生物检材中苯丙胺类兴奋剂和氯胺酮的LC-MS/MS分析

目的建立生物检材中苯丙胺类兴奋剂和氯胺酮的液相色谱-串联质谱(LC-MS/MS)分析方法。方法生物检材包括血液、尿液和毛发,采用稀释法和液液提取的前处理方法,应用两个不同的液相柱,优化LC-MS/MS分析方法,并考察了血液和尿液基质的离子抑制作用。结果同时分析苯丙胺和MDA,液相1在3m in内完成,液相2可用于确认分析或复杂基质分离。尿液稀释法检材用量少,前处理简便快速。毛发中苯丙胺类兴奋剂和氯胺酮的最低检测限(LOD)为0.005~0.05ng/mg。对送检案例检材产妇头发和胎毛进行苯丙胺类兴奋剂和氯胺酮的分析。结论本方法可用于生物检材中苯丙胺类兴奋剂和氯胺酮的同时分析,血、尿等生物检材的离子抑制作用是影响本方法灵敏度的主要原因。     

腐败生物检材中多种碱性滥用药物的检测

目的建立腐败生物检材中多种碱性滥用药物的提取、净化和仪器分析方法。方法用环己烷作为提取溶剂液-液萃取,同时采用Bond Elut Certify小柱、甲醇淋洗、二氯甲烷:异丙醇:氨水(78:20:2)洗脱固相萃取分离提取,GC/MS、GC/NPD定性定量分析各种生物检材中的滥用药物。结果从所送死者肝组织、胃组织、心血及胃内容、尿样、各检材中均同时检出吗啡、可待因、舒乐安定和异丙嗪成份,其中肝组织含量分别为吗啡0.094μg/g、可待因0.257μg/g、异丙嗪0.110μg/g,尿液含量分别为吗啡0.334μg/ml、可待因4.054μg/ml、异丙嗪0.066μg/ml,心血含量分别为吗啡0.036μg/ml、可待因0.106μg/g、异丙嗪0.088μg/ml。结论此方法准确、可靠、科学,可以用于法医毒物分析领域体内检材多种碱性药物的检测。     

抗体芯片竞争抑制法检测尿液中吗啡含量

目的建立抗体芯片竞争抑制法检测尿液中吗啡含量的方法。方法将吗啡单克隆抗体固定在用琼脂糖包被的芯片上,与含有吗啡的尿液检材和Cy3荧光标记-吗啡-BSA复合物进行竞争抑制反应,共聚焦扫描仪采集反应图像并进行分析。结果吗啡单克隆抗体及Cy3-吗啡-BSA的最佳浓度是31.25μg/m l、12.50μg/m l,检测线性范围0.01~10ng/m。回收率在91.2%~109.2%之间,尿液检测限为0.02ng/m l。甲基苯丙胺、安非他明与吗啡抗体之间无交叉反应,与可待因有一定的交叉反应。结论抗体芯片竞争抑制法检测尿液中的吗啡含量具有灵敏度高,特异性好、操作简单、高通量等优点,可用于法医毒物检测、戒毒效果监测。     

QuEChERS-LC/MS快速检测兽用麻药“舒泰”

目的本文对兽药"舒泰"中有效成分进行了结构确证,并建立了生物检材中替来他明和唑拉西泮的快速检验方法。方法在血液和尿液的生物检材中,通过加标实验,经QuEChERS萃取后,进行LC/MS对替来他明和唑拉西泮的定性定量检测分析。结果在血液和尿液的生物样品的加标实验中,替来他明的RSD%在0.5%~3.5%,唑拉西泮的RSD在0.5%~1.1%;替来他明的回收率在75.8%~100.3%,唑拉西泮的回收率在68.8%~76.6%,其中血液中替来他明的方法检出限为0.16ng/mL,尿液中为0.20ng/mL,唑拉西泮在血液中的方法检出限0.17ng/mL,尿液中为0.22ng/mL。结论建立的QuEChERS萃取方法,操作流程简便,方法重现性好,只需100μL取样量,更适合于痕量生物检材中替来他明和唑拉西泮的检验分析。     

