Analysis, occurrence, and consumption of substances with abuse potential in Xinjiang, China, from 2021 to 2022.

Understanding the consumption patterns of substances with abuse potential in the population is critical in combating drug crimes in the region. In recent years, wastewater-based drug monitoring has become a complementary tool worldwide. This study aimed to use this approach to understand the long-term consumption patterns of abuse potential substances in Xinjiang, China (2021-2022) and to provide more detailed and practical information on the current system. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was employed to quantify the levels of abuse potential substances in wastewater. Subsequently, the detection rate and contribution rate of the drug concentrations were evaluated through analysis. Eleven of abuse potential substances were detected in this study. The influent concentrations ranged from (0.48 ng/L) to 133.41 ng/L, with dextrorphan having the highest concentration. The highest detection frequency rates were for morphine (82 %), dextrorphan (59 %), 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (43 %), methamphetamine (36 %), and tramadol (24 %). According to a study on wastewater treatment plants (WWTPs) removal efficiency, compared to the total removal efficiency in 2021, the total removal efficiency of WWTP1, WWTP3, and WWTP4 increased in 2022, while WWTP2 decreased slightly, and WWTP5 did not change significantly. Upon examination of the use of 18 selected analytes, it was determined that methadone, 3,4-methylenedioxy methamphetamine, ketamine, and cocaine were the primary substances of abuse in the Xinjiang region. This study identified significant abuse substances in Xinjiang and identified research priorities. Future studies should consider expanding the study site to obtain a comprehensive understanding of the consumption patterns of these substances in Xinjiang.

Ultrahigh-Throughput ESI-MS: Sampling Pushed to Six Samples per Second by Acoustic Ejection Mass Spectrometry.

We present an acoustic ejection mass spectrometry (AEMS) setup for contactless electrospray ionization mass spectrometry (ESI-MS)-based sample injection at a sampling rate faster than current ESI and matrix-assisted laser desorption ionization (MALDI) techniques. For the direct transfer of samples out of 384-well plates into a modified ESI source, an open port interface (OPI) was combined with a modified acoustic droplet ejection (ADE) system. AEMS has the potential to eliminate bottlenecks known from classical MS approaches, such as speed, reproducibility, carryover, ion suppression, as well as sample preparation and consumption. This setup provided a drastically reduced transfer distance between OPI and ESI electrode for optimum throughput performance and broadens the scope of applications for this emerging technique. To simulate label-free applications of drug metabolism and pharmacokinetics (DMPK) analysis and high-throughput screening (HTS) campaigns, two stress tests were performed regarding ion suppression and system endurance in combination with minor sample preparation. The maximum sampling rate was 6 Hz for dextromethorphan and d3-dextrorphan (each 100 nM) for 1152 injections in 63 s at full width at half-maximum (FWHM) of 105 ms and a relative standard deviation (%RSD) of 7.7/7.5% without internal standard correction. Enzyme assay buffer and crude dog plasma caused signal suppression of 51/73% at %RSD of 5.7/6.7% (n = 120). An HTS endurance buffer was used for >25 000 injections with minor OPI pollution and constant signals (%RSD = 8.5%, FWHM of 177 ms ± 8.5%, n = 10 557). The optimized hardware and method setup resulted in high-throughput performance and enables further implementation in a fully automated platform for ESI-MS-based high-throughput screening.

Interrogation of CYP2D6 Structural Variant Alleles Improves the Correlation Between CYP2D6 Genotype and CYP2D6-Mediated Metabolic Activity.

