Metabolic characterization of (1-(5-fluoropentyl)-1H-indol-3-yl)(4-methyl-1-naphthalenyl)-methanone (MAM-2201) using human liver microsomes and cDNA-overexpressed cytochrome P450 enzymes.

MAM-2201 is a synthetic cannabinoid that is increasingly found in recreational drug abusers and cases of severe intoxication. Thus, characterization of the metabolic pathways of MAM-2201 is necessary to predict individual pharmacokinetics and toxicity differences, and to avoid toxic drug-drug interactions. Collectively, 19 phase 1 metabolites of MAM-2201 were identified using liquid chromatography-Orbitrap mass spectrometry following human liver microsomal incubations in the presence of NADPH: 7 hydroxy-MAM-2201 (M1-M7), 4 dihydroxy-MAM-2201 (M8-M11), dihydrodiol-MAM-2201 (M12), N-(5-hydroxypentyl)-MAM-2201 (M13), hydroxy-M13 (M14), N-dealkyl-MAM-2201 (M15), 2 hydroxy-M15 (M16, M17), MAM-2201 N-pentanoic acid (M18), and hydroxy-M18 (M19). On the basis of intrinsic clearance values in human liver microsomes, hydroxy-MAM-2201 (M1), N-(5-hydroxypentyl)-MAM-2201 (M13), and hydroxy-M13 (M14) were the major metabolites. Based on an enzyme kinetics study using human cDNA-expressed cytochrome P450 (CYP) enzymes and an immunoinhibition study using selective CYP antibodies in human liver microsomes, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 enzymes were responsible for MAM-2201 metabolism. The CYP3A4 enzyme played a prominent role in MAM-2201 metabolism, and CYP1A2, CYP2B6, CYP2C8, and CYP2C9 enzymes played major roles in the formation of some metabolites. MAM-2201 is extensively metabolized by multiple CYP enzymes, indicating that MAM-2201 and its metabolites should be used as markers of MAM-2201 abuse and toxicity. Graphical abstract In vitro metabolic pathways of MAM-2201 were characterized in human liver microsomes and recombinant CYPs using LC-HRMS analysis. Total 19 phase I metabolites were identified with predominant contribution of CYP3A4.

AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5'-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography-tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 µM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 µM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 µM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

Rapid and simple determination of psychotropic phenylalkylamine derivatives in urine by direct injection liquid chromatography-electrospray ionization-tandem mass spectrometry.

A direct injection liquid chromatography-electrospray ionization-tandem mass spectrometric method (LC-ESI-MS/MS) was developed and validated for the rapid and simple determination of 13 phenylalkylamine derivatives. Eight deuterium-labeled compounds were prepared for use as internal standards (ISs) to quantify the analytes. Urine samples mixed with ISs were centrifuged, filtered through 0.22 µm filters and then injected directly into the LC-ESI-MS/MS system. The mobile phase was composed of 0.2% formic acid and 2 mM ammonium formate in distilled water and 0.2% formic acid and 2 mM ammonium formate in acetonitrile. The analytical column was a Capcell Pak MG-II C(18) (150 × 2.0 mm i.d., 5 µm, Shiseido). Separation and detection of the analytes were accomplished within 10 min. The linear ranges were 5-750 ng/mL (ephedrine and fenfluramine), 10-750 ng/mL (3,4-methylenedioxyamphetamine, phendimetrazine, methamphetamine, 3,4-methylenedioxyethylamphetamine and benzphetamine), 20-750 ng/mL (norephedrine, amphetamine, phentermine and ketamine) and 30-1000 ng/mL (3,4-methylenedioxymethamphetamine and norketamine), with determination coefficients, R(2) , ≥ 0.9967. The intra-day and inter-day precisions were within 19.1%. The intra-day and inter-day accuracies ranged from -16.0 to 18.7%. The lower limits of quantification for all the analytes were lower than 26.5 ng/mL. The applicability of the method was examined by analyzing urine samples from drug abusers (n = 30).

Simultaneous qualitative and quantitative method using liquid chromatography selected reaction monitoring-triggered quantitation-enhanced data-dependent tandem mass spectrometry for the identification and classification of amphetamine-type stimulant abusers in human urine.

