UPLC-MS/MS检测人血中18种有机磷及氨基甲酸酯类农药

目的建立人血中18种有机磷及氨基甲酸酯类农药超高压液相色谱-串联质谱(UPLC-MS/MS)的检测方法。方法血液中加入乙腈沉淀蛋白,采用Waters BEH C18(1.7μm 2.1×50mm)柱子,流动相为5mmol/L乙酸铵水-甲醇,流速:0.3m L/min;进样量:2μL,电喷雾离子源(ESI),正离子检测,采用多反应监测方式进行定量分析。结果药物最小检测限(LOD)在0.1~40ng/m L之间,定量限(LOQ)在0.5~50ng/m L之间,各药物浓度在定量限到500ng/m L范围内线性良好,回收率均在64.3%~111.9%之间,相对标准偏差为3.9%~10.3%。结论该方法专属性强、灵敏、准确,可以适用于法庭与临床毒物分析。     

Method validation of a survey of thevetia cardiac glycosides in serum samples

A sensitive and specific liquid chromatography tandem mass spectrometry (HPLC-ESI(+)-MS/MS) procedure was developed and validated for the identification and quantification of thevetin B and further cardiac glycosides in human serum. The seeds of Yellow Oleander (Thevetia peruviana) contain cardiac glycosides that can cause serious intoxication. A mixture of six thevetia glycosides was extracted from these seeds and characterized. Thevetin B, isolated and efficiently purified from that mixture, is the main component and can be used as evidence. Solid phase extraction (SPE) proved to be an effective sample preparation method. Digoxin-d3 was used as the internal standard. Although ion suppression occurs, the limit of detection (LOD) is 0.27 ng/ml serum for thevetin B. Recovery is higher than 94%, and accuracy and precision were proficient. Method refinement was carried out with regard to developing a general screening method for cardiac glycosides. The assay is linear over the range of 0.5-8 ng/ml serum. Finally, the method was applied to a case of thevetia seed ingestion.     

Solid-phase microextraction for the determination of cocaine and cocaethylene in human hair by gas chromatography-mass spectrometry

A method for the simultaneous determination of cocaine (COC) and cocaethylene (CE) in human hair was developed, using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) as analytical technique to identify and quantify the drugs. Selected ion monitoring (SIM) mode was used to obtain higher sensitivity. The deuterated-labeled analogues were used as internal standards. The detector response was linear for the drugs studied over the range 0.4-15 ng/mg, with correlation coefficients higher than 0.995. The coefficients of variation oscillated between 0.65% and 14.18% and the accuracy was in the range from 0.73% to 11.20%. The limits of quantitation and detection were found to be acceptable. Finally, this method was applied to 15 hair samples from cocaine users, obtaining positive results in all cases. The mean concentrations were 5.39 ng/mg (range: 0.43-8.98 ng/mg) for cocaine and 1.11 ng/mg (range: 0.42-2.23 ng/mg) for cocaethylene.     

Mixed fatal poisoning caused by taking L-methadone and chloral hydrate

A 42-year-old female drug user who was enrolled in a methadone maintenance program was found dead in her apartment. Cause of death was an intoxication with chloral hydrate and L-methadone. Trichloroethanol (TCE), the primary metabolite of chloral hydrate, was quantified by solid phase microextraction (SPME) and GC/MS in heartblood (27 micrograms/ml) and urine (338 micrograms/ml). D- and L-methadone were differentiated by chiral HPLC, which showed that only L-methadone had been taken. The quantitation of L-methadone and its metabolite EDDP was carried out by GC/MS from heartblood (1300 ng/ml and 86 ng/ml, respectively), urine (5239 ng/ml and 4960 ng/ml, respectively) and gastric contents (159 ng/ml and 122 ng/ml, respectively). The concentrations of both--trichloroethanol and methadone--were in toxic ranges.     

尿液、血液中γ-羟丁酸的气质联用法分析

目的为尿液、血液中γ-羟丁酸(gamma-hydroxybutyricacid,GHB),γ-羟丁酸内酯(gamma-butyrolactone,GBL)和1,4-丁二醇(1,4-butanediol,1,4-BD)的鉴定提供方法和依据。方法100μl尿液或血液以GHBd6为内标,经乙酸乙酯提取、BSTFA衍生化后,用GC／MS法分析。结果测尿液中内源性GHB的线性范围是20-800ng／ml,R2=0.9995,最低检出限为10ng／ml(S／N≥3);测尿液、血液中外源性GHB的线性范围为5-60μg／ml,R2分别为0.9999和0.9928。相对回收率为99％-104％。以所建方法测定了健康志愿者尿液中内源性GHB含量,并考察了健康受试者外源性GHB的代谢情况。结论所建方法准确、便捷、省时、选择性好,适用于法医毒物学鉴定。     

