Analysis of nitrite in adulterated urine samples by capillary electrophoresis

A simple method for analyzing nitrite in urine has been developed to confirm and quantify the amount of nitrite in potentially adulterated urine samples. The method involved separation of nitrite by capillary electrophoresis and direct UV detection at 214 nm. Separation was performed using a bare fused silica capillary and a 25 mM phosphate run buffer at a pH of 7.5. Sample preparation consisted of diluting the urine samples 1:20 with run buffer and internal standard, and centrifuging for 5 min at 2500 rpm. The sample was hydrodynamically injected, then separated using -25 kV with the column maintained at 35 degrees C. The method had upper and lower limits of linearity of 1500 and 80 microg/mL nitrite, respectively, and a limit of detection of 20 microg/mL. The method was evaluated using the National Committee for Clinical Laboratory Standards (NCCLS) protocol (Document EP10-A2), and validated using controls, standards, and authentic urine samples. Ten anions, ClO-, CrO4(-2), NO3-, HCO3-, I-, CH3COO-, F-, SO4-, S2O8(-2), and Cl-, were tested for potential interference with the assay. Interferences with quantitation were noted for only CrO4(-2) and S2O8(-2). High concentrations of Cl- interfered with the chromatography. The method had acceptable accuracy, precision, and specificity.     

Molecular cloning and immunohistochemical localization of rat dipeptidyl peptidase III

Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat liver cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS, and 82000 on non-denatured PAGE and 82000 on SDS-PAGE in the absence or presence of beta-ME. These findings suggest that the enzyme assumes a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Ala-Arg-MCA in a pH range of 7. 5 to 9.5. The K(in), K(cat) and K(cat)/K(m) values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 6.21x10(4) s(-1)M(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 3.62x10(4) s(-1)M(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS, NEM, beta-ME and iodoacetamide. Furthermore, we screened a rat liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG, and we determined the cDNA structure and deduced the amino acid sequence. The cDNA designated as lambdaRDIII-11 is composed of 2640 bp of nucleotides in length and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH in structure, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. These findings suggest that DPP III is a metalloprotease that is probably regulated by SH modification. The DPP III antigen was extensively detected in the cytosol of various rat tissues by the immunohistochemical examination of the protein.     

Abuse of "pheticol" and its interference to the analysis of methamphetamine

The study was to eliminate interference from ephedrine in the analysis of methamphetamine. The extraction procedure for methamphetamine was modified to include an oxidation step (2 ml urine specimen was treated with 0.5 ml of 1 mol/L phosphate buffer (pH 6.8) and 0.5 ml 0.3 mol/L sodium periodate). Results showed that ephedrine could be oxidized in the presence of periodate ions into smaller fragments while leaving methamphetamine intact. It is recommended that specimens be treated with sodium periodate prior to extraction in order to eliminate any interference caused by ephedrine.     

Achiral and chiral quantification of methamphetamine and amphetamine in human urine by semi-micro column high-performance liquid chromatography and fluorescence detection

In this paper, miniaturized achiral and chiral high-performance liquid chromatographic procedures for the determination of methamphetamine and amphetamine in human urine are described. After a simple pretreatment of human urine (i.e., 10 microL of urine or diluted urine were acidified and dried-up under N2 at room temperature) and fluorescence derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)-benzoyl chloride under mild conditions (pH 9.0, 10 min at room temperature), the derivatives were isocratically separated on a semi-micro ODS column with Tris-HCl buffer (0.1 M, pH 7.0): acetonitrile (45 + 55 v/v) at a flow rate of 0.2 mL/min or their enantiomers were separated on a semi-micro OD-RH column with sodium hexafluorophosphate (0.3 M aq.): acetonitrile (44 + 56 v/v) at a flow rate of 0.1 mL/min as the mobile phase. Wide-ranged calibration curves were obtained with detection limits for the achiral and chiral analyses in the atto and femtomol levels, respectively, per injected volume. Satisfactory within- and between-day reproducibility data were obtained with both the methods with the highest relative standard deviation being 9.6%. The methods were applied to the determination of methamphetamine and amphetamine in human urine samples and the concentrations determined by the two methods were well correlated (r = 0.994).     

