High-performance liquid chromatography assay for simultaneous determination of dextromethorphan and its main metabolites in urine and in microsomal preparations.

An HPLC method has been developed and validated for the determination of dextromethorphan, dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan in urine samples. Deconjugated compounds were extracted on silica cartridges using dichloromethane/hexane (95:05, v/v) as an eluent. Chromatographic separation was accomplished on a Phenyl analytical column serially connected with a Nitrile analytical column. The mobile phase consisted of a mixture of an aqueous solution, containing 1.5% acetic acid and 0.1% triethylamine, and acetonitrile (75:25, v/v). Compounds were monitored using a fluorescence detector. Calibration curves were linear over the range investigated (0.2-8.0 microM) with correlation coefficients >0.999. The method was reproducible and precise. Coefficients of variation and deviations from nominal values were both below 10%. For all the analytes, recoveries exceeded 77% and the limits of detection were 0.01 microM. The validated assay proved to be suitable for the determination of DEM metabolic indexes reported to reflect the enzymatic activity of the cytochrome P450s, CYP2D6 and CYP3A, both in vivo, when applied to urine samples from patients, and in vitro, when applied to samples from the incubation of liver microsomes with dextromethorphan.

Liquid chromatographic method for the simultaneous determination of caffeine and fourteen caffeine metabolites in urine.

An HPLC method has been developed for the separation and the determination of caffeine and its metabolites in urine samples using a one extraction-analysis run and UV detection. The compounds were extracted by liquid-liquid extraction using chloroform-isopropylalcohol (85:15, v/v). Chromatographic separation was accomplished on an ODS analytical column with a mobile phase containing 0.05% acetic acid/methylalcohol (92.5:7.5, v/v). Compounds were monitored at 280 nm. The method was validated for the determination of AFMU, 1X, 1U, 17X and 17U caffeine metabolites required to assess the metabolic activity of the enzymes subject to in vivo caffeine testing. The validated assay was applied to urine samples from ten healthy volunteers. The method was proved to be suitable to assess simultaneously the enzymatic activity of cytochrome P450 CYP1A2 and CYP2A6, as well as N-acetyltransferase and xanthine oxidase.

Acetylation polymorphism expression in patients before and after liver transplantation: influence of host/graft genotypes.

The consequences of liver transplantation on NAT2 activity were studied in 58 patients of Caucasian origin and compared with a group control of 119 unrelated healthy individuals of the same ethnic origin. Acetylation phenotypes were determined using caffeine as a probe drug before and repeatedly after liver transplantation. NAT2 genotypes were determined with three separate polymerase chain reactions to detect either the NAT2*4 wild-type allele or the NAT2*5A, NAT2*6A and NAT2*7A mutated alleles, associated with a decrease in NAT2 enzyme activity. In patients, the molar urinary elimination ratio AFMU/(AFMU+1X+1U) appeared more reliable than AFMU/1X for assessing the acetylation phenotype and fitted better with the various haplotypes. The variation of xanthine oxidase activity as measured by the 1U/1X urinary elimination ratio, appeared to be responsible for the poor phenotype prediction from the AFMU/1X ratio in post-transplanted patients. Regardless of the pathologic conditions of the treatment in progress, the genotype of the liver played an overwhelming role in the phenotypic expression of NAT2 compared with the genotype of other organs, where NAT2 was expressed in patients who presented a chimerism after liver transplantation.

Inhibition of caffeine metabolism by 5-methoxypsoralen in patients with psoriasis.

Eight patients with psoriasis were given 200 mg caffeine orally with or without 1.2 mg kg-1 of 5-methoxypsoralen. Blood and urine samples were collected over a 2-day period. During 5-methoxypsoralen coadministration, the apparent volume of distribution of caffeine remained unchanged, but oral clearance (CLp.o.) decreased from 9.5 +/- 3.8 (mean +/- s.d.) to 3.2 +/- 0.51 h-1 (P < 0.01). The area under the plasma concentration-time curve (AUC) increased from 24 +/- 9 to 73 +/- 29 mg 1(-1) h (P < 0.001). This decrease in CLp.o. with increased AUC was consistent with a CYP1A2-dependent inhibition of caffeine N-demethylation which was further supported by significant decreases in the (AFMU+1U+1X)/17U and (AFMU+1U+1X)/17X urinary metabolic ratios.

Stability of debrisoquine (CYP2D6) phenotype in liver transplant patients.

Liver metabolism may be modified after liver transplantation according to the phenotype of the donor and may be influenced by posttransplantation complications. The CYP2D6 phenotype was assessed in 13 patients (group I) before and after liver transplantation using debrisoquine. CYP2D6 activity was also assessed in vitro on microsomes from the liver of the recipients and the donors, using dextromethorphan. Twelve patients were extensive metabolizers both before and after transplantation. One apparently poor metabolizer was transplanted with the liver of another poor metabolizer. The intrinsic clearance of dextromethorphan (CL(int)) measured on recipient liver microsomes was significantly lower than that on donor liver microsomes (p < 0.05). In extensive metabolizers, the debrisoquine metabolic ratio was correlated with CL(int) before (r = 0.78, p < 0.05) and after (r = 0.89, p < 0.0005) transplantation. Debrisoquine phenotype was measured repeatedly in nine additional patients (group II) up to 3 years after liver transplantation. Their phenotype was stable during the follow-up observation, although the variations observed may be clinically relevant. Therefore, no change in CYP2D6 phenotype (extensive/poor metabolizer) was observed because of the liver transplantation, and the debrisoquine log metabolic ratio was largely unaffected by the liver complications observed during the posttransplantation follow-up observation.