Hwakyung Choi

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.

Recent Trends of Drug Abuse and Drug-Associated Deaths in Korea

Abstract: Methamphetamine is the most abused drug in Korea followed by cannabis and opiates. Recent characteristics of the drug problem in Korea include increased drug smuggling from abroad, drug trafficking by organized gangs, varieties of drug smuggling, foreigners engaged in drug smuggling, and spread among drug abusers and areas. New drugs such as MDMA, Yaba, and LSD are found in greater proportion in the seizure records, indicating diversification of smuggled drugs in Korea. In addition, there is a growing tendency for the abuse of common medicines among young people in Korea because they are easily available. Methamphetamine is so seriously abused that fatalities from its overdose have occurred; since 1985, 20 such fatalities have been reported. Many deaths from the abuse of noncontrolled substances, especially dextromethorphan, zipeprol, and carisoprodol, which are taken for their hallucinogenic effects, were also reported. Recently, there was even a fatality related to smuggling of cocaine by body‐packing.

Immobilized β-cyclodextrin as a simple and recyclable method for cholesterol removal in milk

This study was designed to determine the optimum conditions of three different factors (mixing time, mixing temperature, and tube size) in reduction of cholesterol in milk using immobilized β-CD beads. Immobilized β-CD glass beads were prepared at different conditions of silanization and β-CD immobilization reactions. In result, the glass beads (diameter 1 mm) at 20 mM 3-isocyanatopropyltriethoxysilane and 30 mM β-CD without base showed the highest cholesterol removal rate as 41%. Using above immobilized β-CD glass beads, the cholesterol removal rate was 40.2% with 6 h of mixing time in 7 mm diameter tube at 10°C. After cholesterol removal from milk, the glass beads were washed for cholesterol dissociation and reused. In recycling study, the cholesterol removal rate was 41%, which was mostly same as that using new glass beads. These results indicated that cholesterol removal rate was about 40% with β-CD immobilized glass beads, however, the recycling efficiency was almost 100%.

Simultaneous analysis of Δ9-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.

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

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.

Analysis of cannabis in oral fluid specimens by GC-MS with automatic SPE.

Methamphetamine (MA) is the most commonly abused drug in Korea, followed by cannabis. Traditionally, MA analysis is carried out on both urine and hair samples and cannabis analysis in urine samples only. Despite the fact that oral fluid has become increasingly popular as an alternative specimen in the field of driving under the influence of drugs (DUID) and work place drug testing, its application has not been expanded to drug analysis in Korea. Oral fluid is easy to collect and handle and can provide an indication of recent drug abuse. In this study, we present an analytical method using GC-MS to determine tetrahydrocannabinol (THC) and its main metabolite 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid. The validated method was applied to oral fluid samples collected from drug abuse suspects and the results were compared with those in urine. The stability of THC and THC-COOH in oral fluid stored in different containers was also investigated. Oral fluid specimens from 12 drug abuse suspects, submitted by the police, were collected by direct expectoration. The samples were screened with microplate ELISA. For confirmation they were extracted using automated SPE with mixed-mode cation exchange cartridge, derivatized and analyzed by GC-MS using selective ion monitoring (SIM). The concentrations ofTHC and THC-COOH in oral fluid showed a large variation and the results from oral fluid and urine samples from cannabis abusers did not show any correlation. Thus, detailed information about time interval between drug use and sample collection is needed to interpret the oral fluid results properly. In addition, further investigation about the detection time window ofTHC and THC-COOH in oral fluid is required to substitute oral fluid for urine in drug testing.

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.

The dependence of the incorporation of methamphetamine into rat hair on dose, frequency of administration and hair pigmentation

In this paper, the incorporation of methamphetamine (MA) into rat hair was studied. The main purpose of this study was to investigate whether MA can be detected or positive hair results can be obtained in hair of rats administered a single dose of MA. The relationship between dose and frequency of administration and the concentrations of MA and its metabolite, amphetamine (AP), in rat hair were evaluated and the MA and AP concentrations in white and pigmented hair were compared. MA was administered to rats as follows: low dose (0.5mg/kg/day), medium dose (2mg/kg/day) and high dose (10mg/kg/day). The frequency of administration was one time per day for 1, 2, 3, 4, 5, 15 and 30 days. Hair and urine samples were collected from rats and analyzed by gas chromatography/mass spectrometry (GC/MS). MA could be identified in pigmented rat hair when MA was administered for 4 or more days at low daily dose and on day 1 following administration of medium and high daily doses. Positive results for MA were obtained from pigmented rat hair when MA was administered for 30 days at low daily dose, for 4 or more days at medium daily dose, or for 2 or more days at high daily dose. The concentrations of MA and AP found in rat hair were proportional to the dose and frequency of administration. The concentrations of MA and AP in pigmented rat hair were 2-10 times higher than those in white rat hair. The results of this study on the incorporation of MA into rat hair can serve as a model to better understand the incorporation of MA into human hair even though there are differences between animal models and 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.