Amphetamine (AP) and amphetamine-type stimulants, methamphetamine (MA) and N,N-dimethylamphetamine (DMA), are known as central nervous system stimulants, and their abuse throughout the world has recently increased. Since it is difficult to physically distinguish among AP, MA and DMA, analysts may not be aware of what abusers have administered. In this study, following the detection of specific metabolites of AP, MA and DMA as biomarkers in abuser urines, a rapid and sensitive method was developed for the identification and classification of AP-type stimulants abusers. After the simple filtration of the urine samples, the samples were directly analyzed using a liquid chromatography/tandem mass spectrometry system with selected reaction monitoring (SRM)-triggered quantitation-enhanced data-dependent MS/MS (QED-MS/MS) for the simultaneous qualitative and quantitative analysis of p-hydroxy AP, p-hydroxy MA, p-hydroxy DMA, AP, MA, DMA and DMA N-oxide. The determination of p-hydroxy AP, p-hydroxy MA, AP, MA, DMA and DMA N-oxide was accurate and reproducible, with the limits of quantitation of 5 ng/mL in urine. When applied to the urine samples of suspected AP-type stimulants abusers, the abused drugs were precisely identified between MA and DMA abusers.

Gas chromatography-mass spectrometric method for the screening and quantification of illicit drugs and their metabolites in human urine using solid-phase extraction and trimethylsilyl derivatization.

A simple and rapid GC-MS method has been developed for the screening and quantification of many illicit drugs and their metabolites in human urine by using automatic SPE and trimethylsilylation. Sixty illicit drugs, including parent drugs and their metabolites that are possibly abused in Korea, can be monitored by this method. Among them, 24 popularly abused illicit drugs were selected for quantification. Very delicate optimizations were carried out in SPE, trimethylsilylation derivatization, and GC/MS to enable such remarkable achievements. Trimethylsilylated analytes were well separated within 21 min by GC-MS. In the validation results, the LOD of all the analytes were in the range of 2-75 ng/mL. The LOQ of the quantified analytes were in the range of 5-98 ng/mL. The linearity (r(2)) of the quantified analytes ranged 0.990-1.000 in each concentration range between 10 and 1000 ng/mL. The mean recoveries ranged from 62 to 126% at three different concentrations of each analyte. The inter-day and inter-person accuracies were within -13.3 approximately 14.9%, and -10.1 approximately 13.0%, respectively, and the inter-day and inter-person precisions were less than 12.9%. The method was reliable and efficient for the screening and quantification of abused illicit drugs in routine urine analysis.

Validated gas chromatographic-mass spectrometric analysis of urinary cannabinoids purified with a calcium-hardened beta-cyclodextrin polymer.

A comprehensive solid-phase extraction (SPE) technique based on the formation of an inclusion complex between beta-cyclodextrin (betaCD) and cannabinoids including Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) was developed in gas chromatographic-mass spectrometric (GC-MS) analysis. A betaCD/epichlorohydrin copolymer was prepared and then 'hardened' in aqueous solution with 0.3M CaCl(2) to yield a stable particulate copolymer, which was used as a novel SPE sorbent. An internal standard THC-COOH-d(9) was added to urine samples containing 3 cannabinoids and then purified with the hardened betaCD polymer. The cannabinoids were extracted from the hardened betaCD using tetrahydrofuran. Resulting extracts were evaporated and derivatized with MSTFA/NH(4)I/dithioerythritol (500:4:2, v/w/w) and analyzed by GC-MS in selected-ion monitoring (SIM) mode. Overall recoveries ranged from 85% to 102%, with a detection limit of 0.2 microg L(-1) for the three cannabinoids tested. The precision (% CV) and accuracy (% bias) of the assay were 1.2-5.1% and 93-111% in 0.2-50 microg L(-1) calibration range, respectively (r(2)>0.9997). Twenty actual samples positive by fluorescence polarization immunoassay were also quantitatively analyzed. The devised technique based on the calcium-hardened betaCD sorption of cannabinoids and subsequent GC-SIM/MS resulted in better selectivity and extraction efficiency than is possible using the conventional hydrophobicity-based SPE methods.

