Metabolomic study of polyamines in rat urine following intraperitoneal injection of γ-hydroxybutyric acid.

INTRODUCTION: Recently, illegal abuse of γ-hydroxybutyric acid (GHB) has increased in drug-facilitated crimes, but the determination of GHB exposure and intoxication is difficult due to rapid metabolism of GHB. Its biochemical mechanism has not been completely investigated. And a metabolomic study by polyamine profile and pattern analyses was not performed in rat urine following intraperitoneal injection with GHB. OBJECTIVES: Urinary polyamine (PA) profiling by gas chromatography-tandem mass spectrometry was performed to monitor an altered PA according to GHB administration. METHODS: Polyamine profiling analysis by gas chromatography-mass spectrometry combined with star pattern recognition analysis was performed in this study. The multivariate statistical analysis was used to evaluate discrimination among control and GHB administration groups. RESULTS: Six polyamines were determined in control, single and multiple GHB administration groups. Star pattern showed distorted hexagonal shapes with characteristic and readily distinguishable patterns for each group. N1-Acetylspermine (p < 0.001), putrescine (p < 0.006), N1-acetylspermidine (p < 0.009), and spermine (p < 0.027) were significantly increased in single administration group but were significantly lower in the multiple administration group than in the control group. N1-Acetylspermine was the main polyamine for discrimination among control, single and multiple administration groups. Spermine showed similar levels in single and multiple administration groups. CONCLUSIONS: The polyamine metabolic pattern was monitored in GHB administration groups. N1-Acetylspermine and spermine were evaluated as potential biomarkers of GHB exposure and addiction.

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.

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.

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.

Monitoring of altered amino acid metabolic pattern in rat urine following intraperitoneal injection with γ-hydroxybutyric acid.

INTRODUCTION: γ-Hydroxybutyric acid is a well-known prescription medicine that is used for the clinical treatment of alcohol dependence and narcolepsy. However, the biochemical mechanism underlying γ-hydroxybutyric acid intoxication remains unclear, and metabolomic amino acid profiling and pattern analyses have not been attempted following treatment with γ-hydroxybutyric acid. OBJECTIVES: We carried out urinary amino acid profiling and pattern analyses in rats to determine the biochemical events associated with altered amino acid metabolism and biomarker detection of intoxication following treatment with γ-hydroxybutyric acid. METHODS: Metabolic profiling analysis of amino acids in rat urine samples was performed as ethoxycarbonyl/tert-butyldimethylsilyl derivatives by gas chromatography-mass spectrometry following intraperitoneal administration of γ-hydroxybutyric acid once per day for 1 and 10 consecutive days. RESULTS: A total of 28 amino acids were positively identified in urine samples from the control, single and multiple groups treated with γ-hydroxybutyric acid. Their levels from the single and multiple treated groups were normalized to the corresponding mean control values. The star graphic pattern of the amino acids was characteristic and readily distinguishable for each group owing to its distorted nonacosagonal shape. In the principle component analysis, we monitored phenylalanine, glutamic acid, aspartic acid, asparagine, and methionine as contributing factors that discriminated the three groups. CONCLUSION: The present metabolomic study may explain the altered metabolism of amino acids following administration, and intoxication with γ-hydroxybutyric acid.

LC-MS-MS method for mitragynine and 7-hydroxymitragynine in hair and its application in authentic hair samples of suspected kratom abusers.

Kratom is a natural psychoactive product known primarily in Southeast Asia, including Thailand, Malaysia etc. It is also known as krathom, kakuam, ithang, thom (Thailand), biak-biak, ketum (Malaysia) and mambog (Philippines), and is sometimes used as an opium substitute. It is stimulant at doses of 1-5g, analgesic at doses of 5-15g, and euphoric and sedative at doses above 15g. Mitragynine is the most abundant indole compound in kratom (Mitragyna speciosa) and is metabolized in humans to 7-hydroxymitragynine, the more active metabolite. Adverse effects include seizures, nausea, vomiting, diarrhoea, tachycardia, restlessness, tremors, hallucinations and death. There are few studies on the analytical method for the detection of mitragynine and 7-hydroxymitragynine in hair. Therefore, this study proposes a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the analysis of kratom in hair. Hair samples were first weighed to approximately 10 mg and washed with methanol. Then the washed hair samples were cut into pieces and incubated in methanol with stirring and heating (16h/38℃). Extracts were then analysed by LC-MS-MS. This method was validated by determining the limit of detection (LOD), limit of quantification (LOQ), linearity, intra- and inter-day accuracy and precision, recovery and matrix effects. The intra- and inter-day precision (CV%) and accuracy (bias%) were within ±20%, which was considered acceptable. Using this newly developed LC-MS-MS method, the simultaneous detection of mitragynine and 7-hydroxymitragynine in six authentic hair samples was achieved to provide the direct evidence of kratom use in the past. Mitragynine concentrations ranged from 16.0 to 2,067 pg/mg (mean 905.3 pg/mg), and 7-hydroxymitragynine concentrations ranged from 0.34 to 15 pg/mg (mean 7.4 pg/mg) in six authentic hair samples from kratom abusers. This may be due to the higher sensitivity of the LOD in this study, with values of 0.05 pg/mg for mitragynine and 0.2 pg/mg for 7-hydroxymitragynine in hair, respectively.