Quantitation using GC-TOF-MS: example of bromazepam

Time-of-flight mass spectrometry (TOF-MS) offers new perspectives for forensic toxicology. Qualitative and quantitative analyses of a mixture of three selected benzodiazepines (diazepam, nordazepam and bromazepam) were used to compare gas chromatography (GC-TOF-MS, quadrupole GC-MS, GC-ECD) and liquid chromatography (HPLC-DAD) data. Method validation parameters like LOD, LOQ, S/N-ratios reflect the capabilities of GC-TOF-MS. Five-point calibrations for bromazepam in human peripheral blood (50, 100, 160, 200, 300 ng/ml) using medazepam as internal standard (1000 ng/ml) were performed. The calibrations using GC-TOF-MS (using the fragments of m/z 236 and 288), GC-ECD (dual system) and HPLC-DAD (at 235 nm) all showed correlation coefficients close or superior to 0.99. Quadrupole GC-MS data was not used in the comparison of extracted samples due to the low sensitivity in the full scan mode. Two analyses of real cases concerning bromazepam are presented. In the first case, the presence or absence of bromazepam could not be established with both HPLC-DAD and GC-ECD due to background signals. The extracted ion chromatograms and spectrum traces after the analysis with the GC-TOF-MS could clearly excluded the presence of bromazepam. The second case illustrates the quantitation of bromazepam, where both HPLC-DAD and GC-ECD were unable to give satisfactory results, again due to interfering background signals. The analyses performed on the GC-TOF-MS-system demonstrated high sensitivity and also high selectivity due to the high quality of mass spectra obtained. The advantages of GC-TOF-MS make it a promising analytical technique for forensic toxicology.     

Fatal versus non-fatal heroin "overdose": blood morphine concentrations with fatal outcome in comparison to those of intoxicated drivers

The study was performed to distinguish fatal from non-fatal blood concentrations of morphine. For this purpose, blood levels of free morphine and total morphine (free morphine plus morphine conjugates) in 207 cases of heroin-related deaths were compared to those in 27 drivers surviving opiate intoxication. The majority of both survivors and non-survivors were found to show a concomitant use of depressants including alcohol or stimulants. Blood morphine levels in both groups varied widely, with a large area of overlap between survivors (free morphine: 0-128 ng/ml, total morphine: 10-2,110 ng/ml) and non-survivors (free morphine: 0-2,800 ng/ml, total morphine: 33-5,000 ng/ml). Five (18.5%) survivors and 87 (42.0%) non-survivors exhibit intoxication only by morphine. In these cases, too, both groups overlapped (survivors-free morphine: 28-93 ng/ml, total morphine: 230-1,451 ng/ml; non-survivors-free morphine: 0-2,800 ng/ml, total morphine: 119-4,660 ng/ml). Although the blood levels of free or total morphine do not allow a reliable prediction of survival versus non-survival, the ratio of free/total morphine may be a criterion to distinguish lethal versus survived intoxication. The mean of the ratio of free to total morphine for all lethal cases (N=207) was 0.293, for those that survived (N=27) 0.135, in cases of intoxication only by morphine 0.250 (N=87) and 0.080 (N=5), respectively. Applying a cut-off of 0.12 for free/total morphine and performing ROC analyses, fatal outcome can be predicted in 80% of the cases correctly, whereas 16% of the survivors were classified as dead. Nevertheless, in this study, all cases with a blood concentration of 200 ng/ml and more of free morphine displayed a fatal outcome.     

Detection of methamphetamine and amphetamine in a single human hair by gas chromatography/chemical ionization mass spectrometry

A detailed procedure of an extremely sensitive method for quantitation of methamphetamine and amphetamine in human hair by gas chromatography (GC)/chemical ionization (CI) mass spectrometry (MS) is presented. N-methylbenzylamine was used as an internal standard. The samples, after extraction with an organic solvent, were derivatized with trifluoroacetic anhydride before the GC/MS analysis. Quantitation was made with quasi-molecular ions of the derivatives by selected ion monitoring in the CI mode. The detection limit was about 10 pg in an injected volume. The high sensitivity enabled us to measure both stimulants in a single human hair in actual cases.     

