An UPLC-MS/MS method for the determination of valproic acid in blood of a fatal intoxication case

Valproic acid (VPA) has been used as an anticonvulsant for the treatment of epilepsy. The authors present a fatal case involving a 45-year-old female, found dead lying in bed with empty tablets of Diplexil^® next to her. She was a chronic alcoholic and epileptic who had been under psychiatric treatment, having repeatedly demonstrated intent to commit suicide.A rapid method was developed and validated to determine VPA in blood by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) with electrospray ionization source in negative ion mode.The method involved sample treatment with phosphoric acid followed by solid-phase extraction. Chromatographic separation was achieved using an Acquity UPLC^® BEH (2.1 × 50 mm id, 1.7 μm) column and a mobile phase containing ammonium acetate and acetonitrile, at a 0.5 mL/min flow rate. Detection and quantification of VPA was achieved using multiple reaction monitoring (MRM). The MS/MS transitions used for monitoring were m/z 143.1–143.1 for valproic acid and m/z 296.1–205.0 for hydrochlorothiazide used as an internal standard (IS).The limit of quantification (LOQ) was 0.5 μg/mL and the method was linear in the concentration range of 0.5–100 μg/mL. The coefficients of variation obtained for accuracy and precision were less than 10% and the mean recovery was 95% for the three concentrations levels studied (5 μg/mL, 10 μg/mL and 50 μg/mL). Toxicological results showed high concentration of VPA (556 μg/mL) and therapeutic concentrations of tiapride, mirtazapine, oxazepam and nordiazepam. Blood sample analysis also revealed the presence of ethanol at a concentration of 1.34 g/L.A specific, selective and sensitive method for the determination of VPA in blood was developed and can be used in routine forensic investigation. Toxicological results led the pathologist to rule that death was due to an intoxication caused by the simultaneous ingestion of high VPA concentrations and alcohol, with a suicidal legal-medical etiology.     

用灯盏乙素定量人血浆中异灯盏乙素的LC/MS/MS方法探讨

目的探讨用灯盏乙素定量人血浆中异灯盏乙素的LC/MS/MS方法。方法用AgiLent SB-C18柱(4.6 mm×150 mm,5μm),流动相为甲醇∶水∶甲酸(55∶43∶2)。采用电喷雾离子源(ESI),以选择反应监测(SRM)方式对灯盏乙素和异灯盏乙素及其苷元进行一级和二级质谱分析,离子反应分别为m/z 463→287和m/z 287→123。结果根据所得色谱和质谱数据,推断灯盏乙素可能的代谢产物和异灯盏乙素的结构,并测定人血浆中异灯盏乙素的浓度。结论用灯盏乙素定量异灯盏乙素的方法是可行的。     

Development of a preparation method to produce a single sample that can be applied to both LC–MS/MS and GC–MS for the screening of postmortem specimens

Simple and efficient extraction methods have been developed for the screening of a wide array of drugs in postmortem autopsy specimens. Acidic and basic compounds were targeted with two extraction methods that can be applied to both GC–MS and LC–MS/MS instrumentation. The extractions were achieved by utilizing lipid-removal and solid-phase extraction cartridges while carefully monitoring the pH of the samples to ensure the adequate removal of interfering substances like lipids and amino acid derivatives. These methods were applied to actual autopsy cases, with 94 and 124 compounds detected by GC–MS and LC–MS/MS, respectively. The developed methods could easily be incorporated into a forensic laboratory’s daily routine for screening many different compounds from postmortem samples.     