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.

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.

Comprehensive evaluation of drug cases in Seoul and its metropolitan areas - 2022.

In order to prepare a response strategy for future drug analyses, the number and results of drug cases handled by the Seoul Institute of National Forensic Service were comprehensively evaluated, with a focus on Seoul and its metropolitan areas. In 2022, the Seoul Institute received approximately 12,150 requests for drug testing related to drug abuse and possession, and the urine samples were tested for approximately 16,000 drug species. The most frequently requested test was for cannabis (Δ-9-THC and Δ-8-THC), followed by methamphetamine, MDMA, ketamine, and synthetic cannabinoids. ADB-5'Br-BUTINACA and propyl butylone were newly emerging substances in 2022. These results were consistent with the main drug detection findings of the confiscated materials. During this period, 24 cases of drug-related deaths were reported, of which 6 were suspected to be the result of acute overdose poisoning caused by methamphetamine, MDMA, fentanyl, and heroin. In addition to the controlled substances regulated by the Narcotics Control Act, new psychoactive substances are being found to be circulating, and various measures are required to address this issue. This study is expected to improve future drug analyses methods and assist in establishing drug policies, and responding to future investigations.

Detection of thiocyanate in postmortem blood of a fatal potassium thiocyanate-poisoning patient using gas chromatography-mass spectrometry.

Thiocyanate is an inorganic compound used in industrial applications. Here, we report a case of suicidal death due to acute thiocyanate overdose. A 44-year-old man who consumed an unknown amount of thiocyanate solution was transferred to the emergency room and died 2 h after admission. An autopsy was performed 2 days after death. General toxicological analysis of blood using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry found no drug or alcohol. Quantification using GC-MS post-derivatization with pentafluorobenzyl bromide revealed 2,290 and 1,920 mg/L of thiocyanate in the heart and femoral blood samples, respectively. Thus, the cause of death was attributed to thiocyanate overdose. This study provides useful information for the interpretation of thiocyanate-related fatalities.

Determination of etomidate and etomidate acid in hair using liquid chromatography-tandem mass spectrometry.

Etomidate, with efficacy similar to that of propofol, has been used as a propofol substitute because propofol is a designated narcotic drug, and an increase in the frequency of illegal distribution and misuse has been reported in Korea. Previous analytical studies on etomidate used blood and urine. For long-term use and timing estimation, a method for etomidate analysis using hair should be developed. Therefore, in this study, an analytical method using LC-MS/MS was developed to determine etomidate and its major metabolite in hair. Human hair samples were segmented after washing to eliminate possible contaminants on the hair and stirred with methanol. The LC-MS/MS conditions were optimized, and the chromatographic separation time was 10 min. Selectivity, linearity, limit of detection, limit of quantification, precision, accuracy, recovery, process efficiency, matrix effect, and stability were evaluated to validate the analytical method. The calibration curves ranged from 0.25 to 50 pg/mg for etomidate and 2-250 pg/mg for etomidate acid; the coefficients of determination were higher than 0.997. The intra- and inter-assay precision results for all the compounds were <15% and satisfied at recovery, process efficiency, matrix effect, and stability. In addition, this method was applied to the hair of 4 rats which are administered with etomidate to evaluate. The etomidate concentrations in the rat hair ranged from 2.60 to 8.50 pg/mg, and the etomidate acid concentrations were 2.06-7.13 pg/mg. Thus, this method can be used as basic data for monitoring etomidate in hair.

Monitoring urinary testosterone and epitestosterone levels, and their ratio, in Korean chemical castration subjects using liquid chromatography-tandem mass spectrometry.

