Gas chromatography with mass-selective detector in testing blood for amitriptyline and nortriptyline

The article describes the method of simultaneous detection of amitriptiline and nortriptilin in cadaveric blood using gas chromatography with mass-selective detection. Preparation includes liquid-liquid extraction and derivation with trifluoroacetic anhydride. Nortriptilin threshold of detectability in the blood is 0.02 mcg/ml, amitriptilin one--0.05 mcg/ml. The range of detectability--0.05-3.0 mcg/ml for both compounds. Maximal error of the compounds detectability was under 12.9% for concentrations 0.10 mcg/ml and 10.6% for concentrations 2.0 mcg/ml. The method was tested on expert material in forensic chemical examinations.     

Use of microcolon gradient high performance liquid chromatography for urine dimedrol assay in combined poisoning

A technique of a rapid urine dimedrol assay by gradient microcolon high performance liquid chromatography is proposed. Dimedrol is detected selectively in the presence of large amounts of medicinal and narcotic substances metabolites. Threshold of detectability was 0.005 mcg/ml, optimal range of quantitation -0.02 - 1 mcg/ml, extraction degree in sampling -85%. It was found that concentration of dimedrol in the urine obtained for chemicotoxicological tests ranges from 0.01 to 2.5 mcg/ml. Establishment of dimedrol intake by presence of its metabolites was studied.     

尿中氯喹定性定量分析方法的研究

建立了人尿中氯喹的定性定量分析方法,2ml尿样用2ml×2环己烷：乙酸乙酯（8：2）提取净化后,60℃水浴室气吹干,残留物定容溶解后,气相色谱分析,氯喹的保留时间为9．44min。方法最低检测限为200ng／ml,回收率为87．0％,RSD＝7．9％（n＝5）,在0～50μg／ml浓度范围内,有良好的线性关系：A＝1778．9＋13686C,r＝0．999。方法同时可用于血中氯喹的分析。附一例应用报告,测得尿中氯喹的含量为0．745mg／ml,血中氯喹的含量为3．68μg／ml。尿液中同时检出氯喹的N－去单已基代谢物。定性结果经质谱法验证。     

Nicotine and cotinine levels in body fluids of smokers who committed suicide

Cigarette smoking is associated with a higher risk for suicide. The present study was conducted on the hypothesis that suicide smokers show higher nicotine and cotinine levels in blood and urine than non-suicide smokers. We determined nicotine and cotinine levels in blood and urine of 87 deceased individuals (18 suicides and 69 non-suicides) by gas chromatography. The smoking rate was 77.8% for individuals who committed suicide and 42.0% for those who did not commit suicide. Average nicotine and cotinine levels in blood were significantly higher in the suicide smokers than in the non-suicide smokers (nicotine: 93.2+/-46.6 ng/ml versus 25.8+/-14.4 ng/ml, p<0.0001 and cotinine: 378+/-235 ng/ml versus 201+/-137 ng/ml, p<0.005). Average levels of urinary nicotine and cotinine were also significantly higher in the suicide smokers than in the non-suicide smokers (nicotine: 1980+/-2210 ng/ml versus 394+/-376 ng/ml, p<0.005 and cotinine: 1170+/-1330 ng/ml versus 414+/-290 ng/ml, p<0.05). Twenty-six decedents were intoxicated with alcohol, and they included 7 suicides (7 smokers) and 19 non-suicides (15 smokers). Our data suggest that cigarette smokers who commit suicide smoke more heavily than other cigarette smokers.     

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.     

The detection of isoniazide in the cadaveric blood and plasma by high performance liquid chromatography with a diode matrix detector

The authors describe the analytical procedure for the rapid detection of isoniazide in the cadaveric blood and plasma using high performance liquid chromatography with a diode matrix detector (HPLC-DAD). The method is designed for toxicological and forensic medical analyses. The preliminary preparation of the samples includes protein precipitation with trichloroacetic acid, formation of isoniazide derivatives with cinnamic aldehyde, and chromatographic analysis on an Eclipse XDB-C18 column with registration of absorption at a wavelength of 340 nm. The mobile phase is a mixture of methanol and 0.05 M ammonium acetate buffer (50:50 v/v), pH 3.8. The detection limit is 0.5 mcg/ml in plasma and 1 mcg/ml in blood hemolizate. The qualitative determination is possible in a concentration range from 2 to 200 mcg/ml. Mean square deviation does not exceed 12% in a concentration interval from 2 to 50 mcg/ml.     

