Development and validation of a liquid chromatography–mass spectrometry assay for hair analysis of methylphenidate

Methylphenidate (MPH) is a phenethylamine derivative used in the treatment of childhood attention-deficit hyperactivity disorder. MPH is biotransformed in the body by the hydrolysis of the methyl ester linkage to its metabolite, ritalinic acid. Whereas both compounds are usually measured in plasma and urine, preliminary observations show that only the parent compound is present in hair from treated individuals.     

Development and validation of a liquid chromatography-mass spectrometry assay for hair analysis of methylphenidate

Examination of various SIM cards and smart card devices indicates that data may be retained in SIM card memory structures even after heating to temperatures up to 450 degrees C, which the National Institute of Standards and Technology (NIST) has determined to be approximately the maximum average sustained temperature at desk height in a house fire. However, in many cases, and certainly for temperatures greater than 450 degrees C, the SIM card chip has suffered structural or mechanical damage that renders simple probing or rewiring ineffective. Nevertheless, this has not necessarily affected the data, which is stored as charge in floating gates, and alternative methods for directly accessing the stored charge may be applicable.     

液相色谱/质谱法检测血液中的艾司佐匹克隆

目的建立手性分离-液相色谱/质谱法,用于分离检测血液中艾司佐匹克隆。方法血液经HLB小柱固相萃取后,采用手性分离柱,用电喷雾正离子模式离子化、多反应监测模式检测艾司佐匹克隆,外标法定量。结果采用本文方法可有效分离佐匹克隆的左旋、右旋异构体,血中艾司佐匹克隆在1～100ng/mL范围内线性关系良好,相关系数为0.996 4,最低检出限为0.8ng/mL,回收率为76%～88%,日内与日间精密度均小于8%。结论本文所建方法简便、快速、分离度好,适用于血液中艾司佐匹克隆的检测。     

Tandem mass spectrometry: a helpful tool in hair analysis for the forensic expert

The Bavarian State Bureau of Investigation in Munich has the exclusive responsibility for investigation of criminal acts. One considerable expertise is that of hair analysis. According to the legal system in Germany, there is a special interest when some clients' hair tested positive for illicit drugs. An accused with a lot of drugs in his hair will be treated as a supposed addict and will be guaranteed extenuating circumstances. The instrumentation used for hair analysis is a powerful analytical tool: a Varian 3400 gas chromatograph linked to a Finnigan Tandem-MS (TSQ 700). The methanol extraction method is used for the detection of illegal drugs and metabolites: amphetamine, methamphetamine, MDA, MDMA (ecstasy), MDE, MBDB, methadone, THC, EDDP (metabolite of methadone), cocaine, benzoylecgonine, cocaethylene, opiates (dihydrocodeine, codeine, heroin, 6-monoacetylmorphine, morphine, acetylcodeine). For the detection of 9-carboxy-THC by negative chemical ionization the hair sample is hydrolyzed under alkaline conditions. Solid-phase extraction is used for clean-up. The LOQ for the determination of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic-acid is 0.16 pg/mg hair. An unsurpassed combination for rendering an expert opinion based on hair analysis may be: a forensic expert using diligence and experience, coupled with the performance of a sophisticated analytical instrument.     

液相色谱-串联质谱法检测血液中的美西律

目的建立测定血液中美西律(mexiletine)的液相色谱-串联质谱联用法(LC-MS/MS)。方法采用简便的乙腈蛋白沉淀法对血液进行预处理,应用Allure PFP Propyl液相柱分离,用电喷雾正离子模式离子化,多反应监测模式对美西律进行分析。结果美西律与内标纳洛酮分离良好,在0.02～10.00μg/mL内线性关系良好,相关系数为0.9999,回归方程为y=0.0283x-0.0151,日内与日间精密度的RSD均小于15%,最低检测限为0.01μg/mL。结论建立的LC-MS/MS方法简单、灵敏、可靠,可同时适用于美西律临床药物监测和法医毒物分析的需要。     

Rapid determination of ketamine in urine by liquid chromatography-tandem mass spectrometry for a high throughput laboratory

In recent years, the abuse of ketamine had gained popularity in rave parties in Hong Kong. The Urinalysis Unit of the Government Laboratory of Hong Kong faced a tremendous increase in workload for ketamine analysis. The number of tests performed rose from 10 in 1999 to 15,000 in 2002. As a fully validated immunoassay test for ketamine was not available in the market, most laboratories analyzed ketamine by chromatographic techniques after liquid-liquid extraction. However, these methods reported in the literature are not suitable for high throughput laboratories. Hence, a rapid screening/confirmation method for ketamine by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with a detection limit of 5 ng/ml was developed. After automated solid-phase extraction (SPE), the urine extract was analyzed for ketamine by a 2.5-min chromatographic run, the estimated recovery was 89% and the precision was 11% R.S.D. at 20 ng/ml. With the aid of an in-house developed computer program, the results were presented in spreadsheet format for easy checking. The method has been applied to our laboratory for routine ketamine analysis and a maximum of 200 samples per day can be achieved.     

