Analysis of quazepam and its metabolites in human urine by gas chromatography-mass spectrometry: application to a forensic case.

A sensitive method for the simultaneous determination of quazepam and two of its metabolites, 2-oxoquazepam and 3-hydroxy-2-oxoquazepam, in human urine was developed using gas chromatography-mass spectrometry (GC/MS) with an Rtx-5MS capillary column. The quazepam and its metabolites were extracted from human urine using a simple solid-phase extraction Oasis(®) HLB cartridge column, and the 3-hydroxy-2-oxoquazepam was derivatised using BSTFA/1%TMCS and pyridine at 60 °C for 30 min. The mass spectrometric detection of the analytes was performed in the full scan mode, m/z 60-480, and selected ion monitoring (SIM) mode, m/z 386, for quazepam; m/z 342, for 2-oxoquazepam; m/z 429, for 3-hydroxy-2-oxoquazepam-TMS; and m/z 284, for alprazolam-d5 (internal standard), by electron ionization. The calibration curves of quazepam and its metabolites in urine showed good linearity in the concentration range of 2.5-500 ng/0.2 ml of urine. The average recoveries of quazepam and its metabolites from 0.2 ml of urine containing 500 ng and 50 ng of each drug were 71-83% and 88-90%, respectively. The limits of detection of quazepam, 2-oxoquazepam and 3-hydroxy-2-quazepam in urine by the selected ion monitoring mode were 0.096-0.37 ng/ml. This method would be applicable to other forensic biological materials containing low concentrations of quazepam and its metabolites.

Metabolic interaction between ethanol, high-dose alprazolam and its two main metabolites using human liver microsomes in vitro.

Alprazolam is widely used as a short-acting antidepressant and anxiolytic agent and its effect appears at very low doses while ethanol is used as a social drug worldwide. Sometimes, toxic interactions occur following combined administration of these two drugs. In this study we have investigated the interaction between ethanol and high-dose alprazolam using human liver microsomes in vitro. The interaction effects between ethanol and alprazolam were examined by a mixed-function oxidation reaction using a human liver microsomal preparation. Alprazolam and its two main metabolites (alpha-hydroxyalprazolam: alpha-OH alprazolam, 4-hydroxyalprazolam: 4-OH alprazolam) were measured by HPLC/UV. The production of 4-OH alprazolam, one main metabolite of alprazolam, was weakly inhibited by higher dose of ethanol, but not alpha-OH alprazolam. These results using a human liver microsomal preparation show that the production of 4-OH alprazolam is weakly inhibited by ethanol but not alpha-OH alprazolam. Toxic levels may be reached by simultaneous administration of ethanol and high-dose alprazolam.

Simple and simultaneous determination for 12 phenothiazines in human serum by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of the 12 phenothiazines (chlorpromazine, fluphenazine, levomepromazine, perazine, perphenazine, prochlorperazine, profenamine, promethazine, propericiazine, thioproperazine, thioridazine and trifluoperazine) in human serum using HPLC/UV. The separation was achieved using a C(18) reversed-phase column (250 mm x 4.6 mm I.D., particle size 5 microm, Inersil ODS-SP). The mobile phase, consisting of acetonitrile-methanol-30 mM NaH(2)PO(4) (pH 5.6) (300:200:500, v/v/v), was delivered at a flow rate of 0.9 mL/min and UV detection was carried out at 250 nm. The recoveries of the 12 phenothiazines spiked into serum samples were 87.6-99.8%. Regression equations for the 12 phenothiazines showed excellent linearity, with detection limits of 3.2-5.5 ng/mL for serum. The inter-day and intra-day coefficients of variation for serum samples were commonly below 8.8%. The selectivity, accuracy and precision of this method are satisfactory for clinical and forensic purposes. This sensitive and selective method offers the opportunity for simultaneous screening and quantification of almost all phenothiazines available in Japan for the purposes of clinical and forensic applications.

