The Homocysteine and Metabolic Syndrome: A Mendelian Randomization Study.

Homocysteine (Hcy) is well known to be increased in the metabolic syndrome (MetS) incidence. However, it remains unclear whether the relationship is causal or not. Recently, Mendelian Randomization (MR) has been popularly used to assess the causal influence. In this study, we adopted MR to investigate the causal influence of Hcy on MetS in adults using three independent cohorts. We considered one-sample MR and two-sample MR. We analyzed one-sample MR in 5902 individuals (2090 MetS cases and 3812 controls) from the KARE and two-sample MR from the HEXA (676 cases and 3017 controls) and CAVAS (1052 cases and 764 controls) datasets to evaluate whether genetically increased Hcy level influences the risk of MetS. In observation studies, the odds of MetS increased with higher Hcy concentrations (odds ratio (OR) 1.17, 95%CI 1.12-1.22, p < 0.01). One-sample MR was performed using two-stage least-squares regression, with an MTHFR C677T and weighted Hcy generic risk score as an instrument. Two-sample MR was performed with five genetic variants (rs12567136, rs1801133, rs2336377, rs1624230, and rs1836883) by GWAS data as the instrumental variables. For sensitivity analysis, weighted median and MR-Egger regression were used. Using one-sample MR, we found an increased risk of MetS (OR 2.07 per 1-SD Hcy increase). Two-sample MR supported that increased Hcy was significantly associated with increased MetS risk by using the inverse variance weighted (IVW) method (beta 0.723, SE 0.119, and p < 0.001), the weighted median regression method (beta 0.734, SE 0.097, and p < 0.001), and the MR-Egger method (beta 2.073, SE 0.843, and p = 0.014) in meta-analysis. The MR-Egger slope showed no evidence of pleiotropic effects (intercept -0.097, p = 0.107). In conclusion, this study represented the MR approach and elucidates the significant relationship between Hcy and the risk of MetS in the Korean population.

Concentrations of THC, CBD, and CBN in commercial hemp seeds and hempseed oil sold in Korea.

Hemp seeds and hempseed oil are marketed on- and off-line as health foods and cosmetics and have been reported to have high nutrient contents. However, because of the various side effects of cannabinoids, especially △9-tetrahydrocannabinol (THC), many countries regulate upper limits for THC in products, which creates the need for analytical techniques capable of measuring THC, cannabidiol (CBD), and cannabinol (CBN) levels in commercial hemp seeds and hempseed oil. In the present study, hemp seed and hempseed oil extracts obtained by methanol extraction, were analyzed by gas chromatography-mass spectrometry (GC/MS). Validation of the technique used was performed using calibration curves and by determining LODs, LOQs, specificities, selectivities, and intra- and inter-day precision and accuracies. In addition, matrix effects, process efficiencies, recoveries, and sample stabilities were investigated. In hemp seeds, as determined using the fully optimized method THC concentrations ranged from 0.06 to 5.91 µg/g, CBD concentrations from 0.32 to 25.55 µg/g, and CBN concentrations from 0.01 to 1.50 µg/g; CBN/THC ratios ranged from 0.1 to 1.60, and CBD/THC ratios from 0.11 to 62.56. Furthermore, the (THC + CBN)/CBD ratio of most hemp seed samples was less than one. In hempseed oil, THC concentrations ranged from 0.3 to 19.73 µg/mL, CBD concentrations from 6.66 to 63.40 µg/mL, CBN concentrations from 0.11 to 2.31 µg/mL, CBN/THC ratios from 0.12 to 0.42, and CBD/THC ratios from 3.21 to 22.50. Furthermore, (THC + CBN)/CBD ratios in all hempseed oil samples were less than one. The optimized methanol extraction-GC/MS technique was found to be satisfactory for determining THC, CBD, and CBN concentrations in hemp seeds and hempseed oil.

Correction to: Differentiation of endogenous and exogenous γ-Hydroxybutyrate in rat and human urine by GC/C/IRMS.