LC/MS/MS分析方法测定人血浆中匹多莫德的浓度

目的建立LC/MS/MS测定人血浆中匹多莫德的浓度。方法以舒巴坦为内标,采用甲醇-1%甲酸水溶液(10∶90,v/v)为流动相,以SymmetrySheildC18(3.5μm,20cm×2.1mm)色谱柱为分析柱,通过电喷雾电离源(ESI),以正离子多离子反应监测(MRM)方式进行检测。用于定量分析的离子反应分别为m/z245.2→m/z134.0(匹多莫德),m/z234.0→m/z124.2(舒巴坦)。结果匹多莫德线性范围0.010~20.0mg·L-1,定量下限为0.010mg·L-1(n=5)。日内、日间的RSD均<6.7%,平均回收率>90%。结论本法专属性强,样品处理方便,灵敏度高,适用于匹多莫德临床药动学研究。     

液相色谱-串联质谱法测定人血浆中拉米夫定浓度

 目的 试图建立测定人血浆中拉米夫定浓度的液相色谱-串联质谱法.方法 以吉西他滨为内标,内标法定量.流动相:甲醇-10 mmol/L乙酸铵(12: 88,v/v);质谱采用离子喷雾离子源,扫描方式为多反应监测(MRM),用于定量分析的离子反应分别为m/z 230m/z 111.9(拉米夫定)和m/z 263.8m/z 111.9(吉西他滨);药动学参数采用DAS2.0软件处理获得.结果 拉米夫定和内标吉西他滨的保留时间分别为2.9 min和2.4 min;拉米夫定的线性范围为0.0100~2.0000 μg/ml ,r=0.9996,回归方程:Y=0.0032X+0.0003,最低检测浓度为0.0100 μg/ml;提取回收率为95%～105%范围内;日内日间精密度RSD<6%.结论 此法适合人体血浆拉米夫定浓度的监测及生物利用度研究,结果准确、可靠.     

HPLC-MS/MS法快速测定人血浆中氟哌噻吨浓度及生物等效性研究

目的建立测定人血浆中氟哌噻吨浓度的高效液相串联质谱法（HPLC-MS/MS）,用于氟哌噻吨美利曲辛片的生物等效性研究。方法以Agilent ZORBAX Eclipse Plus C18（4.6mm×150mm,5μm）为色谱柱,流动相为乙腈（含1%甲酸）：0.02mol/L甲酸铵水溶液（80：20,V：V）,流速：0.8ml/min;柱温：40℃,以醋酸乙酯：二氯甲烷（4：1,V：V）为提取剂。样品经电喷雾离子源正离子化后,通过三重四级杆串联质谱仪,采用选择性反应监测（SRM）对氟哌噻吨（m/z435.2→305.1）和氯普噻吨（m/z316.2→231.1）进行测定。结果氟哌噻吨的高（1μg/L）、中（0.5μg/L）、低（0.05μg/L）3个浓度的平均回收率分别为101.01%、96.19%和106.63%,日内（n=5）、日间（n=3）RSD均小于15%;分析方法的最低定量限为0.025μg/L。线性范围为：0.025～2.5μg/L,回归方程为：F=0.6625ρ＋0.0049,r=0.997（n=7）,权重为1/ρ2。结论该方法灵敏、准确、简单、快速,可用于临床血浓监测和药动学研究。     

LC／MS／MS法测定中国健康志愿者血浆中瑞舒伐他汀浓度及药动学

目的 建立人血浆中瑞舒伐他汀的液相色谱-质谱-质谱联用测定方法，研究中国健康人体药代动力学。方法 以氢氯噻嗪为内标物，采用液相色谱-质谱-质谱联用法，电喷雾电离源选择性正离子峰检测。测30名健康男性志愿者单剂量口服瑞舒伐他汀钙片的体内血药浓度，获得药动学参数。结果 口服瑞舒伐他汀钙片5、10、20mg后的主要药代学参数：达峰时间Tmax分别为（3．10±0．99）、（2．00±0．82）、（3．40±1．58）h；峰值血浆浓度Cmax分别为（8．32±2．44）、（14．8±3、97）、（20．1±5．0）ng·ml^-1；药时曲线下面积AUC0-∞分别为（77．0±22．6）、（153±38）、（270±61）ng·h·ml^-1；AUCo．1分别为（75．8±22．0）、（151±37）、（258±62）ng·h·ml^-1；T1／2分别为（13．0±3．9）、（12．5±3．5）、（18．5±4．9）h。结论 受试者口服瑞舒伐他汀钙片后，在人体内表现为线性药代学特征。     