Identification of alprazolam in hair in two cases of drug-facilitated incidents

The use of a drug to modify a person's behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault, robbery) has caused alarm by the general public. Among the drugs that can be used, alprazolam (Xanax), an anxiolytic benzodiazepine, has been seldom observed. To document two cases involving this drug, we have developed an approach based on hair testing by LC-MS/MS. After pH 8.4 buffer incubation and extraction with methylene chloride/diethyl ether (80/20, v/v), hair extracts were separated on a XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Alprazolam and diazepam-d5, used as internal standard, were detected by electrospray tandem mass spectrometry. In the first criminal case, alprazolam tested positive in two consecutive 2 cm hair segments at 4.9 and 2.4 pg/mg, from a 12-year-old girl, assaulted by her father who had sedated her three or four times. In the other case, alprazolam was detected in four consecutive 1cm hair segments at 3.1-0.4 pg/mg, obtained from an adolescent who had been forced to prostitute herself.     

Colchicine poisoning: case report of two suicides

Colchicine overdose is uncommon but potentially life threatening because of the high toxicity of the drug. Poisoning by colchicine may occur following ingestion of medication used in acute attacks of gout and inflammatory diseases. We describe two cases involving suicide by the ingestion of medications marketed in France. In case 1, only heart blood was taken after body external examination. In case 2 an autopsy was performed and heart blood, urine, gastric contents and bile were taken for toxicological analysis. Colchicine was assayed in biological specimens by an HPLC-DAD method, after extraction by dichloromethane at pH 8, adding prazepam as internal standard (IS). Analyses were performed on a Symetry C-8 column. Mobile phase was a gradient of acetonitrile/pH 3.8 phosphate buffer. Colchicine is eluted at 13.1 min and the method is linear for blood, urine and bile over the range 4-1000 ng/mL. LOQ is 4 ng/mL. The concentrations of colchicine detected are: case 1: heart blood 13 ng/mL; case 2: heart blood 66 ng/mL, urine 500 ng/mL, gastric content 12 ng/mL, bile 5632 ng/mL. Our findings are in the range of lethal concentrations previously described, but there is no correlation with the amount of ingested drug. Even after massive overdose, it could be impossible to detect colchicine in blood, and as there is a widespread enterohepatic recirculation before excretion in bile and feces, bile is the target sample to analyse. We conclude in both cases that the cause of death was suicide with colchicine. It appears very important to perform an autopsy in order to obtain bile, urine, heart blood and femoral blood.     

Identification of lorazepam and sildenafil as examples for the application of LC/ionspray-MS and MS-MS with mass spectra library searching in forensic toxicology

A mass spectra (MS) library using in-source collision induced dissociation (ESI-CID) as well as a tandem-mass spectra (MS-MS) library with product ion spectra of drugs has recently been developed with a triple-quadrupole ionspray mass spectrometer [1,2]. For the ESI-CID MS library, single-quadrupole mode and for the MS-MS library triple-quadrupole mode have been used. These mass spectra libraries were applied successfully for the general-unknown screening for drugs and metabolites in serum and urine with liquid-chromatography-mass spectrometry (LC-MS) using a PE/SCIEX API 365 with a turboionspray source. As examples, the identification of lorazepam and lorazepam-glucuronide in a serum extract and the identification of sildenafil and alkyloxidated sildenafil in urine are presented here.     

A single-column procedure on Bond Elut Certify for systematic toxicological analysis of drugs in plasma and urine.

A single-column solid-phase extraction procedure was developed for the screening of acidic, neutral, and basic drugs from plasma. The recoveries of all 25 tested drugs exceeded 82%. After the plasma had been diluted with phosphate buffer (pH 6.0), the drugs were extracted using a single Bond Elut Certify column. The acidic and most of the neutral drugs were eluted by acetone/chloroform (1:1) and the basic drugs were eluted by 2% ammoniated ethyl acetate. Some neutral drugs appeared in both fractions. The two fractions were collected separately and evaporated until approximately 100 microL of solvent remained in the tube. Both fractions were analyzed separately on a gas chromatograph equipped with a wide-bore capillary column and a flame ionization detector. The procedure could also be used for urine samples.     