In Europe, chemical castration has been adopted as a treatment for paraphilia since the 1930s. Among the various chemical castration agents, luteinizing hormone-releasing hormone (LHRH) agonists are now used widely because of their effectiveness and safety. In South Korea, a legislation of chemical castration to control the sexual impulses of sexual offenders was enforced in July 2011. Most of these subjects are treated with leuprorelin acetate, an LHRH agonist, for chemical castration. Despite this, there are few studies that address the long-term influence of LHRH agonists on testosterone (T) and epitestosterone (E) levels in chemical castration subjects. In order to analyze the urinary levels of T in chemical castration subjects, whose T levels are extremely low, we developed and validated an analytical method for the detection of both T and E in human urine using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The urine samples were hydrolyzed, extracted, and analyzed by LC-MS/MS with electrospray ionization in the positive-ion mode. The limits of detection were 0.02 ng/mL and the limits of quantitation were 0.05 ng/mL, which provided great sensitivity. The established method was applied to urine samples from chemical castration subjects and healthy male volunteers. The chemical castration subjects showed significantly lower urinary T levels than the control subjects. In addition, the urinary E levels were also lower in the chemical castration subjects; however, the T/E ratios were constant and did not show a notable decrease because of the simultaneous decrease in both urinary T and E. The urinary T levels and T/E ratio did not exceed the doping control criteria for exogenous T ingestion for any subject. This study shows the trend of urinary T and E levels in long-term treated chemical castration subjects by establishing a highly sensitive LC-MS/MS method, that provides useful information for monitoring chemical castration.

Determination of boldenone in postmortem specimens including blood and urine samples using LC-MS/MS.

Boldenone (BOLD), one of androgenic anabolic steroids (AAS), although banned in humans, is still available illegally. AAS abuse has previously been associated with various cardiovascular adverse events including acute myocardial infarction, arrhythmia, and sudden death. In this study, the concentration of BOLD was determined in postmortem specimens from the corpse of a human male who intentionally injected BOLD undecylenate into his shoulder muscle. In addition, the endogenous levels of BOLD in the blood and urine samples of young human males have been reported. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with solid-phase extraction (SPE) was developed and validated for the analysis of BOLD in blood, muscular tissue and urine samples. The validation parameters including linearity, accuracy, precision, matrix effect, and recovery were satisfactory. The concentrations of BOLD in the blood of 20 young human males who didn't take BOLD were under the limit of quantitation (LOQ, 0.5 ng/mL). Additionally, the mean level of BOLD in the urine samples was 3.19 ± 1.65 ng/mL (range: 0.37˜6.02 ng/mL). The concentrations of BOLD in the victim's blood from the femoral vein and heart were 140.44 and 25.74 ng/mL, respectively. On the other hand, those in the muscular tissue from the injection site and the urine sample were 142.3 ng/g and 3474 ng/mL, respectively.

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.

Effect of alpha-tocopherol and deferoxamine on methamphetamine-induced neurotoxicity.

Methamphetamine (MA)-induced dopaminergic neurotoxicity is believed to be associated with the increased formation of free radicals. This study examined the effect of alpha-tocopherol (alpha-TC), a scavenger of reactive oxygen species, and deferoxamine (DFO), an iron chelator, on the MA-induced neurotoxicity. Male rats were treated with MA (10 mg/kg, every 2 h for four injections). The rat received either alpha-TC (20 mg/kg) intraperitoneally for 3 days and 30 min prior to MA administration or DFO (50 mg/kg) subcutaneously 30 min before MA administration. The concentrations of dopamine (DA), serotonin and their metabolites decreased significantly after MA administration, which was inhibited by the alpha-TC and DFO pretreatment. alpha-TC and DFO attenuated the MA-induced hyperthermia as well as the alterations in the locomotor activity. The level of lipid peroxidation was higher and the reduced glutathione concentration was lower in the MA-treated rats. These changes were significantly attenuated by alpha-TC and DFO. This suggests that alpha-TC and DFO ameliorate the MA-induced neuronal damage by decreasing the level of oxidative stress.

Standardization of method for the analysis of dextromethorphan in urine.

