7-[125I]iodoclonazepam: purification by high-performance liquid chromatography for use in a very sensitive benzodiazepine radioimmunoassay of broad specificity

The purification of 7-[125I]iodoclonazepam by high-performance liquid chromatography (HPLC) for use in a very sensitive benzodiazepine radioimmunoassay is described. A silica column is used with a non-aqueous eluent and sequential ultra-violet and gamma-ray detection. A commercially available antiserum is used at a dilution of 1:1000. Blood samples are diluted 10-fold with buffer before analysis and only 25 microliters of diluted sample are required per assay tube. Benzodiazepines, but not the radiolabel, appear to be bound by blood proteins in competition with the antiserum and so, if undiluted blood is assayed, erroneously low results are obtained. The minimal sample requirement and the high sensitivity of the assay described here largely avoid this problem while maintaining acceptable detection limits. For diazepam, the detection limit is 2.5 ng/ml in blood or urine (after correction for the initial 10-fold dilution) and therapeutic or sub-therapeutic levels of many other benzodiazepines can be detected. In practice, the assay is reliable, simple to perform and extremely economical.     

Evaluation of a commercial radioimmunoassay kit for the detection of lysergide (LSD) in serum, whole blood, urine and stomach contents

The Diagnostic Products Corporation Coat-A-Count radioimmunoassay kit for LSD in urine has been evaluated for use in forensic toxicology with a variety of sample types. The cut-offs (defined as the mean response of blank samples plus three standard deviation) for LSD in serum, haemolysed whole blood, urine and stomach contents were 0.06, 0.050-0.055, 0.18 and 0.18 ng/ml, respectively. Preliminary extraction of LSD from the samples is not usually necessary. The precision of the analysis and the recoveries from spiked samples were satisfactory. The cross-reactivities of 2-oxo-LSD, lysergic acid methyl propylamide, lysergic acid monoethylamide and nor-LSD were estimated to be 11,6,2 and 1% respectively relative to LSD (100%).     

Headspace solid phase microextraction for the gas chromatographic analysis of methyl-parathion in post-mortem human samples. Application in a suicide case by intravenous injection

A simple and rapid procedure for the determination of methyl-parathion (m-p) in post-mortem biological samples was developed using headspace solid phase microextraction (SPME) and gas chromatography (GC) with nitrogen-phosphorous detection (NPD). Methyl-parathion was extracted on 85 microm polyacrylate SPME fiber. Salt addition, extraction temperature, and extraction time were optimized to enhance the sensitivity of the method. The linearity (y = 0.0473x - 0.0113, R2 = 0.9992) and the dynamic range (0.1-40 microg/ml) were found very satisfactory. The recoveries of methyl-parathion were found to be 46% in spiked human whole blood, 53% in spiked homogenized liver tissue, and 54% in spiked homogenized kidney tissue compared with samples prepared in water. The coefficients of variations for 2, 4, and 20 microg/ml of methyl-parathion in blood ranged from 0.9 to 5.1%, whereas the detection limit of the method was satisfactory (1 ng/ml in aqueous samples, 50 ng/ml in whole blood). The developed procedure was applied to post-mortem biological samples from a 21-year-old woman fatally poisoned (suicide) by intravenous injection of methyl-parathion. The intact insecticide was found in the post-mortem blood at a concentration of 24 microg/ml. No methyl-parathion was detected in the liver, kidneys, and gastric contents.     

The determination of lysergide (LSD) in urine by high-performance liquid chromatography-isotope dilution mass spectrometry (IDMS)

The use of isotope dilution mass spectrometry (IDMS) has been investigated for the forensic confirmation of lysergic acid diethylamide (LSD) in urine by LC-MS. The advantages of using a deuterated analog of LSD as an internal standard over methysergide are discussed. This study includes a comparison of the electrospray mass spectra of LSD, LSD-d3 and methysergide, and discusses the choice of suitable ions for use in selected ion monitoring (SIM) mode. An IDMS method is presented for the LC-MS confirmation of LSD in urine, with a limit of quantification (LOQ) of 0.5 ng/mL, reflecting the forensic requirement at this laboratory. Under some circumstances the LOQ can be improved to 0.1 ng/mL. This method is linear in the range tested (up to 10 ng/mL LSD in urine) and has been validated in terms of accuracy and precision.     