Simultaneous determination of amphetamine-type stimulants and cannabinoids in fingernails by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometric (GC-MS) method was developed and validated for the simultaneous detection and quantification of four amphetamine-type stimulants (amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA)) and two cannabinoids (Delta9-tetrahydrocannabinol (Delta9-THC) and 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH)) in fingernails. Fingernail clippings (30 mg) were washed with distilled water and methanol, and then incubated in 1.0 M sodium hydroxide at 95 degrees C for 30 min. The compounds of interest were isolated by liquid-liquid extraction followed by derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) at 70 degrees C for 15 min. The derivatized compounds were analyzed by GC-MS in the selective ion monitoring (SIM) mode. The linear ranges were 0.1-15.0 ng/mg for AP, 0.2-15.0 ng/mg for MDA, Delta9-THC and THCCOOH, and 0.2-30.0 ng/mg for MA and MDMA, with good correlation coefficients (r2 > 0.9991). The intra-day, inter-day, and inter-person precisions were within 10.6%, 6.3%, and 5.3%, respectively. The intra-day, inter-day and inter-person accuracies were between -6.1 and 5.0%, -6.2 and 5.7%, and -6.4 and 5.6%, respectively. The limits of detection (LODs) and quantification (LOQs) for each compound were lower than 0.056 and 0.2 ng/mg, respectively. The recoveries were in the range of 74.0-94.8%. Positive GC-MS results were obtained from specimens of nine suspected MA or cannabis abusers. The concentration ranges of MA, AP, and THCCOOH were 0.10-1.41, 0.12-2.64, and 0.20 ng/mg, respectively. Based on these results, the method proved to be effective for the simultaneous qualification and quantification of amphetamine-type stimulants and cannabinoids in fingernails.

Simultaneous determination of methamphetamine, 3,4-methylenedioxy-N-methylamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, N,N-dimethylamphetamine, and their metabolites in urine by liquid chromatography-electrospray ionization-tandem mass spectrometry.

A liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method was developed and validated for the simultaneous detection and quantification of seven amphetamine derivatives (amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxy-N-amphetamine (MDA), 3,4-methylenedioxy-N-methamphetamine (MDMA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), N,N-dimethylamphetamine (DMA), and N,N-dimethylamphetamine-N-oxide (DMANO)) in human urine. Seven deuterium-labeled compounds were prepared for use as internal standards to quantify the analytes. One milliliter of urine was combined with 1 mL of 0.2 M sodium carbonate buffer solution (pH 9.0) before solid phase extraction (SPE). An Oasis HLB SPE column followed by chromatographic separation on a Capcell Pak C18 MG-II column (150 x 2.0 mm I.D., 5 microm) and electrospray mass spectrometry with multiple reaction monitoring were used for selective and sensitive detection. The use of ammonium formate (5 mM, pH adjusted to 4.0 with formic acid, Solvent A) and acetonitrile (Solvent B) as the mobile phase at a flow rate of 230 microL/min was found to be the most effective for the separation. The linear ranges were 5.0-1000 ng/mL for AP, MDA, MDMA, MDEA, DMA, and DMANO and 10.0-1000 ng/mL for MA, with good correlation coefficients (r2 > 0.996). The intra-day, inter-day, and interperson precisions were within 14.6%, 12.1% and 15.5%, respectively. The intra-day, inter-day, and interperson accuracies were between -11.6 and 9.0%, -7.9 and 2.3%, and -13.2 and 4.3%, respectively. The limits of detection (LODs) for each analytical compound were lower than 1.95 ng/mL. The recovery ranged from 72.3 to 103.3%. The applicability of the developed method was examined by analyzing several urine samples from confirmed drug abusers.

Simultaneous Determination of Amphetamine-Related New Psychoactive Substances in Urine by Gas Chromatography-Mass Spectrometry.