Dextromethorphan (DMP), an antitussive, is one of the most popular drugs among the younger generation in Korea. It usually is taken for its hallucinogenic properties and overdoses have been responsible for the fatalities that have been reported frequently. To control the abuse of DMP, the authorities restricted its use through classifying it as a controlled drug on October 2003. The purpose of this study is to provide a standard method for the analysis of DMP and its main metabolite, dextrorphan (DTP) in biological specimens. At first we established a standard operating procedure (SOP) for DMP/DTP in urine, and a method validation was performed. We also quantified DMP from 16 drug abuser's urine samples all of which were positive in the screening test for DMP. For the detection of DMP/DTP, urine samples were adjusted with 6N NaOH (pH 11) and extracted with ethylacetate. Thin layer chromatography was used as the screening test, and the final identification for DMP/DTP was used by GC/MS. The ions (m/z 271 for DMP, m/z 257 for DTP and m/z 86 for lidocaine as internal standard) were extracted from the full scan mass spectrum and were used for quantification. The selectivity, linearity of calibration, accuracy, within- and between day precision, limit of detection and quantification, recovery and stability were examined as parts of the method validation. Extracted calibration curves were linear from 100 to 2000 ng/mL for DMP and DTP with correlation coefficients better than 0.999. Limit detection was 50 ng/mL for DMP and DTP. Within-run precision (%CV) for DMP and DTP at three different concentrations (100, 500 and 1000 ng/mL) was 6.10-18.85%, and between-run precision was 1.70-7.86% for DMP and DTP. Absolute recovery for DMP and DTP was 57-74%, and relative recovery (extraction efficiency) was 80-89%. For 16 drug abuser's urine samples, the concentrations of DMP and DTP were 0.16-52.63 and 0.41-23.75 microg/mL, respectively. Method validation is an important requirement in the practice of chemical analysis, and it will be particularly useful in verifying the reliability of analytical results in the field of forensic science.

Distribution of organochlorines and PCB congeners in Korean human tissues.

In order to investigate the residual amounts of organochlorines and polychlorinated biphenyls (PCBs) in Korean human tissues (blood, adipose tissue, liver, kidney cortex, and lung), the samples were collected from the autopsied cadavers of 40 men and 40 women (from teens to seventies of age). Alpha-BHC, beta-BHC, gamma-BHC, delta-BHC, p,p'-DDT, p,p'-DDD, p,p'-DDE, endrin, dieldein, aldrin, and 7 marker PCBs (28, 52, 101, 118, 138, 153, and 180) were determined in human tissues. The levels of organochlorines and PCB congeners indicated that they have been widely distributed in Korean human body. Positive correlations in terms of age were observed for the following cases: p,p'-DDE, p,p'-DDT, Sigma-DDT, PCB 118, PCB 138, PCB 153, and Sigma-PCB in the adipose tissue, and p,p'-DDE in the lung. Concentration of these compounds showed a significant age-related increase. Accumulation of these compounds in aged people revealed that these compounds were more slowly eliminated in our environment and risk assessment was necessary for further proper action. Significant differences in the levels of PCBs between genders were found for PCB 118 in the adipose tissue and PCB 138 in the liver. Positive correlation coefficients between tissues were detected with p,p'-DDE and p-BHC.

Determination of XLR-11 and its metabolites in hair by liquid chromatography-tandem mass spectrometry.

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 disposition of synthetic cannabinoids including cyclopropylindoles (UR-144 and XLR-11) and their metabolites in hair. XLR-11 has been widely abused in South Korea recently. Identification of metabolites in hair can be an important proof of synthetic cannabinoids use because it can exclude the possibility of passive smoke exposure. In this study, we described a quantitative analytical method of XLR-11 and its metabolites (UR-144, UR-144 N-5-hydroxypentyl metabolite, UR-144 N-4-hydroxypentyl metabolite, UR-144 N-pentanoic acid metabolite and XLR-11 N-4-hydroxypentyl metabolite) in hair by liquid chromatography with ESI-MS/MS. The target analytes were extracted with methanol from washed and cut hair samples and the extracts were evaporated, filtered and analyzed by LC-MS/MS with electrospray ion source in positive-ionization mode. JWH-018-d9 and JWH-018 N-5-hydroxypentyl metabolite-d5 were used as internal standards. Chromatographic separation was completed within 15 min. No interferences were detected in 10 blank hair samples. In intra- and inter-assay precision and accuracy study, CV (%) and bias (%) were below 12. The limit of detection (LOD) was 0.1∼2 pg/mg and the limit of quantification (LOQ) was 0.2-2 pg/mg, respectively. The validation results proved that the method was selective, accurate and precise with acceptable linearity within calibration range. No significant variation was observed by different sources of matrices. This method was applied to hair samples from 14 individual suspects of XLR-11 use. In this result, XLR-11, UR-144, UR-144 N-5-hydroxypentyl metabolite and UR-144 N-pentanoic acid metabolite, XLR-11 N-4-hydroxypentyl metabolite were detected. The concentration of XLR-11 as a parent drug was much higher than other metabolites. UR-144 N-5-hydroxy metabolite and UR-144 N-pentanoic acid were detected mainly in the authentic hair samples from suspected of XLR-11 use. UR-144 N-4- hydroxypentyl metabolite was not detected in all cases.