QTRAP LC-MS/MS方法分析头发中氯胺酮结构类似物

目的 建立头发中氯胺酮结构类似物的液相色谱-四极杆/线性离子阱质谱（QTRAP LC-MS/MS）的检测方法。方法 将洗净晾干的20 mg头发加入1 mL提取液冷冻研磨后冰浴超声提取,离心取上清液过滤膜后,经ACQUITY UPLC?HSS T3色谱柱分离,采用多反应监测模式同时测定10种氯胺酮结构类似物。以该方法分析20例阳性头发样本中乙基氟胺酮、去甲氟胺酮和替来他明的含量。结果 头发中10种氯胺酮结构类似物在0.01～2.00ng/mg范围内线性关系良好,相关系数> 0.99,回收率为89.1%～106.1%,基质效应为88.3%～106.0%。20例阳性头发样本中乙基氟胺酮的含量范围为0.02～8.35 ng/mg,平均值1.59 ng/mg,中位值0.40 ng/mg;去甲氟胺酮的含量范围为0.01～0.94 ng/mg,平均值0.28 ng/mg,中位值0.19 ng/mg;替来他明的含量范围为0.02～10.93 ng/mg,平均值2.69 ng/mg,中位值2.11 ng/mg。结论 本方法简便、高效、可靠,适用于头发中氯胺酮结构类似物的检验。样本数据为氯胺酮结构类...     

UPLC-MS/MS分析尿液中阿片类化合物

目的 建立尿液中同时分析可待因(codeine,COD)、6-单乙酰吗啡(6-monoacetylmorphine,6-MAM)、吗啡(morphine,MOR)、吗啡-3-葡萄糖醛酸苷(morphine-3-glucuronide,M3G)和吗啡-6-葡萄糖醛酸苷(morphine-6-glucuronide,M6G)的超高效液相色谱-串联质谱(UPLC-MS/MS)方法.方法 以吗啡-d3(MOR-d3)和吗啡-3-葡萄糖醛酸苷-d3(M3G-d3)为内标,尿液用乙腈沉淀蛋白后,过SiroccoTM蛋白沉淀板,UPLC-MS/MS法分离检测.结果 尿液中COD和MAM检出限为0.2 ng/Ml,定量限为0.5 ng/Ml;MOR、M3G和M6G检出限为0.5 ng/Ml,定量限为1 ng/Ml;线性相关系数r≥0.999 7;日内精密度和日间精密度均在10%以内;回收率70.0%～98.3%,基质效应50.5%～99.0%.结论 所建方法简便、快速、准确,可以满足法庭毒物分析的需要.     

UPLC-MS/MS检验尿液中7种芬太尼类物质

目的建立尿液中7种芬太尼类物质的液质分析方法,检测4-氟丁酰芬太尼、乙酰芬太尼、丙烯酰芬太尼、呋喃芬太尼、异丁酰芬太尼、奥芬太尼、戊酰芬太尼等7种常见毒品。方法生物检材经过处理,应用超快速液相色谱法分离,采用高灵敏度的多离子监测模式(MRM)分析,并考察了方法的专属性,基质效应,回收率和检出限。结果尿液中各成分在浓度1ng/mL～500ng/mL范围内线性关系良好,方法回收率为85.7%～102.0%,基质效应为85.3%～98.5%,检出限浓度均为0.5ng/mL。结论所建立的方法灵敏度高,重现性好,准确性高,专属性好,检材的内源性杂质不干扰分析物测定,可在芬太尼类物质的司法检验鉴定及相关研究中应用。     

QTRAP LC-MS/MS方法分析头发中氟胺酮及其代谢物的研究

目的建立头发中氟胺酮及其代谢物的液相色谱-四极杆/线性离子阱质谱(QTRAP LC-MS/MS)检测方法并分析头发样本中氟胺酮含量范围。方法将洗净的20mg头发样本加入2mL提取液研磨后超声提取,离心取上清液过滤膜后,采用多反应监测模式测定氟胺酮及其代谢物,并以该方法分析了50例样本中氟胺酮的含量。基于氟胺酮、氯胺酮结构的相似性,参考氯胺酮代谢物去甲氯胺酮的质谱裂解途径,对氟胺酮主要代谢物进行推断。结果氟胺酮在浓度范围0.004ng/mg～2ng/mg内线性良好;方法检出限为0.001ng/mg;在0.05、0.20、1.00 ng/mg 3个添加水平的回收率为90.2%～94.4%。50例阳性样本中氟胺酮含量在0.2ng/mg以上46例占比92%,含量最高值92.56ng/mg、平均值15.32ng/mg、中位值5.34ng/mg,反映了氟胺酮较为严重的滥用形势。结论本方法简便、高效、可靠,适用于头发中氟胺酮及其代谢物的鉴定。样本数据为氟胺酮列管后鉴定及阈值确定提供了参考。     

