尿样中苯骈二氮杂Zhu类药物筛选分析研究

目的建立尿样中苯骈二氮杂类药物两种筛选分析方法.方法用GC/ECD、GC/MS直接测定苯骈二氮杂类药物原体和GC/ECD、GC/MS测定1,4-苯骈二氮杂类药物的酸水解产物苯甲酮同系物.结果GC/ECD直接测定苯骈二氮杂类药物方法,大部分药物的回收率为60%～90%,线性范围为20～200ng/ml尿,线性相关系数大于0.99,最低检出限达0.5ng/ml～10ng/ml.结论所建两种方法各有其特点又可相互补充,已成功地应用于司法鉴定实践.     

Time of drug elimination in chronic drug abusers. Case study of 52 patients in a "low-step" detoxification ward

The elimination time of illicit drugs and their metabolites is of both clinical and forensic interest. In order to determine the elimination time for various drugs and their metabolites we recruited 52 volunteers in a protected, low-step detoxification program. Blood samples were taken from each volunteer for the first 7 days, daily, urine sample for the first 3 weeks, daily. Urine was analyzed using a fluorescence-polarization immunoassay (FPIA) and gas chromatography/mass spectrometry (GC/MS), serum using GC/MS. The elimination times of the drugs and/or their metabolites in urine and serum as well as the tolerance intervals/confidence intervals were determined. Due to the sometimes extremely high initial concentrations and low cut-off values, a few of the volunteers had markedly longer elimination times than those described in the literature. The cut-off values were as follows: barbiturates II (200ng/ml), cannabinoids (20ng/ml), cocaine metabolites (300ng/ml), opiates (200ng/ml). GC/MS detected the following maximum elimination times: total morphine in urine up to 270.3h, total morphine and free morphine in serum up to 121.3h, monoacetylmorphine in urine up to 34.5h, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in urine up to 433.5h, THC-COOH in serum up to 74.3h, total codeine in urine up to 123h, free codeine in urine up to 97.5h, total codeine in serum up to 29h, free codeine in serum up to 6.3h, total dihydrocodeine (DHC) in urine up to 314.8h, free DHC in urine up to 273.3h, total and free DHC in serum up to 50.1h. Cocaine and its metabolites were largely undetectable in the present study.     

The comparison of toluene determination between headspace-solid phase microextraction and headspace methods in glue-sniffer's blood and urine samples

An accurate and simple method was developed to determine the level of toluene in urine and blood quantitatively by using the gas chromatography/mass spectrometry (GC/MS) with headspace--solid phase microextraction (HS-SPME) technique. An assembly of SPME with a replaceable extraction fiber, coated with 100 microm polydimethylsiloxane, was used. The detection limit of toluene in blood and urine with HS-SPME technique was 10 times higher than that with headspace (HS) technique. To compare the HS-SPME with HS technique for the determination of toluene in biological fluids, blood and urine samples from glue sniffers were analyzed by both methods. The level of toluene by the two techniques was highly correlated: the correlation coefficient (r2) between the two sets of values were 0.98 and 0.96 in urine and blood, respectively.     

尿中苯丙胺、甲基苯丙胺、MDA和MDMA的固相微萃取和GC/MS/SIM测定

目的研究固相微萃取(SPME)用于尿中苯丙胺(AMP)、甲基苯丙胺(MET)、3,4-亚甲二氧基苯丙胺(MDA)和3,4-亚甲二氧基甲基苯丙胺(MDMA)的提取。方法样品调节至碱性和用盐饱和后用顶空SPME,内标为MET-d5。萃取纤维为100μm聚二甲基硅氧烷(PDMS)。用气质联用选择离子检测(GC/MS/SIM)。结果0.2μg/ml加标尿样,AMP、MET、MDA和MDMA的富集倍数分别为22,60,13和47。检出限(S/N=3)为0.4～9.5ng/ml。线性范围为0.05～1μg/ml。0.2、0.5和1.0μg/ml加标尿样,相对回收率77.9%～112.4%,变异系数2.7%～18.0%(n=5)。用该方法分析5个案件样品,和常规液液萃取结果接近。结论顶空SPME法用于尿中AMP、MET、MDA和MDMA等化合物的分析,无需有机溶剂,富集效率高,提取-富集-进样一体化,简单方便实用。     

