Development of a Column-Switching HPLC-MS/MS Method and Clinical Application for Determination of Ethyl Glucuronide in Hair in Conjunction with AUDIT for Detecting High-Risk Alcohol Consumption.

It is critical to assess the severity of alcohol consumption in certain diseases such as alcohol liver disease and alcohol addiction. Ethyl glucuronide (EtG) is a highly stable metabolite of ethanol in hair; thus, it was proposed as a long-term monitoring marker for alcohol consumption. Therefore, an HPLC-MS/MS method for EtG in hair was developed and applied to a clinical setting to assess the relevance of the EtG concentration and/or the Alcohol Use Disorders Identification Test (AUDIT) score to high-risk alcohol consumption. EtG was extracted from 10 mg of hair using water and analyzed using on-line sample purification coupled to HPLC-MS/MS. The diagnostic performances of the EtG concentration and/or the AUDIT score for detecting high-risk alcohol consumption were statistically evaluated between alcohol addicts (n = 44) and average alcohol users (n = 19). The on-line sample purification resulted in labor-saving with smaller sample amount. Both the EtG concentrations (4.0⁻587.4 pg/mg vs. 12.9⁻74.9 pg/mg) and the AUDIT scores (4⁻40 vs. 5⁻28) obtained from the alcohol addicts were significantly higher than those from the average alcohol users. The performance evaluation demonstrated that the integration score of the EtG concentration and the AUDIT score increased diagnostic performance for high-risk alcohol consumption.

Simultaneous determination of 18 abused opioids and metabolites in human hair using LC-MS/MS and illegal opioids abuse proven by hair analysis.

Natural and synthetic opioids have efficient analgesic activity but can also be addictive. Thus, the determination of opioids and their metabolites in biological specimens is of interest in clinical and forensic toxicology laboratories. The analysis of drugs in hair provides valuable information on previous chronic drug use and has been successfully applied to the diagnosis of drug abuse, tolerance, compliance and gestational drug exposure. Despite the abuse of prescription opioids along with heroin and other illegal opiates, few studies have been conducted on the simultaneous determination of the broad range of opioids covering those drugs in hair. In the present study, an analytical method for the simultaneous detection in hair of 18 opioids and metabolites considered to have a high abuse risk based on the results of urine drug screening was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the purpose of clinical and forensic applications. The drugs and metabolites were extracted from hair using methanol and analyzed using LC-MS/MS. The validation results proved that the method was selective, accurate and precise with acceptable linearity within calibration ranges. No significant variation was observed by different sources of matrices. The limits of detection and the limits of quantification ranged from 0.05 to 0.25ng/10mg hair and from 0.05 to 0.5ng/10mg hair, respectively. The developed method was successfully applied to 15 hair samples from opioids users. This method will be very useful for monitoring the inappropriate use of opioid drugs.

Screening of multiple drugs of abuse and metabolites in urine using LC/MS/MS with polarity switching electrospray ionization.

A recent trend in urine drug testing in forensic and clinical toxicology has been the simultaneous determination of different chemical groups of target drugs, which are selected based on their local popularity. Rapid multiple drug analysis, made possible by the use of liquid chromatography-tandem mass spectrometry (LC/MS/MS), has become more widely used, especially in workplace drug testing. Therefore, in the present study, a method for simultaneously analyzing 35 drugs of abuse and relevant metabolites that are most prevalent in Korea, using LC/MS/MS with polarity switching electrospray ionization, was developed and validated. The drugs and metabolites in urine were extracted by using mixed mode strong cation exchange polymeric solid phase extraction cartridges after enzymatic hydrolysis and were then injected into the LC/MS/MS system. The validation results for selectivity, linearity, intra- and inter-assay precision and accuracy for this method were satisfactory, while the results for matrix effects and recovery showed significant variance among the urine samples from different sources. The limits of detection ranged from 0.1 to 10 ng/ml and the limits of quantification were from 1 to 10 ng/ml. To reduce the matrix effects in authentic samples, two different quantitative approaches were compared: quantification using calibration standards prepared by the drug-free pooled urine matrix and quantification using the standard addition. Of these, the latter method was found to be the most suitable. The method developed in this study will be very useful for forensic and clinical toxicology laboratories to adopt for monitoring the inappropriate use of controlled drugs.