Analysis of fentanyl and sufentanil in hair by GC/MS/MS

Fentanyl and sufentanil are potent narcotic analgesics used only in hospitals as anaesthetic agents. The dependence potential of fentanyl is known. As they are given in doses at the microgram level and their elimination half-life is in the order of a few hours, detection in body fluids is possible only for a short time after administration. Radioimmunological methods are the only ones capable of detecting fentanyl in hair, as normal GC/MS methods for hair analysis are not sensitive enough to detect the drugs after doses in the order of micrograms. We therefore chose GC/MS/MS to analyse fentanyl and sufentanil in two cases where the drugs were given under controlled conditions over several days. The concentration was in the order of less than 100 pg/mg hair.     

Detection of ethyl glucuronide in dried human blood using LC-MS/MS

Ethyl glucuronide, as a direct metabolite of ethanol degradation, has proven useful as a long-term marker in many forensic applications. The inability to determine ethyl glucuronide in dried blood left a missing link in many investigations. Here, we describe a new method based on mass spectrometry in a Pauli-type ion trap in order to determine this substance in dried blood samples.     

中毒患者血液芬那露GC/MS的检测及其在临床救治中的作用

目的 利用气相色谱-质谱(GC/MS)仪建立一种快速检测中毒患者血液芬那露的方法.方法 采用乙酸乙酯液-液法提取血液芬那露,进样行GC/Ms检测,获取血液芬那露检测的线性方程、回收率、最低检测限及精密度.GC/MS配HP-5MS毛细管色谱柱(长度30m,直径0.25mm,膜厚0.25μm).高纯氦气作载气,流速1ml/min.1μl进样,分流比10∶1.气相色谱升温程序为160 ℃,保持2min,以10℃/min速率升温至280℃,保持10min.进样口、离子源(EI)、四极杆、传输线温度分别为250℃、230℃、150℃和280℃.EI电压70eV.结果 乙酸乙酯提取芬那露的回收率为82.1%,RSD=6.6%,n=5;芬那露线性方程为y=13 852x 140 588,r=0.998 2;日内精密度RSD为2.6 %,日间精密度RSD为4.9%;检测限全扫描法(m/z 30～280)为4μg/L,选择离子法(m/z 98,152,154)为0.1μg/L.结论 所建方法 简单快速,选择性好,可用于临床芬那露中毒患者血液浓度的检测.     

液质联用法测定龙胆水煎剂中三种环烯醚萜的含量

目的利用液质联用（LC／MS／MS）方法测定龙胆水煎剂中环烯醚萜类化合物龙胆苦苷、獐牙菜苦苷和马钱苷的含量。方法色谱柱为J’Sphere ODS-H80（150mm×2．0mm，4μm）；流动相为甲醇-10mmol·L^-1醋酸铵溶液（30：70）；流速为200μl／min。样品经电喷雾离子源（ESI）正离子化后，通过三级四极杆串联质谱仪，采用多反应离子监测方式测定样品中龙胆苦苷（m／z 357．2-195．3）、獐牙菜苦苷（m／z 392．1→195．2）和马钱苷（m／z 408．4→229．1）浓度。结果在该分析条件下，3种化合物能够同时进行检测。龙胆苦苷浓度在200～5000ng·ml^-1范围内线性关系良好，回归方程为Y=1．26×10^3X＋2．27×10^3，r=0．9983；獐牙菜苦苷浓度在20-500ng·ml^-1范围内线性关系良好，回归方程为Y=360X＋144，r=0．9991；马钱苷浓度在20-500ng·ml^-1范围内线性关系良好，回归方程为Y=782X＋42．6，r=0．9994。对照品峰面积精密度RSD分别为龙胆苦苷2．52％，獐牙菜苦苷2．77％和马钱苷2．89％；平均加样回收率分别为龙胆苦苷98．2％，獐牙菜苦苷99．1％和马钱苷99．7％。结论该方法灵敏、准确、特异性强。     