The above article was published online with incorrect author names. The right spelling should be Dong-Hun Lee instead of Donghun Lee, Sanggil Choe instead of Sanggil Choi. The correct names are presented here. The original article has been corrected.

Complementary approach for accurate determination of carbon isotopic compositions in γ-hydroxybutyric acid using gas chromatography/combustion-isotope ratio mass spectrometry.

RATIONALE: γ-Hydroxybutyric acid (GHB) is a naturally endogenous neurotransmitter that is popular as a recreational drug due to its sedative, hypnotic, and euphoric effects. GHB derived from endogenous production or exogenous ingestion has been effectively discriminated by carbon isotopic compositions (δ13 C values) through gas chromatography/combustion-isotope ratio mass spectrometry (GC/C-IRMS). However, an unintended uncertainty of isotopic signatures caused by a wide range of GHB quantities remains unsolved when using only single-isotope corrections of the di-TMS derivative. METHODS: The δ13 C values of the original GHB standard were first determined by elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). The δ13 C values of silylated GHB in concentrations from 10 to 500 ppm were determined by GC/C-IRMS. With respect to the silylated reaction products, the correction of δ13 C values for the introduced carbons was calculated from a stoichiometric mass balance equation. RESULTS: The results showed a significant quantity-dependent trend in δ13 C values of introduced carbon (δ13 Cdi-TMS values) with increased GHB standard concentrations (r2 = 0.70, p <0.05). We applied a logarithmic equation to determine isotopic data in low-GHB urine specimens from five healthy female volunteers. The δ13 CGHB values in urine samples corrected with quantity-dependent δ13 Cdi-TMS values were different by an average of 2.7‰ from those corrected with single δ13 Cdi-TMS values (p <0.05). CONCLUSIONS: Our results suggest that the overall residual amount-dependent isotope fractionation should be mathematically corrected by the logarithmic function and this may improve the reliability of isotopic analysis to evaluate the origin of GHB before applying the approach to routine toxicological and forensic studies.

Differentiation of endogenous and exogenous γ-Hydroxybutyrate in rat and human urine by GC/C/IRMS.

Gamma (γ)-hydroxybutyric acid (GHB) has been reported to be an endogenous compound in the mammalian brain. It used to treat symptoms of alcohol, opioid, and drug withdrawal and cataplexy of narcolepsy. However, it is often used for criminal purposes because it is colorless, tasteless, and has short half-life. For this reason, there is a need for a method of distinguishing between endogenous and exogenous GHB administration. Therefore, urine from rat before administration of GHB and GHB urine after the single intraperitoneal injection of GHB as 30 mg/100 g were collected from Sprague-Dawley rats (7 weeks old, 10 males and females). Negative control urine, urine from individuals suspected of taking GHB, and urine from victims who were GHB-involved crime were collected. In urine samples, GHB was extracted with two-step SPE and collected fraction was derivatized and analyzed by GC/MS and GC/C/IRMS. In GC/MS and GC/C/IRMS analysis of rat urine, there was a statistically significant difference between urine from rat before administration of GHB and GHB rat urine (p < 0.05). In GC/MS analysis of human urine samples, there was no significant difference among human urine groups (negative control, suspects' urine, and victims' urine), but in GC/C/IRMS analysis of human urine samples, there was a statistically significant difference among human urine groups (p = 0.0001). Through these results, GC/C/IRMS can be more effective tool to identify endogenous and exogenous GHB in urine than GC/MS. This study can build a drug management system in forensic investigation agency and offer interpretation method to forensic science and court.

The correlation between concentrations of zolpidem and benzodiazepines in segmental hair samples and use patterns.