A fentanyl fatality involving midazolam

A case is presented of a 35-year-old black African male anesthesiology resident, found dead in his apartment. At the scene a syringe, butterfly intravenous line and a bottle of Versed (Midazolam) were recovered. A comprehensive screen for common drugs of abuse and therapeutic agents failed to detect any drugs in blood and urine. The blood ethanol concentration was 0.06 g/dl. A GC/MS SIM assay for midazolam was developed. A sub-therapeutic midazolam blood concentration of 7.5 ng/ml was detected and concentrations (ng/ml or ng/g) in bile, urine, and liver were 3.3, 7.5, and 96, respectively. The syringe fluid was then analyzed and found to contain only fentanyl, midazolam was absent. The blood fentanyl concentration was 4.9 ng/ml which is consistent with those reported in fentanyl fatalities. Fentanyl concentrations (ng/ml or ng/g) in bile, urine, and liver were 8.8, 5.0, and 5.9, respectively. The cause of death was ruled to be fentanyl intoxication and the manner of death undetermined.     

用气相色谱法/电子捕获检测器测定尿液中的三唑仑

建立了用气相色谱／电子捕获检测器测定尿液中三唑仑含量的方法。2ml尿样在破性条件下用2ml×2氯仿提取净化后,60℃水浴下用空气吹干,残留物用环己烷定容溶解后,进气相色谱仪分析,三唑仑的保留时间为10．74min。最低检测限为0．5ng／ml,回收率为95．98％,变异系数为7．85％（n＝5）。在2～50ng／ml浓度范围内有良好的线性关系：A＝－67．9＋570．IC,r＝0．9939。     

血尿肝中毒鼠强硅藻土提取GC／NPD检测法

目的建立硅藻土提取气相色谱测定血、尿、肝中毒鼠强的方法。方法原尿液、血液用水稀释、肝匀浆用6％高氯酸沉淀蛋白的上清液倒入硅藻土小柱中，血和尿用苯洗脱，肝用三氯甲烷洗脱，挥干洗脱液，用甲醇定容至0．1ml。结果血提取率98．4％，尿提取率95．6%，肝提取率98．1％。相对标准偏差低于3．2％，检出限低于20ng／ml（g）。结论该法简便、快速，提取率高，适合作为常规毒物分析方法。     

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。     

Metal and metalloid multi-elementary ICP-MS validation in whole blood, plasma, urine and hair. Reference values

Four multi-elementary metal and metalloid quantification methods using inductively coupled plasma mass spectrometry (ICP-MS) were developed and validated in human whole blood, plasma, urine and hair by means of a single preparation procedure for each sample. The ICP-MS measurements were performed using a Thermo Elemental X7CCT series and PlasmaLab software without a dynamic reaction cell. With this procedure 27-32 elements can be simultaneously quantified in biological matrices: Li, Be, B, Al, V, Cr, Mn, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Mo, Pd, Ag, Cd, Sn, Sb, Te, Ba, W, Pt, Hg, Tl, Pb, Bi, U. Whole blood, plasma and urine samples (0.4 ml each) were diluted with purified water, acid, triton X100 and butanol. Rhodium was used as internal standard. The urine sample results were corrected for enzymatic creatinine determination. Twenty-five milligrams hair samples were acid mineralized after a decontamination procedure and diluted as previously described for biological fluids. To be validated, each element had to show linearity with a correlation coefficient higher than 0.99. The intra-assay and inter-assay inaccuracy, measured as the variation coefficient, were below 5 and 10% respectively. Global performance was assessed by a quality control program. Our laboratory is a registered participant of the Institut National de Santé Publique du Québec (Sainte-Foy, Canada) inter-laboratory comparison program for whole blood, urine, and beard hair of non-occupationally exposed individuals spiked with selected elements. In our study multi-element metal and metalloid analysis was assessed for 27 elements in whole blood, 27 elements in plasma, 30 elements in urine and 32 elements in hair, from 0 to 25, or 250 to 1000 ng/ml, depending on the element. Quantification limits ranged from 0.002 ng/ml (U) to 8.1 ng/ml (Al) for whole blood, from 0.002 ng/ml (U) to 7.7 ng/ml (Al) for plasma, from 0.001 ng/ml (U) to 2.2 ng/ml (Se) for urine, and from 0.2 pg/mg (Tl) to 0.5 ng/mg (B) for hair. Normal values were determined in whole blood (n=100), plasma (n=100), urine (n=100), and hair (n=45) of healthy volunteers, leading to approximately 10,000 analyses. All results are presented and discussed. Clinical toxicology and forensic toxicology applications are also reported. ICP-MS has made significant advances in the field of clinical biology, particularly in toxicological analysis. This is due to the use of extremely effective equipment that permits better clinical and forensic toxicological analysis of metal and metalloid status of each individual patient.     