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 an automated data processing method for sample to sample comparison of seized methamphetamines.

The information about the sources of supply, trafficking routes, distribution patterns and conspiracy links can be obtained from methamphetamine profiling. The precursor and synthetic method for the clandestine manufacture can be estimated from the analysis of minor impurities contained in methamphetamine. Also, the similarity between samples can be evaluated using the peaks that appear in chromatograms. In South Korea, methamphetamine was the most popular drug but the total seized amount of methamphetamine whole through the country was very small. Therefore, it would be more important to find the links between samples than the other uses of methamphetamine profiling. Many Asian countries including Japan and South Korea have been using the method developed by National Research Institute of Police Science of Japan. The method used gas chromatography-flame ionization detector (GC-FID), DB-5 column and four internal standards. It was developed to increase the amount of impurities and minimize the amount of methamphetamine. After GC-FID analysis, the raw data have to be processed. The data processing steps are very complex and require a lot of time and effort. In this study, Microsoft Visual Basic Application (VBA) modules were developed to handle these data processing steps. This module collected the results from the data into an Excel file and then corrected the retention time shift and response deviation generated from the sample preparation and instruments analysis. The developed modules were tested for their performance using 10 samples from 5 different cases. The processed results were analyzed with Pearson correlation coefficient for similarity assessment and the correlation coefficient of the two samples from the same case was more than 0.99. When the modules were applied to 131 seized methamphetamine samples, four samples from two different cases were found to have the common origin and the chromatograms of the four samples were appeared visually identical. The developed VBA modules could process raw data of GC-FID very quickly and easily. Also, they could assess the similarity between samples by peak pattern recognition using whole peaks without spectral identification of each peak that appeared in the chromatogram. The results collectively suggest that the modules would be useful tools to augment similarity assessment between seized methamphetamine samples.

Genetic and chemical components analysis of Papaver setigerum naturalized in Korea.

Of the 110 species of genus Papaver, only Papaver somniferum and P. setigerum are controlled poppies in Korea. All poppy samples share similar morphology therefore it is important to check if they contain controlled substances such as morphine and codeine for forensic purpose. Since the alkaloid content of Papaver plants varies according to their growing stage, chemical components analysis alone is not enough to identify exact species. In 2010, hundreds of poppy plants suspected to be P. somniferum were found in Jeju Island, South Korea. They had a slightly different but overall similar appearance to P. somniferum. Using GC-MS analysis, codeine, rhoeadine, papaverine, protopine, noscapine, setigeridine and trace amounts of morphine were detected in these samples. Although their chemical components were different from what has been described in literatures for P. setigerum, they could be assumed to be P. setigerum based on their morphological features and GC-MS results. Also, chromosome numbers using their seeds showed 2n=44 and the numbers were in accordance with those of P. setigerum. Nucleotide substitution or insertion/deletion of ITS (internal transcribed spacer), 18S rRNA (ribosomal RNA), rbcL (large subunit of ribulose 1,5-bisphosphate carboxylase), trnL-trnF IGS (intergenic spacer), trnL intron and psbA-trnH were assessed as universal genetic markers for P. setigerum. Also, genetic analysis using six target genes involved in the biosynthesis of benzylisoquinoline alkaloids, including TYDC (tyrosine/dopa decarboxylase), SAT (salutaridinol-7-O-acetyltransferase), BBE (berberine bridge enzyme), COR (codeinone reductase), CYP80B1 ((S)-N-methylcoclaurine 3'-hydroxylase) and NCS (norcoclaurine synthase) were tested as Papaver-specific genetic markers by the existence of their PCR products. From the results, the sequences of the 6 universal genetic markers and 6 Papaver-specific genetic markers for P. setigerum were identified and then Genbank accession numbers of them were registered in NCBI. Also, the trnL intron and psbA-trnH nucleic acid sequences of the 7 Papaver species were identified and registered.