Quantitative Analysis of Gamma‐Hydroxybutyrate at Endogenous Concentrations in Hair using Liquid Chromatography Tandem Mass Spectrometry

Abstract: A method capable of quantifying endogenous concentrations of gamma‐hydroxybutyrate (GHB) in human head hair was developed and validated using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Hair was digested under alkaline conditions, and GHB was isolated using liquid–liquid extraction. LC/MS/MS was performed using atmospheric pressure chemical ionization in the negative mode, multiple reaction monitoring, and deuterated internal standard (GHB‐D₆). Linearity was observed between 0.1 and 100 ng/mg GHB (R² = 1.000). The limits of detection and quantitation in human hair were 0.2 and 0.4 ng/mg, respectively. Accuracy at 2 ng/mg and 10 ng/mg was determined to be 97% and 94%, and intra‐assay CVs at these concentrations were 5.2% and 7.4% (n = 4). Beta‐hydroxybutyrate (BHB), alpha‐hydroxybutyrate, gamma‐butyrolactone, and 1,4‐butanediol did not produce an interference, and there was negligible ion suppression or enhancement from the matrix.     

Intoxication due to 1,4-butanediol

We report a case of intoxication resulting from the ingestion of a liquid, sold in the illicit market as "liquid ecstasy," which was found to contain 1,4-butanediol, a metabolic precursor of gamma-hydroxybutiric acid (GHB). Identification of the substance in the liquid was performed by gas chromatography-mass spectrometry (GC-MS).The toxicological analysis of blood, urine and gastric content of the victim was performed by immunoassay and gas chromatography with nitrogen-phosphorus detection as screening techniques and by means of GC-MS for confirmation and quantitation of 1,4-butanediol and GHB. The following drug concentrations were found: 82 microg/ml (blood), 401 microg/ml (urine) and 7.4 microg/ml (gastric content) for 1,4-butanediol and 103 microg/ml (blood), 430.0 microg/ml (urine) for GHB. In addition to these, other drugs detected and their blood concentration found in this case were methylenedioxymethylamphetamine (MDMA) 0.23 microg/ml and its metabolite methylenedioxyphenylamphetamine (MDA) 0.10 microg/ml. In the urine, a concentration of 0.10 microg/ml of benzoylecgonine was also found.     

血中毒鼠强的固相萃取和GC-NPD法测定

目的 研究人全血中毒鼠强的固相革取(SPE)。方法 用Bond Elute C18固相萃取柱萃取,GC-NPD检测,以甲基对硫磷为色谱内标(CS)。结果 全血中加标0.5μg/ml,毒鼠强回收率为92.5％,变异系数2.3％(CV,n=4)。在0.5～10μg/ml的浓度范围内,线性相关系数为0.9994。检出限(S/N=3)和定量限(S/N=10)分别为6ng/ml和20ng/ml。同一根萃取柱连续使用6次未见性能明显下降(CV=3.6%)。结论 本文方法适用于毒鼠强中毒的全血测定。     

Quantitative analysis of tropane alkaloids in biological materials by gas chromatography-mass spectrometry

A simple and rapid method for quantitation of tropane alkaloids in biological materials has been developed using an Extrelut column with gas chromatography-mass spectrometry (GC-MS). Biological materials (serum and urine) were mixed with a borate buffer and then applied to an Extrelut column. The adsorbed tropane alkaloids were eluted with dichloromethane before a GC-MS analysis. Atropine-d(3) was used as an internal standard. The extracted tropane alkaloids were converted to trimethylsilyl derivatives prior to GC analysis, to improve the instability of tropane alkaloids from heating and the property of them for a GC column. The recoveries of the compounds, which had been spiked to biological materials, were more than 80%. The GC separation of the derivatives from endogenous impurities was generally satisfactory with the use of a semi-polar capillary column. Tropane alkaloids showed excellent linearity in the range of 10-5000 ng/ml and the limit of detection was 5.0 ng/ml for biological materials. The present method is simple and more rapid than those previously reported, and was applied to a poisoning case. It is useful for the routine analysis of tropane alkaloids in cases of suspected tropane alkaloids poisoning.     