The aim of this study was to investigate the correlation between histories of zolpidem and benzodiazepines use and their concentrations in hair as determined by segmental hair analysis, that is, by analyzing hair samples taken 0-1, 1-2, 2-3, 3-4, 4-5, and 5-6cm etc. and 0-3cm from the scalp, and whole hair. Of the 23 hair samples examined, 18 were collected from patients in a rehabilitation program and five were from patients that had taken zolpidem only once by prescription. All 23 patients provided written informed consent after reviewing the research plan, described their zolpidem and benzodiazepines use histories accurately, and provided hair samples, which were weighed, washed, cut into lengths of <1mm, and extracted in 100% methanol for 16h (diazepam-d5 was used as an internal standard). Extracts were evaporated under reduced pressure and reconstituted with aqueous methanol (1:1 v/v). These extracts (10µL) were analyzed by Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS). The method used was validated by determining LOD, LOQ, calibration curves, intra- and inter-accuracies, precisions, matrix effects, process efficiencies, extraction efficiencies, and processed sample stabilities. Five hundred and ninety-five 1cm hair segments showed 61.59% positive probability and 86.71% negative probability of quality correlation between zolpidem and benzodiazepines use and concentrations in hair. Good qualitative correlations were observed between drug use and detection in hair. False positivity and false negativity were very low. Of the hair samples taken from patients in a rehabilitation program, subject nos. 4, 5, and 12 had correlation coefficients of 0.68, 0.54 and 0.71, respectively, for relationships between zolpidem use and concentration of zolpidem in hair. For the 5 patients taking only a single dose of zolpidem (10mg), the average zolpidem concentrations in hair were 20, 15 and 40pg/mg after 5, 30 and 60 days, respectively. This study shows a relationship between history of zolpidem and benzodiazepines use and their concentrations in 1cm hair segment.

Tentative identification of in vitro metabolites of 5-APDB, a synthetic benzofuran, by LC-Q/TOF-MS.

5-(2-Aminopropyl)-2,3-dihydrobenzofuran (5-APDB) is a designer drug of phenethylamine and amphetamine class. In this study, the in vitro metabolism of 5-APDB was investigated in rat and human liver microsomes and human hepatocytes to characterize its metabolites. 5-APDB was incubated with microsomes or hepatocytes, and the reaction mixture was analyzed using liquid chromatography-quadrupole time-of-flight with tandem mass spectrometry (LC-Q/TOF-MS). 5-APDB was metabolized to yield three metabolites (M1, M2 and M3). These metabolites were structurally characterized on the basis of accurate mass analysis and MS/MS fragmentation patterns. Metabolite M1 and M2 were identified as hydroxylated metabolites in the benzofuran moiety; M3 was a reduced metabolite which may be generated from M1 or M2 via dehydration. These results provide evidence for the in vivo 5-APDB metabolism, and would be forensically useful for the detection of 5-APDB and its metabolites in biological samples.

Relationship between methamphetamine use history and segmental hair analysis findings of MA users.

The aim of this study was to investigate the relationship between methamphetamine (MA) use history and segmental hair analysis (1 and 3cm sections) and whole hair analysis results in Korean MA users in rehabilitation programs. Hair samples were collected from 26 Korean MA users. Eleven of the 26 subjects used cannabis with MA and two used cocaine, opiates, and MDMA with MA. Self-reported single dose of MA from the 26 subjects ranged from 0.03 to 0.5g/one time. Concentrations of MA and its metabolite amphetamine (AP) in hair were determined by gas chromatography mass spectrometry (GC/MS) after derivatization. The method used was well validated. Qualitative analysis from all 1cm sections (n=154) revealed a good correlation between positive or negative results for MA in hair and self-reported MA use (69.48%, n=107). In detail, MA results were positive in 66 hair specimens of MA users who reported administering MA, and MA results were negative in 41 hair specimens of MA users who denied MA administration in the corresponding month. Test results were false-negative in 10.39% (n=16) of hair specimens and false-positive in 20.13% (n=31) of hair specimens. In false positive cases, it is considered that after MA cessation it continued to be accumulated in hair still, while in false negative cases, self-reported histories showed a small amount of MA use or MA use 5-7 months previously. In terms of quantitative analysis, the concentrations of MA in 1 and 3cm long hair segments and in whole hair samples ranged from 1.03 to 184.98 (mean 22.01), 2.26 to 89.33 (mean 18.71), and 0.91 to 124.49 (mean 15.24)ng/mg, respectively. Ten subjects showed a good correlation between MA use and MA concentration in hair. Correlation coefficient (r) of 7 among 10 subjects ranged from 0.71 to 0.98 (mean 0.85). Four subjects showed a low correlation between MA use and MA concentration in hair. Correlation coefficient (r) of 4 subjects ranged from 0.36 to 0.55. Eleven subjects showed a poor correlation between MA use and MA concentration in hair. Correlation between MA use and MA concentration in hair of remaining one subject could not be determined or calculated. In this study, the correlation between accurate MA use histories obtained by psychiatrists and well-trained counselors and MA concentrations in hair was shown. This report provides objective scientific findings that should considerably aid the interpretation of forensic results and of the results of trials related to MA use.