The cytochrome P450 2D6 (CYP2D6) gene locus is challenging to accurately genotype due to numerous single nucleotide variants and complex structural variation. Our goal was to determine whether the CYP2D6 genotype-phenotype correlation is improved when diplotype assignments incorporate structural variation, identified by the bioinformatics tool Stargazer, with next-generation sequencing data. Using CYP2D6 activity measured with substrates dextromethorphan and metoprolol, activity score explained 40% and 34% of variability in metabolite formation rates, respectively, when diplotype calls incorporated structural variation, increasing from 36% and 31%, respectively, when diplotypes did not incorporate structural variation. We also investigated whether the revised Clinical Pharmacogenetics Implementation Consortium (CPIC) recommendations for translating genotype to phenotype improve CYP2D6 activity predictions over the current system. Although the revised recommendations do not improve the correlation between activity score and CYP2D6 activity, perhaps because of low frequency of the CYP2D6*10 allele, the correlation with metabolizer phenotype group was significantly improved for both substrates. We also measured the function of seven rare coding variants: one (A449D) exhibited decreased (44%) and another (R474Q) increased (127%) activity compared with reference CYP2D6.1 protein. Allele-specific analysis found that A449D is part of a novel CYP2D6*4 suballele, CYP2D6*4.028. The novel haplotype containing R474Q was designated CYP2D6*138 by PharmVar; another novel haplotype containing R365H was designated CYP2D6*139. Accuracy of CYP2D6 phenotype prediction is improved when the CYP2D6 gene locus is interrogated using next-generation sequencing coupled with structural variation analysis. Additionally, revised CPIC genotype to phenotype translation recommendations provides an improvement in assigning CYP2D6 activity.

Analysis of Dextromethorphan and Three Metabolites in Decomposed Skeletal Tissues by UPLC-QToF-MS: Comparison of Acute and Repeated Drug Exposures.

Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) analysis of dextromethorphan (DXM) and its metabolites-dextrorphan, 3-methoxymorphinan (3-MEM) and 3-hydroxymorphinan-in skeletal remains of rats exposed to DXM under different dosing patterns is described. Rats (n = 20) received DXM in one of four dosing patterns: acute (ACU1 or ACU2-100 or 200 mg/kg, i.p.; n = 5, respectively) or repeated (REP1 or REP2-3 doses of 25 or 50 mg/kg, i.p., 30 min apart; n = 5, respectively). Drug-free animals (n = 5) served as negative controls. Following euthanasia, the animals decomposed to skeleton outdoors. Bones were sorted by animal and skeletal element (vertebra, femur, pelvis, tibia, rib and skull), washed, air-dried and pulverized prior to dynamic methanolic drug extraction, filtration/pass-through extraction and analysis by UPLC-QToF-MS in positive electrospray ionization mode. Analyte levels (expressed as mass-normalized response ratios, RR/m) differed significantly between ACU1 and ACU2 (Mann-Whitney (MW), P < 0.05) in all skeletal elements for all analytes investigated, and between REP1 and REP2 in most skeletal elements for 3-MEM and 3-HOM, but in all skeletal elements for DXM. Between ACU1 and ACU2, and between REP1 and REP2, analyte level ratios (RRi/RRj) differed significantly (MW, P < 0.05) in 3/6 to 6/6 skeletal elements, depending on the ratios concerned, with no analyte level ratio differing significantly between both ACU1 vs ACU2 and REP1 vs REP2. Kruskal-Wallis (KW) analysis showed skeletal element to be a main effect for all analyte levels and analyte level ratios in all ACU and REP groups examined (P < 0.05). For data pooled only according to exposure pattern, KW analysis showed dose pattern to be a main effect for both analyte levels and analyte level ratios (P < 0.05). These data illustrate a dependence of these measures on dose, dose pattern and skeletal element, suggesting that some exposure patterns may be distinguished by toxicological analysis of bone.

A novel and simple LC-MS/MS quantitative method for dextromethorphan and dextrorphan in oral fluid.

Aim: To develop and validate a simple method using LC-MS/MS for determination of dextromethorphan (DXM) and dextrorphan (DT) in human oral fluid. Results: Following protein precipitation, chromatographic separation used a phenyl column with isocratic elution (1 ml/min) of 10 mM ammonium-formate buffer and acetonitrile (65:35; v/v) with 0.1% formic acid. Retention times were 2.6 min for DT and 5 min for DXM. Total run time was 7 min. The intra- and inter-assay deviations (accuracy) for DT (1-100 ng/ml) and DXM (5-1000 ng/ml) ranged from -13.6 to 8.8% and -9.6 to 5.7%, respectively. Precision variations were ≤7.5%. Matrix effect was ≤11.8%. Conclusion: This method may prove helpful for quantification of DT and DXM in oral fluid for either clinical or toxicological purposes.

UPLC-MS/MS analysis of dextromethorphan-O-demethylation kinetics in rat brain microsomes.

Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. The reaction was stopped, and the proteins were precipitated by the addition of acetonitrile, containing internal standard (d3-DXT). After centrifugation, supernatant (2 µL) was injected onto a UPLC, C18 column with gradient elution. Analytes were quantitated using triple-quadrupole MS/MS with electrospray ionization in positive ion mode. The assay, which was validated for accuracy and precision in the linear range of 0.25 nM to 100 nM DXT, has a lower limit of quantitation of 0.125 fmol on the column. Using our optimized incubation and quantitation methods, we were able to reduce the incubation volume (25 µL), microsomal protein amount (5 µg), and incubation time (20 min), compared with reported methods. The method was successfully applied to estimation of the Michaelis-Menten (MM) kinetic parameters of dextromethorphan-O-demethylase activity in the rat brain microsomes (mean ±â€¯SD, n = 4), which showed a maximum velocity of 2.24 ±â€¯0.42 pmol/min/mg and a MM constant of 282 ±â€¯62 µM. It is concluded that by requiring far less biological material and time, our method represents a significant improvement over the existing techniques for investigation of CYP2D activity in rat brain microsomes.

Use of Hybrid Capillary Tube Apparatus on 400 MHz NMR for Quantitation of Crucial Low-Quantity Metabolites Using aSICCO Signal.

Metabolites of new chemical entities can influence safety and efficacy of a molecule and often times need to be quantified in preclinical studies. However, synthetic standards of metabolites are very rarely available in early discovery. Alternate approaches such as biosynthesis need to be explored to generate these metabolites. Assessing the quantity and purity of these small amounts of metabolites with a nondestructive analytical procedure becomes crucial. Quantitative NMR becomes the method of choice for these samples. Recent advances in high-field NMR (>500 MHz) with the use of cryoprobe technology have helped to improve sensitivity for analysis of small microgram quantity of such samples. However, this type of NMR instrumentation is not routinely available in all laboratories. To analyze microgram quantities of metabolites on a routine basis with lower-resolution 400 MHz NMR instrument fitted with a broad band fluorine observe room temperature probe, a novel hybrid capillary tube setup was developed. To quantitate the metabolite in the sample, an artificial signal insertion for calculation of concentration observed (aSICCO) method that introduces an internally calibrated mathematical signal was used after acquiring the NMR spectrum. The linearity of aSICCO signal was established using ibuprofen as a model analyte. The limit of quantification of this procedure was 0.8 mM with 10 K scans that could be improved further with the increase in the number of scans. This procedure was used to quantify three metabolites-phenytoin from fosphenytoin, dextrophan from dextromethorphan, and 4-OH-diclofenac from diclofenac-and is suitable for minibiosynthesis of metabolites from in vitro systems.

Analysis of Dextromethorphan and Dextrorphan in Skeletal Remains Following Differential Microclimate Exposure: Comparison of Acute vs. Repeated Drug Exposure.

Analysis of dextromethorphan (DXM) and its metabolite dextrorphan (DXT) in skeletal remains of rats following acute (ACU, 75 mg/kg, IP, n = 10) or three repeated (REP, 25 mg/kg, IP, n = 10, 40-min interval) doses of DXM is described. Following dosing and euthanasia, rats decomposed outdoors to skeleton in two different microclimate environments (n = 5 ACU and n = 5 REP at each site): Site A (shaded forest microenvironment) and Site B (rocky substrate exposed to direct sunlight, 600 m from Site A). Two drug-free rats at each site served as negative controls. Skeletal elements (vertebrae, ribs, pelvic girdles, femora, tibiae, skulls and scapulae) were recovered, pulverized and underwent methanolic microwave assisted extraction (MAE). Extracts were analyzed by GC-MS following clean-up by solid-phase extraction (SPE). Drug levels, expressed as mass-normalized response ratios and the ratios of DXT and DXM levels (RRDXT/RRDXM) were compared between drug exposures, microclimate sites, and across skeletal elements. DXM levels differed significantly (P < 0.05) between corresponding bone elements across exposure groups (5/7-site A; 4/7-site B), but no significant differences in DXT levels were observed between corresponding elements. RRDXT/RRDXM differed significantly (P < 0.05) between corresponding bone elements across exposure groups (6/7-site A; 5/7-site B). No significant differences were observed in levels of DXM, DXT or RRDXT/RRDXM between corresponding elements from either group between sites. When data from all bone elements was pooled, levels of DXM and RRDXT/RRDXM differed significantly between exposure groups at each site, while those of DXT did not. For both exposure groups, comparison of pooled data between sites showed no significant differences in levels of DXM, DXT or RRDXT/RRDXM. Different decomposition microclimates did not impede the discrimination of DXM exposure patterns from the analyses of DXM, DXT and RRDXT/RRDXM in bone samples.