Despite the efforts to prevent the spread of new psychoactive substances (NPS) such as synthetic amphetamine derivatives, it is apparent that newer types of NPS are still emerging on the market in recent years. Due to high potential for their abuse, reliable analytical methods are required to determine these substances in biological samples. The objective of this study was to develop and validate the gas chromatography-mass spectrometric (GC-MS) method for the simultaneous determination of 13 amphetamine-related NPS (amphetamine; AP, 4-fluoroamphetamine; 4FA, methamphamine; MA, 4-fluoromethamphetamine; 4FMA, 4-chloroamphetamine; 4CA, para-methoxyamphetamine; PMA, 4-chloromethamphetamine; 4CMA, 6-(2-aminopropyl)benzofuran; 6APB, 4-methylenedioxyamphetamine; MDA, para-methoxymethamphetamine; PMMA, 6-(2-methylaminopropyl)benzofuran; 6MAPB, 3,4-methylenedioxymethamphetamine; MDMA, 5,6-methylenedioxy-2-aminoindane; MDAI) in urine. The analytes were extracted at pH 7.4 by liquid-liquid extraction prior to their trifluoroacetyl derivatives and then analyzed by GC-MS. The validation parameters included selectivity, linearity, lower limits of quantification (LLOQ), intra and interday precision and accuracy, recovery and stability. The linear ranges were 2-100 ng/mL for AP, 4FA, 4FMA, 4CA, PMA, 6APB, MDA, and MDAI, 2-250 ng/mL for 4CMA, PMMA, and 6MAPB and 25-1,000 ng/mL for MA and MDMA, with acceptable coefficients of determination (r2 > 0.9963). The intra and interday precision were within 11.9 and 12.5%, while the intra and interday accuracies ranged from -10.6% to 13.0% and -11.0% to 6.8% for the nominal concentration at all studied levels, respectively. The LLOQs for each analyte were 2.0-25 ng/mL. The recoveries ranged from 69.3% to 96.4%. The short- and long-term variations of the analytes in urine were lower than 8.5 and 12.7%, indicating that the analytes are stable at least for 16 h at room temperature and for 7 days at 4°C, respectively. The applicability of the method was examined by analyzing urine samples from drug abusers and was determined to be effective for detecting multiple drug use.

Inhibition of cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferases by MAM-2201 in human liver microsomes.

MAM-2201, a synthetic cannabinoid, is a potent agonist of the cannabinoid receptors and is increasingly used as an illicit recreational drug. The inhibitory effects of MAM-2201 on major drug-metabolizing enzymes such as cytochrome P450s (CYPs) and uridine 5'-diphospho-glucuronosyltransferases (UGTs) have not yet been investigated although it is widely abused, sometimes in combination with other drugs. We evaluated the inhibitory effects of MAM-2201 on eight major human CYPs (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six UGTs (UGTs 1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) of pooled human liver microsomes; we thus explored potential MAM-2201-induced drug interactions. MAM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, and UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, with K i values of 5.6, 5.4 and 5.0 µM, respectively. MAM-2201 exhibited mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-de-ethylation with K i and k inact values of 1.0 µM and 0.0738 min-1, respectively. In human liver microsomes, MAM-2201 (50 µM) negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7. Based on these in vitro results, we conclude that MAM-2201 has the potential to trigger in vivo pharmacokinetic drug interactions when co-administered with substrates of CYP2C8, CYP2C9, CYP3A4, and UGT1A3.

Rapid analysis of drugs of abuse and their metabolites in human urine using dilute and shoot liquid chromatography-tandem mass spectrometry.

Liquid chromatography-tandem mass spectrometric method for analysis of 113 abuse drugs and their metabolites in human urine was developed and validated. A simple sample clean-up procedure using the "dilute and shoot" approach, followed by reversed phase separation, provided a fast and reliable method for routine analysis. Drugs were separated in a Capcell Pak MG-III C18 column using a gradient elution of 1 mM ammonium formate with 0.1% formic acid in water and acetonitrile. The total time for analysis was 32 min. The multiple reaction monitoring mode using two transitions (e.g., quantifier and qualifier) was optimized for both identification and determination. The calibration curves for each analyte were linear over the concentration ranges of 1-100, 5-100, or 10-100 ng/mL using 400 µL of human urine sample with the coefficient of determination above 0.9921. The coefficient of variation and accuracy for the intra- and inter-assays of the tested drugs at three QC levels were 1.1-14.6 and 86.7-106.8%, respectively. The present method was successfully applied to the analysis of forensic urine samples obtained from 17 drug abusers. This method is useful for the rapid and accurate determination of multiple drug abuse with a small amount of urine in forensic and clinical toxicology.

