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In this study, an automated online micro-solid-phase extraction (μSPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection of metabolites of cannabidiol (CBD), Δ

A liquid chromatography–tandem mass spectrometry (LC–MS-MS) method was developed for 25 antipsychotic drugs and their metabolite in urine for monitoring medication compliance of mentally disordered criminals on probation. Target compounds were extracted with a solid-phase extraction technique using a newly developed hydrophilic-lipophilic balanced sorbent to remove interferences and minimize the matrix effect (ME). Extracted sample was injected into the LC–MS-MS with an electrospray ionization source in positive mode and multiple-reaction monitoring mode. The analytes were separated and detected within 10 minutes using a reversed-phase column with a gradient elution method using 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phase. The validation parameters were evaluated as follows: selectivity, limit of detection, lower limit of quantification (LLOQ), linearity, accuracy and precision, stability, dilution integrity, recovery (RE), ME and process efficiency (PE). The LLOQs were 0.1 to 2.0 ng/mL, and determination coefficients of the calibration curve were above 0.9943 over the concentration ranges. The intra-and inter-day accuracy ranged from −10.4% to 9.9% and from −9.6% to 9.4%, while the intra-and inter-day precision were within 10.7% and 9.9%. The bench-top and long-term stability ranged from 92.1% to 109.5% and 88.7% to 111.6%, respectively. The reproducibility of auto-sampler stability was

Alcohol use disorder (AUD) is a major risk factor for numerous social concerns worldwide. In the Republic of Korea, the court imposes compulsory medication treatment on criminals diagnosed with AUD who are on probation. The purpose of the treatment is to reduce the number of repeat offenses committed by AUD-afflicted criminals. In this study, a method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine ethyl glucuronide (EtG), acamprosate (ACAM), naltrexone (NTX), and 6β-naltrexol (6BNT) in urine was developed and validated to monitor the medication compliance and alcohol use of probationers diagnosed with AUD. A zirconia-based hybrid solid-phase extraction technique was introduced to increase the sensitivity toward target analytes having significantly differing pKa values while minimizing the matrix effect of the urine sample. The pretreated urine samples were analyzed by LC-MS/MS performed in a polarity-switching electrospray ionization mode via a three-period multiple-reaction monitoring method. All the target analytes were separated and detected within 6.5 min with an Xselect HSS T3 column and gradient elution using water containing 0.02% formic acid and methanol as the mobile phase. The lower limits of quantification of EtG, ACAM, NTX, and 6BNT were 10.0, 4.0, 0.4, and 0.2 ng mL

Since cannabis and amphetamine-type stimulants (ATS) are drugs of abuse used alone and concurrently worldwide, it is important to analyze them simultaneously. However, there are no reports of analytical method for the simultaneous extraction of these compounds and metabolites in hair samples of suspected drug abusers, due to the different chemical properties of acidic and lipophilic cannabinoids, and basic and hydrophilic ATS. We developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the quantification of cannabidiol (CBD), cannabinol (CBN), (-)-trans-Δ