GC／MS检测尿中4种苯氧羧酸类除草剂

目的建立尿中2，4-D、2，4-DP、MCPA、MCPP等4种苯氧羧酸类除草剂的分析方法；方法液液提取分离，乙醚为提取液，DCPA为内标，硫酸正丁醇酯化衍生化，气质联用分析法分析；结果尿中4种除草剂添加样品的相对回收率在80％以上，检测限都在5ng／ml以下，对中毒兔尿样进行了分析；结论对实际发生的中毒案件分析有足够的灵敏度。     

中毒致死者体内芬氟拉明的GC/NPD,GC/MS法测定

建立了生物检材中芬氟拉明的定性定量分析方法。体液及脏器组织经有机溶剂提取后 ,用GC/MS法进行药物筛选、定性 ,生物检材中的芬氟拉明浓度用4 -苯丁胺作内标、GC/NPD法测定。测得芬氟拉明中毒致死者的血液、尿液、肝等组织中浓度分别为7.8μg/ml、64.2μg/ml、31.3μg/g。并对尸体解剖所见及方法可行性进行讨论     

Screening and determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry

Benzodiazepines are one of the most widely prescribed drugs for the treatment of a wide spectrum of clinical disorders. They are used as anticonvulsants, anxiolytics, hypnotics or muscle relaxants with different duration of action. In this paper, a simple and sensitive method for the determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry (GC/MS) is described. The drugs spiked in whole blood were extracted with an Oasis HLB solid-phase extraction cartridge (Waters), which contains a copolymer designed to have a hydrophilic-lipophilic balance. GC/MS analysis was performed using a Shimadzu QP-5000 equipped with a BPX5 capillary column (15 mx0.32 mm I.D., film thickness 0.25 microm, SGE). Nineteen benzodiazepines and two thienodiazepines were well separated from each other on their SIM chromatograms and also on the TIC with the exception of oxazolam to cloxazolam separation. The blank extract from whole blood gave no peaks that interfered with all benzodiazepines and thienodiazepines on the chromatogram. The calibration curves for selected benzodiazepines with fludiazepam as an internal standard showed excellent linearity over the concentration range 5-500 ng/ml blood with a correlation coefficients of >0.995. The detection limits ranged from 0.2 to 20 ng/ml blood. The method is simple and sensitive for the determination of benzodiazepines in whole blood and seems to be useful in the practice of forensic science.     

Validation of a high performance liquid chromatographic method for alpha amanitin determination in urine

The objective of the present study was to develop and validate a liquid chromatographic method with electrochemical detection to measure alpha amanitin concentrations in urine after sample pretreatment with double mechanism (reversed phase/cation exchange) solid-phase extraction cartridges. The urine samples (10 ml) were purified and concentrated to 1 ml with elimination of matrix contaminants. The extracts were then separated by isocratic reversed-phase chromatography using a C18 column (4.6 mm×25 cm) with a mobile phase composed of 0.005 M phosphate buffer (pH 7.2) and acetonitrile (90:10). Coulometric detection was performed by applying an oxidation potential of +500 mV to a porous graphite electrode in a low-volume analytical cell. The limit of quantitation was 10 ng/ml with a signal-to-noise ratio=25. The linearity studied on spiked urine was satisfactory (r=0.9966) from 10 ng/ml to 200 ng/ml. The average extraction recovery of alpha amanitin was 78%, determined using spiked urine samples ranging from 10–300 ng/ml. The intra-assay precision was checked at 10, 50 and 100 ng/ml levels (n=10) in spiked urine samples, with resulting coefficients of variation of 3.6%, 2% and 1.5%, respectively.     