Analysis of Dextromethorphan and Dextrorphan in Skeletal Remains Following Decomposition in Different Microclimate Conditions.

The effects of decomposition microclimate on the distribution of dextromethorphan (DXM) and dextrorphan (DXT) in skeletonized remains of rats acutely exposed to DXM were examined. Animals (n = 10) received DXM (75 mg/kg, i.p.), were euthanized 30 min post-dose and immediately allowed to decompose at either Site A (shaded forest microenvironment on a grass-covered soil substrate) or Site B (rocky substrate exposed to direct sunlight, 600 m from Site A). Ambient temperature and relative humidity were automatically recorded 3 cm above rats at each site. Skeletal elements (vertebral columns, ribs, pelvic girdles, femora, tibiae, humeri and scapulae) were harvested, and analyzed using microwave assisted extraction, microplate solid phase extraction, and GC/MS. Drug levels, expressed as mass-normalized response ratios, and the ratios of DXT and DXM levels were compared across bones and between microclimate sites. No significant differences in DXT levels or metabolite/parent ratios were observed between sites or across bones. Only femoral DXM levels differed significantly between microclimate sites. For pooled data, microclimate was not observed to significantly affect analyte levels, nor the ratio of levels of DXT and DXM. These data suggest that microclimate conditions do not influence DXM and metabolite distribution in skeletal remains.

Analysis of dextromethorphan and dextrorphan in decomposed skeletal tissues by microwave assisted extraction, microplate solid-phase extraction and gas chromatography- mass spectrometry (MAE-MPSPE-GCMS).

Analysis of decomposed skeletal tissues for dextromethorphan (DXM) and dextrorphan (DXT) using microwave assisted extraction (MAE), microplate solid-phase extraction (MPSPE) and gas chromatography-mass spectrometry (GC-MS) is described. Rats (n = 3) received 100 mg/kg DXM (i.p.) and were euthanized by CO2 asphyxiation roughly 20 min post-dose. Remains decomposed to skeleton outdoors and vertebral bones were recovered, cleaned, and pulverized. Pulverized bone underwent MAE using methanol as an extraction solvent in a closed microwave system, followed by MPSPE and GC-MS. Analyte stability under MAE conditions was assessed and found to be stable for at least 60 min irradiation time. The majority (>90%) of each analyte was recovered after 15 min. The MPSPE-GCMS method was fit to a quadratic response (R(2) > 0.99), over the concentration range 10-10 000 ng⋅mL(-1) , with coefficients of variation <20% in triplicate analysis. The MPSPE-GCMS method displayed a limit of detection of 10 ng⋅mL(-1) for both analytes. Following MAE for 60 min (80 °C, 1200 W), MPSPE-GCMS analysis of vertebral bone of DXM-exposed rats detected both analytes in all samples (DXM: 0.9-1.5 µg⋅g(-1) ; DXT: 0.5-1.8 µg⋅g(-1) ).

A validated SIM GC/MS method for the simultaneous determination of dextromethorphan and its metabolites dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan in biological matrices and its application to in vitro CYP2D6 and CYP3A4 inhibition study.

Dextromethorphan is used as a probe drug for assessing CYP2D6 and CYP3A4 activity in vivo and in vitro. A SIM GC/MS method without derivatization for the simultaneous determination of dextromethorphan and its metabolites, dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan, in human plasma, urine and in vitro incubation matrix was developed and validated. Calibration curves indicated good linearity with a coefficient of variation (r) better than 0.995. The lower limit of quantitation was found to be 10 ng/mL for all analytes in all matrices. Intra-day and inter-day precision for dextromethorphan and its metabolites was better than 9.02 and 9.91%, respectively and accuracy ranged between 91.76 and 106.27%. Recovery for dextromethorphan, its metabolites and internal standard levallorphan was greater than 72.68%. The method has been successfully applied for the in vitro inhibition of metabolism of dextromethorphan by CYP2D6 and CYP3A4 using known inhibitors of CYPs such as quinidine and verapamil.