An LC-MS/MS method for the determination of five erectile dysfunction drugs and their selected metabolites in hair.

The abuse of sildenafil and its analogous, accelerated by their inappropriate or illegal distribution, is a serious social issue globally. However, no studies have been conducted to monitor these drugs simultaneously in hair, which can provide valuable information on chronic drug use. In the present study, an LC-MS/MS method was developed for the simultaneous determination in hair of five erectile dysfunction drugs having a high risk for abuse (mirodenafil, sildenafil, tadalafil, udenafil and vardenafil) and their selected metabolites (SK3541, desmethylsildenafil, DA8164 and desethylvardenafil). The novel method was fully validated after optimizing matrix effects and extraction efficiency. The optimized sample preparation included acidic methanol extraction followed by solid phase extraction using C18 mixed mode strong cation exchange polymeric cartridges. The prepared samples were analyzed by LC-MS/MS with electrospray ion source in the positive ionization mode. The validation results proved the method to be selective, sensitive, accurate and precise, with acceptable linearity within calibration ranges. LODs ranged from 0.05 (DA8164) to 1 ng/10 mg hair (tadalafil). LOQs were 1 ng/10 mg hair except for DA8164 and vardenafil, of which they were 2.5 ng/10 mg hair. No significant variations were observed by different sources of matrices in both human and rat hair, except for tadalafil, for which a stable isotope-labeled internal standard was effective. The animal study suggested hair pigmentation was a major factor for the incorporation of the drugs and metabolites into hair. However, a wide variation of the sildenafil-to-desmethylsildenafil ratios was observed in human hair samples. The developed method will be very useful for monitoring the abuse of erectile dysfunction drugs for both legal and public health aspects.

Quantitative determination of 11-nor-9-carboxy-tetrahydrocannabinol in hair by column switching LC-ESI-MS(3).

Hair analysis has been regarded as an alternative method to urine analysis in forensic and criminal cases. Cannabis (marijuana) is one of the most widely used drugs in the world and it has been controlled in South Korea since 1976. Identification of 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) in hair can be an important proof of cannabis use because it can exclude the possibility of passive cannabis smoke exposure. In this study, we described a quantitative method of THCCOOH in hair using simple liquid-liquid extraction (LLE), selective column switching liquid chromatography with electrospray ionization (ESI)-MS(3). For the column switching system three columns (precolumn, trap column and analytical column) were used. Valve switch from the precolumn to the trap column was set from 3.0 to 4.0 min because THCCOOH appeared around 3.5 min with this precolumn. After 4.0 min the valve was switched to the original position and the analytes in the trap column were eluted onto the analytical column. Resolution occurred in this column and eluted into the ESI-MS(3) system. The internal standard was THCCOOH-d3. We used ESI-negative-MS(3) transition of ions at m/z 343 to 299 to 245 (343/299/245) and m/z 346 to 302 to 248 (346/302/248) for quantification of THCCOOH and THCCOOH-d3, respectively. The validation results of selectivity, matrix effect, recovery, linearity, precision and accuracy, and processed sample stability were satisfactory. The limit of detection (LOD) was 0.05 pg/mg and the limit of quantification (LOQ) was 0.10 pg/mg. The range of concentration of THCCOOH from 98 authentic human hair was 0.13-15.75 pg/mg. This method was successfully applied in the analysis of authentic human hair samples.