Simultaneous determination of eight catechins and four theaflavins in bottled tea by liquid chromatography-tandem mass spectrometry for forensic analysis

Tea, and particularly bottled tea, is widely consumed worldwide and is often encountered at crime scenes in poisoning cases or used in place of urine in drug abuse monitoring. Tea is a rich source of polyphenols, such as catechins and theaflavins, and these compounds are useful for identification of trace quantities of tea samples. However, information on the contents of catechins and theaflavins in bottled tea is limited. In this study, a method was developed for simultaneous analysis of eight catechins and four theaflavins in tea using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of these polyphenols were determined in bottled black, oolong, and green teas after a simple pretreatment process by the standard addition method. The developed LC-MS/MS method was rapid and all tested polyphenol compounds were separated within ~14 min. All tea types contained all the catechins, at varying concentrations, but not all the theaflavins were present in all the tea types. This indicates that the theaflavin composition reflects the degree of the fermentation and could be used for discrimination among different types of tea. All the green tea samples contained all eight catechins; however, the concentrations of these compounds varied among the tea samples. Principal component analysis and hierarchical cluster analysis were useful for discrimination of samples. It has been unclear whether the variations of chemical components are useful for forensic discrimination. Our results demonstrate that, in addition to identification of tea varieties, catechins and theaflavins can be used for the discrimination of bottled tea samples.     

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。     

生物检材中吗啡类生物碱的LC-MS／MS分析

目的针对滥用药物分析鉴定实践中亟待解决的问题,开展LC-MS/MS分析生物检材中吗啡类生物碱的应用研究。方法满足不同的鉴定需要,分别建立血液、尿液、唾液和头发等生物检材的样品前处理方法,确定同时分析海洛因、单乙酰吗啡、吗啡、可待因、乙酰可待因、二氢可待因酮和氢吗啡酮等吗啡类生物碱的LC-MS/MS方法。将方法应用于实际案例。结果所建立的方法对吗啡类生物碱分离良好。尿液稀释法、尿液提取法和头发中吗啡的最低检测限(LOD)分别为10ng/mL、0.01ng/mL和0.01ng/mg。结论所建立的方法简便、快速、特异性强、灵敏度高。目标物中加入二氢可待因酮和氢吗啡酮扩大了方法的实用范围。     

Determination and validation of tetrodotoxin in human whole blood using hydrophilic interaction liquid chromatography-tandem mass spectroscopy and its application

A sensitive analytical method was developed for the quantitative determination of tetrodotoxin (TTX), a powerful sodium channel blocker, in human postmortem whole blood. The sample mixture was cleaned up using cation exchange SPE catridge after protein precipitation by methanol and then separated on a PC-HILIC (phosphorylcholine hydrophilic interaction liquid chromatography) column (150 mm × 2.0mm i.d., 5 μm) using a isocratic elution of 1% acetic acid and acetonitrile. The identification of TTX was performed on tandem mass spectrometry with electrospray ionization interface in positive ion mode. The retention time of voglibose (internal standard) and TTX was 5.1 and 6.0 min, respectively. TTX and internal standard (voglibose) were monitored and quantitated using the ion transitions: the respective precursor to product ion combinations, m/z 320/302 for TTX and m/z 268/92 for voglibose in the multiple reaction monitoring (MRM) mode. The recovery of TTX and voglibose was 61.4% and 62.8%, respectively and the good accuracy (97.7-103.9%), linearity (2-1200 ng/mL) and reproducibility were shown in this method. The limit of detection and limit of quantification were 0.32 ng/mL and 1.08 ng/mL, respectively. This method was applied in the case of three fishermen who were poisoned (including one death) by unknown fish on their boat in October 2010. In this case, the levels of TTX were 27.2, 30.0 and 29.7 ng/mL in heart blood, peripheral blood and serum of a victim, were 3.1 and 12.1 ng/mL in peripheral blood and 3.9 and 12.8 ng/mL in serum of two survivors, respectively.     