Simple analysis of arylamide herbicides in serum using headspace-solid phase microextraction and GC/MS

A simple and sensitive method for analysis of four arylamide herbicides (butachlor, propanil, diphenamide and propyzamide) in serum was developed using a headspace–solid phase microextraction (SPME) and a gas chromatograph–mass spectrometer (GC–MS). A vial containing a serum sample and sodium chloride was heated at 90°C. The extraction fiber of the SPME was exposed for 45 min in the headspace of the vial. The compounds adsorbed on the fiber were desorbed by exposing the fiber in the injection port of the GC–MS. The calibration curves, using an internal standard method, demonstrated good linearity throughout the concentration range from 0.25 to 10.0 μg/ml. Propyzamide was used for an internal standard. The limit of detection was 0.10, 0.05, and 0.25 μg/ml for butachlor, diphenamide, and propanil, respectively. No interferences were found, and the time for analysis was 60 min for one sample. In addition, this proposed method was applied to a suicide case in which the patient ingested Kusanon A^®, a herbicide. Propanil, which was the main ingredient in the herbicide, was detected in the eight serum samples collected from the patient during the hospitalization at the concentration range from 26.7 to 1.1 μg/ml.     

A simple gas chromatographic identification and determination of 11 CNS stimulants in biological samples. Application on a fatality involving phendimetrazine

A method is presented for the simultaneous identification and quantification of several CNS stimulants, including amphetamine in plasma and urine by GC/FID using mephentermine as an internal standard. No derivation is necessary and after a single alkaline extraction, GC analysis for the 11 compounds tested is achieved in 23 min. The lower limit of detectability was found to be 4 ng/ml for amphetamine in plasma. This method is sensitive, reproducible, selective and applicable in forensic and clinical toxicological analyses. Toxicological findings, after a fatality involving phendimetrazine are presented as an application of the procedure.     

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.     

SPME与GC/MS相结合检验尿液中的氯胺酮

目的应用固相微萃取与气相色谱-质谱联用技术分析尿液中的氯胺酮。方法对影响SPME萃取效果的萃取头类型、萃取时间、解析时间、离子强度等因素条件进行优化,分析尿中氯胺酮。结果在0.5~2.5μg/m l范围内线性关系良好,线性相关系数为0.995 6;最小检出限为2.5μg/L。结论该方法具有预处理简单、分析速度快、灵敏度高等优点,适合实际案件尿样的检验。     

Detection of THCCOOH in hair by MSD-NCI after HPLC clean-up

Regular consumption of cannabis can easily be detected by examination of hair for tetrahydrocannabinol, cannabinol, and cannabidiol. Although several studies have demonstrated that after contamination with smoke or treatment with THC containing shampoos THC is not detectable, or only in small traces, the detection of 11-nor-9-carboxy-delta 9-tetrahydrocannabinol (THCCOOH) should be offered to prove the consumption and metabolisation of THC. Up to now this confirmation was only available using tandem MS techniques combined with negative chemical ionisation. A new method using a normal quadrupole GC/MS is described. The lack of expensive instruments has to be paid for by a costly and time consuming extraction and clean-up. After the sample has been digested by 2 M NaOH at 95 degrees C and the neutralised liquid has been extracted with a mixture of n-hexane and ethyl acetate the dried residue is reconstituted in acetonitrile-methanol-0.01 M sulfuric acid (49:21:30, v/v/v) and the cannabinoids separated by HPLC. Each fraction is collected over 1 min. Another extraction with n-hexane-ethyl acetate is followed by evaporation, derivatisation, and GC/MS determination. The calibration with THCCOOH spiked hair led to a LOD of 0.3 pg/mg and a LOQ of 1.1 pg/mg.     

The detection of psilocin in human urine

Pharmacokinetic studies of psilocybin in humans have shown the rapid dephosphorylation of psilocybin to psilocin with further conversion to 4-hydroxy-tryptophole (4HT) and 4-hydroxyindole-3-acetic acid (4HIAA) in plasma. Our study shows that psilocin also undergoes conjugation and can be found in the urine as the psilocin-glucuronide conjugate. Recoveries after enzymatic hydrolysis of the urine with beta-glucuronidase (Helix Pomatia or E. Coli) when compared to non-hydrolyzed urine confirmed the presence of the glucuronide. Detection of psilocin from hydrolyzed and extracted samples was optimized for GC/MS by derivatization with MSTFA. The method developed allows for the detection of psilocin in urine with a limit of quantitation of 10 ng/mL, based on 5 mL of spiked urine. Using this method, our laboratory has confirmed the presence of psilocin in 6 out of 8 urine samples, with concentrations ranging from 10 ng/mL to greater than 200 ng/mL. Before implementation of the hydrolysis and derivatization steps, our limit of detection was 200 ng/mL, based on spiked urine standards. No case samples were positive without hydrolysis and derivatization.     