Plasma,oral fluid and sweat wipe ecstasy concentrations in controlled and real life conditions

In a double-blind placebo controlled study on psychomotor skills important for car driving (Study 1), a 75 mg dose of +/- 3,4-methylenedioxymethamphetamine (MDMA) was administered orally to 12 healthy volunteers who were known to be recreational MDMA-users. Toxicokinetic data were gathered by analysis of blood, urine, oral fluid and sweat wipes collected during the first 5h after administration. Resultant plasma concentrations varied from 21 to 295 ng/ml, with an average peak concentration of 178 ng/ml observed between 2 and 4h after administration. MDA concentrations never exceeded 20 ng/ml. Corresponding MDMA concentrations in oral fluid, as measured with a specific LC-MS/MS method (which required only 50 microl of oral fluid), generally exceeded those in plasma and peaked at an average concentration of 1215 ng/ml. A substantial intra- and inter-subject variability was observed with this matrix, and values ranged from 50 to 6982 ng/ml MDMA. Somewhat surprisingly, even 4-5h after ingestion, the MDMA levels in sweat only averaged 25 ng/wipe. In addition to this controlled study, data were collected from 19 MDMA-users who participated in a driving simulator study (Study 2), comparing sober non-drug conditions with MDMA-only and multiple drug use conditions. In this particular study, urine samples were used for general drug screening and oral fluid was collected as an alternative to blood sampling. Analysis of oral fluid samples by LC-MS/MS revealed an average MDMA/MDEA concentration of 1121 ng/ml in the MDMA-only condition, with large inter-subject variability. This was also the case in the multiple drug condition, where generally, significantly higher concentrations of MDMA, MDEA and/or amphetamine were detected in the oral fluid samples. Urine screening revealed the presence of combinations such as MDMA, MDEA, amph, cannabis, cocaine, LSD and psilocine in the multiple-drug condition.     

Instability of pancuronium in postmortem blood and liver taken after a fatal intramuscular Pavulon injection

The present study was designed to determine the stability of pancuronium in postmortem blood and liver during storage. Results were obtained using the method by Kerskes et al. [C.H.M. Kerskes, K.J. Lusthof, P.G.M. Zweipfenning, J.P. Franke, The detection and identification of quaternary nitrogen muscle relaxants in biological fluids and tissues by ion-trap LC-ESI-MS, J. Anal. Toxicol. 26 (2002) 29-34.], modified and validated in our laboratory. Target analytes were isolated after enzymatic hydrolysis followed by solid phase extraction (BondElut C18 column). Internal standardisation was carried out using laudanosine and the target ions were monitored by LC-ESI-MS (monitoring ions m/z 358 for IS and 286 for pancuronium). Materials were taken from a 46-year-old woman, who had been found dead. A syringe (2 ml) and an empty ampoule of Pavulon (4 mg/2 mL) were found in her hand. The residual volume of fluid in the syringe was 0.7 ml. An autopsy was performed six days after death. It revealed a needle mark on the left thigh. Postmortem materials (muscle from the injection site, blood and liver) and the syringe with fluid were stored for four months in a freezer at -20 degrees C. The initial pancuronium concentrations were 81 ng/mL in blood and 532 ng/g in liver. The analyte was stable when stored at -20 degrees C in blood even up to seven months. In liver samples its concentrations were variable. Pancuronium in blood stored at 20 degrees C underwent degradation very rapidly. After three months of storage these blood samples had concentrations not greater about 10% of the initial value. The degradation patterns of pancuronium depended on temperature and the biological matrix.     

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.     

高效液相色谱法测定人血清中安眠酮及其苯甲基羟化代谢物的浓度

本文建立了用反相高效液相色谱法(RP-HPLC)测定人血清中安眠酮及其苯甲基羟化代谢物-2-甲基-3[2-(羟甲基)-苯基]-4(3H)喹唑酮(Ⅰ)的浓度。安眠酮浓度在1-35μg/ml范围内呈直线相关(相关系数r=0.9980;回归方程y=0.06324x—0.1029),方法回收率平均为102.08±9.987(SD)％(n=5),检出限为1ng。其代谢物Ⅰ按原型安眠酮的线性浓度测定其相对量。本法为测定中毒者体内安眠酮及其代谢物Ⅰ的血浓度提供可行的手段。     

Stability study of the designer drugs "MDA, MDMA and MDEA" in water, serum, whole blood, and urine under various storage temperatures.