Solid-phase extraction and analysis of 20 antidepressant drugs in human plasma by LC/MS with SSI method.

A simultaneous determination of 20 antidepressant drugs (imipramine, amitriptyline, desipramine, trimipramine, nortriptyline, clomipramine, amoxapine, lofepramine, dosulepin, maprotiline, mianserin, setiptiline, trazodone, fluvoxamine, paroxetine, milnacipran, sulpiride, tandspirone, methylphenidate and melitracen) in human plasma was developed using LC/MS with sonic spray ionization (SSI) method. These drugs showed good separation and sensitivity by LC-MS using an Inertsil C-8 column with methanol:10mM ammonium acetate (pH 5.0):acetonitrile (70:20:10) as mobile phase at 0.10 mL/min at 35 degrees C. Solid-phase extraction of these drugs added to the human plasma was performed with an Oasis HLB cartridge column. Recovery and limit of detection of these drugs were between 69 and 102% and between 0.03 and 0.63 microg/mL, respectively. The present procedure offers an easier and more convenient screening method for antidepressants, and will be useful for forensic toxicology investigations.

Simultaneous determination of three local anesthetic drugs from the pipecoloxylidide group in human serum by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous analysis of the local anesthetic amide drugs, bupivacaine, mepivacaine and ropivacaine, belonging to the pipecoloxylidide group using a C(18) reversed-phase column (150 x 4.6 mm I.D.) filled with 5-microm particles and attached to a UV detector. The mobile phase was composed of acetonitrile-methanol-30 mM NaH(2)PO(4) (pH 5.6) (100:100:300, v/v/v) and the flow rate was 1ml/min. The absorbance of the eluate was monitored at 210 nm. The retention times of the three compounds were: 4.6 min (mepivacaine), 9.7min (ropivacaine) and 16.4 min (bupivacaine). With this sample preparation method, good and consistent recoveries of the three compounds were obtained: 88-91% for mepivacaine, 87-89% for ropivacaine and 88-91% for bupivacaine. The limit of quantification for three compounds in human serum was 2 ng/ml for mepivacaine, 5 ng/ml for bupivacaine and ropivacaine. This method may be useful in clinical and forensic applications for the determination or identification of the local anesthetic drugs: bupivacaine, mepivacaine or ropivacaine.

Effects of premedication medicines on the formation of the CYP3A4-dependent metabolite of ropivacaine, 2', 6'-Pipecoloxylidide, on human liver microsomes in vitro.

Ropivacaine is a relatively new amide-type local anaesthetic, mainly used for surgery and postoperative pain relief. In this study we have investigated the interaction between the CYP3A4 metabolite of ropivacaine, 2',6'-pipecoloxylidide (PPX), and premedication with, i.e., psychotropic and antianxiety agents (diazepam, midazolam), hypnotics (thiamylal), local anaesthetics (lidocaine), depolarizing muscular relaxants (vecuronium), antihypertensive (clonidine) and H(2)-receptor antagonist (cimetidine) using human liver microsomes in vitro. The effects of the interaction between PPX and premedications were examined using a human liver microsomal preparation in vitro. The concentrations of ropivacaine and PPX were determined by HPLC with UV detection. The apparent Michaelis-Menten constant (Km) and the maximal velocity of total metabolic formation (V(max)) of PPX, the main metabolite of ropivacaine in human liver microsomes, were 17.7 (microM, mean) and 711 (nmol/min./mg protein, mean), respectively. Five premedications (diazepam, lidocaine, cimetidine, vecuronium and clonidine) did not inhibit ropivacaine metabolism in human liver microsomes at concentrations within the therapeutic range. However, midazolam and thiamylal weakly inhibited ropivacaine metabolism in competitive manner (IC(50) 7.8 microM and 250 microM, respectively). The results show lack of interaction between ropivacaine and seven premedication medicines within the therapeutic range of ropivacaine using human liver microsomes in vitro.

A fatal poisoning with 5-methoxy-N,N-diisopropyltryptamine, Foxy.

