Baohua Shen

Segmental hair analysis using liquid chromatography–tandem mass spectrometry after a single dose of benzodiazepines

In China, benzodiazepines are the most frequently observed compounds in cases of drug-facilitated crime. Sensitive, specific, and reproducible methods for the quantitative determination of 18 benzodiazepines in hair have been developed using LC-MS/MS. Fourteen volunteers had ingested a single 1-6mg estazolam tablet. Hair was collected 1 month after administration. All the proximal segments were positive for estazolam. With increased dosage, estazolam can be detected in the 2-4cm segments in some subjects hair. Even some of 4-6cm segments were positive. Hair analysis was applied to two authentic criminal cases. Full-length hair samples collected 5 weeks after the offense were cut into segments of 2cm from the root, analyzed and quantified. The clonazepam concentrations measured in the first two segments for V#1 and V#2 were 15.47 and 11.93pg/mg, respectively. However, both the 4-6cm and the 6-8cm segment of V1# remained positive, while those of V#2 were negative. It needs more substantial guidelines to use segmental hair analysis in drug-facilitated crime.

Detection of antidepressant and antipsychotic drugs in human hair

The presence of therapeutic drugs and their metabolites in the hair of psychiatric patients was investigated using gas chromatography (GC)-mass spectroscopy (MS)-electron ionization (EI) and GC-MS-chemical ionization (CI). In hair samples tested from 35 subjects, carbamazepine, amitriptyline, doxepin, trihexyphenidyl, chlorpromazine, chlorprothixene, trifluoperazine, clozapine and haloperidol were detected, with maximal concentrations of 22.5, 57.7, 183.3, 15.6, 68.2, 30.0, 36.8, 59.2 and 20.1 ng/mg of hair sample, respectively. Chlorpromazine and clozapine concentrations in the hair were found to be dependent on the dosage used and their correlation coefficients were 0.8047 (P<0.001, n=16) and 0.7097 (P<0.001, n=16), respectively. Segmental analysis demonstrated that there was a correlation between the history of subjects drug exposure and the distribution of drug along the hair shaft. Our results also show that drug analysis in hair may provide useful information about drug treatment and the history of usage, and that drugs can be detected in normally kept hair for at least 16 months after intake.

Rapid screening of drugs of abuse in human urine by high-performance liquid chromatography coupled with high resolution and high mass accuracy hybrid linear ion trap-Orbitrap mass spectrometry

A novel analytical toxicology method has been developed for the analysis of drugs of abuse in human urine by using a high resolution and high mass accuracy hybrid linear ion trap-Orbitrap mass spectrometer (LTQ-Orbitrap-MS). This method allows for the detection of different drugs of abuse, including amphetamines, cocaine, opiate alkaloids, cannabinoids, hallucinogens and their metabolites. After solid-phase extraction with Oasis HLB cartridges, spiked urine samples were analysed by HPLC/LTQ-Orbitrap-MS using an electrospray interface in positive ionisation mode, with resolving power of 30,000 full width at half maximum (FWHM). Gradient elution off of a Hypersil Gold PFP column (50mm×2.1mm) allowed to resolve 65 target compounds and 3 internal standards in a total chromatographic run time of 20min. Validation of this method consisted of confirmation of identity, selectivity, linearity, limit of detection (LOD), lowest limits of quantification (LLOQ), accuracy, precision, extraction recovery and matrix effect. The regression coefficients (r(2)) for the calibration curves (LLOQ - 100ng/mL) in the study were ≥0.99. The LODs for 65 validated compounds were better than 5ng/ml except for 4 compounds. The relative standard deviation (RSD), which was used to estimate repeatability at three concentrations, was always less than 15%. The recovery of extraction and matrix effects were above 50 and 70%, respectively. Mass accuracy was always better than 2ppm, corresponding to a maximum mass error of 0.8 millimass units (mmu). The accurate masses of characteristic fragments were obtained by collisional experiments for a more reliable identification of the analytes. Automated data analysis and reporting were performed using ToxID software with an exact mass database. This procedure was then successfully applied to analyse drugs of abuse in a real urine sample from subject who was assumed to be drug addict.

Physiological concentrations of anabolic steroids in human hair.

