Determination of urinary metabolites of cannabidiol, Δ8-tetrahydrocannabinol, and Δ9-tetrahydrocannabinol by automated online µSPE-LC-MS/MS method.

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), Δ8-tetrahydrocannabinol (Δ8-THC), and Δ9-tetrahydrocannabinol (Δ9-THC), particularly 7-carboxy- cannabidiol (7-COOH-CBD), 11-nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THCCOOH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH), and 11-nor-9-carboxy-Δ9- tetrahydrocannabinol-glucuronide (Δ9-THCCOOH-glu) in urine. An instrument top sample preparation (ITSP) cartridge was introduced to increase the sensitivity toward analytes and decrease the matrix effect of the urine. LC-MS/MS analysis was performed in the multiple-reaction monitoring mode, and the analytes were separated using an Acquity UPLC HSS T3 (2.1 × 100 mm, 1.8 µm) column and gradient elution with water containing 0.05 % acetic acid and methanol as the mobile phase. The calibration range was 0.5-200 ng/mL for all the analytes, with a correlation coefficient (r) of ≥0.996 and a weighting factor of 1/x2. The limits of detection for 7-COOH-CBD, Δ8-THCCOOH, Δ9-THCCOOH, and Δ9-THCCOOH-glu were 0.06, 0.02, 0.03, and 0.1 ng/mL, respectively. The intra- and inter-day accuracy ranged from -8.0 to 6.2 % and -7.3 to 7.8 % with a precision of ≤7.2 % and ≤6.2 %, respectively. The method was also validated for selectivity, recovery, matrix effect, stability, and dilution integrity. The developed method was successfully applied to the analysis of 78 urine samples, and 7-COOH-CBD, Δ8-THCCOOH, Δ9-THCCOOH, and Δ9-THCCOOH-glu were detected in 54 urine samples at normalized concentrations of 1.1, 0.6-939.1, 0.9-2595.0, and 1.3-527.6 ng/mg creatinine, respectively.

Derivatization-assisted LC-MS/MS method for simultaneous quantification of endogenous gamma-hydroxybutyric acid and its metabolic precursors and products in human urine.

The accurate, precise, and robust quantification of endogenous biomarkers is a challenging task because of the presence of significantly low levels of endogenous compounds in biological samples, the absence of analyte-free matrix-matched calibrators, and sample instability due to in-vitro production or degradation of the analytes. Gamma-hydroxybutyric acid (GHB), a compound often used in drug-facilitated crimes, is a human neurotransmitter produced during both the biosynthesis and metabolism of gamma-aminobutyric acid (GABA). Evidently, proving GHB intoxication through the quantification of GHB and its metabolites in biological samples is not straightforward. This study aimed to develop a sensitive and accurate quantitative method for the simultaneous determination of endogenous GHB and its metabolic precursors and products (glutamic acid, GABA, succinic acid, 2,4-dihydroxybutyric acid, 3,4-dihydroxybutyric acid, glycolic acid, and succinylcarnitine) in human urine using LC-MS/MS. For this purpose, chemical derivatization with benzoyl chloride was employed to improve the sensitivity to glutamic acid and GABA. Synthetic urine was used to prepare calibrators, and the validity of this approach was fully demonstrated, particularly focusing on the instability issues. The validation results proved the method to be selective, sensitive, accurate, and precise, with acceptable linearity within calibration ranges. Moreover, our results regarding the in-vitro production or degradation of metabolites highlight the effects of handling and storage conditions of urine samples. Finally, this effective analytical method is expected to be useful in studying the relationship between GHB intoxication and metabolic alterations and, thus, discovering practical biomarkers for GHB ingestion.

Discrimination of Human Urine from Animal Urine Using 1H-NMR.

Urine was most commonly used biological sample in drug test. To create a false-negative test result, some drug abusers were reported to submit animal urine instead of their own. So, the purpose of this study was to compare and differentiate human from animal urine (Rat 370, Pig 12, Horse 10, Cat 8, Dog 13, Cow 10, Monkey 10) samples through the uses of quantitative 1H-NMR and to find biomarkers that can be used for the discrimination of human urine from animal urine. The 1H-NMR spectroscopy was employed and metabolomic analysis with multivariate statistics was carried out. Human urine samples and animal urine samples showed different patterns in metabolites profile and several characteristic metabolites were identified.

In Vitro Metabolism of Flucetosulfuron by Human Liver Microsomes.

To investigate herbicide metabolism, human liver microsomes were incubated with threo- and erythro-isomers of flucetosulfuron. Each isomer produced one metabolite; the metabolites were unambiguously identified as enzymatic hydrolysis products by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These metabolites were synthesized, producing white solids characterized using LC-MS/MS and nuclear magnetic resonance spectroscopy (1H and 13C). Using specific esterase inhibitors and activators, carboxylesterases and cholinesterases were demonstrated to be involved in flucetosulfuron metabolism. Under optimized metabolic conditions, the kinetic parameters for metabolite formation from threo-flucetosulfuron and erythro-flucetosulfuron were: Vmax, 151.41 and 134.38 nmol/min/mg protein, respectively; Km, 2957.37 and 2798.53 µM, respectively; and CLint, 51.20 and 48.02 µL/min/mg microsomes respectively. No significant kinetic differences were observed between the two isomers. These results indicated that the primary metabolic pathway for both flucetosulfuron isomers in human liver microsomes involves hydrolysis, catalyzed by carboxylesterase and cholinesterase.

Screening method for the detection of methamphetamine in hair using fluorescence polarization immunoassay.

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.

(1S,2S)-1-Methylamino-1-phenyl-2-chloropropane: Route specific marker impurity of methamphetamine synthesized from ephedrine via chloroephedrine.

Identification of route specific marker impurities of (S)-(+)-methamphetamine can provide us with very useful information for (S)-(+)-methamphetamine abuse criminal investigation. (1S,2S)-(+)-Chloropseudoephedrine and (1R,2S)-(-)-chloroephedrine are well known impurities of (S)-(+)-methamphetamine synthesized by metal catalyzed hydrogenation of (1R,2S)-(-)-ephedrine or (1S,2S)-(+)-pseudoephedrine. In this report (1S,2S)-1-methylamino-1-phenyl-2-chloropropane is identified as a route specific marker impurity from metal catalyzed hydrogenation method for the synthesis of (1R,2S)-(-)-ephedrine or (1S,2S)-(+)-pseudoephedrine via its chloro-derivative. (1S,2S)-1-Methylamino-1-phenyl-2-chloropropane is a ring-opening compound of cis-1,2-dimethyl-3-phenylaziridine by reacting with HCl in high temperature condition of GC inlet.

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.

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.

The impurity characteristics of methamphetamine synthesized by Emde and Nagai method.

Impurity profiling and classification of seized methamphetamine may play an important role in the interpretation of analytical results, the determination of the synthetic method employed, and the criminal investigations of drug traffic routes. Our study is focused on classifying seized methamphetamine samples according to the groups sorted by the types and quantities of impurities present in illicit methamphetamine samples. The samples (100mg) were dissolved in 2 mL of potassium phosphate buffer (pH 7.0), extracted with 200 microL of ethyl acetate under basic condition, and then analyzed by gas chromatography-mass spectrometry (GC-MS) with a DB-1 capillary column (30 m x 0.25 mm i.d., 0.25 microm). Five impurities are used as criteria for the classification of seized methamphetamine samples by Emde and Nagai method. A total of fifty-two samples of seized methamphetamine were analyzed by GC-MS and classified by five organic impurities, and then sorted into four groups, which are Nagai type, Emde Type, Undetermined I type, and Undetermined II type.