Development of a target component extraction method from GC-MS data with an in-house program for metabolite profiling.

After gas chromatography-mass spectrometry (GC-MS) analysis, data processing, including retention time correction, spectral deconvolution, peak alignment, and normalization prior to statistical analysis, is an important step in metabolomics. Several commercial or free software packages have been introduced for data processing, but most of them are vendor dependent. To design a simple method for Agilent GC/MS data processing, we developed an in-house program, "CompExtractor", using Microsoft Visual Basic. We tailored the macro modules of an Agilent Chemstation and implanted them in the program. To verify the performance of CompExtractor processing, 30 samples from the three species of the genus Papaver were analyzed with Agilent 5973 MSD GC-MS. The results of CompExtractor processing were compared with those of AMDIS-SpectConnect processing by hierarchical cluster analysis (HCA) and principal component analysis (PCA). The two methods showed good classification according to their species in HCA. The PC1+PC2 scores were 54.32-63.62% for AMDIS-SpectConnect and 56.65-85.92% for CompExtractor in PCA. Although the CompExtractor processing method is an Agilent GC-MS-specific application and the target compounds must be selected first, it can extract the target compounds more precisely in the raw data file with batch mode and simultaneously assemble the matrix text file.

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.

Simultaneous analysis of psychotropic phenylalkylamines in oral fluid by GC-MS with automated SPE and its application to legal cases.

Phenylalkylamine derivatives, such as methamphetamine (MA), amphetamine (AM), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), phentermine (PT), fenfluramine (FFA) and phenmetrazine (PM), and ketamine (KT) are widely abused recreational or anorectic drugs in Korea and are regulated under the Controlled Substance Act in Korea. Phenylalkylamines and ketamine analysis is normally performed using both urine and hair samples but there is no established method for the simultaneous analysis of all these phenylalkylamines and ketamine in oral fluids. Oral fluid is easy to collect/handle and can provide an indication of recent drug abuse. In this study, to confirm the presence of phenylalkylamine derivatives and ketamine in oral fluid after screening with an immunoassay, an analytical method using automated solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) was developed and fully validated according to international guidelines. The applicability of the assay was demonstrated by analyzing of authentic oral fluid samples and the results of oral fluid analysis were compared with those in urine and hair to to evaluate the feasibility of oral fluid in forensic cases. The recovery of phenylalkylamines and ketamine from oral fluid collection devices was also assessed. Oral fluid specimens from 23 drug abuse suspects submitted by the police were collected using Salivette (Sarstedt, Nümbrecht, Germany), Quantisal (Immunalysis, Pomona, CA) or direct expectoration. The samples were screened using a biochip array analyzer (Evidence Investigator, Randox, Antrim, UK). For confirmation, the samples were analyzed by GC-MS in selected-ion monitoring (SIM) mode after extraction using automated SPE (RapidTrace, Zymark, MA, USA) with a mixed-mode cation exchange cartridge (CLEAN SCREEN, 130 mg/3 ml, UCT, PA, USA) and derivatization with trifluoroacetic anhydride (TFA). The results from the immunoassay were consistent with those from GC-MS. Twenty oral fluid samples gave positive results for MA, AM, PT and/or PM among the 23 cases, which gave positive results in urine and/or hair. Although large variations in the MA, AM, PT and PM concentrations were observed in three different specimens, the oral fluid specimen was useful for demonstrating phenylalkylamines and ketamine abuse as an alternative specimen for urine.

A comparative study on the concentrations of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in head and pubic hair.