Doping with endogenous anabolic steroids is one of the most serious issues in sports today. The measurement of anabolic steroid levels in human hair is necessary in order to distinguish between pharmaceutical steroids and natural steroids. This is the first investigation into the physiological concentrations of anabolic steroids in human hair in Chinese subjects. A gas chromatography-tandem mass spectrometry (GC/MS/MS) method was developed for the simultaneous identification and quantitation of five endogenous anabolic steroids (testosterone, epitestosterone, androsterone, etiocholanolone and dehydroepiandrosterone) in hair. After basic hydrolysis, hair samples were extracted with diethyl ether, derivatized and then detected using GC/MS/MS in the multiple-reaction monitoring mode (MRM). The one precursor/two product ion transitions for each anabolic steroid were monitored. The limits of detection for the five endogenous anabolic steroids were in the 0.1-0.2 pg/mg range. All analytes showed good linearity and the extraction recoveries were 74.6-104.5%. Within-day and between-day precisions were less than 20%. This method was applied to the analysis of testosterone, epitestosterone, androsterone, etiocholanolone, and dehydroepiandrosterone in human hair. Full-length hair samples were taken at the skin surface from the vertex of 39 males, 30 females and 11 children from China. None of the subjects were professional athletes. Testosterone and dehydroepiandrosterone were detected in all the hair segments. The physiological concentrations of testosterone were in the range 0.8-24.2 pg/mg, 0.1-16.8 pg/mg and 0.2-11.5 pg/mg in males, females and children, respectively, however, the mean values of dehydroepiandrosterone were much higher than the concentrations of testosterone. These data are suitable reference values and are the basis for the interpretation of results from investigations into the abuse of endogenous anabolic steroids.

Determination of ethyl glucuronide in hair samples of Chinese people by protein precipitation (PPT) and large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC/MS/MS)

Ethyl glucuronide (EtG) has been shown to be a suitable marker of excessive alcohol consumption. Determination of EtG in hair samples may help to differentiate social drinkers from alcoholics, and this testing can be widely used in forensic science, treatment programs, workplaces, military bases as well as driving ability test to provide legal proof of drinking. A method for determination of EtG in hair samples using large volume injection-gas chromatography-tandem mass spectrometry (LVI-GC/MS/MS) was developed and validated. Hair samples (in 1 mL deionized water) were ultrasonicated for 1h and incubated overnight; these samples were then deproteinated to remove impurities and derivatisated with 15 μL of pyridine and 30 μL of BSTFA. EtG was detected using GC/MS/MS in multiple-reaction monitoring mode. This method exhibited good linearity: y=0.0036 x+0.0437, R²=0.9993, the limit of detection and the limit of quantification were 5 pg/mg and 10 pg/mg, respectively. The extraction recoveries were more than 60%, and the inter-day and intra-day relative standard deviations (RSD) were less than 15%. This method has been applied to the analysis of EtG in hair samples from 21 Chinese subjects. The results for samples obtained from all of those who were teetotallers were negative, and the results for the other 15 samples ranged from 10 to 78 pg/mg, except for one negative sample. These data are the basis for interpretation of alcohol abuse.

Disappearance of 6-acetylmorphine, morphine and codeine from human scalp hair after discontinuation of opiate abuse

Opiates continue to be used at high rates in East and Southeast Asia. Hair analysis for drugs of abuse has been developed into a powerful and widely used tool in forensic and clinical toxicology. Specifically, testing the proximal segment of scalp hair to confirm morphine (MOR) positive urine samples could solve the poppy seed problem. Human scalp hair grows approximately 1cm per month and can therefore reflect a retrospective timeline of drug exposure. This study is the first to investigate the disappearance of 6-acetylmorphine (6-AM), MOR and codeine (COD) from human scalp hair after the discontinuation of drug use. Thirty-two healthy women (ages 21-51 years) with a known history of heroin abuse, who went to a rehabilitation centre and ceased consuming heroin (for 4-5 months), were recruited into the study. A pharmacokinetic analysis in seven individual hair segments was performed using a first-order kinetic. Assuming a rate of hair growth of 1cm/month, the mean hair elimination half-lives of 6-AM, MOR and COD were 0.88 months (95% CI, 0.74-1.03), 0.73 months (95% CI, 0.64-0.81), and 0.61 months (95% CI, 0.54-0.69), respectively. Our results suggest that to evaluate the discontinuation of opiate abuse after a 6-month period of abstinence, the results from a 3-cm proximal hair segment should be free of 6-AM at the proposed 0.2 ng/mg cutoff level. This finding should become the basis for the interpretation of results from segmental hair analyses in the evaluation of drug abstinence.

Chiral separation and determination of R/S-methamphetamine and its metabolite R/S-amphetamine in urine using LC-MS/MS.

Methamphetamine (MA) and amphetamine (AM) are widely abused drugs. Differentiation of MA and/or AM abuse from therapeutic ingestion of MA and/or AM or one of their precursor drugs is therefore of relevance in clinical and forensic toxicology. The aim of the study was to develop a simple, rapid, and accurate method for the chiral separation and determination of R/S-MA and R/S-AM in urine using liquid chromatography electrospray ionization tandem mass spectrometry operating in the positive ion multiple-reaction monitoring (MRM) mode. 20 μL of urine was diluted 500 times and 20 μL was injected. The chromatographic system consisted of a Chirobiotic™ V2 column (2.1 mm × 250 mm, 5 μm), and the mobile phase was methanol containing 0.1% (v/v) glacial acetic acid and 0.02% (v/v) ammonium hydroxide. The method was fully validated through assessments of its linearity (0.05-50.00 mg/L, r(2)>0.994 for all analytes), and LOQ (0.05 mg/L for all analytes). No matrix effect was observed. The method was successfully applied to 86 urine samples from suspected MA abusers. Only the S-isomers of MA and AM were detected in 72 samples. The concentrations of R-MA ranged from below the LOQ to 13.76 mg/L in 14 urine samples with both enantiomers of MA and/or AM. Pure S-MA is the most common found analyte in urine and principally used by abusers.