Enzyme immunochemical detection of the use of hashish and confirmation by thin-layer chromatography

The EMIT cannabinoid assay was used for screening blood and urine after smoking tetrahydrocannabinol (THC; 10 mg) or ingestion of THC (30 mg). Cannabinoid levels in urine remain detectable up to 1 week. Confirmation was done by adsorption of the THC carboxylic acid onto a C18 extraction column and elution with acetone and TLC. The method is simple and sensitive and is applicable with common laboratory equipment. The detection limit is 10 ng/ml, using 10 ml urine.     

度冷丁滥用者尿中原体及其代谢产物的分析研究

本研究利用ＧＣ／ＭＳ（ＥＩ．ＰＣＩ）技术，在度冷丁滥用者尿中确认了６种代谢产物。并建立了ＧＣ／ＦＩＤ分析度冷丁及其代谢产物的方法。方法线性范围为０．１～２０μｇ／ｍｌ，变异系数小于１０％，最小检出量为５０ｎｇ／ｍｌ，适用于度冷丁成瘾者尿中含量的测定。     

The development and evaluation of a 125I radioimmunoassay for the measurement of LSD in body fluids

A radioimmunoassay (RIA) has been developed for the direct detection of LSD in biological fluids. The radiotracer, (+)-2-[125I]iodo-LSD, allows the use of gamma-counting rather than the liquid scintillation counting currently used for existing 3H radioimmunoassays. The assay is specific for LSD and very closely related compounds. It is inexpensive, sensitive, simple to use and small volumes of samples (50 microliter) can be assayed directly without the need for any time-consuming extraction procedures. The cut-off levels are 1.2 ng/ml in blood and 3.0 ng/ml in urine. The results obtained using the 125I assay described in this work compare very favourably with those obtained using the 3H assay currently used by Home Office Forensic Science Laboratories. The advantages of the assay make it a most appropriate method for the routine screening of LSD in biological samples of forensic interest.     

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.     

顶空固相微萃取与气质联用快速检测尿液中氯胺酮及去甲基氯胺酮

目的建立利用顶空固相微萃取（HS／SPME）结合气相色谱／质谱联用技术（GC／MS）快速检测吸毒人员尿液中氯胺酮（KT）及其主要代谢物去甲基氯胺酮（NK）的方法。方法样品瓶中加入尿样、6mol／L氢氧化钠溶液、固体氯化钠、SKF525A（内标），85℃下加热搅拌，用100μm聚二甲基硅氧烷（PDMS）萃取头顶空萃取10min，GC／MS（EI-SIM）检测。结果尿液中NK和KT浓度在0．1～2．0μg／ml范围内呈现线性关系，相关系数分别为（r^2）0．9991和0．9945，检测限（D／N=3）分别为0．87ng／ml和2．76ng／ml，定量限（S／N=10）分别为2．90ng／ml和18．52ng／ml。1ml尿液加标600ng，NK回收率在85．5％～110．1％，RSD〈13．2％（n=6）；KT回收率在77．5％～109．6％，RSD〈511．99％（n=6）。结论建立的方法简单、快速、灵敏、准确，适合尿液等生物检材中NK及KT的快速定性定量分析。     