Development of a comprehensive spectral library of sildenafil and related active analogues using LC-QTOF-MS and its application for screening counterfeit pharmaceuticals.

The abuse or misuse of forged erectile-dysfunction drugs, containing phosphodiesterase type 5 inhibitors (e.g. sildenafil), is a serious issue globally. Therefore, the detection of sildenafil and related active analogues in counterfeit pharmaceuticals or the differentiation between counterfeit and authentic drugs has been performed with a variety of analytical techniques. Recently, a liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS)-based in-house library, consisting of accurate mass ion fragmentation information and retention times, was effectively applied to screen a large number of compounds in field of forensic toxicology. However, a comprehensive LC-QTOF-MS spectral library of sildenafil and related active analogues has not yet been reported. In the present study, a spectral library of 40 compounds of sildenafil and related analogues was developed with accurate mass spectra and retention times using LC-QTOF-MS, and applied to screen nine marketed counterfeit products. The in-house library successfully identified sildenafil, dimethylsildenafil, hydroxyhomosildenafil, demethylhongdenafil, pseudovardenafil and vardenafil in the samples. Our LC-QTOF-MS-based spectral library search is considered a powerful approach for identifying sildenafil and related active analogues in counterfeit pharmaceuticals.

Simultaneous determination of five naphthoylindole-based synthetic cannabinoids and metabolites and their deposition in human and rat hair.

The continuing appearance of new synthetic cannabinoids has been a major issue in the field of forensic and clinical toxicology. In response to that, analytical methods for synthetic cannabinoids have been increasingly established in a variety of biological matrices. Since most of synthetic cannabinoids with structure similarity share some enzymatic metabolites, making the interpretation of analytical results and the discovery of the parent drug actually ingested very complicated, the investigation on metabolites of the first generation of synthetic cannabinoids with their relatively short side chains in chemical structure could be more important. Therefore, in the present study, we developed the analytical method for AM-2201, JWH-122 and MAM-2201 with JWH-018 as a precursor and their monohydroxylated metabolites in hair matrix. Also, using a rat model, AM-2201 and its monohydroxylated metabolites were identified and then the ratios of metabolite-to-parent drug were estimated to be used as criteria on external contamination. All analytes were extracted with methanol from washed and cut hair samples and the extracts were injected into LC-MS/MS with electrospray ion source in the positive ionization mode. Matrix effect and recovery were evaluated in hair matrices and no significant variations were observed. The validation results for precision and accuracy were satisfactory in both human and rat hair. The LOD and LOQ were 0.5 pg/10mg and 1.0 pg/10mg in human hair and 0.5 pg/20mg and 1.0 pg/20mg in pigmented and non-pigmented rat hair, respectively. Additionally, as a result of the animal study, there were not significant differences in the effect of pigmentation on the distribution of AM-2201 and its monohydroxylated metabolites in hair. Wide variations were observed for the concentrations of the naphthoylindole-based synthetic cannabinoids and metabolites in authentic hair samples from nine cases; those were 0.4-59.2 pg/mg for JWH-018, 0.1-0.8 pg/mg for JWH-073, 1.7-739.0 pg/mg for AM-2201, 0.1-402.0 pg/mg for JWH-122, 0.2-276.0 pg/mg for MAM-2201, 0.2-1.1 pg/mg for JWH-018 N-COOH, 0.3-37.2 pg/mg for JWH-018 N-5-OH, 0.3 pg/mg for JWH-073 N-COOH, 0.4 pg/mg for AM-2201 N-4-OH, 0.2-3.1 pg/mg for AM-2201 N-6-OHindole and 0.1-3.5 pg/mg for JWH-122 N-5-OH. This quantitative LC-MS/MS analytical method for five naphthoylindole-based synthetic cannabinoids and their metabolites was very useful to be applied to authentic hair samples, of which their analytical results suggested the incorporation of synthetic cannabinoids in the hair matrix and provided the information on ingested parent drugs.