Fatal intoxication involving 2-methyl AP-237

Novel synthetic opioids contribute considerably to the opioid epidemic, especially with the frequent emergence of structurally similar compounds. This case report describes a fatal intoxication involving 2-methyl AP-237. A 54-year-old Caucasian male was found deceased from an apparent drug overdose. A plastic container labeled “2MAP” and a cut straw were found in the decedent's backpack at the scene. A white substance found in the container tested positive for fentanyl by field testing. According to his medical history, the decedent was treated for a drug overdose 3 years prior to his death. With no diagnostic findings at autopsy, the case was submitted for toxicological analysis. An unknown substance was detected in peripheral blood and urine using gas chromatography with nitrogen phosphorous detection (GC-NPD). Further testing was conducted using gas chromatography–mass spectrometry (GC–MS) and liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) which confirmed the presence of 2-methyl AP-237 and potential metabolites in blood and urine. Quantitation by GC-NPD revealed concentrations of 2-methyl AP-237 in blood and urine at 480 ng/mL and 4200 ng/mL, respectively. The toxicological analysis also identified and quantitated alprazolam in the blood at 55 ng/mL. Additionally, the metabolism of 2-methyl AP-237 was investigated and three hydroxylated metabolites were identified in peripheral blood and urine. Limited literature is available for the detection and quantitation of 2-methyl AP-237 in postmortem specimens. Given the toxicological findings with unremarkable autopsy findings, this case is an example of a fatal intoxication involving 2-methyl AP-237.     

HPLC／MS检测生物样品中的丁丙诺啡

目的建立了生物样品中丁丙诺啡的高效液相色谱-电喷雾串联质谱检测方法。方法样品经固相萃取提取净化、反相液相色谱分离后进行质谱检测，根据保留时间及特征离子进行定性分析，以母离子m／z468进行定量分析。结果在10-500ng／ml（ng／g）范围内峰面积与质量浓度的线性关系良好（r^2〉0．993）。在50、100、500ng／ml（ng／g）3个添加水平，尿、血、肝中丁丙诺啡的平均回收率为74％～94％，日内测定结果的相对标准偏差小于8％，日间测定结果的相对标准偏差小于10％。结论该方法简单、灵敏，特异性强，适用于生物样品中丁丙诺啡的分析检验。     

Quantitation of human genomic DNA through amplification of the amelogenin locus

An alternate method for quantitation of human genomic DNA is presented. Quantitative template amplification technology (abbreviated "Q-TAT") estimates the quantity of human DNA present in an extract by comparing fluorescence in X and Y amplicons produced from unknowns with fluorescence in a standard curve amplified from known quantities of reference DNA. Q-TAT utilizes PCR and electrophoresis with fluorescent detection/quantitation, precluding the need for new instrumentation, methodology, or quality assurance associated with slot-blot or real-time PCR. In a comparison study incorporating shared samples, Q-TAT was found to be more sensitive than widely used slot-blot methods but somewhat less sensitive than real-time PCR. Among samples containing DNA concentrations ranging from 100 pg/microL to 2-4 ng/microL, Q-TAT produced DNA concentration estimates that agreed reasonably well with either Quantiblot or real-time PCR. Q-TAT was reproducible with a typical coincidence of variation of about 35%. Quantitation of human DNA in this study involved summing fluorescence in X and Y amplicons in unknowns and quantitation standards. However, analyzing fluorescence in X and Y amplicons individually could allow estimates of male and female DNA present in mixtures to be made. Moreover, since X and Y amplicons exhibit sizes of 210 and 216 bp, respectively, the integrity as well as the concentration of the genomic DNA template can be assessed. Q-TAT represents an alternate method useful for the quantitation of human genomic DNA prior to amplification of STR loci used for identity testing purposes. The method uses existing equipment and procedures in conjunction with a well-characterized DNA standard to produce concentration estimates for unknowns that reliably produce STR profiles suitable for analysis.     

Postmortem determination of the biological distribution of sufentanil and midazolam after an acute intoxication

A case is presented of a death caused by self-injection of sufentanil and midazolam. Biological fluids and tissues were analyzed for midazolam by high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) and for sufentanil by GC/MS. Midazolam was extracted from basified fluids or tissues homogenated with n-butyl chloride and analyzed by HPLC by using a phosphate buffer: acetonitrile (60:40) mobile phase on a mu-Bondapak C18 column at 240 nm. Sufentanil was extracted from basified fluids and tissue homogenates with hexane:ethanol (19:1). GC/MS methodology for both compounds consisted of chromatographic separation on a 15-m by 0.25-mm inside diameter (ID) DB-5 (1.0-micron-thick film) bonded phase fused silica capillary column with helium carrier (29 cm/s) splitless injection at 260 degrees C; column 200 degrees C (0.8 min) 10 degrees C/min to 270 degrees C; and electron ionization and multiple ion detection for midazolam (m/z 310), methaqualone (IS, m/z 235), sufentanil (m/z 289), and fentanyl (IS, m/z 245). Sufentanil concentrations were: blood 1.1 ng/mL, urine 1.3 ng/mL, vitreous humor 1.2 ng/mL, liver 1.75 ng/g, and kidney 5.5 ng/g. Midazolam concentrations were: blood 50 ng/mL, urine 300 ng/mL, liver 930 ng/g, and kidney 290 ng/g. Cause of death was attributed to an acute sufentanil/midazolam intoxication and manner of death a suicide.     