鉴定科学LC-MS／MS多反应监测筛选分析血液中132种毒药物

目的建立血液中常见毒药物的LC-MS/MS筛选分析方法。方法采用多反应监测(MRM)模式,以目标物的保留时间和母离子／子离子对为指标进行筛选分析。建立LC-MS/MS 库和定性认定准则,并考察了方法的有效性。结果多反应监测筛选体系兼具有选择离子扫描的曼敏度和二级质谱的特征性,血液中132种毒药物筛选分析的最低检出限为0．1- 10ng/mL．其中83％的检出限≤lng/mL。鉴定应用表明方法的检出率和可靠性良好。结论该筛选分析方法能满足法医毒物学和临床毒物学检测中毒或治疗浓度目标物的需要,且能扩展至其它生物检材。     

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。     

The detection of hydromorphone in urine specimens with high morphine concentrations

A previous study suggested that small amounts of morphine are metabolically converted to hydromorphone. In the present study, morphine positive urine specimens obtained from a postmortem laboratory and a random urinalysis program were tested for morphine, codeine, hydromorphone, hydrocodone, oxymorphone, and oxycodone to assess the possibility that small amounts of hydromorphone are produced from the metabolism of morphine. The opioids were analyzed by gas chromatography-mass spectrometry as their respective trimethylsilyl derivatives following solid phase extraction. The limit of detection for hydromorphone was 5 ng/mL. A total of 73 morphine positive urine specimens were analyzed, with morphine concentrations ranging from 131 to 297,000 ng/mL. Hydromorphone was present at a concentration > or =5 ng/mL in 36 of these specimens at concentrations ranging from 0.02% to 12% of the morphine concentration. Hydrocodone was not detected in these specimens at the assay detection limit of 25 ng/mL. These results support earlier work suggesting that the detection of hydromorphone in urine specimens does not necessarily mean that exogenous hydromorphone or hydrocodone was used.     

固相萃取／LC-MS／MS测定尿液中吗啡类药物

目的 建立尿液中吗啡类药物的固相萃取／LC—MS／MS方法。方法采用OASIS MCX3cc（60mg）固相萃取柱进行提取,应用LC—MS／MS方法进行检测,运用保留时间和MRM方式对尿液中吗啡类药物及其代谢物进行定性定量分析。结果磷酸盐缓冲液pH4．0时,海洛因、6-MAM、可待因、吗啡、M3G的固相萃取回收率分别达64．33％-70．21％,96．95％～117．57％,83．60％～123．63％,68．82％～91．03％,94．64％～107．33％;最低检测限（LOD）分别为5、10、5、5、2pg,线性范围0．005～10μg／mL;相关系数分别为0．9998、0．9958、0．9992、0．9994、0．9997。结论本文所建方法,适用于尿液中吗啡类药物的分析。     

吗啡及其代谢物在藏毒致死者体内分布初探

目的采用固相萃取、液相色谱一串联质谱（LC-MS／MS）检验方法,考察吗啡和葡萄糖醛酸吗啡（M3G）在一例体内藏毒致急性死亡者体内分布情况。方法提取死者心血、尿、胃内容物、肝、肾、脑等15种检材,经Waters HLB小柱固相萃取后,C18色谱柱分离,采用电喷雾电离（ESI）、多反应监测模式（MRM）检测目标化合物。结果所建方法在0．0l～101μg／mL浓度范围内线性关系良好,提取回收率大于75％。结果显示总吗啡含量（游离态＋结合态）在胃内容物中最高,其次是尿、‘肾,在心血、胃组织、肺和腺体中居中,脑组织和心脏含量最低。结论本例检验结果验证了胃内容物、尿液和肾脏等是该类中毒案件的理想检材,其分布规律也可作为体内毒品分析实验依据。     

基质辅助激光解吸飞行时间质谱分析生物样品中甲基苯丙胺

目的建立尿样和头发中甲基苯丙胺的基质辅助激光解吸飞行时间质谱（matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS）分析方法。方法尿样采用液液提取,头发经0.1mol/L盐酸水解后采用液液提取,以碳纳米管为基质应用MALDI-TOF-MS法检测。结果尿样中甲基苯丙胺的最低检测限（LOD）为0.5μg/mL,线线范围为线性范围为0.5～100μg/mL（R2=0.9970）;毛发中甲基苯丙胺的最低检测限（LOD）为0.4ng/mg,线性范围为0.4～60ng/mg（R2=0.9976）,对送检案例中尿样和头发检材进行检测,效果良好。结论本方法适用于尿样和头发中甲基苯丙胺的分析,与传统气相色谱质谱联用和液相色谱-质谱联用相比,分析速度更快,适合大批量样品同时分析。     