Fast confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine by LC/MS/MS using negative atmospheric-pressure chemical ionisation (APCI)

A fast method using automated solid-phase extraction (SPE) and short-column liquid-chromatography coupled to tandem mass-spectrometry (LC/MS/MS) with negative atmospheric-pressure chemical ionisation (APCI) has been developed for the confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine samples. This highly specific method which combines chromatographic separation and MS/MS-analysis can be used for the confirmation of positive immunoassay results with a NIDA cut-off of 15ng/ml. The conjugates of THC-COOH were hydrolysed prior to SPE, and a standard SPE was performed using C18-SPE columns. No derivatisation of the extracts was needed as in GC/MS analysis, and the LC run-time was 6.5min by gradient elution with a retention time of 2.4min. Linearity of calibration was obtained in the range between 0 and 500ng/ml (correlation coefficient R(2)=0.998). Using linear regression (0-50ng/ml) the limit of detection (LOD) was 2.0ng/ml and the limit of quantitation (LOQ) was 5.1ng/ml; day-to-day reproducibility and precision were tested at 15 and 250ng/ml and were 13.4ng/ml+/-3.3% and 255.8ng/ml+/-4.5%, respectively.     

The detection of acetylcodeine and 6-acetylmorphine in opiate positive urines

Acetylcodeine (AC), an impurity of illicit heroin synthesis, was investigated as a urinary biomarker for detection of illicit heroin use. One hundred criminal justice urine specimens that had been confirmed positive by GC/MS for morphine at concentrations >5000 ng/ml were analyzed for AC, 6-acetylmorphine (6AM), codeine, norcodeine and morphine. The GC/MS analysis was performed by solid phase extraction and derivatization with propionic anhydride. Total codeine and morphine concentrations were determined by acid hydrolysis and liquid/liquid extraction. AC was detected in 37 samples at concentrations ranging from 2 to 290 ng/ml (median, 11 ng/ml). 6AM was also present in these samples at concentrations ranging from 49 to 12 600 ng/ml (median, 740 ng/ml). Of the 63 specimens negative for AC, 36 were positive for 6AM at concentrations ranging from 12 to 4600 ng/ml (median, 124 ng/ml). When detected, the AC concentrations were an average of 2.2% (0.25 to 10.2%) of the 6AM concentrations. There was a positive relationship between AC concentrations and 6AM concentrations (r=0.878). Due to its very low concentration in urine, AC was found to be a much less reliable biomarker for illicit heroin use than 6AM in workplace or criminal justice urine screening programs. However, AC detection could play an important role in determining if addicts in heroin maintenance programs are supplementing their supervised diacetylmorphine doses with illicit heroin.     

A rapid and convenient LC/MS method for routine identification of methamphetamine/dimethylamphetamine and their metabolites in urine

A rapid and sensitive LC/MS method was developed for the simultaneous analysis of N,N-dimethylamphetamine (DMA), N,N-dimethylamphetamine N-oxide (DMANO), methylamphetamine (MA) and amphetamine (A) in urine samples. Employing an Alltech C18 column for solid phase extraction followed by LC/MS analysis using an Alltech Platinum EPS C18 column with a mixture of ammonium formate (0.01 M, pH 3) and acetonitrile (77:23, v/v) as mobile phase at a flow rate of 0.2 mL/min, simultaneous identification and quantitation of A, MA, DMA and DMANO in urine can be achieved using a 5-min chromatographic run. The calibration ranges were 0.10-3.0 micro g/mL for DMANO, 0.05-3.0 micro g/mL for DMA and 0.05-5.0 micro g/mL for both MA and A. The intra-, inter-day precision and accuracy for all analytes, spiked at three different concentrations in quality control samples, were in the ranges of 1.7-8.6, 4.1-10.0, -11.6 to 12.9%, respectively. The newly developed method was applied to the analysis of urine samples obtained from 118 suspected MA/DMA abusers, with the presence of MA confirmed in their urine samples under the drug-use surveillance program. Of these 118 samples, 43 were found to contain DMANO and 11 with both DMANO and DMA.     