Sample preparation is an important step in the isolation of target compounds from complex matrices to perform their reliable and accurate analysis. Hair samples are commonly pulverized or processed as fine cut, depending on preference, before extraction by techniques such as solid-phase extraction (SPE), liquid&ndash, liquid extraction, and other methods. In this study, a method based on hybrid solid-phase extraction (hybridSPE) and gas chromatography&ndash, mass spectrometry (GC&ndash, MS) was developed and validated for the determination of methamphetamine (MA) and amphetamine (AP) in hair. The hair samples were mechanically pulverized after washing with de-ionized water and acetone. The samples were then sonicated in methanol at 50 &deg, C for 1 h and centrifuged at 50,000&times, g for 3 min. The supernatants were transferred onto the hybridSPE cartridge and extracted using 1 mL of 0.05 M methanolic hydrogen chloride. The combined solutions were evaporated to dryness, derivatized using pentafluoropropionic anhydride, and analyzed by GC&ndash, MS. Excellent linearity (R2 &gt, 0.9998) was achieved in the ranges of 0.05&ndash, 5.0 ng/mg for AP and 0.1&ndash, 10.0 ng/mg for MA. The recovery was 83.4&ndash, 96.8%. The intra- and inter-day accuracies were &minus, 9.4% to 5.5% and &minus, 5.1% to 3.1%, while the intra- and inter-day precisions were within 8.3% and 6.7%, respectively. The limits of detections were 0.016 ng/mg for AP and 0.031 ng/mg for MA. The validated hybridSPE method was applied to dyed hair for MA and AP extraction and compared to a methanol extraction method currently being used in our laboratory. The results showed that an additional hybridSPE step improved the recovery by 5.7% for low-concentration quality control (QC) samples and by 24.1% for high-concentration QC samples. Additionally, the hybridSPE method was compared to polymeric reversed-phase SPE methods, and the absolute recoveries for hybridSPE were 50% and 20% greater for AP (1.5 ng/mg) and MA (3.0 ng/mg), respectively. In short, the hybridSPE technique was shown to minimize the matrix effects, improving GC&ndash, MS analysis of hair. Based on the results, the proposed method proved to be effective for the selective determination of MA and AP in hair samples.

Although urine and blood samples have been conventionally used for testing zolpidem (ZPD), a sedative-hypnotic, these matrices have limited application because they have a relatively short detection period and can be used only in case of recent drug exposure. Therefore, it is necessary to use an alternative biological sample to obtain the evidence of ZPD misuse. Herein, a liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for the determination of ZPD and its metabolites, zolpidem phenyl-4-carboxylic acid (ZPCA) and zolpidem 6-carboxylic acid (ZCA), in hair to resolve the above-mentioned problems. Mechanical pulverization of hair, methanol extraction with sonication, and the zirconia-based hybrid solid–phase extraction technique were used for obtaining improved extraction efficiency and effective sample purification. The treated hair sample was analyzed using the LC–MS/MS method with the electrospray ionization source in positive and multiple-reaction monitoring modes. The target analytes were separated and detected within 8 min using an Xselect HSS T3 column. Gradient elution was performed using 5 mM ammonium formate and acetonitrile. The lower limit of quantification of ZPD, ZPCA, and ZCA were 1.0, 0.5, and 1.0 pg mg−1, respectively. The calibration ranges were 1.0–1000.0 pg mg−1 for ZPD, 0.5–200.0 pg mg−1 for ZPCA, and 1.0–200.0 pg mg−1 for ZCA, with the determination coefficients (r2 ≥ 0.9986). The intraday accuracy and precision ranged from −7.1 to 9.0% and within 6.5%, respectively, and the interday accuracy and precision ranged from −6.1 to 7.9% and within 5.4%, respectively. The recovery, matrix effect, and process efficiency were 65.2–96.6%, 64.6–106.5%, and 44.3–100.5%, respectively, with the relative standard deviation of 4.0–5.0%. The developed method was successfully applied to analyze 13 forensic hair samples of ZPD abusers, and the concentration ratios of ZPD and its two main metabolites (ZPCA and ZCA) in the ZPD-positive samples were also presented. These results revealed that ZPCA and ZCA were not easily incorporated into hair, and demonstrated that their analysis in hair samples requires the employed method to have picogram-level sensitivity. Therefore, the developed method was suitable for simultaneous analysis of ZPD, ZPCA, and ZCA in hair samples, and it could provide clear evidence for illegal ZPD administration, including ZPD-facilitated sexual assault.