Development and validation of the Cozart DDS oral fluid collection device

The testing of oral fluid for drugs of abuse has increased significantly over recent years and is now commonplace in drug rehabilitation clinics, the workplace, prisons and custody suites. The global problem of identifying drugged drivers has also led to an increase in oral fluid testing at the roadside. The main requirements for the implementation of roadside drug testing are a rapid sample collection time, collection of a known sample volume and recovery of drugs from the collection device. We report here the development of the Cozart^® DDS oral fluid collector, an oral fluid collector that combines rapid and adequate sample collection with satisfactory drug recovery. Oral fluid was collected from drug users (n = 134) and drug-free individuals (n = 137), using the Cozart^® DDS oral fluid collector. The mean time for the completion of collection (full coloration of the sample presence indicator) was 34 s for drug-free individuals and 44 s for drug users. The average volume collected was 0.34 mL (n = 271). No chemical stimulant (to induce salivation) was used to achieve the collection times observed in either the drug-free or the drug-taking sample populations. Drugs were extracted from the collector using the Cozart^® DDS buffer and drug recovery was determined by Cozart^® enzyme immunoassays. The recovery studies showed that for amphetamine, Δ⁹THC, cocaine, methadone, methamphetamine, morphine and temazepam over 90% of the drug in the sample was eluted from the collector. The Cozart^® DDS oral fluid collector provides a reliable mechanism for the collection of oral fluid at the roadside that achieves the rapid collection times required.     

Hair analysis for diphenhydramine after surreptitious administration to a child

Diphenhydramine is one of the first effective antihistamine agents to have been discovered. The compound is also used for its sedative and antiemetic effects. The first case involving repetitive sedation linked to the use of diphenhydramine as a drug-facilitated crime and subsequent impairment of a 9-year-old female victim is reported. Due to the long delay between the alleged crime and clinical examination, collection of blood or urine was of little value. Hence, the laboratory developed an original approach based on hair testing by LC-MS/MS. A single strand of hair from the victim was sampled about 7 weeks after the last suspected administration and was cut into small segments. After cutting into small pieces, about 20 mg of hair per segment was incubated overnight in a phosphate buffer (pH 8.4). The aqueous phase was extracted with 5 ml of a mixture of methylene chloride/diethyl ether (80/20), in presence of diazepam-d5, used as internal standard (IS). The hair extract was separated on an XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 256.2-152.1 and 167.1 and m/z 289.9-154.0 for diphenhydramine and the IS, respectively. In the hair of the child, diphenhydramine was detected at concentrations in the range 33-39 pg/mg, depending on the segment.     

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.     

GC-MS分析尿液中甲卡西酮

目的建立尿液中甲卡西酮的气相色谱-质谱(gas chromatography-mass spectrometry,GC-MS)分析方法。方法在尿液中加入内标双苯戊二氨酯(SKF525A)和pH=9的缓冲溶液,用乙酸乙酯提取,提取液在50℃氮气流下挥干,残余物用甲醇溶解,用GC-MS分析。结果尿液中甲卡西酮在0.02~2.00μg/m L质量浓度范围内线性关系良好,线性方程为y=0.301 9 x+0.018 9(r=0.999 2),检出限为0.01μg/m L。尿液中甲卡西酮回收率为96.4%~99.2%,日内精密度为5.8%~7.6%,日间精密度为6.0%~8.1%。结论该方法操作简便、灵敏度高,可用于司法鉴定实践尿液样品中甲卡西酮的分析。     

