdotal reports of low benzoylecgonine (BE) concentrations (usually much less than 100 ng/mL) being found in urine of race horses. In order to protect horsemen against harsh penalties associated with the presence of trace amounts of BE in horse urine as a result of environmental contamination, in February 2005 the Illinois Racing Board issued new medication rules that established the threshold level of 150 ng/mL for BE in equine urine. The penalties associated with this rule provide for increasing fines ($250, $500, $1000) with successive positive reports against a trainer for levels of BE below 150 ng/mL. A total of 19,315 urine samples were collected over the 2-year period of time from winning horses (both harness and thoroughbred) at race tracks in Illinois for routine drug screening (ELISA). The presence of BE was confirmed by GC/MS in 28 urine samples (0.14%). The concentration range for BE in harness horses (21 detections) was < 5-91 ng/mL, and for thoroughbred (seven detections) was 7-52 ng/mL. To date, the laboratory has not reported concentrations of BE that exceed the established threshold concentration of 150 ng/mL.     

Occupational exposure to cocaine involving crime lab personnel.

The possibility of exposure to cocaine as a result of analyzing it or handling material contaminated by it has been a major concern of laboratory personnel. Several different work environments and simulated situations were examined to assess the likelihood of this type of exposure occurring. Urine specimens were collected and evaluated for cocaine and benzoylecgonine using the Syva ETS System (EMIT). Each specimen was analyzed for the two substances using gas chromatography/mass spectrometry (GC/MS). Urine specimens of laboratory-management personnel not working with drug samples showed no trace of cocaine or benzoylecgonine. A urinary benzoylecgonine level of 227 ng/mL was found in the specimen from one narcotics criminalist who was working on a routine case of 2 kilos of cocaine hydrochloride in the Narcotics Laboratory. A maximal urinary benzoylecgonine concentration of 1570 ng/mL was determined in the urine specimen from one narcotics criminalist who was sampling a case containing 50 kilos of cocaine hydrochloride over a period of 3 h. Decreasing the levels of airborne cocaine dust appears to minimize the amount of cocaine absorbed by the criminalists. Gloves, face masks, and goggles prove to be effective in minimizing exposure.     

Evaluation of immunoassay methods for the screening of cocaine metabolites in urine

Immunoassay kits for urine cocaine (and metabolite) screening, obtained from two commercial sources, were examined for correlation of their results, expressed in terms of equivalent benzoylecgonine concentration, with the gas chromatography/mass spectrometry (GC/MS) concentration of benzoylecgonine. The correlation coefficients obtained, based on 62 (out of a total sample population of 3295) highly relevant samples, were 0.467 and 0.766 for Abuscreen (ARIA) and TDx (TDX), respectively. The preliminary screen cutoff values, which correspond to 150 ng/mL benzoylecgonine (as determined by GC/MS), were calculated based on the resulting regression equations and found to be 380 and 190 ng/mL for ARIA and TDX, respectively. With these cutoff values, ARIA generates 5 false negatives and 16 unconfirmed presumptive positives, while TDX results in 3 false negatives and 6 unconfirmed presumptive positives.     

Concentrations of cocaine and its major metabolite benzoylecgonine in blood samples from apprehended drivers in Sweden

Cocaine and its major metabolite benzoylecgonine (BZE) were determined in blood samples from people arrested in Sweden for driving under the influence of drugs (DUID) over a 5-year period (2000-2004). Venous blood or urine if available, was subjected to a broad toxicological screening analysis for cannabis, cocaine metabolite, amphetamines, opiates and the major benzodiazepines. Verification and quantitative analysis of cocaine and BZE in blood was done by gas chromatography-mass spectrometry (GC-MS) at limits of quantitation (LOQ) of 0.02mg/L for both substances. Over the study period 26,567 blood samples were analyzed and cocaine and/or BZE were verified in 795 cases (3%). The motorists using cocaine were predominantly men (>96%) with an average age of 28.3+/-7.1 years (+/-standard deviation, S.D.). The concentration of cocaine was below LOQ in 574 cases although BZE was determined at mean, median and highest concentrations of 0.19mg/L, 0.12mg/L and 1.3mg/L, respectively. In 221 cases, cocaine and BZE were together in the blood samples at mean and (median) concentrations of 0.076mg/L (0.05mg/L) and 0.859mg/L (0.70mg/L), respectively. The concentrations of BZE were always higher than the parent drug; mean BZE/cocaine ratio 14.2 (median 10.9) range 1-55. Cocaine and BZE were the only psychoactive substances reported in N=61 cases at mean (median) and highest concentrations of 0.095 (0.07) and 0.5mg/L for cocaine and 1.01 (0.70) and 3.1mg/L for BZE. Typical signs of drug influence noted by the arresting police officers included bloodshot and glossy eyes, agitation, difficulty in sitting still and incoherent speech.