Analysis of anabolic steroids in hair: time courses in guinea pigs.

Sensitive, specific, and reproducible methods for the quantitative determination of eight anabolic steroids in guinea pig hair have been developed using LC/MS/MS and GC/MS/MS. Methyltestosterone, stanozolol, methandienone, nandrolone, trenbolone, boldenone, methenolone and DHEA were administered intraperitoneally in guinea pigs. After the first injection, black hair segments were collected on shaved areas of skin. The analysis of these segments revealed the distribution of anabolic steroids in the guinea pig hair. The major components in hair are the parent anabolic steroids. The time courses of the concentrations of the steroids in hair (except methenolone, which does not deposit in hair) demonstrated that the peak concentrations were reached on days 2-4, except stanozolol, which peaked on day 10 after administration. The concentrations in hair appeared to be related to the physicochemical properties of the drug compound and to the dosage. These studies on the distribution of drugs in the hair shaft and on the time course of their concentration changes provide information relevant to the optimal time and method of collecting hair samples. Such studies also provide basic data that will be useful in the application of hair analysis in the control of doping and in the interpretation of results.

The prevalence of drugs in motor vehicle accidents and traffic violations in Shanghai and neighboring cities

The objective of this study was to determine the prevalence of psychoactive drug use among motor vehicle drivers in Shanghai and its neighboring cities. We selected 10,002 drivers involved in a traffic accident or violation between 2007 and 2008 in Shanghai, Suzhou and Wuxi. We checked for the presence of psychoactive drugs from blood samples using liquid chromatography tandem mass spectrometry (LC-MS-MS). Of the 10,002 drivers, 10.5% tested positive for drugs (excluding alcohol). Cold medicines were the most frequently detected drugs including chlorpheniramine (4.78%), pseudoephedrine (2.15%) and paracetamol (1.32%). The use of multiple cold medicines was common. Illegal drugs such as methamphetamine (0.15%), ketamine (0.03%) and MDMA (0.01%) were also detected. The prevalence of psychoactive drugs among drivers involved in traffic accidents or violations in Shanghai and its neighboring cities was lower compared to previous reports in Europe. Furthermore, cannabis--which has been reported to be the most widely used psychoactive drug after alcohol--was not commonly encountered among Shanghai drivers.

A rapid and accurate UPLC/MS/MS method for the simultaneous determination of zolpidem and its main metabolites in biological fluids and its application in a forensic context

Zolpidem (ZPD) is an imidazopyridine derivative used as a new type of hypnotic and is commonly used in drug-facilitated crimes. A rapid, sensitive, and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) assay method for the simultaneous determination of zolpidem and its main metabolites zolpidem 6-carboxylic acid (ZCA) and zolpidem phenyl-4-carboxylic acid (ZPCA) in biological fluids was developed and validated. Aliquots of 0.1mL blood or urine specimens were used for the analysis, and zolpidem and its metabolites were extracted in a single step using acetonitrile (containing 0.1% formic acid) precipitation. The supernatant was then dried, and 100μL methanol was added. The separation was performed on an Acquity™ UPLC HSS T3 (100mm×2.1mm, 1.8μm) analytical column by API 4000Q ultra-performance liquid chromatography-tandem mass spectrometry. Positive ionisation tandem MS detection in the multiple reaction monitoring (MRM) mode was used. The mobile phases consisted of either acetonitrile or water containing 20mmol/L ammonium acetate and 0.1% formic acid, and the flow rate was 0.5mL/min. The chromatographic separation time was 4min, and calibration curves for human blood were generated over the range of 0.1-300ng/mL for ZPD, 0.1-500ng/mL for ZPCA and 0.1-200ng/mL for ZCA. For urine, the linear range was 0.1-600ng/mL for ZPD and ZPCA, and 0.1-300ng/mL for ZCA. The limit of detection was 0.05ng/mL and the limit of quantitation was 0.1ng/mL for ZPD, ZCA and ZPCA. The linear correlation coefficients were greater than 0.9995. Both the inter-day and intra-day precisions were less than 15%, the recoveries were in the range of 70.0-98.3%, the matrix effects were approximately 79.5-99.0%, and the process efficiency was between 60.7% and 94.4%. This method allowed for the determination of zolpidem and its metabolites in human blood and urine and may be applied to forensic toxicological analyses.