GC／MS法测定人血浆中地芬尼多药物浓度及其在药代动力学研究中的应用

目的建立GC/MS法测定人血浆中地芬尼多的含量,用于药代动力学研究中血药浓度的测定。方法液-液萃取法进行血样前处理,分析采用（15 m×0.25 mm×0.25μm）DB-17MS毛细管柱,载气为氦气,载气流速为1.0 ml/m in;进样口温度250℃,炉温升程：100℃→280℃（按25℃/m in的速度升温,且在280℃保持1 min）再升至300℃（按50℃/min的速度升温,且在300℃保持1.6 m in）;MS源温度230℃,四级杆温度150℃,电子轰击能量为70 eV。结果地芬尼多及内标的保留时间分别为7.8 min及7.97 min,其定量碎片基峰分别为m/z98和138。地芬尼多在1300 ng/ml浓度范围内,浓度与响应呈良好的线性关系（r2=0.999）;定量下限为1 ng/ml,准确度在82%104%范围内;地芬尼多的提取回收率为72.7%90.0%;批内和批间RSD分别在2.7%和3.9%以内,准确度在101.5%114.1%范围内;地芬尼多人血浆样品在-20℃保存10 d及在室温放置24 h稳定;其甲醇溶液在4℃冰箱保存21 d稳定。结论本研究建立的GC/MS方法测定人血浆中地芬尼多浓度,特异性好、灵敏度高,适用于盐酸地芬尼多口崩片在健康受试者体内的药代动力学研究。     

Evaluation of a solid-phase extraction procedure for the simultaneous determination of morphine, 6-monoacetylmorphine,codeine and dihydrocodeine in plasma and whole blood by GC/MS

Different procedures of solid-phase extraction were re-examined and a new solid-phase extraction procedure was developed using gas chromatography-mass spectrometry for the simultaneous detection and quantitation of morphine, 6-monoacetylmorphine, codeine and dihydrocodeine in plasma and whole blood. The effects of different types of sorbent and buffer solutions on the recoveries and purity of the extracts were also studied. Some preparation techniques on whole blood samples were also investigated. The method developed using Chromabond C18 (100) with spiked plasma samples had good recoveries for all opiates of interest: morphine 93.1% ± 7.4%, 6-monoacetylmorphine 68.0% ± 6.7%, codeine 77.0% ±8.3% and dihydrocodeine 67.9% ± 8.4%. The detection limit of all compounds was less than 5 g/L. The blank plasma showed no interfering peaks in the GC/MS-analysis.     

HPLC-MS/MS法测定大鼠血浆中LH14的含量

目的建立SD大鼠血浆中LH14的测定方法。方法以CS55为内标,采用HPLC-MS/MS法测定,以Ulti-mate C18色谱柱(50 mm×2.1 mm,3μm)为分析柱,甲醇-水为流动相,梯度洗脱,流速为0.2 ml/min,柱温为25℃,进样量5μl。结果在2～500 ng/ml浓度范围内具有良好的线性关系,线性方程为:Y=0.0333+0.0043X,R2=0.9928(n=8)。精密度与准确度均符合生物样品的测定要求,最低定量浓度为2 ng/ml。结论该检测方法简便、准确、专属性强,能够满足LH14在SD大鼠体内药代动力学研究的需要。     

Direct detection of serum psilocin glucuronide by LC/MS and LC/MS/MS: time-courses of total and free (unconjugated) psilocin concentrations in serum specimens of a “magic mushroom” user

Psilocin glucuronide (PCG) was directly identified in serum specimens of a “magic mushroom” user by liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS, together with the free (unconjugated) psilocin (PC). A major part of serum PC existed in the conjugated form. To quantify the total (conjugated plus free) PC in serum, enzymatic hydrolysis conditions were optimized using the user’s urine as the source of PCG; PCG in serum could be completely hydrolyzed by Escherichia coli β-glucuronidase. Using the established procedure, both total and free PC in the serum specimens of the user collected at various intervals were quantified. For the first specimen collected 5h after magic mushroom ingestion, 71.0 ng/ml of total PC and 13.3 ng/ml of free PC were detected. The ratio of free PC to total PC decreased with time after ingestion. The β-glucuronidase treatment of serum was found to clearly extend the detectable period of the serum PC; PC could be detected even 52 h after ingestion of magic mushroom.     