Quantitative analysis of multiple illicit drugs in preserved oral fluid by solid-phase extraction and liquid chromatography-tandem mass spectrometry

We present a validated method for the simultaneous analysis of basic drugs which comprises a sample clean-up step, using mixed-mode solid-phase extraction (SPE), followed by LC-MS/MS analysis. Deuterated analogues for all of the analytes of interest were used for quantitation. The applied HPLC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions for the non-deuterated analogues. Oral fluid was collected with the Intercept, a FDA approved sampling device that is used on a large scale in the US for workplace drug testing. However, this collection system contains some ingredients (stabilizers and preservatives) that can cause substantial interferences, e.g. ion suppression or enhancement during LC-MS/MS analysis, in the absence of suitable sample pre-treatment. The use of the SPE was demonstrated to be highly effective and led to significant decreases in the interferences. Extraction was found to be both reproducible and efficient with recoveries >76% for all of the analytes. Furthermore, the processed samples were demonstrated to be stable for 48 h, except for cocaine and benzoylecgonine, where a slight negative trend was observed, but did not compromise the quantitation. In all cases the method was linear over the range investigated (2-200 microg/L) with an excellent intra-assay and inter-assay precision (coefficients of variation <10% in most cases) for QC samples spiked at a concentration of 4, 12 and 100 microg/L. Limits of quantitation were estimated to be at 2 microg/L with limits of detection ranging from 0.2 to 0.5 microg/L, which meets the requirements of SAMHSA for oral fluid testing in the workplace. The method was subsequently applied to the analysis of Intercept samples collected at the roadside by the police, and to determine MDMA and MDA levels in oral fluid samples from a controlled study.     

超高效液相色谱-MS/MS法测定血中11种苯丙胺类物质

目的应用超高效液相色谱-质谱法对全血中11种苯丙胺类毒品进行定量测定。方法全血样品经1%(v/v)甲酸-乙腈提取,采用Ostra磷脂过滤板净化处理,使用ACQUITY UPLC BEH Phenyl(100mm×2.1mm,1.7μm)色谱柱,以0.3%(v/v)甲酸溶液-乙腈为流动相进行梯度洗脱。在多反应监测模式下测定全血样本中苯丙胺、甲基苯丙胺、二亚甲基双氧苯丙胺、替苯丙胺、3,4-亚甲基二氧基乙基苯丙胺、N-甲基-1-(3,4-亚甲二氧基苯基)-2-丁胺、副甲氧基甲基苯丙胺、麻黄碱、甲基麻黄碱、卡西酮、甲卡西酮,并进行方法学考察。结果 11种苯丙胺类物质的检出限(S/N≥3)为0.01~0.4ng/m L,在0.5~50μg/L范围内线性关系良好(r0.999);回收率在75.8%~103.4%之间,相对标准偏差在1.6%~13.0%之间。结论本文建立的超高效液相色谱-质谱法快速、简便、灵敏,适用于中毒案件检验及吸毒人员排查。     

Identification of ten corticosteroids in human hair by liquid chromatography-ionspray mass spectrometry

This paper describes a screening procedure based upon high-performance liquid chromatography-ionspray mass spectrometry for the identification of ten corticosteroids in human hair: triamcinolone, prednisolone, prednisone, methylprednisolone, cortisone, cortisol, beta- and dexamethasone, flumethasone and beclomethasone. Hair strands were washed in methylene chloride, pulverized in a ball mill and 50 mg of the powdered hair were incubated in 1 ml Soerensen buffer, pH 7.6 for 16 h at 40 degrees C, in presence of 50 ng cortisol-d3 used as internal standard. Purification of the incubation medium was achieved on SPE C18 Isolute extraction columns. The eluates were evaporated to dryness and resuspended in 30 microliters MeOH before analysis by HPLC-IS-MS in positive and negative modes of detection. The validation parameters were found satisfactory for a corticosteroid screening procedure. The correlation coefficient of the calibration curve ranged from 0.939 to 0.997, showing linearity between 0.1 and 10 ng/mg, excepted for beclomethasone which was between 0.2 and 10 ng/mg. Extraction recovery at 4 ng/mg ranged from 43.2 to 85.7%. Repeatability (CV values) at 4 ng/mg ranged from 6.1 to 17.5%. The limits of detection ranged from 0.03 to 0.17 ng/mg for a signal-to-noise ratio of 2. The detection of prednisone and beclomethasone in three hair specimens obtained from forensic and clinical cases have documented corticosteroids incorporation into human hair.     