In this study, the concentrations of 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in pubic, axillary and beard hair were measured and the correlation between the concentrations of THCCOOH in head and pubic hair from same cannabis users were evaluated. The papers on body hair analysis for THCCOOH were rarely found although police officers submit body hair as a complimentary specimen to forensic laboratories in case cannabis users had no hair. Head, pubic, axillary, and beard hair were collected. All hair samples were cut into 0.5mm segments and decontaminated with methanol, digested with 1 mL of 1M NaOH at 85 °C for 30 min and extracted in 2 mL of n-hexane:ethyl acetate (9:1) two times after adding 1 mL of 0.1N sodium acetate buffer (pH = 4.5) and 200 µL of acetic acid followed by derivatization with 50 µL of PFPA and 25 µL of PFPOH for 30 min at 70 °C. The extracts were analyzed using gas chromatography tandem mass spectrometry operating in negative chemical ionization mode (GC/MS/MS-NCI). We determined the concentrations of THCCOOH in both pubic and head hair. The concentrations of THCCOOH in pubic hair were higher than those in head hair. We also evaluated the concentrations of THCCOOH in body hair (pubic, axillary and beard hair) and head hair according to the positive/negative urine test results. There was no statistically significant difference in the concentrations of THCCOOH in head and body hair according to urine results.

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.

Species identification of Papaver by metabolite profiling.

Papaver somniferum L. and Papaver setigerum D.C. are controlled as opium poppy in Korea because they contain narcotic substances such as morphine and codeine. It is one of the critical issues whether the plants similar to opium poppy in shape are controlled plants or not. There are more than 110 species in the genus Papaver worldwide and about 10 species in Korea. As the morphological features of some species are very similar and the alkaloid contents and the ratios among the major alkaloids vary even within the same species, it is often difficult to identify the exact species by the morphological features and/or major alkaloids analysis. To develop a new method that uses metabolite profiling for species discrimination between P. somniferum, Papaver rhoeas and P. setigerum, the gas chromatography/mass spectrometry (GC-MS) data of the alkaline extract were processed with in-house Microsoft Visual Basic(®) modules and the chemical information was analyzed through multivariate statistical analyses such as Hierarchical cluster analysis (HCA), principal component analysis (PCA) and discriminant analysis (DA). The GC-MS results combined with multivariate analysis demonstrated that the metabolite profiling was an efficient technique for the classification and this method will provide a powerful tool for the identification of Korean Papaver species.

A study on the concentrations of 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in hair root and whole hair.

In this study, we investigated the patterns of cannabis users (n=412) according to their sex, age, and the results of urinalysis and hair analysis, and classified the concentrations of THCCOOH in hair into three categories to examine the levels of cannabis use. We also compared the concentrations of THCCOOH in hair root, hair without the hair root and whole hair and examined the relationship among them according to the results of urinalysis. The hair samples were washed, digested with 1ml of 1M NaOH at 85°C for 30min and extracted with 2ml of n-hexane:ethyl acetate (9:1) two times after adding 1ml of 0.1N sodium acetate buffer (pH 4.5) and 200µl of acetic acid. The final mixture was derivatized with 50µl of PFPA and 25µl of PFPOH for 30min at 70°C. The solution was evaporated, and the residue was reconstituted in 40µl of ethyl acetate and transferred to an autosampler vial. One microlitre was injected into the GC/MS/MS-NCI system. The concentrations of THCCOOH ranged from 0.06 to 33.44pg/mg (mean 2.96; median 1.32) in hair from cannabis users who had positive urine results and ranged from 0.05 to 7.24pg/mg (mean 1.35; median 0.37) in hair from cannabis users who had negative urine results. The average concentration of THCCOOH in hair from cannabis users who had positive urine results was higher than that from cannabis users who had negative urine results. Male cannabis users in their forties were predominant. We classified the concentrations of THCCOOH in hair into three groups (low, medium and high), and could use the grouping of THCCOOH in hair as a guide for determining the level of use. The low, medium and high concentration ranges for THCCOOH in hair were 0.05-0.24, 0.25-2.60 and 2.63-33.44pg/mg, respectively. We also investigated 28 hair samples with the root. The highest concentrations of THCCOOH were seen in the hair root from 18 out of the 28 hair samples. The average concentrations of THCCOOH in hair root, hair without hair root and whole hair from cannabis users who had positive urine results were higher than those who had negative urine results.