A controlled study was undertaken to determine the stability of the designer drugs MDA, MDMA and MDEA in pooled serum, whole blood, water and urine samples over a period of 21 weeks. The concentrations of the individual designer drugs in the various matrices were monitored over time, in the dark at various temperatures (-20, 4 or 20 degrees C), for a low (+/- 6 ng/ml for water, serum and whole blood and +/- 150 ng/ml for urine) and a high concentration level (+/- 550 ng/ml for water, serum and whole blood and +/- 2500 ng/ml for urine). Compound concentrations were measured using a validated HPLC assay with fluorescence detection. Our study demonstrated no significant loss of the designer drugs in water and urine at any of the investigated temperatures for 21 weeks. The same results were observed in serum for up to 17 weeks, and up to 5 weeks in whole blood. After that time, the compounds could no longer be analyzed due to matrix degradation, especially in the low concentration samples that were stored at room temperature. This study demonstrates that the designer drugs, MDA, MDMA and MDEA are stable when stored at -20 degrees C for 21 weeks, even in haemolysed whole blood.     

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.     

Validation of the Cozart Amphetamine Microplate EIA for the analysis of amphetamines in oral fluid

The purpose of this study was to determine the performance characteristics of the Cozart Amphetamine Microplate EIA for detecting amphetamine in oral fluid. Oral fluid samples were collected using the Cozart RapiScan Collection System from 135 volunteer donors from drug treatment clinics. A further 35 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory using the Cozart Amphetamine Microplate EIA and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen until analysis by GC-MS. The intra-assay precision for the Cozart Amphetamine Microplate EIA for amphetamine in oral fluid over forty assays was 2.74-7.1% CV (within assay) and 3.4-7.0% CV (within day). A total of 78 samples were positive for various amphetamines and related designer drugs. The Cozart Amphetamine Microplate EIA, using a cutoff of 45 ng/ml amphetamine equivalents in neat oral fluid, had a sensitivity of 91.7+/-3.3% and a specificity of 95.9+/-1.9% versus GC-MS using a cutoff of 30 ng/ml. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.     

HS/GC/ECD分析生物检材样品中的氰化物

目的 建立生物样品中氰化物的衍生化定性定量分析方法。方法 用氯胺T衍生化,HS/GC/ECD分析衍生物CICN。结果 在1ml血中,添加0.2μg氰化钾,回收率为84.6％,RSD为6.39％;在1g肝中添加0.5μg氰化钾,回收率为67.3％,RSD为5.05％;血中检出限为5ng/ml。结论 所建方法能定性定量分析生物样品中的氰化物。     

Amphetamine, cocaine and cannabinoids use among truck drivers on the roads in the State of Sao Paulo, Brazil

Lysergic acid diethylamide (LSD) is a potent hallucinogen, active at very low dosage and its determination in body fluids in a forensic context may present some difficulties, even more so in hair. A dedicated liquid chromatography-electrospray-tandem mass spectrometry (LC-ES-MS/MS) assay in hair was used to document the case of a 24-year-old man found dead after a party. Briefly, after a decontamination step, a 50mg sample of the victim's pubic hair was cut into small pieces (<1mm length), and incubated overnight in 3mL of phosphate buffer pH 5 at room temperature. After a liquid-liquid extraction (dichloromethane/ether), the extract was analyzed using a LC-ES-MS/MS method exhibiting a limit of quantification of 0.5pg/mg for LSD. A LSD concentration of 0.66pg/mg of pubic hair was observed. However, this result remains difficult to interpret owing to the concomitant LSD presence in the victim's post mortem blood and urine, the lack of previously reported LSD concentrations in hair, and the absence of data about LSD incorporation and stability in pubic hair.     

Dual-label time-resolved fluoroimmunoassay of psychopharmaceuticals and stimulants in serum

A new method to measure two different drugs simultaneously by time-resolved fluoroimmunoassay (TR-FIA) has been developed. In the TR-FIA reported here, psychopharmaceuticals [chlorpromazine (CPZ) and desipramine (DSP)] and methamphetamine (MA) contained in serum are assayed by a combined use of a new europium (Eu) chelate and a samarium (Sm) chelate, as labels. The drug concentrations were determined by the competition between a labeled antigen with Eu(3+) or biotin and a sample antigen. A microtiter plate coated with a mixture of rabbit IgGs (anti-MA and anti-CPZ or anti-MA and anti-DSP) was used. In the assay of MA and CPZ, Eu(3+) labeled MA-bovine serum albumin conjugate (MA-BSA) and biotinylated CPZ-BSA were added to the well with their non-labeled standard solutions or samples. MA was assayed by measuring the fluorescence intensity of Eu(3+) at 615 nm. After incubation of the Sm(3+) labeled streptavidin, CPZ was assayed by measuring the fluorescence of Sm(3+) at 643 nm. In the assay of MA and DSP, Eu(3+) labeled DSP-BSA and biotinylated MA-BSA were used. In our dual-assay, the minimum detection limits of these drugs were 1ng/ml for MA, 10 ng/ml for CZP and 10 ng/ml for DSP. Since the simultaneous detection of different drugs by TR-FIA is time and sample saving, the method can be employed in rapid and sensitive screening tests.     