苯并二氮类药物及其主要代谢物的薄层色谱分析

目的建立饮料、血、尿中 11种常见苯并二氮类药物及尿中 7种主要代谢物的薄层色谱分析法(HPTLC)。方法分析物采用GDX10 1树脂进行固相萃取 ,乙醚作为洗脱溶剂。苯 :丙酮 (10∶6)等作为展开体系 ,改良碘化铋钾显色。结果所建方法绝对灵敏度 0 3～ 0 6μg,尿检出限 0 4～ 1 0 μg/ml、血检出限 0 6～ 1 0 μg/ml、饮料检出限 0 4～ 0 8μg/ml。结论HPTLC法简便、快速 ,适合作为常规毒物分析方法     

Benzodiazepine findings in blood and urine by gas chromatography and immunoassay

Gas chromatography (GC) and immunoassay techniques applied to blood and urine specimens were compared for the screening of benzodiazepines in postmortem forensic toxicology. Five hundred and six such successive postmortem cases in which both urine and peripheral blood was sent for toxicological analysis by the medical examiners were selected. The urine specimens were tested by the Emit((R)) d.a.u. Benzodiazepine Assay, and in parallel, the blood and urine specimens were screened for benzodiazepine drugs and their metabolites by an established automated dual-column GC method. The lowest number of positives (153) was obtained when immunoassay was performed without enzyme hydrolysis. When urine samples were hydrolysed before immunoassay, the number of positives increased to 175. The highest number of positives (200) was obtained in urine by GC, and the screening of blood by GC yielded 185 quantitative results. Despite the urine GC screening produced the most positives, the quantitative screening of the blood by GC appears to be the most efficient approach in postmortem forensic toxicology, considering the fact that although urine findings confirm the presence of the drug, quantitative results in urine are irrelevant to acute toxicity.     

Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS. Application to the determination of MDMA after low ecstasy intake

A rapid and sensitive method using LC-MS/MS triple stage quadrupole for the determination of traces of amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"), 3,4-methylenedioxyethamphetamine (MDEA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair, blood and urine has been developed and validated. Chromatography was carried out on an Uptisphere ODB C(18) 5 microm, 2.1 mm x 150 mm column (Interchim, France) with a gradient of acetonitrile and formate 2 mM pH 3.0 buffer. Urine and blood were extracted with Toxitube A (Varian, France). Segmented scalp hair was treated by incubation 15 min at 80 degrees C in NaOH 1M before liquid-liquid extraction with hexane/ethyl acetate (2/1, v/v). The limits of quantification (LOQ) in blood and urine were at 0.1 ng/mL for all analytes. In hair, LOQ was <5 pg/mg for MA, MDMA, MDEA and MBDB, at 14.7 pg/mg for AP and 15.7 pg/mg for MDA. Calibration curves were linear in the range 0.1-50 ng/mL in blood and urine; in the range 5-500 pg/mg for MA, MDMA, MDEA and MBDB, and 20-500 pg/mg for AP and MDA. Inter-day precisions were <13% for all analytes in all matrices. Accuracy was <20% in blood and urine at 1 and 50 ng/mL and <10% in hair at 20 and 250 pg/mg. This method was applied to the determination of MDMA in a forensic case of single administration of ecstasy to a 16-year-old female without her knowledge during a party. She suffered from hyperactivity, sweating and agitation. A first sample of urine was collected a few hours after (T+12h) and tested positive to amphetamines by immunoassay by a clinical laboratory. Blood and urine were sampled for forensic purposes at day 8 (D+8) and scalp hair at day 60 (D+60). No MDMA was detected in blood, but urine and hair were tested positive, respectively at 0.42 ng/mL and at 22 pg/mg in hair only in the segment corresponding to the period of the offence, while no MDA was detectable. This method allows the detection of MDMA up to 8 days in urine after single intake.     

Determination of methadone in human hair by radioimmunoassay

The concentrations of methadone in human hair were measured. The washed hair was cut in 1-mm pieces approximately, then incubated overnight at 45 degrees C with 0.1 m HCl. The extracts were alcalized by 1 m NaOH and diluted by phosphate buffer, pH 7.4. The methadone concentrations were determined by radioimmunoassay. The method is simple, rapid, and practicable for routine determination.     