Deposition of JWH-018, JWH-073 and their metabolites in hair and effect of hair pigmentation.

Analysis of drugs in hair is often used as a routine method to obtain detailed information about drug ingestion. However, few studies have been conducted on deposition of synthetic cannabinoids and metabolites in hair. The first purpose of this study was to establish and validate an analytical method for detection of JWH-018, JWH-073, and their metabolites in hair, by use of UHPLC-MS-MS, for forensic application. The second purpose was to investigate the distribution of synthetic cannabinoids metabolites in hair and the effect of hair pigmentation, by use of an animal model. For this, JWH-073 was chosen as a representative synthetic cannabinoid. Finally, the developed method was applied to hair samples from 18 individuals suspected of synthetic cannabinoids use. JWH-018, JWH-073, and their metabolites were extracted from hair with methanol. The extract was then filtered and analyzed by UHPLC-MS-MS with an electrospray ion source in positive-ionization mode. Validation proved the method was selective, sensitive, accurate, and precise, with acceptable linearity within the calibration ranges. No significant variations were observed when different sources of both human and rat hair were used. The animal study demonstrated that JWH-073 N-COOH M was the major metabolite of JWH-073 in rat hair, and hair pigmentation did not have a significant effect on incorporation of JWH-073 and its metabolites into hair. In the analysis of 18 authentic hair samples, only JWH-018, JWH-018 N-5-OH M, and JWH-073 were detected, with wide variation in concentrations.

Quantitative analysis of propofol-glucuronide in hair as a marker for propofol abuse.

The inappropriate or illegal use of propofol has recently come to the fore as a serious social issue in South Korea. Thus, in spite of its superior potency as a therapeutic drug, propofol was classified as a controlled drug under the purview of Narcotics Control Law in South Korea in February of 2011. Accordingly, the determination of propofol and/or its metabolites in biological specimens is required to prove ingestion. Therefore, to demonstrate chronic ingestion, a quantitative analytical method for propofol-glucuronide in hair was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method was applied to measure propofol-glucuronide in hair samples from 23 propofol abuse suspects and in both pigmented and nonpigmented hair from rats which had ingested propofol. Propofol-glucuronide in hair was extracted in methanol and then filtered and analyzed by LC-MS/MS with electrospray ionization in negative mode. The validation results of selectivity, matrix effect, recovery, linearity, precision and accuracy, and processed sample stability were satisfactory. The limit of detection was 20 pg/10 mg hair and the limit of quantification was 50 pg/10 mg hair. The concentration range of propofol-glucuronide in hair segments from 23 propofol abuse suspects was shown up to 1,410 pg/mg. The animal study demonstrated that the presence of melanin did not affect the deposition of propofol-glucuronide in hair. Thus, we propose propofol-glucuronide in hair as a marker for propofol abuse. This method will be very useful for monitoring the inappropriate use of propofol for both legal and public health aspects.

Illegal use of benzodiazepines and/or zolpidem proved by hair analysis.

The abuse and misuse of benzodiazepines and zolpidem are widespread internationally. Their illegal distribution has raised their abuse to a serious level, and they are often misused in crimes. In the present study, 18 cases involving the illegal use of benzodiazepines and/or zolpidem were proved by hair analysis. The drugs were extracted from the hair samples using methanol and analyzed using LC-MS/MS. The cases were classified according to case history: five of illegal use in medical staff, eight through inappropriate or illegal distribution, and five related to drug-facilitated crimes. Among the 18 cases, zolpidem was identified in eight, alprazolam in seven, diazepam in six, and clonazepam in four. The drug concentrations ranged from

Development of a simultaneous analytical method for selected anorectics, methamphetamine, MDMA, and their metabolites in hair using LC-MS/MS to prove anorectics abuse.