Development and validation of ELISA and GC-MS procedures for the quantification of dextromethorphan and its main metabolite dextrorphan in urine and oral fluid.

The development of a highly sensitive enzyme-linked immunosorbent assay and gas chromatography-mass spectrometry confirmation method for the detection of dextromethorphan and its major metabolite dextrorphan in urine and oral fluid is described. For the screening assay, the intraday precision was less than 8% for urine and less than 5% for oral fluid. The interday precision was less than 10% for both drugs in urine and oral fluid. For the confirmatory procedure, both inter- and intraday precision was less than 5% for both matrices. The detection limit for both methods was 1 ng/mL. The quantifying ions chosen from the full scan mass spectra were m/z 271 for dextromethorphan, m/z 329 for dextrorphan, and m/z 332 for tri-deuterated dextrorphan-d(3). A high recovery yield (> 93%) from the Quantisal oral fluid collection device was achieved, and the drugs were stable in the collection device for at least 10 days at room temperature. The extracted drugs from both matrices were stable for at least 48 h while kept at room temperature. Both screening and confirmatory procedures were applied to authentic urine and oral fluid specimens obtained from volunteers following therapeutic ingestion of dextromethorphan.

High-throughput cytochrome P450 inhibition assays using laser diode thermal desorption-atmospheric pressure chemical ionization-tandem mass spectrometry.

This paper describes the development of a high-throughput method for the analysis of cytochrome P450 (CYP) inhibition assay incubation samples using laser diode thermal desorption interfaced with atmospheric pressure chemical ionization mass spectrometry (LDTD-APCI-MS). Data for the CYP isoforms 3A4, 2D6, 2C9, and 1A2 from competitive inhibition assays are shown. The potential for inhibition of the CYP isoforms was measured by monitoring the level of the metabolites 6beta-hydroxytestosterone (3A4), dextrorphan (2D6), 4'-hydroxydiclofenac (2C9), and acetaminophen (1A2) formed in the presence of drug candidates using an eight-point titration. The analytical method involves plating of the inhibition samples on specially designed 96-well plates with stainless steel bottoms, followed by direct analysis using the LDTD source. Validation of the LDTD-MS method was performed by testing for interferences, reproducibility, dynamic range, ion suppression, and the ability of the source to produce comparable results to previously validated LC-MS methods. IC50 values for each CYP isoform using 33 different test compounds showed excellent agreement between LDTD-APCI-MS and LC-MS methods and literature values where available. Assay analysis time using the LDTD-APCI source is reduced to less than 30 min for a single 96-well plate compared to greater than 10 h using the LC-MS method. The LDTD-APCI-MS and LC-MS methods and results are compared and limitations and future potential for LDTD-APCI-MS are discussed.

Validation of a liquid chromatography-mass spectrometry method to assess the metabolism of dextromethorphan in rat everted gut sacs.

A rapid, sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method was developed for the simultaneous assay of dextromethorphan and its metabolites in tissue culture medium and its intestinal metabolism studied with the rat everted gut sac model. The method was validated in the concentration range of 0.1-2.5 microM (27.1 ng/mL-0.677 microg/mL) for dextromethorphan and 0.005-0.5 microM for dextrorphan and 3-methoxymorphinan (1.28 ng/mL-0.128 microg/mL) and 3-hydroxymorphinan (1.22 ng/mL-0.122 microg/mL). The limits of quantification (LOQ) were 0.0025 microM (12.5 fmoles, 3.4 pg, 5 microL injected) for dextromethorphan; 0.0025 microM for dextrorphan, 3-methoxymorphinan (24.9 fmoles, 6.4 pg injected), and 3-hydroxymorphinan (25.1 fmoles, 6.1 pg injected) with 10 microL injected. The detection of dextrorphan and 3-methoxymorphinan showed that both the P450 isoforms CYP3A and 2D were active in the intestinal mucosa and metabolised dextromethorphan during its passage across the mucosa.