Evaluation of the One-Step ELISA kit for the detection of buprenorphine in urine, blood, and hair specimens

A solid-phase enzyme immunoassay involving microtiter plates was recently proposed by International Diagnostic Systems corporation (IDS) to screen for buprenorphine in human serum. The performance of the kit led us to investigate its applicability in other biological matrices such as urine or blood, and also hair specimens. Low concentrations of buprenorphine were detected with the ELISA test and confirmed by HPLC/MS (buprenorphine concentrations measured by HPLC/MS: 0.3 ng/mL in urine, 0.2 ng/mL in blood, and 40 pg/mg in hair). The intra-assay precision values were 8.7% at 1 ng/mL of urine (n = 8), 11.5% at 2 ng/mL in serum (n = 8), and 11.5% at 250 pg/mg of hair (n = 8), respectively. The immunoassay had no cross-reactivity with dihydrocodeine, ethylmorphine, 6-monoacetylmorphine, pholcodine, propoxyphene, dextromoramide, dextrometorphan at 1 and 10 mg/L, or codeine, morphine, methadone, and its metabolite EDDP. A 1% cross-reactivity was measured for a norbuprenorphine concentration of 50 ng/mL. Finally, the immunoassay was validated by comparing authentic specimens results with those of a validated HPLC/MS method. From the 136 urine samples tested, 93 were positive (68.4%) after the ELISA screening test (cutoff: 0.5 ng/mL) and confirmed by HPLC/MS (buprenorphine concentrations: 0.3-2036 ng/mL). From the 108 blood or serum samples screened, 27 were positive (25%) after the ELISA test with a cutoff value of 0.5 ng/mL (buprenorphine concentrations: 0.2-13.3 ng/mL). Eighteen hair specimens were positive (72%) after the screening (cutoff: 10 pg/mg) and confirmed by LC/MS (buprenorphine concentrations: 40-360 pg/mg). The ELISA method produced false positive results in less than 21% of the cases, but no false negative results were observed with the immunological test. Four potential adulterants (hypochloride 50 mL/L, sodium nitrite 50 g/L, liquid soap 50 mL/L, and sodium chloride 50 g/L) that were added to 10 positive urine specimens (buprenorphine concentrations in the range 5.3-15.6 ng/mL), did not cause a false negative response by the immunoassay.     

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。     

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS. Application to the determination of MDMA after low ecstasy intake

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C(18) 5 microm, 2.1 mm x 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 degrees C in NaOH 1M before liquid-liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1-50 ng/mL in blood and urine; in the range 5-500 pg/mg for MA, MDMA, MDEA and MBDB, and 20-500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T+12h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D+8) and scalp hair at day 60 (D+60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.     

Heroin, morphine, and hydromorphone determination in postmortem material by high performance liquid chromatography

A procedure has been developed for the simultaneous determination of heroin, morphine, and hydromorphone from postmortem tissues by reversed phase high performance liquid chromatography (HPLC) using electrochemical detection. This method permits the direct determination of unmetabolized heroin from antemortem or postmortem urine as evidence of illegal drug use. Presumptive confirmation of heroin was based on the ability to hydrolyze the HPLC heroin fraction to morphine. Heroin was also confirmed in urine by gas chromatographic/mass spectroscopic (GC/MS) analysis of the HPLC fraction. Analysis of postmortem blood, gastric contents, urine, and injection site tissues have revealed the presence of morphine and hydromorphone, while heroin has only been identified in urine.     

Poppy seed consumption and toxicological analysis of blood and urine samples

Poppy seeds contain morphine in different amounts. Reported concentrations are up to 294 mg morphine/kg poppy seeds. Since penalties based on Street Traffic Law (parapgraph 24a StVG) in Germany (administrative offence) require definitive proof of morphine in blood samples, and the "Grenzwertkommission" in consultation with the Ministry of Transportation recommended a threshold of free morphine of 10 ng/mL, the question arose whether the consumption of poppy seeds can lead to a blood concentrations equal or higher than 10 ng/mL of free morphine. Therefore, five volunteers ate poppy seed products (50 mg morphine/kg poppy seeds). In urine, all on-site tests were enzyme immunologically positive for opiates and were positive to morphine by GC/MS. All the blood samples were negative to morphine by EIA and to free morphine by GC/MS. However, after hydrolysis, morphine was detected by GC/MS in all cases. Accordingly, in Germany, penalties based on parapgraph 24a StVG are not likely to cause road users any concerns should they have consumed poppy seeds. Driver Licensing Authorities, however, should be advised of this problem to avoid unjustified legal measures.