Simultaneous analysis of Δ(9)-tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol in hair without different sample preparation and derivatization by gas chromatography-tandem mass spectrometry.

The present study describes a gas chromatography/tandem mass spectrometry-negative ion chemical ionization assay (GC/MS/MS-NCI) for simultaneous analysis of Δ(9)-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) in hair. Each hair sample, of approximately 20mg, was weighed and the sample was dissolved in 1ml of 1M sodium hydroxide (30min at 85°C) in the presence of THC-d(3) and THCCOOH-d(3). For the analysis of THC, hair samples were extracted with n-hexane:ethyl acetate (9:1) two times; acetic acid and sodium acetate buffer were added for the analysis of THCCOOH, and hair samples were re-extracted with n-hexane:ethyl acetate (9:1) two times. The extracts were then derivatized with pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH). This method allowed the analysis of THC and THCCOOH using the GC/MS/MS-NCI assay. This method was also fully validated and applied to hair specimens (n=54) collected from known cannabis users whose urine test results were positive. The concentrations of THC and THCCOOH in hair ranged from 7.52 to 60.41ng/mg and from 0.10 to 11.68pg/mg, respectively. In this paper, we simultaneously measured THC and THCCOOH in human hair using GC/MS/MS-NCI without requiring different sample preparation and derivatization procedures. The analytical sensitivity for THCCOOH in hair was good, while that for THC in hair needs to be improved in further study.

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.

Establishment of the measurement uncertainty of 11-nor-D9-tetrahydrocannabinol-9-carboxylic acid in hair.

The quantitative analysis of 11-nor-D(9)-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in hair requires a sensitive method to detect a low-pg level. Before applying the method to real hair samples, the method was validated; in this study, we examined the uncertainty obtained from around the cut-off level of THCCOOH in hair. We calculated the measurement uncertainty (MU) of THCCOOH in hair as follows: specification of the measurand, identification of parameters using "cause and effect" diagrams, quantification of the uncertainty contributions using three factors, the uncertainty of weighing the hair sample, the uncertainty from calibrators and the calibration curve, and the uncertainty of the method precision. Finally, we calculated the degrees of freedom and the expanded uncertainty (EU). The concentration of THCCOOH in the hair sample with its EU was (0.60 ± 0.1) × 10(-4)ng/mg. The relative uncertainty percent for the measurand 0.60 × 10(-4)ng was 9.13%. In this study, we also selected different concentrations of THCCOOH in real hair samples and then calculated the EU, the relative standard uncertainty (RSU) of the concentration of THCCOOH in the test sample [u(r)(c0)], the relative uncertainty percent, and the effective degree of freedom (v(eff)). When the concentrations of THCCOOH approached the cut-off level, u(r)(c0) and the relative uncertainty percent increased but absolute EU and v(eff) decreased.

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.

Thebaine in hair as a marker for chronic use of illegal opium poppy substances.

Opium poppy products are often illegally used for both recreational and medicinal purposes. In order to demonstrate the ingestion of opium poppy substances, morphine, codeine and their metabolites have been identified. However, morphine and codeine also originate from the ingestion of therapeutic drugs. Therefore, thebaine, one of the main opium alkaloids, in hair was suggested as a marker for chronic use of illegal opium poppy substances in the present study. First, thebaine was included in the analyte list of our routine analytical method for the simultaneous quantification of codeine, morphine, norcodeine, normorphine and 6-acetylmorphine (6-AM) in hair, which was fully validated previously. Then, the incorporation of thebaine and other opiates into hair and the effect of hair pigmentation were examined using lean Zucker rats with both dark grey and white hair on the same body. Thebaine was also measured in hair samples from actual cases of opium poppy substance use. Consequently, thebaine in hair was demonstrated as a marker of chronic use of illegal opium poppy substances using an animal study and actual cases. Thebaine and other opiates were successfully measured in pigmented hair from rats that ingested raw opium suspension. Moreover, thebaine identified in hair excluded possibility of ingestion of pharmaceutical opiates in actual cases.