Metabolic activity of dextromethorphan O-demethylation in healthy Japanese volunteers carrying duplicated CYP2D6 genes: duplicated allele of CYP2D6*10 does not increase CYP2D6 metabolic activity.

BACKGROUND: This study was designed to assess the metabolic activities of dextromethorphan O-demethylation in healthy Japanese subjects carrying duplicated CYP2D6 alleles, CYP2D6*1 x 2, CYP2D6*2 x 2 or CYP2D6*10 x 2. METHODS: Forty-one unrelated healthy Japanese subjects containing carriers who had previously been genotyped as CYP2D6*1 x 2/*2, CYP2D6*1/*2 x 2, and CYP2D6*10/*10 x 2 were phenotyped with dextromethorphan. RESULTS: The metabolic ratios of dextromethorphan/dextrorphan in subjects with CYP2D6*1 x 2/*2 or CYP2D6*1/*2 x 2 were lower than those in subjects with CYP2D6*1/*2, while the metabolic ratios in subjects with CYP2D6*10/*10 x 2, as well as homozygotes for CYP2D6*10, were significantly (P<0.01) higher than those in homozygotes for CYP2D6*1. CONCLUSIONS: The results suggested that carriers with three functional CYP2D6 genes, CYP2D6*1 x 2/*2 or CYP2D6*1/*2 x 2, are ultrarapid metabolizer phenotypes in Japanese. The results also suggested that there is no gene-dose effect with the dextromethorphan O-demethylation activities between carriers with two and three CYP2D6*10 mutated genes per genome. Therefore, CYP2D6*10 x 2 may play an important role for the treatment of Japanese patients as well as CYP2D6*10 which is mainly responsible for the intermediate metabolizers in Japanese.

A rapid electron-capture gas chromatographic procedure for the analysis of p-hydroxymephenytoin.

An electron-capture gas chromatographic procedure was developed for detection and quantification of p-hydroxymephenytoin (OHMEP), a metabolite of S-mephenytoin, in human liver microsomal preparations. OHMEP was derivatized with pentafluorobenzoyl chloride (PFBC) under basic aqueous conditions prior to analysis on a gas chromatograph equipped with a capillary column and an electron-capture detector. Dextrorophan was carried through the procedure as internal standard. The structure of the PFB derivative was confirmed using combined gas chromatography-mass spectrometry (GC-MS). The procedure is rapid and reproducible and produces a stable derivative that has excellent chromatographic properties. The limit of detection was less than 5 ng/ml, and the method was applied to extracts of human liver microsomes, which had been incubated with S-mephenytoin [a probe substrate for cytochrome P450 (CYP) 2C19].

Characterization of cytochrome P-450 2D1 activity in rat brain: high-affinity kinetics for dextromethorphan.

We investigated the enzymatic function, stability, and regional distribution of rat brain cytochrome P-450 (CYP) 2D1 activity. CYP2D1 is the homolog of human CYP2D6, a genetically variable enzyme that activates or inactivates many clinical drugs acting on the central nervous system (e.g., antidepressants, monoamine oxidase inhibitors, serotonin uptake inhibitors, and neuroleptics), drugs of abuse (e.g., amphetamine and codeine), neurotoxins (e.g., 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1,2,3, 4-tetrahydroquinoline), and endogenous neurochemicals (e.g., tryptamine). The CYP2D family has been identified in rodent, canine, and primate brain. Conversion of dextromethorphan to dextrorphan by rat brain membranes was assayed by HPLC and was dependent on NADPH, protein concentration, and incubation time. Significant loss of activity was observed in some homogenizing buffers and after freezing of whole tissues or membrane preparations. Dextromethorphan (0.5-640 microM) metabolism was mediated by high- and low-affinity enzyme systems; K(m1) was 2.7 +/- 2.6 and K(m2) was 757 +/- 156 microM (n = 3 rats, mean +/- S.E.). The enzyme activity was significantly (p <.01) and stereoselectively inhibited by CYP2D1 inhibitors quinine and quinidine (not by CYP2C or CYP3A inhibitors), and by anti-CYP2D6 peptide antiserum (not by anti-CYP2C, -CYP2B, or -CYP3A antibodies). The enzymatic activity demonstrated significant brain regional variation (n = 10 regions, p <.001). These data characterize CYP2D1-mediated dextromethorphan metabolism in rat brain and suggest that localized metabolism of other CYP2D1 substrates (drugs, neurotoxins, and possibly endogenous compounds) within the brain will occur. In humans, CYP2D6 is genetically polymorphic; the variable expression of brain CYP2D6 may result in interindividual differences in central drug and neurotoxin metabolism, possibly contributing to interindividual differences in drug effects and neurotoxicity.