A fatal overdose involving case by 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is reported. 5-MeO-DIPT and its two metabolites, 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT) and 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT), were identified by LC-MS. The level of 5-MeO-DIPT, 5-OH-DIPT and 5-MeO-NIPT in blood and urine was 0.412, 0.327 and 0.020 microg/ml, and 1.67, 27.0 and 0.32 microg/ml, respectively. These blood and urine levels were higher than published data for such poisoning.

Effect of propofol on ropivacaine metabolism in human liver microsomes.

A combination of the general anesthetic propofol and epidural anesthesia with a local anesthetic is widely used. The metabolism of ropivacaine and that of lidocaine are mediated by similar P450 isoforms. Previously, propofol was found to inhibit the metabolism of lidocaine in vitro. Here we investigated whether propofol inhibits the metabolism of ropivacaine using human liver microsomes in vitro. Ropivacaine (6.0 micromol.l(-1)) as the substrate and propofol (1-100 micromol.l(-1)) were reacted together using human microsomes. The concentrations of ropivacaine and its major metabolite 2',6'-pipecoloxylidide (PPX) were measured using high-performance liquid chromatography. The metabolic activity of ropivacaine was reflected in the production of PPX. The inhibitory effects of propofol on ropivacaine metabolism were observed to be dose-dependent. The IC50 of propofol was 34.9 micromol.l(-1). Propofol shows a competitive inhibitory effect on the metabolism of ropivacaine (i.e., PPX production mediated by CYP3A4) in human CYP systems in vitro.

Metabolism of the psychotomimetic tryptamine derivative 5-methoxy-N,N-diisopropyltryptamine in humans: identification and quantification of its urinary metabolites.

The urinary metabolites of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in humans have been investigated by analyzing urine specimens from its users. For the unequivocal identification and accurate quantification of its major metabolites, careful analyses were conducted by gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and liquid chromatography-tandem mass spectrometry, using authentic standards of each metabolite synthesized. Three major metabolic pathways were revealed as follows: 1) side chain degradation by O-demethylation to form 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT), which would be partly conjugated to its sulfate and glucuronide; 2) direct hydroxylation on position 6 of the aromatic ring of 5-MeO-DIPT, and/or methylation of the hydroxyl group on position 5 after hydroxylation on position 6 of the aromatic ring of 5-OH-DIPT, to produce 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine (6-OH-5-MeO-DIPT), followed by conjugation to its sulfate and glucuronide; and 3) side chain degradation by N-deisopropylation, to the corresponding secondary amine 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT). Of these metabolites, which retain structural characteristics of the parent drug, 5-OH-DIPT and 6-OH-5-MeO-DIPT were found to be more abundant than 5-MeO-NIPT. Although the parent drug 5-MeO-DIPT was detectable even 35 h after dosing, no trace of its N-oxide was detected in any of the specimens examined.

CYP2C19 genotype affects diazepam pharmacokinetics and emergence from general anesthesia.