Owing to the tight control of methamphetamine, it is presumed that phentermine, an amphetamine-type anorectic, has recently been considered a supplement for methamphetamine abusers in Korea. In addition, the abuse of other anorectics obtained by inappropriate means has become a social issue. Hair is a useful specimen to prove chronic drug use. Therefore, an analytical method for the simultaneous detection of phentermine, phendimetrazine, amfepramone, fenfluramine, mazindol, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA), as well as their metabolites, which covers the major amphetamines and anorectic agents in Korea, in hair was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using 1 % HCl in methanol and then filtered and analyzed by LC-MS/MS with electrospray ionization in positive mode. The validation results for selectivity, linearity, matrix effect, recovery, process efficiency, intra- and interassay precision and accuracy, and processed sample stability were satisfactory. The limits of detection ranged from 0.025 to 1 ng/10 mg hair and the limits of quantification were 0.25 ng/10 mg hair for every analyte except mazindol and phentermine, for which they were 10 ng/10 mg hair. The method was successfully applied for the segmental determination of selected anorectics, methamphetamine, MDMA, and their metabolites in hair from 39 drug suspects. Among the anorectics, phentermine and/or phendimetrazine were identified with or without methamphetamine in the hair samples. Closer supervision of the inappropriate use of anorectics is necessary. Also, hair analysis is useful for monitoring the abuse potential of unnoticed drugs.

Determination of illegally abused sedative-hypnotics in hair samples from drug offenders.

As several sedative-hypnotics are distributed illegally and are available domestically through media like the internet, their abuse is becoming a serious social problem. In the present study, four legal cases involving abuse of diazepam, midazolam, and/or zolpidem were proved by hair analysis using a simultaneous quantification method for the determination of diazepam (and its metabolites), lorazepam, midazolam, and zolpidem, which are often illegally abused in Korea, in hair that was developed and validated. Drugs and metabolites in hair were extracted using methanol followed by solid-phase extraction. The extracts were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed using gas chromatography-mass spectrometry in selected ion monitoring mode. The validation parameters of the method, including selectivity, linearity, limits of detection and quantification (LOQ), recovery, intra- and interassay precision and accuracy, and processed sample stability, were satisfactory. Moreover, the developed method was successfully applied to actual cases. In case 1, which involved a pop singer who was detained for suspected drug abuse, the concentrations of diazepam and nordiazepam were 5.7 and 2.0 ng/mg in nonpigmented hair and 6.6 and 1.8 ng/mg in pigmented hair, respectively. In case 2, 0.4 ng/mg zolpidem was detected in hair from a drug abuser who purchased illegally through the internet, and 0.2 ng/mg midazolam was detected in hair from an illegal drug seller in case 3. In case 4, diazepam (lower than the LOQ), nordiazepam (0.7 ng/mg), and zolpidem (0.7 ng/mg) were detected in hair from a medical doctor who abused drugs using forged prescriptions.

Feasibility of rat hair as a quality control material for the determination of methamphetamine and amphetamine in human hair.

A quality control material (QCM) is a necessity in hair drug analysis, but it is not always easy to have an authentic hair sample containing various target drugs and metabolites. In the present study, the feasibility of rat hair as a QCM was examined for its application in the determination of methamphetamine (MA) and amphetamine (AP) in human hair. MA was administered to lean Zucker rats, from which only pigmented hair was collected for the preparation of a QCM. The rat hair was then washed, homogenized and finally bottled. Both homogeneity and stability were examined in order to demonstrate the suitability of rat hair as a QCM in hair drug analysis. The concentrations of MA and AP in each bottle were determined using extraction with 1% HCl in methanol at 38°C followed by gas chromatography/mass spectrometry after derivatization with trifluoroacetic anhydride. Furthermore, the prepared QCM was used in an inter-laboratory quality assurance program. In the homogeneity test, no significant difference was observed between bottles of the QCM. The statistical results also showed no significant trends in stability for three months at room temperature. An inter-laboratory quality assurance program was also performed successfully using this material. Thus, rat hair will be useful as an alternative QCM sample for the determination of a variety of drugs and their metabolites in human hair.