Solid phase extraction method for rapid isolation and clean-up of some synthetic pyrethroid insecticides from human urine and plasma.

A simple and rapid method for the isolation of seven synthetic pyrethroid insecticides (methothrin, fenpropathrin, cyhalothrin, permethrin, cypermethrin, fenvalerate, deltamethrin) with a solid phase extraction (SPE), utilizing Sep-Pak C18 cartridges, from human urine and plasma is presented. The detection of the insecticides was performed using a wide bore capillary gas chromatograph (GC) with flame ionization detection (FID). The insecticide-containing samples mixed with 70% methanol were directly applied to the cartridges and eluted with 2 ml chloroform. The recoveries using the cartridges were between 90-102% for urine and 81-93% for plasma. Mixing samples with 70% methanol prior to extraction, seems very useful for the screening of synthetic pyrethroid insecticides.     

A validated SPME-GC-MS method for simultaneous quantification of club drugs in human urine

A solid-phase microextraction-gas chromatographic-mass spectrometric (SPME-GC-MS) method has been developed and validated for measuring four club drugs in human urine. These drugs include gamma-hydroxybutyrate (GHB), ketamine (KET), methamphetamine (MAMP), and methylenedioxymethamphetamine (MDMA). These drugs are referred to as 'club drugs' because of their prevalence at parties and raves. Deuterium labeled internal standards for each of the four drugs was included in the assay to aid in quantitation. The drugs were spiked into human urine and derivatized using pyridine and hexylchloroformate to make them suitable for GC-MS analysis. The SPME conditions of extraction time/temperature and desorption time/temperature were optimized to yield the highest peak area for each of the four drugs. The final SPME parameters included a 90 degrees C extraction for 20min with a 1min desorption in the GC injector at 225 degrees C using a splitless injection. All SPME work was done using a 100microm PDMS fiber by Supelco. The ratio of pyridine to hexylchloroformate for derivatization was also optimized. The GC separation was carried out on a VF-5ht column by Varian (30m, 0.25mm i.d., 0.10microm film thickness) using a temperature program of 150-270 degrees C at 10 degrees C/min. The instrument used was a ThermoFinnigan Trace GC-Polaris Q interfaced with a LEAP CombiPal autosampler. The data was collected by using extracted ion chromatograms of marker m/z values for each drug from the total ion chromatograms (TIC) (full scan mode). Calibration curves with R(2)>0.99 were generated each day using the peak area ratios (peak area drug/peak area internal standard) versus concentration. The validated method resulted in intra-day and inter-day precision (% R.S.D.) of less than 15% and a % error of less than 15% for four concentrations in the range of 0.05-20microg/mL (MAMP) and 0.10-20microg/mL (GHB, KET, and MDMA). This method has the advantage of an easy sample preparation with acceptable accuracy and precision for the simultaneous quantification of these four drugs of abuse and shows no interference from the urine matrix.     

固相萃取-气相色谱-质谱法检验人血浆中的右美托咪定

目的采用固相萃取结合气相色谱-质谱法(SPE-GC/MS)检验人血浆中盐酸右美托咪定。方法采用SPE提取血浆,用GC-MS/MS方法测定。结果盐酸右美托咪定在0.2μg/m L~5.0μg/m L范围内与峰面积呈现良好的线性关系(r2=0.999 1),检出限为20.0ng/m L,回收率为86.1%~91.5%,日内日间精密度均小于7.86%。结论本方法操作简单,结果准确,可以作为测定人血浆中右美托咪定的方法。