血中安定及其代谢物的酶水解研究

目的考察木瓜蛋白酶水解血中安定及其代谢物蛋白结合物的酶解条件,提高安定的提取率。方法采用正交试验确定酶解的最优条件,检材经蛋白酶水解,固相萃取后,应用LC-MS／MS方法进行检测,运用保留时间和MRM（多离子反应监测）方式来对血中安定及其代谢物进行定性定量分析。结果安定、去甲安定、去甲异安定、羟基安定和去甲羟基安定的最佳酶解条件分别为55℃,2．5h,pH7．0,8000U;50℃,1h,pH7．5,8000U;50℃,1h,pH7．5,8000U;50℃,1．5h,pH7．5,8000U;50℃,1．5h,pH7．5,8000U。结论酶水解后的血液中安定及其代谢物检出量明显增加。     

Analysis of biofluids for flunitrazepam and metabolites by electrospray liquid chromatography/mass spectrometry

A rapid and sensitive liquid chromatography/electrospray ionization mass spectrometry (LC/MS) procedure has been developed for the analysis of biofluids containing flunitrazepam and its metabolites. Specimens were spiked with deuterated analogs of the analytes. Urine specimens were enzymatically hydrolyzed and blood specimens were untreated. Extractions were carried out using CleanScreen DAU SPE cartridges. The drugs were separated on a C18 column using a methanol-water-ammonium hydroxide (60:40: 0.03 v/v) mobile phase. After determination of base peaks using full scan mass spectrometry, the mass spectrometry method was optimized to operate in selected-ion monitoring (SIM) mode for the base peak of each analyte. Positive findings were confirmed by LC/MS/MS using the same mobile phase and column. This analytical procedure allows for the detection of low levels of flunitrazepam and metabolites in biofluids. It is useful for ascertaining the role of flunitrazepam in cases of drug-facilitated sexual assault.     

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。     

LC-MS/MS-MRM筛选血液中22种有毒生物碱成分

目的建立液相色谱-串联质谱法对血液中的22种常见有毒生物碱成分进行筛选分析。方法血液以丁丙诺菲为内标经液液提取后,用液相色谱-串联质谱仪以电喷雾电离(ESI )、多反应监测(MRM)方式进行分析。结果以化合物的保留时间、两对母离子/子离子对定性,最低检出限为0.1～20ng/mL。结论该方法选择性佳、灵敏度高,适用于法医毒物分析和临床毒物分析中有毒生物碱的分析。     

超高效液相色谱-串联质谱法同时测定尿液中16种抗生素

目的 建立同时分析尿液中16种抗生素的超高效液相色谱-串联质谱(UPLC-MS、MS)方法.方法 以哌拉西林为内标.尿样中的目标化合物经Oasis HLB固相萃取柱富集、纯化,利用ZORBAX SB-C18色谱柱,以0.1%的甲酸溶液-乙腈为流动相经梯度洗脱分离,采用多反应监测(MRM)模式进行分析.结果各化合物的最低...     

A quantitative method for simultaneous determination of 5-methoxy-N,N-diisopropyltryptamine and its metabolites in urine using liquid chromatography-electrospray ionization-tandem mass spectrometry

5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is a designer hallucinogen derived from tryptamine and is reportedly abused and involved in criminal activities. For the detection of 5-MeO-DIPT use, a liquid chromatography-tandem mass spectrometric method for 5-MeO-DIPT and its metabolites, 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT) and 5-methoxy-N,N-isopropyltryptamine (5-MeO-IPT) was developed and validated in rat urine. The urine samples were pretreated by protein precipitation with acetonitrile and introduced into a BDS HYPERSIL C(18) column (50 × 2.0 mm, 5 μm) for chromatographic separation. Mobile phases consisted of methanol, water, and 1% formic acid, and gradient elution was used at a flow rate of 0.2 mL/min. For the MS detection, multiple-reaction monitoring analysis was adopted. The linear range was 0.01-10 μg/mL, and the lower limit of quantification was 10 ng/mL for all analytes. The intra- and interday accuracies and precisions met the criteria (<15%). The developed method was successfully applied to the drug-treated rat urine.     

液相色谱一高分辨质谱联用技术在毒物筛选分析中的应用

毒物种类的广泛性、目标化合物的不确定性以及检材的多样性、特殊性,使得毒物筛选技术一直受到法医毒物分析工作者的重视.液相色谱-高分辨质谱联用技术凭借液相色谱强大的分离功能及高分辨质谱超高的质量分辨率和精确分子质量测定功能,结合一级、二级谱库匹配以及同位素离子丰度比,在化合物的筛选定性方面表现出强大的优势.本文综述了液相色...