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。     

Determination of lysergic acid diethylamide in body fluids by high-performance liquid chromatography and fluorescence detection--a more sensitive method suitable for routine use

A new method for determination of lysergic acid diethylamide (LSD) in body fluids by high-performance liquid chromatography and fluorescence detection was developed based on previously published methods. The new method is suitable for confirmation of samples tested positive by immunoassay, avoiding loss of LSD by absorption to surfaces. The reduced loss of LSD results in improved sensitivity. This is achieved by adding ethylene glycol to the samples, which cover glass surfaces. This principle can similarly be used to improve analysis of other drugs. Body fluids for analysis included urine and whole blood. An internal standard was applied for quantification of LSD. The new method offers satisfying precision data and has a detection limit of less than 0.05 ng/nL.     

Gas chromatographic/mass spectrometric determination of lysergic acid diethylamide (LSD) in serum samples

A sensitive method for the detection and quantification of lysergic acid diethylamide (LSD) in serum samples is described. After liquid-liquid extraction the trimethylsilyl derivative of LSD is detected by gas chromatography — mass spectrometry. Experiments with spiked samples resulted in a recovery of 76%, the coefficient of variation was 9.3%. Excellent linearity was obtained over the range 0.1–10 ng ml⁻¹. Additionally experiments demonstrating the light sensitivity of LSD are presented together with casuistics.     

LC／MS／MS法测定生物组织中百草枯

目的建立LC／MS／MS检测生物体液中百草枯方法。方法弱阳离子交换固相萃取小柱提取剂，应用LC／MS／MS法对生物样品中百草枯进行定性定量分析。结果经该方法测得百草枯的最小检出限为10ng／ml血（S／N≥3），线性范围为0．02～20μg／ml。结论该方法快速、灵敏、准确，适用于生物检材中百草枯的分析。     

Analysis of LSD in human body fluids and hair samples applying ImmunElute columns

Immunoaffinity extraction units (LSD ImmunElute) are commercially available for the analysis of lysergic acid diethylamide (LSD) in urine. The ImmunElute resin contains immobilized monoclonal antibodies to LSD. We applied the ImmunElute procedure to serum and also to human hair samples. For hair analysis the samples were first extracted with methanol under sonication. The extracts were then purified using the ImmunElute resin. LSD analysis was carried out with HPLC and fluorescence detection. The immunoaffinity extraction provides highly purified extracts for chromatographic analysis. The limit of detection (signal-to-noise ratio = 3) has been determined to be < 50 pg regardless of which sample material was used. The procedure was applied to authentic hair samples from drug abusers (n = 11). One of these samples tested positive with an amount of 110 pg LSD in 112 mg extracted hair corresponding to a concentration of 1 pg/mg.     

Validation of an ion-trap gas chromatographic-mass spectrometric method for the determination of cocaine and metabolites and cocaethylene in post mortem whole blood

The coingestion of cocaine (COC) and ethanol is a very frequent occurrence and is known to increase the risk of morbidity and mortality. The formation occurs of a transesterification product, the cocaethylene (CE), which is even more toxic than cocaine. In order to study the role of ethanol as an agent of interaction in lethal cocaine intoxication, and to establish its influence in post mortem cocaine concentrations, an ion-trap gas chromatographic-mass spectrometric method (GC-MS) was validated to quantify simultaneously the agent and its biotransformation products, benzoylecgonine (BE), ecgoninemethylester (EME) and the 'biomarker' of the interaction, the CE present in whole blood. Deuterated internal standards were added to 2 ml of post mortem whole blood and extracted in Bond Elut Certify columns. The residues were evaporated and derivatized with N-methyl-N-t-butyldimethylsilyltrifluoroacetamide (MTBSTFA). Detection was performed by electron impact ionization. The monitored ions were m/z 82/85 for EME-tert-butyldimethylsilyl (TBDMS)/EME-d3-TBDMS; m/z 182/185 for COC/COC-d3; m/z 196/199 for CE/CE-d3 and m/z 282/285 for BE-TBDMS/BE-d3-TBDMS. The limits of detection and quantification were found to be 25 ng and 50 ng ml(-1), respectively, for COC and CE, and 50 and 100 ng ml(-1) for BE and EME. Accuracy was different for each of the compounds, varying from 65 to 98%. The dynamic range of the assay was 50-2000 ng ml(-1).