OBJECTIVES: Diazepam is widely used to relieve preoperative anxiety in patients. The objective of this study was to investigate the effects of polymorphism in CYP2C19 and the effects of CYP3A4 messenger ribonucleic acid (mRNA) content in blood on recovery from general anesthesia and on diazepam pharmacokinetics. METHODS: Sixty-three Japanese patients were classified into the following 3 genotype (phenotype) groups on the basis of polymerase chain reaction-restriction fragment length polymorphism analysis of CYP2C19 polymorphism: no variants, *1/*1 (extensive metabolizer [EM]); 1 variant, *1/*2 or *1/*3 (intermediate metabolizer [IM]); and 2 variants, *2/*2, *2/*3, or *3/*3 (poor metabolizer [PM]). We assessed the effects of these polymorphisms and of CYP3A4 mRNA content in the lymphocytes on the patients' recovery from general anesthesia. RESULTS: CYP2C19 genotyping analysis in the 63 subjects showed that 32%, 46%, and 22% of subjects were classified into the EM, IM, and PM groups, respectively. The PM subjects showed a larger area under the curve representing the concentration of diazepam over a 24-hour period (AUC(0-24)) (2088 +/- 378 ng/mL.h(-1), P = .0259), lower clearance of diazepam (0.049 +/- 0.009 L.h(-1).kg(-1), P = .0287), and longer emergence time (median, 18 minutes; 25th-75th percentile range, 13-21 minutes; P < .001) in comparison with subjects in the EM group (AUC(0-24), 1412 +/- 312 ng/mL; clearance, 0.074 +/- 0.018 L.h(-1).kg(-1); and emergence time, 10 minutes, 8-12 minutes [median and 25th-75th percentile range]). The IM group also showed a longer emergence time (median, 13 minutes; 25th-75th percentile range, 9-20 minutes; P < .001) and a larger variation in this parameter in comparison with the EM group. The distributions of the CYP2C19 genotype were significantly different between the 2 groups (rapid emergence <20 minutes, slow emergence >20 minutes) (P = .0148). The mean value of the CYP3A4 mRNA level in the slow-emergence group (mean +/- SD, 4.80 +/- 3.99 x10(-10)) was significantly lower than that of the rapid-emergence group (mean +/- SD, 12.50 +/- 11.90 x10(-10)) (P = .0315). However, there was no significant correlation between emergence time and CYP3A4 mRNA levels (r = 0.239, P = .0601). CONCLUSION: We found that the CYP2C19 genotype affects diazepam pharmacokinetics and emergence from general anesthesia and that the slow-emergence group possesses lower levels of CYP3A4 mRNA than are found in the rapid-emergence group.

A study of the in vitro interaction between ethanol, and triazolam and its two metabolites using human liver microsomes.

AIM: Triazolam is widely used as an ultrashort-acting anxiolytic drug and hypnosedatives and its effect appears at very low doses. Ethanol is used as a social drug worldwide. Sometimes, toxic interactions occur following combined administration of these two drugs. In this study, we have investigated the interaction between alcohol and triazolam in vitro. METHODS: The interaction effects between alcohol and triazolam were examined by a mixed-function oxidation reaction using a human liver microsomal preparation. Triazolam and its two metabolites (alpha-hydroxytriazolam: alpha-OH triazolam, 4-hydroxytriazolam: 4-OH triazolam) were measured by HPLC/UV. RESULTS: The production of alpha-OH triazolam and 4-OH triazolam was shown to be weakly inhibited by 13-29% (p < 0.05) and 8-14%,respectively, by ethanol (20-80 mM). CONCLUSIONS: These results using a human liver microsomal preparation show that the formation of both metabolites of triazolam is weakly inhibited by ethanol. Toxic levels may be reached by simultaneous administration of ethanol and triazolam.

Premedication medicines do not cause drug metabolic interaction with propofol using human liver microsomes in vitro.

OBJECTIVE: Propofol (2,6-diisopropylphenol) is widely used for anesthetic induction as well as for chronic sedation in intensive care units. In this study, we investigated the interaction between propofol and premedications, i.e., psychotropic and antianxiety agents (diazepam, midazolam), hypnotics (thiamylal), local anesthetics (lidocaine), depolarizing muscular relaxants (vecuronium), an antihypertensive (clonidine) and an H2-receptor antagonist (cimetidine) using human liver microsomes in vitro. METHODS: The interaction effects between propofol and premedications were examined using human liver microsomal preparation in vitro. The concentration of propofol was determined by HPLC with UV detection. RESULTS: The apparent Michaelis-Menten constant (Km) and the maximal velocity of total metabolic formation (Vmax) of propofol in human liver microsomes were 123 microM and 26.1 micromol/min per milligram of mg protein, respectively. Seven premedications (diazepam, midazolam, thiamylal, lidocaine, cimetidine, vecuronium, and clonidine) did not inhibit propofol metabolism in human liver microsomes at concentrations within the therapeutic range. CONCLUSIONS: These results showed no interactions between propofol and seven premedication drugs within the therapeutic range of propofol using human liver microsomes in vitro.