Determination of phentermine, N-hydroxyphentermine and mephentermine in urine using dilute and shoot liquid chromatography-tandem mass spectrometry.

Nonmedical use of prescription stimulants such as phentermine (PT) has been regulated by law enforcement authorities due to its euphorigenic and relaxing effects. Due to high potential for its abuse, reliable analytical methods were required to detect and identify PT and its metabolite in biological samples. Thus a dilute and shoot liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for simultaneous determination of PT, N-hydroxyphentermine (NHOPT) and mephentermine (MPT) in urine. A 5µL aliquot of diluted urine was injected into the LC-MS/MS system. Chromatographic separation was performed by reversed-phase C18 column with gradient elution for all analytes within 5min. Identification and quantification were based on multiple reaction monitoring (MRM) detection. Linear least-squares regression with a 1/x(2) weighting factor was used to generate a calibration curve and the assay was linear from 50 to 15000ng/mL (PT and MPT) and 5 to 750ng/mL (NHOPT). The intra- and inter-day precisions were within 8.9% while the intra- and inter-day accuracies ranged from -6.2% to 11.2%. The limits of quantification were 3.5ng/mL (PT), 1.5ng/mL (NHOPT) and 1.0ng/mL (MPT). Method validation requirements for selectivity, dilution integrity, matrix effect and stability were satisfied. The applicability of the developed method was examined by analyzing urine samples from drug abusers.

In vitro metabolism of a novel synthetic cannabinoid, EAM-2201, in human liver microsomes and human recombinant cytochrome P450s.

In vitro metabolism of a new synthetic cannabinoid, EAM-2201, has been investigated with human liver microsomes and major cDNA-expressed cytochrome P450 (CYP) isozymes using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Incubation of EAM-2201 with human liver microsomes in the presence of NADPH resulted in the formation of 37 metabolites, including nine hydroxy-EAM-2201 (M1-M9), five dihydroxy-EAM-2201 (M10-M14), dihydrodiol-EAM-2201 (M15), oxidative defluorinated EAM-2201 (M16), two hydroxy-M16 (M17 and M18), three dihydroxy-M16 (M19-M21), N-dealkyl-EAM-2201 (M22), two hydroxy-M22 (M23 and M24), dihydroxy-M22 (M25), EAM-2201 N-pentanoic acid (M26), hydroxy-M26 (M27), dehydro-EAM-2201 (M28), hydroxy-M28 (M29), seven dihydroxy-M28 (M30-M36), and oxidative defluorinated hydroxy-M28 (M37). Multiple CYPs, including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2J2, 3A4, and 3A5, were involved in the metabolism of EAM-2201. In conclusion, EAM-2201 is extensively metabolized by CYPs and its metabolites can be used as an indicator of EAM-2201 abuse.

Simultaneous Measurement of Serum Chemical Castration Agents and Testosterone Levels Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Chemical castration involves administration of drugs to prevent pathological sexual behavior, reduce abnormal sexual drive and treat hormone-dependent cancers. Various drugs have been used for chemical castration; however, substantial interindividual variability and side effects are often observed. In this study, we proposed a useful monitoring method for the application of chemical castration agents using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS). Testosterone, cyproterone acetate, medroxyprogesterone, goserelin acetate, leuprolide acetate and triptorelin acetate were analyzed by UPLC-MS-MS. The target drugs were extracted from serum samples by double protein precipitation using methanol. Testosterone-1,2-d2 and buserelin acetate were used as internal standards. Parameters of analytical performance were evaluated, including imprecision, linearity, ion suppression and detection capabilities. Testosterone measurements were compared with the results of immunoassays. Serum specimens from 51 subjects who underwent chemical castration were analyzed. All drugs and testosterone were well extracted and separated using our method. The method was essentially free from potential interferences and ion suppression. Within-run and between-run imprecision values were <15%. The lower limits of quantification were 0.125 and 0.5-1.0 ng/mL for testosterone and other drugs, respectively. Good correlations with pre-existing immunoassays for testosterone measurement were observed. Sera from subjects who underwent androgen deprivation therapy showed variable levels of drugs. We successfully developed a UPLC-MS-MS-based monitoring method for chemical castration. The performance of our method was generally acceptable. This method may provide a novel monitoring strategy for chemical castration to enhance expected effects while reducing unwanted side effects.