禁用药物β_2-激动剂的分析研究

采用灵敏度很高的气相色谱 /负离子化学电离质谱 (GC/NICIMS)对 9种国际奥委会禁用药物 β2 -激动剂进行分析研究 ,比较了不同衍生化方法得到的产物在NICIMS上的响应。确定了五氟丙酸酐衍生化产物有最佳的灵敏度 ,适合此类药物的检测要求。重点对克伦特罗尿样进行了检测 ,其结果符合实际工作的需要     

水中主要毒剂GC／MS／SIM方法的研究

目的：建立同时分析水中７种主要毒剂沙林、梭曼，塔崩，ＧＦ，Ｖｘ、俄罗斯Ｖｘ和芥子气的气－质联用选择离子监测（ＧＳ／ＭＳ／ＳＩＭ）分析方法。为饮用水样的毒剂检测提供切实可行的分析手段。     

A fatal clomipramine intoxication case of a chronic alcoholic patient: application of postmortem hair analysis method of clomipramine and ethyl glucuronide using LC/APCI/MS

Toxicological investigations of postmortem specimens of a 26-year-old man were performed with the use of LC/APCI/MS. They revealed in the blood of the deceased clomipramine (9.49 microg/g) and its main metabolite norclomipramine (1.10 microg/g) at concentrations explaining the fatal outcome. The presence of these xenobiotics in a 12-cm-long strand of hair (clomipramine, 7.60 ng/mg in I segment; 4.19 ng/mg in II segment; 1.86 ng/mg in III segment; norclomipramine, 5.71 ng/mg in I segment; 9.71 ng/mg in II segment; 4.13 ng/mg in III segment) confirmed the fact obtained from the medical history that the deceased had been receiving clomipramine as an antidepressant for 1 year prior to his death. The analysis demonstrated ethanol in autopsy blood (2.5mg/ml) and urine (3.2mg/ml); ethyl glucuronide as a marker of chronic alcohol abuse was detected in the deceased's hair (0.44 ng/mg in I segment; 0.07 ng/mg in II segment; n.d. in III segment). These findings may suggest the contribution of alcohol in the mechanism of drug-ethanol interaction, which in consequence might have affected the biotransformation of clomipramine in the final period of his life and evoked the ultimate toxic effect.     

HPLC-MS/MS法测定人血浆中左西孟旦

目的建立快速、灵敏的HPLC-MS/MS法测定人血浆中的左西孟旦浓度。方法以氢氯噻嗪为内标,血浆样品经甲基叔丁基醚沉淀蛋白,以甲醇为流动相,采用C18色谱柱进行分离,电喷雾离子化源,以多重反应监测方式进行负离子检测。结果测定血浆中左西孟旦方法的线性范围为0.5～1200 ng/ml,定量下限可达0.5 ng/ml。结论本方法专属性好,准确、快速,适用于左西孟旦注射液的临床药代动力学研究。     

莫达非尼R-和S-对映体的LC-MS/MS定量检测以及在Caco-2单层细胞模型上的转运特性

目的建立准确、灵敏、可靠的LC-MS/MS法,定量测定莫达非尼对映体,考察其在Caco-2细胞单层模型上的转运。方法选用资生堂C8(50 mm×2.1 mm,3μm)色谱柱及API4000液相色谱-质谱联用仪,用于定量分析的离子反应分别为m/z 274.4→165.1(莫达非尼)和m/z 391.2→265.1(CS55)。结果在本实验条件下,建立范围为1～1000 ng/ml的定量方法,批内、批间精密度<15%(n=5)。结论建立一种快速、准确、灵敏度高、专属性好,可用于莫达非尼对映体在Caco-2单层细胞模型转运研究的方法。