Validation of a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair using LC-MS/MS and its application to human and rat hair.

Benzodiazepines and zolpidem are controlled in many countries due to their inherent adverse effects of a high degree of tolerance and dependence. Recently, as some of these drugs have become distributed illegally and available through media such as the Internet, their abuse is becoming a serious social problem. Hair is a useful specimen to prove chronic drug use. In the present study, a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using methanol, filtered and injected on the LC-MS/MS. The following validation parameters of the method were satisfactory: selectivity, linearity, matrix effect, recovery, process efficiency, intra- and inter-assay precision and accuracy and processed sample stability. The limit of detection (LOD) and the limit of quantification (LOQ) were the total drug detected from the sample. The LODs ranged from 0.005 ng (zolpidem) to 0.5 ng (bromazepam and chlordiazepoxide) and the LOQs were 0.25 ng in every analyte except for bromazepam and chlordiazepoxide, for which they were 0.5 ng. The developed method was successfully applied to five legal cases involving use of benzodiazepines and zolpidem and to an animal study on drug incorporation into hair. Diazepam and its three metabolites, as well as lorazepam, were detected in hair from both the multiple- and single-dose administration groups of lean Zucker rats. The concentration of diazepam was higher than those of its metabolites in both dark grey and white hair from the multiple-dose administration groups, with the mean concentration ranges from 0.16 to 0.51 ng/mg and from 0.10 to 0.24 ng/mg, respectively. The mean concentration ranges of lorazepam were from 0.05 to 0.37 ng/mg in dark grey hair and from 0.11 to 0.45 ng/mg in white hair from the multiple-dose administration groups. Hair pigmentation did not have any significant effect on the degree of the deposition of drugs and their metabolites in hair.

Analysis of pubic hair as an alternative specimen to scalp hair: a contamination issue.

Pubic hair is often analyzed as an alternative to scalp hair to prove previous drug use. However, urine is a potential source of external contamination. In the present study, the concentrations of methamphetamine (MA) and amphetamine (AP) in both scalp and pubic hair from illegal MA users were compared. Furthermore, in order to investigate the external contamination of pubic hair by urine, MA and AP absorbed into pubic hair that had been contaminated with authentic urine from a MA user were measured using a previously validated method. The effect of shampoo-wash on the contaminated pubic hair was also examined. However, no correlation was found in the MA and AP concentrations between scalp and pubic hair from illegal MA users. As the number of contamination events by authentic urine increased, the concentrations of MA and AP in pubic hair increased. Both MA and AP were detected in the first methanol washes of the contaminated hair samples but were not detected in the second methanol washes. As the number of shampoo-washes of the contaminated pubic hair increased, the concentrations of MA and AP gradually decreased. Even though pubic hair can be used as an alternative to scalp hair to prove previous drug use, it should be avoided when estimating drug use history. It should be also noted that higher quantitative results in pubic hair do not necessarily represent heavier drug use.

Detection of phentermine in hair samples from drug suspects.

Phentermine (PT) has been widely used as an anti-obesity drug. This drug has to be used with caution due to its close resemblance with amphetamines in its structure and toxicity profile. Recently, PT is in distribution by illegal modes and is found to be available through sources such as the internet, thus their misuse and/or abuse is threatening to be a serious social issue. In the present study, 32 cases of drug suspects were observed for PT abuse, detected using hair samples for drug analysis. PT and other amphetamines, such as methamphetamine (MA), amphetamine (AP), 3,4-methylenedioxyamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA), were extracted using 1% HCl in methanol for 20 h at 38°C. The extracts were derivatized with trifluoroacetic anhydride (TFAA) and analyzed using gas chromatography/mass spectrometry (GC/MS). Among the 32 cases of PT abuse, MA and its main metabolite, AP were identified in seven cases and MDMA and its main metabolite, MDA were detected in two other cases.