Since cannabis and amphetamine-type stimulants (ATS) are drugs of abuse used alone and concurrently worldwide, it is important to analyze them simultaneously. However, there are no reports of analytical method for the simultaneous extraction of these compounds and metabolites in hair samples of suspected drug abusers, due to the different chemical properties of acidic and lipophilic cannabinoids, and basic and hydrophilic ATS. We developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the quantification of cannabidiol (CBD), cannabinol (CBN), (−)-trans-Δ9-tetrahydrocannabinol (THC), THC metabolites such as 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) and THC-COOH-glucuronide (THC-COOH-glu), and six ATS of amphetamine, methamphetamine, phentermine, methylenedioxyamphetamine, methylenedioxymethamphetamine, and methylenedioxyethylamphetamine in the least amounts of human hair samples. The pulverized hair samples (10 mg) were extracted with 1 mL of 0.5% formic acid in methanol three times, and the supernatants were evaporated in a stream of nitrogen gas. The residue was dissolved and the aliquot was analyzed by LC-HRMS using positive electrospray ionization and the parallel reaction monitoring mode. The lower limits of quantitation were 0.1 pg mg−1 for THC-COOH and THC-COOH-glu, 4 pg mg−1 for CBD, CBN, and THC, and 10 pg mg−1 for the six ATS. Linearity, accuracy, precision, matrix effect, recovery, and post-preparation stability for the 11 analytes were fully validated. The present method was successfully applied to the determination of 11 analytes in hair samples of 10 suspected drug abusers.

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.

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.

A rapid and simple gas chromatography–mass spectrometry (GC–MS) method was developed and validated for the simultaneous detection and quantification of five psychotropic phenylalkylamines (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and norketamine) in toenails. After external decontamination, nail clippings were mechanically pulverized with a bead mill and then incubated in methanol under ultrasonication at 50°C for 1 h. The resulting solutions were evaporated to dryness, derivatized, and analyzed by GC–MS. The intra- and inter-day precisions were within 10.7% and 13.9%, respectively. The intra- and inter-day accuracies were −4.2% to 5.0% and −2.4% to 8.4%, respectively. Limits of detection and quantification for each analyte were lower than 0.024 and 0.08 ng/mg, respectively. The recoveries were in the range of 80.6–87.5%. The results indicated that the proposed method is a simple, rapid, accurate, and precise for quantification of five phenylalkylamines in nails. The method was successfully applied to the simultaneous detection and quantification of phenylalkylamines in nail samples of possible drug abusers.

We described an estimation of measurement uncertainty in quantitative analysis of 11-nor-9-carboxy--tetrahydrocannabinol (THC-COOH), the metabolite of -tetrahydrocannabinol, in hair samples by using the bead-assisted liquid-liquid extraction and gas chromatography-tandem mass spectrometric (GC-NCI-MS/MS) detection. Traceability of measurement was established through the use of reference materials, calibrated volumetric tubes, volume measuring devices, and measuring instruments. The analytical results were compared and the different contributions to the uncertainty were evaluated. Inter-day variation was performed by using statistical analysis of several indicative factors. Measurement uncertainty associated with the analyte in real forensic hair samples were estimated using QC data. The major factor of contribution to combined standard uncertainty was inter-day repeatability, while those associated with preparation of analytical standard and also sample of weight were insignificant considering the degree of contribution. Relative uncertainty of relative extended standard uncertainty divided into the measured concentration of the analyte was 17% in a hair sample. The uncertainty of result evaluation will be invaluable to improve quality of the analysis.

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.

For the evaluation of our laboratory`s 11-nor--tetrahydrocannabinol-9-carboxylic acid (THCCOOH) urinalysis test, THCCOOH urinalysis test was carried out with Certified Reference Material (CRM). The used CRM was THCCOOH in freeze-dried urine produced by NIST as Standard Reference Material 1507b. Comparing the estimated value of our laboratory with CRM, the results was coincided in the confidence level of approximately 95%.

A fast, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantitation of propofol glucuronide in human hair has been developed and validated. Propofol glucuronide was extracted from 10mg of hair using a simple methanol extraction method, with recovery greater than 91% at 3 quality control samples (15, 100, 4000 pg/mg). A reversed phase column (C8) was used to analyze and the mobile phase was composed of ammonium formate and acetonitrile gradient at a flow rate of 0.2 mL/min. The lower limit of quantitation (LLOQ) was 5 pg/mg and the assay was linear to 5000 pg/mg. The intra- and inter-day precision (% CV, coefficient of variation) ranged from 1.26 to 4.50% while the accuracy (% RE, relative error) were -4.24 to 4.4%. The matrix effects were monitored at 3 different concentrations and the %CV of the results for these concentrations was less than 10.6%. Propofol glucuronide was stable during processing and analysis in human hair. The procedure was validated and applied to the analysis of hair samples in human subjects previously administered in propofol.