固相微萃取-气相色谱质谱法测定血浆中的氯氮平浓度

目的建立固相微萃取-气相色谱质谱法测定人血浆中氯氮平浓度的方法。方法以固相微萃取法提取血浆中的氯氮平,萃取头为100μm聚二甲基硅氧烷,洛沙平作内标,用气相色谱质谱选择离子法进行检测。结果本文建立的方法在5~2000 ng/ml浓度范围内呈线性关系,检测限为0.1 ng/ml(信噪比>3),低、中、高浓度(100、500、1000 ng/ml)平均相对回收率分别为98.6%、94.6%和94.6%,日内、日间RSD分别小于7.4%和7.1%。结论本文建立的固相微萃取-气相色谱质谱法灵敏度高、准确度好、操作简便,适用于氯氮平急性中毒案件的检测。     

An analytical method for the rapid screening of organophosphate pesticides in human biological samples and foodstuffs

Gas chromatography with nitrogen/phosphorus sensitive detection (GC/PND) and electron impact mass spectrometry (GC/MS) with selected ion monitoring provides a simple, rapid and sensitive method for the determination of organophosphate pesticides (OPs). A selective single-step extraction of 23 different OPs in urine, blood, serum and food samples (baby food, soft drinks and instant soups suspected of contamination from a blackmailing scare) is described. The OPs were extracted with 1ml toluene (with and without addition of mevinphos as internal standard), using a 0.7ml aliquot of urine, blood or serum sample. Food samples (0.2g) were homogenised with water (0.5ml) before extraction. An amount of 1microl of the toluene phase (extraction supernatant) was analysed directly by GC/PND and GC/MS.The method was validated using spiked human serum. The OPs were mixed with serum containing 10mg/ml disodium ethane diamine tetraacetic acid disodium salt (EDTA disodium salt) and stored up to 10 days at 4 and -20 degrees C, respectively. The recovery rates of OPs in freshly spiked human plasma ranged between 50% (dimethoate) and 133% (dialifos). OPs in plasma proved to be stable at -20 degrees C. Their levels decreased only slightly after storage at 4 degrees C.     

A novel method for cyanide quantification in human whole blood using ion chromatography with amperometric detection and its application to cyanide intoxication cases

Cyanide is a highly toxic agent that has been frequently used for suicide in South Korea. It is also used in various industrial fields, such as metal plating, in which many accidental cyanide intoxications have occurred. To overcome the disadvantages of conventional cyanide analysis methods, a simple and fast method for the analysis of cyanide in whole blood using ion chromatography (IC) with amperometric detection was developed in this study. Whole blood samples were deproteinized, diluted, and analyzed using an IC–amperometric detection system. The limits of detection and quantitation were 0.1 and 0.2 mg/L, respectively. The method showed good linearity in the range of 0.2 to 50 mg/L with R² > 0.99. The intra- and inter-assay precision and accuracy values were <10%. The established method was successfully applied to analyze whole blood samples from three cyanide intoxication cases.     

The interpretation of cocaine and benzoylecgonine concentrations in postmortem cases.

This study examined cocaine and benzoylecgonine concentrations in 100 consecutive deaths where either compound was identified in blood or urine specimens to determine whether any relationship between these concentrations and cause of death can be found. Forty-seven of the 100 cases were deaths attributed to cocaine, narcotic or combined cocaine and narcotic intoxication. There were 13 cases of cocaine intoxication where no psychoactive substance other than ethanol was detected. The mean cocaine concentration in these deaths was 908 ng/ml; three cases had cocaine concentrations greater than 2000 ng/ml, while the other ten cases had cocaine concentrations less than or equal to 700 ng/ml. The mean cocaine concentration in non-cocaine deaths where no psychoactive substance other than ethanol was detected was 146 ng/ml. This difference was not statistically significant. However, the average blood benzoylecgonine concentration in the 13 cocaine deaths was significantly higher than in the 19 non-cocaine deaths. A review of combined cocaine and narcotic deaths suggest that the narcotic is the main causative agent in these deaths.