Analysis of dextrorphan, a metabolite of dextromethorphan, using gas chromatography with electron-capture detection.

Dextromethorphan, a constituent of many over-the-counter cough syrups, is used as a probe drug for phenotyping subjects for their cytochrome P450 2D6 (CYP2D6) enzyme activity and for measuring CYP2D6 activity of preparations such as microsomes. In such studies, formation of the metabolite dextrorphan is used as indicator of the activity of this CYP enzyme. The present report describes an electron-capture gas chromatographic procedure developed for detection and quantification of dextrorphan in human liver microsomal preparations in vitro. After basification of the incubation mixture, dextrorphan was derivatized with pentafluorobenzoyl chloride under aqueous conditions prior to analysis on a gas chromatograph equipped with a capillary column, an electron capture detector, and a printer-integrator. Para-hydroxymephenytoin was carried through the procedure as internal standard. The procedure, which involves the derivatization of dextrorphan under aqueous conditions, is rapid and involves the use of the relatively economical procedure of electron-capture gas chromatography. The derivative is stable and possesses excellent chromatographic properties.

Identification with liquid chromatography-ionspray mass spectrometry of the metabolites of the enantiomers N-methyl dextrorphan and N-methyl levorphanol after rat liver perfusion.

To gather more information on stereochemical factors in the hepatic disposition of organic cations, mass spectrometry coupled to liquid chromatography was used to determine the identity of the metabolites excreted in bile after isolated rat liver perfusions with the quaternary ammonium derivatives of the enantiomeric drugs dextrorphan and levorphanol. Ionspray mass spectrometry was chosen for its soft ionization and absence of thermal degradation of labile compounds. The drugs were labelled with a stable (2H) isotope and mixed with unlabelled drugs to create an artificial isotope pattern in the mass spectrum and facilitate the recognition of unknown metabolites. In mass spectra that were recorded under normal conditions, fragmentation was absent and metabolites of N-methyl dextrorphan and N-methyl levorphanol were visible as parent-ion 'doublets'. Collision-induced fragmentation studies were performed to support the identification of the metabolites. For N-methyl dextrorphan the glucuronide, the glutathione conjugate and the glucuronide of the N-demethylated metabolite were found in bile. For N-methyl levorphanol the glucuronide, the glutathione conjugate, the sulphate conjugate and the glucuronide of a hydroxylated N-methyl levorphanol were excreted in bile. Thus a remarkable stereoselectivity occurs in the metabolism of these quaternary ammonium compounds in the rat liver.

Simultaneous determination of dextromethorphan and three metabolites in plasma and urine using high-performance liquid chromatography with application to their disposition in man.

A simple, sensitive, and reproducible high-performance liquid chromatrography assay is described for the simultaneous determination of dextromethophan, dextrorphan, 3-hydroxymorphinan, and 3-methoxymorphinan in plasma and urine. A conventional solvent-solvent extraction procedure was used for the isolation of the analytes from plasma and urine samples. The compounds were separated on a cyano column (150 x 4.6 mm, 5-micron particle size) using a mobile phase of acetonitrile/triethylamine/distilled water (17:0.06:82.94, vol/vol), pH 3.0, and then were measured by fluorescence detection. Calibration curves in the range 2-200 ng/ml for plasma and 0.05-10 micrograms/ml for urine were linear and passed through the origin. The precision and accuracy were greater than 90% and the lowest detectable concentrations were 0.5 ng/ml for 3-hydroxymorphinan and 3-methoxymorphinan and 1 ng/ml for dextromethorphan and dextrophan in plasma. The utility of this method is demonstrated in a preliminary study of dextromethorphan metabolism and pharmacokinetics in man.