A hair screening method has been developed for the detection of methamphetamine using an immunoassay analyzer (AxSYM) with a fluorescence polarization immunoassay (FPIA) technique. The method consisted of washing, cutting and digesting a hair sample (5 mg) with an enzymatic digestion solution. The digested hair sample was centrifuged, and then an aliquot of the supernatant was used to conduct the screening. The results obtained from FPIA, in most cases, showed concentrations above 70.0 ng/mL of methamphetamine for hair samples that contained 0.5 ng/mg of methamphetamine, determined by gas chromatography-mass spectrometry (GC-MS). The percent sensitivity, defined as the true positive rate of screened and confirmed results, and the percent specificity, defined as the true negative rate of screened and confirmed results, of the FPIA screening method were 100.0 and 96.7% (false positive rate of 3.3%), respectively, when the threshold level for FPIA analysis was set at 70.0 ng/mL (n = 60).The correlation coefficient (r) for the linear relationship between FPIA and GC-MS results was 0.91 in real hair samples. The recommended amount of hair sample was found to be 5.0 mg for FPIA screening analysis when the concentration of methamphetamine in hair samples determined by GC-MS was found to be more than 0.5 ng/mg. The method developed in this study was reliable and effective for the screening of methamphetamine in routine hair analysis.

A rapid and simple gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the simultaneous detection and quantification of five psychotropic phenylalkylamines (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and norketamine) in toenails. After external decontamination, nail clippings were mechanically pulverized with a bead mill and then incubated in methanol under ultrasonication at 50°C for 1 h. The resulting solutions were evaporated to dryness, derivatized, and analyzed by GC-MS. The intra- and inter-day precisions were within 10.7% and 13.9%, respectively. The intra- and inter-day accuracies were -4.2% to 5.0% and -2.4% to 8.4%, respectively. Limits of detection and quantification for each analyte were lower than 0.024 and 0.08 ng/mg, respectively. The recoveries were in the range of 80.6-87.5%. The results indicated that the proposed method is a simple, rapid, accurate, and precise for quantification of five phenylalkylamines in nails. The method was successfully applied to the simultaneous detection and quantification of phenylalkylamines in nail samples of possible drug abusers.

A gas chromatography-negative ion chemical ionization tandem mass spectrometric (GC-NCI-MS/MS) method was developed and validated for the determination of 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in human hair. After decontamination, hair samples were weighed (25mg), mechanically pulverized with a bead mill, and incubated in 0.7 mL of 1.0M sodium hydroxide at 95 °C for 30 min. Bead-assisted liquid-liquid extraction was performed with n-hexane:ethyl acetate (9:1, v/v), a method developed in our laboratory. The extract was evaporated to dryness, derivatized with pentafluoropropanol and pentafluoropropionic anhydride, and analyzed by GC-MS/MS in the negative ion chemical ionization mode using methane as the reagent gas. The linear ranges were 0.05-10.0 pg/mg for THC-COOH with the coefficient of determination (r(2) = 0.9976). The intra-day and inter-day precisions were within 1.7 and 13.8%, respectively. The intra-day and inter-day accuracies were -4.8 to 10.0% and -3.9 to 3.8%, respectively. The limit of detection and quantification were 0.015 and 0.05 pg/mg, respectively. The recoveries were in the range of 79.4-87.1%. The results indicate that the proposed method is simple, rapid, accurate, and precise for determination of THC-COOH in hair. The method identified THC-COOH in hair specimens from suspected marijuana abusers.

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.

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 (r20.9991). 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.