Gas chromatography-high-resolution mass spectrometric method for determination of methamphetamine and its major metabolite amphetamine in human hair.

Gas chromatography-high-resolution mass spectrometric (GC-HRMS) method is presented for the qualitative and quantitative analysis of methamphetamine (MA) and its major metabolite, amphetamine (AMP), in human hair. The method procedure involves decontamination of hair with distilled water and acetone, acidic hydrolysis and extraction in the presence of the internal standard, and GC-HRMS selective ion monitoring (SIM) analysis. The limits of detection (LOD) were 9 pg/mg for MA and 21 pg/mg for AMP using a 30-mg hair sample, and the SIM responses were linear with coefficients of correlation ranged from 0.9998 to 0.9999. The recoveries were found to be 91.1-92.3%. By using HRMS (resolution of 5000), detection sensitivity is improved because of the elimination of the biological background, and the LODs for MA and AMP were 2.4-4.4 times lower than those of low-resolution MS. The GC-HRMS method was successfully applied to the analysis of cosmetically treated hair, which is difficult to analyze with the conventional method.

Simultaneous determination of cannabidiol, cannabinol, and delta9-tetrahydrocannabinol in human hair by gas chromatography-mass spectrometry.

An analytical method was developed for evaluating the cannabidiol (CBD), cannabinol (CBN), and delta9-tetrahydrocannabinol (delta9-THC) level in human hair using gas chromatography-mass spectrometry (GC-MS). Hair samples (50 mg) were washed with isopropyl alcohol and cut into small fragments (< 1 mm). After adding a deuterated internal standard, the hair samples were incubated in 1.0 M NaOH for 10 min at 95 degrees C. The analytes from the resulting hydrolyzed samples were extracted using a mixture of n-hexane-ethyl acetate (75:25, v/v). The extracts were then evaporated, derivatized, and injected into the GC-MS. The recovery ranges of CBD, CBN, and delta9-THC at three concentration levels were 37.9-94.5% with good correlation coefficients (r2 >0.9989). The intra-day precision and accuracy ranged from -9.4% to 17.7%, and the inter-day precision and accuracy ranged from -15.5% to 14.5%, respectively. The limits of detection (LOD) for CBD, CBN, and delta9-THC were 0.005, 0.002, and 0.006 ng/mg, respectively. The applicability of this method of analyzing the hair samples from cannabis abusers was demonstrated.

Rapid determination of benzodiazepines, zolpidem and their metabolites in urine using direct injection liquid chromatography-tandem mass spectrometry.

Benzodiazepines and zolpidem are generally prescribed as sedative, hypnotics, anxiolytics or anticonvulsants. These drugs, however, are frequently misused in drug-facilitated crime. Therefore, a rapid and simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for identification and quantification of benzodiazepines, zolpidem and their metabolites in urine using deuterium labeled internal standards (IS). Urine samples (120 µL) mixed with 80 µL of the IS solution were centrifuged. An aliquot (5 µL) of the sample solution was directly injected into the LC-MS/MS system for analysis. The mobile phases consisted of water and acetonitrile containing 2mM ammonium trifluoroacetate and 0.2% acetic acid. The analytical column was a Zorbax SB-C18 (100 mm × 2.1 mm i.d., 3.5 µm, Agilent). The separation and detection of 18 analytes were achieved within 10 min. Calibration curves were linear over the concentration ranges of 0.5-20 ng/mL (zolpidem), 1.0-40 ng/mL (flurazepam and temazepam), 2.5-100 ng/mL (7-aminoclonazepam, 1-hydroxymidazolam, midazolam, flunitrazepam and alprazolam), 5.0-200 ng/mL (zolpidem phenyl-4-carboxylic acid, α-hydroxyalprazolam, oxazepam, nordiazepam, triazolam, diazepam and α-hydroxytriazolam), 10-400 ng/mL (lorazepam and desalkylflurazepam) and 10-100 ng/mL (N-desmethylflunitrazepam) with the coefficients of determination (r(2)) above 0.9971. The dilution integrity of the analytes was examined for supplementation of short linear range. Dilution precision and accuracy were tested using two, four and ten-folds dilutions and they ranged from 3.7 to 14.4% and -12.8 to 12.5%, respectively. The process efficiency for this method was 63.0-104.6%. Intra- and inter-day precisions were less than 11.8% and 9.1%, while intra- and inter-day accuracies were less than -10.0 to 8.2%, respectively. The lower limits of quantification were lower than 10 ng/mL for each analyte. The applicability of the developed method was successfully verified with human urine samples from drug users (n=21). Direct urine sample injection and optimized mobile phases were introduced for simple sample preparation and high-sensitivity with the desired separation.