Identification of human skin from a tissue fragment by detection of squamous cell carcinoma-related antigen using an enzyme immunoassay

A new method of identifying human skin from a tissue fragment by detection of squamous cell carcinoma-related (SCC) antigen, using an enzyme immunoassay, was developed. When an extract was prepared from 0.1 g human skin homogenized with 1 ml of phosphate buffered saline, this method was able to detect SCC antigen in extracts diluted 10²-fold. There was no difference in the detection limit between individuals. Species specificity was good, and there was no cross reaction observed with skins from animals. Our method could also discriminate between skin and other organs or tissues, except for esophagus and lung. A practical case to which this method was applied is presented.     

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.     

Prevalence and blood concentrations of benzodiazepines and opioids in opioid-positive death investigations in Ontario,Canada, from 2017 to 2021

The aim of this study was to investigate the incidence of benzodiazepines in opioid-positive death investigations, including trends in frequency and combination of drugs, as well as demographic data and blood concentrations, where available. Additionally, naloxone concentrations in polysubstance compared to opioid-only cases were analyzed. This was a retrospective study that consisted of all post-mortem toxicology cases in Ontario, Canada, from January 01, 2017, to December 31, 2021, with an opioid finding in any analyzed autopsy specimen. There were 11,033 death investigations identified. The overall rate of benzodiazepine co-involvement was 54.5%. Males accounted for the majority of cases (71%), and the most affected age group was 30- to 39-year-olds. The most frequently detected opioid was fentanyl and the most frequently detected benzodiazepine was etizolam, which was also the most frequently observed opioid/benzodiazepine combination. Findings related to differences in concentrations of opioids when naloxone was also present were mostly non-significant, except for methadone. The rate of benzodiazepine detection with opioids grew faster than opioid detections overall, potentially due to the increasingly toxic drug supply. Detection of novel psychoactive drugs fluctuated more unpredictably than opioids and benzodiazepines associated with clinical use. These findings can help inform policy decisions by public health agencies in exploring harm reduction efforts, for example, education and drug-checking services.     

Roadside detection of impairment under the influence of ketamine--evaluation of ketamine impairment symptoms with reference to its concentration in oral fluid and urine

Although there are many roadside testing devices available for the screening of abused drugs, none of them can be used for the detection of ketamine, a popular abused drug in Hong Kong. In connection to local drug driving legislation, effective roadside detection of ketamine in suspected drug-impaired drivers has to be established. According to the drug evaluation and classification program (DEC), ketamine is classified in the phencyclidine (PCP) category. However, no study has been performed regarding the signs and symptoms exhibited by users under the influence of ketamine. In a study to develop a protocol for effective roadside detection of drug-impaired drivers, 62 volunteers exiting from discos were assessed using field impairment tests (FIT) that included measurements of three vital signs (i.e. body temperature, pulse rate and blood pressure), three eye examinations [pupil size, lack of convergence (LOC) and horizontal gaze nystagmus (HGN)] and four divided attention tests (Romberg, one-leg stand, finger-to-nose and walk-and-turn tests). Subsequent laboratory analysis of oral fluid and urine samples from the participants revealed the presence of common abused drugs in both the urine and oral fluid samples of 55 subjects. The remaining 7 subjects with no drug in their oral fluid samples were used as drug-free subjects. In addition, 10 volunteers from the laboratory who were regarded as drug-free subjects were also assessed using the same FIT. Among the 62 volunteers, 39 of them were detected with ketamine in their oral fluid. Of these ketamine users, 21 of them (54%) with only ketamine found in their oral fluid samples while the rest (18 subjects) of them had other drugs (i.e. MA, MDMA, benzodiazepines and/or THC) in addition to ketamine. Of the 21 ketamine-only users, 15 of them (71%) were successfully identified by FIT. It was found that when salivary ketamine concentrations were greater than 300 ng/mL, signs of impairment became evident, with over 90% detection rate using the FIT. By comparing the FIT observations on the 21 ketamine-only users with the drug-free subjects, the typical signs and symptoms observable for subjects under the influence of ketamine included LOC, HGN, elevated pulse rate and in general, failing the divided attention tests, especially the walk-and-turn and one-leg stand.