A rapid method for the extraction of cocaine and benzoylecgonine from body fluids

A method has been developed for the rapid extraction of cocaine and benzoylecgonine from post-mortem blood and urine samples. Solid phase columns containing C8 packing material gave clean, emulsion free extracts from body fluids. The total time for each extraction was approx. 10 min and the system has the capacity for the simultaneous extraction of up to 10 samples. Urine can be extracted directly, blood samples were sonicated before extraction to allow for their easy passage through the columns. The method gave excellent recoveries of cocaine (98-100%) from spiked samples at concentrations of between 50 ng/ml and 10 micrograms/ml. Analysis of the extracts was by high performance liquid chromatography (HPLC).     

QuEChERS-LC/MS快速检测兽用麻药“舒泰”

目的本文对兽药"舒泰"中有效成分进行了结构确证,并建立了生物检材中替来他明和唑拉西泮的快速检验方法。方法在血液和尿液的生物检材中,通过加标实验,经QuEChERS萃取后,进行LC/MS对替来他明和唑拉西泮的定性定量检测分析。结果在血液和尿液的生物样品的加标实验中,替来他明的RSD%在0.5%~3.5%,唑拉西泮的RSD在0.5%~1.1%;替来他明的回收率在75.8%~100.3%,唑拉西泮的回收率在68.8%~76.6%,其中血液中替来他明的方法检出限为0.16ng/mL,尿液中为0.20ng/mL,唑拉西泮在血液中的方法检出限0.17ng/mL,尿液中为0.22ng/mL。结论建立的QuEChERS萃取方法,操作流程简便,方法重现性好,只需100μL取样量,更适合于痕量生物检材中替来他明和唑拉西泮的检验分析。     

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.     

LC-MS/MS analysis of pholedrine in a fatal intoxication case

Pholedrine (4'-hydroxymethamphetamine) is a cardiovascular agent exerting hypertensive and adrenergic effects. High doses may cause a drop in the peripheral circulation blood flow and increase blood pressure, heart rate and body temperature up to a state of central respiratory paralysis. A 15-year-old girl who suffered from heavy agitation and hallucinations was admitted to the intensive care unit in a comatose state. The clinical findings included a maximum heart rate of 170 bpm and a body temperature of 43.8 degrees C. Resuscitation measures were in vain and abandoned after approximately 2h. A toxicological emergency analysis using GC/MS revealed a considerable amount of pholedrine in blood and urine. A method for determining pholedrine in human body fluids utilizing high-performance liquid chromatography (HPLC)/tandem mass spectrometry (LC-MS/MS) with a turbo ion-spray source was developed, using D11-methamphetamine and D5-methylenedioxymethamphetamine as internal standards. Samples were prepared by SPE extraction using SPEC-C18AR/MP3((R)) columns, which yielded the best extraction recovery (67%). Chromatographic separation was achieved at pH 5 on an RP-18 stationary phase applying gradient elution from 50 to 70% of B (methanol/acetonitrile 3/1 (v/v), 0.02% acetic acid) in A (5mM ammonium acetate/acetonitrile 95/5 (v/v), 0.02% acetic acid). Supra-pure acetic acid was added to the post-column effluent with a flow rate of 0.2 microl/min to optimize ionization. Detection was carried out in the positive ionization, multiple reaction monitoring (MRM) mode. The chromatograms showed no interference from other substances. The limit of detection (LOD, S/N=3) of pholedrine was 0.8 ng/ml and its lower limit of quantification (LLOQ, S/N=10) 3ng/ml. The calibration curve was linear (r=0.999) in the range 1-100 ng/ml. Samples with higher concentrations were diluted to suit the working range. The intra-day R.S.D. between 5 and 80 ng/ml were 3.8-8.7% and the inter-day R.S.D. between 5 and 100 ng/ml were 6.7-10.7%. The pholedrine concentrations in blood and urine collected when the girl was still alive were 16.1 microg/ml (R.S.D. 10.5%) and 1120 microg/ml (R.S.D. 8%), respectively. In post-mortem samples, they were 23.0 microg/ml (R.S.D. 5.1%) in heart blood and 27.3 microg/g (R.S.D. 6.6%) in the liver.