Propofol inhibits lidocaine metabolism in human and rat liver microsomes.

PURPOSE: When two drugs are metabolized by similar P450 isoforms, one drug inhibits the metabolism of the other when both the present. The metabolism of lidocaine and propofol can be mediated by similar P450 isoforms. Therefore, we investigated the relationship in the metabolism between lidocaine and propofol in both rat and human liver microsomal P450 (CYP) systems in vitro. METHODS: (1) Propofol, 4 micro g.ml(-1), as the substrate and lidocaine (between 0.5 and 8 micro g.ml(-1)) and (2) lidocaine, 4.7 micro g.ml(-1), as the substrate and propofol (between 0.5 and 40 micro g.ml(-1)) were reacted separately with human and rat microsomes. The concentrations of lidocaine, its major metabolite (monoethylglycinexylidide, MEGX) and propofol were measured using high-pressure liquid chromatography. The metabolism of lidocaine was presented as a reaction activity (MEGX/lidocaine). RESULTS: The dose-dependent inhibitory effects of propofol on lidocaine metabolism were observed in both the human and rat groups. The IC50 (the concentration producing 50% maximal inhibition) of propofol was 5.0 micro g.ml(-1) and 0.70 micro g.ml(-1) in the human and the rat groups, respectively. The propofol concentration of 5.0 micro g.ml(-1) is within the range of clinical doses for humans. On the other hand, lidocaine did not change propofol metabolism. CONCLUSION: Propofol possesses a dose-dependent inhibitory effect on the metabolism of lidocaine in both human and rat CYP systems in vitro.

Simultaneous determination of flunitrazepam and 7-aminoflunitrazepam in human serum by ion trap gas chromatography-tandem mass spectrometry.

A method for the determination of flunitrazepam (FNZ) and 7-aminoflunitrazepam (7-AFNZ) in human serum was developed with ion trap gas chromatography (GC)-tandem mass spectrometry. The 7-AFNZ was derivatizated with 50 microl trifluoroacetic anhydride (TFAA), 60 degrees C-20 min. EI mass spectra and tandem mass spectra of FNZ and 7-AFNZ-TFA were m/z 238, 239, 266, 286, 294, 312, 313(M(+)), m/z 350, 351, 360, 378, 379(M(+)), m/z 238, 239, 240 (precursor ion m/z 286, collision energy 1.5 V), and m/z 239, 254, 264, 336 (precursor ion m/z 351, collision energy 1.8 V), respectively. The detection limits of full scan EI mass spectrometry and tandem mass spectrometry for FNZ and 7-AFNZ in human serum were ca. 200 ng/ml, 60 ng/ml, 15 ng/ml and 1 ng/ml, respectively.

The specific mitochondrial DNA polymorphism found in Klinefelter's syndrome.

Hypervariable segments of mitochondrial DNA (mtDNA) (HV1 and HV2) were analyzed in Klinefelter's syndrome and compared to normal population data. One pair of samples consisting of a Japanese mother and affected son with Klinefelter's syndrome (involved in a criminal case), and seven unrelated DNA samples from Caucasian Klinefelter males (two involved in criminal cases and five diagnosed) were collected in Japan and the United States. The diagnosis of Klinefelter's syndrome was established previously by multiplex XY-STR typing detecting two X alleles and one Y allele in the samples. Haplotype analysis of the mtDNA sequence in Klinefelter males was found to be identical, unique, and specific, as it was not found in the normal population. Astonishingly, family data exhibited that the haplotype of the mtDNA in the son was apparently different from the mother's, suggesting that the mtDNA of Klinefelter male would not be inherited from mother to son. Our data indicate that possible interaction of the sex chromosome and the mtDNA exists, and suggests that the specific mtDNA haplotype could cause the abnormal cell to fertilize and reproduce itself.