Estimation of measurement uncertainty for the quantification of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid and its glucuronide in urine using liquid chromatography-tandem mass spectrometry.

Recently, the estimation of the measurement uncertainty has become a significant issue in the quality control of forensic drug testing. In the present study, the uncertainty of the measurement was calculated for the quantification of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) and its glucuronide conjugate (THC-COOH-glu) in urine using liquid chromatography-tandem mass spectrometry. The procedure was based on liquid-liquid extraction of a volume of urine (800 µL) with ethyl acetate. The sources of uncertainty were identified and classified into four major categories as follows: standard preparation, calibration curve, method precision and bias. The overall contribution of combined standard uncertainty on THC-COOH increased in the order of standard preparation (0.9%), method precision (10.4%), calibration curve (30.3%) and bias (58.4%) and, while calibration curve (53.0%) and bias (40.4%) gave the bigger contributions to the combined standard uncertainty for THC-COOH-glu than method precision and standard preparation, which accounted for 6.3 and 0.3%, respectively. The reliability of a measurement was expressed by stating the expanded uncertainty of the measurement result at 95% confidence level. The concentrations of THC-COOH and THC-COOH-glu in the urine sample with their expanded uncertainties were 10.20 ± 1.14 ng/mL and 25.42 ± 5.01 ng/mL, respectively.

Reference ranges for urinary levels of testosterone and epitestosterone, which may reveal gonadal function, in a Korean male population.

Cannabis, or marijuana, the most commonly used illicit drug in the world, has been shown to be responsible for suppressing the production and secretion of androgens, particularly testosterone. However, despite such findings in animals, the chronic effects of marijuana use on human endocrine systems have proved to be inconsistent. Here, we investigated the reference ranges of urinary levels of testosterone (T) and epitestosterone (E) as well as their metabolic ratio of T/E in a Korean male population (n=337), which would enable an evaluation of abnormal changes in steroid metabolism induced by habitually administered cannabis. The T/E ratio was significantly decreased in the marijuana group (n=18), while the urinary testosterone concentrations were also tended to decrease. This study is the first to provide data for the reference values of two urinary androgens and T/E values among control Korean males, and, furthermore, suggests that the T/E ratio, though not testosterone levels, might be used to understand the suppression of human male gonadal function affected by smoking marijuana.

Stereoselectivity in the cytochrome P450-dependent N-demethylation and flavin monooxygenase-dependent N-oxidation of N,N-dimethylamphetamine.

N,N-dimethylamphetamine (DMA), a methamphetamine (MA) analog, is known as a weak central nervous system stimulant. As DMA possesses a chiral center, we investigated the enantioselective formation of N,N-dimethylamphetamine N-oxide (DMANO) and MA from DMA using human liver microsomes, recombinant cytochrome P450 (CYP) 2D6, and flavin monooxygenases (FMO) 1 and 3. d-DMA was preferentially metabolized to MA, whereas l-DMA was more rapidly transformed to DMANO in human liver microsomes. CYP2D6 showed a preference for catalyzing N-demethylation of d-DMA, and the intrinsic clearance (Clint) ratio of d-isomer to l-isomer was 1.41. FMO1 catalyzed the formation of slightly less d-DMANO than l-DMANO, and the Clint ratio of the D- to L-isomer was 0.78. The reverse was observed for the formation of DMANO by FMO3. However, given the minor contribution of FMO3 compared with FMO1, it would not affect the overall enantioselective formation of DMANO in human liver microsomes. Enantioselectivities in the formation of MA and DMANO in human liver microsomes were consistent with those of CYP2D6 and FMO1, respectively.