A gas chromatography/mass spectrometry method for the determination of sildenafil, vardenafil and tadalafil and their metabolites in human urine.

Sildenafil (SDF), vardenafil (VDF) and tadalafil (TDF) are phosphodiesterase type 5 enzyme inhibitors (PDE5Is), used in the treatment of erectile disorders and to improve breathing efficiency in pulmonary hypertension. The increasing incidence of their use among young athletes has drawn the attention of the anti-doping authorities to the possible abuse of PDE5Is by athletes due to their pharmacological activities. This paper describes a method for the determination in urine of PDE5Is and their metabolites by gas chromatography/mass spectrometry (GC/MS) after liquid/liquid extraction of the analytes from urine and derivatisation to obtain trimethylsilyl derivatives. The metabolic profile was studied on real samples collected from subjects taking PDE5Is (Viagra, Levitra or Cialis); the main urinary metabolites were identified and their MS fragmentation characterized. The sample pre-treatment and GC/MS conditions for the detection of the metabolites have been optimised. A method for their preliminary screening and subsequent confirmation is described that takes into account the general requirements of a routine doping analysis to be used for the screening of large numbers of samples. The main metabolites identified can be included in a general purpose screening method and all the metabolites in a more specific confirmation method. The method developed has been applied for the screening of PDE5Is in 5000 urine samples. Based on the obtained results, the proposed method appears to be of practical use in analytical and forensic toxicology, including doping analysis.

Impact of long-term ethanol consumption on CYP1A2 activity.

Ethanol is a well-known inducer of CYP2E1; whether or not it is an inducer of other cytochromes has not been investigated systematically. The aim of our study was to evaluate the impact of ethanol consumption on the activity of CYP1A2, which has been shown to be influenced by drugs (inhibited or induced). We evaluated CYP1A2 activity by the ratio of the molar urinary concentrations of the three end products of paraxanthine demethylation of caffeine to the molar concentration of a paraxanthine 8-hydroxylation product. This urinary metabolite ratio has previously been shown to correlate with caffeine clearance. The caffeine metabolites were measured in urine collected during the 3 hours after oral administration of 200 mg caffeine. The caffeine test was performed in 12 smokers (> 25 cigarettes/day) and 12 nonsmokers, all of whom were alcoholic inpatients (daily intake > 100 mg absolute ethanol), within the first 3 days of their hospital stay and after 14 days of abstinence from ethanol. In alcoholic patients who were smokers the molar urinary concentration ratio was 3.14 +/- 0.97 before withdrawal and 4.01 +/- 0.92 after 14 days of abstinence from ethanol. In contrast, in alcoholic patients who were nonsmokers it was 2.62 +/- 0.95 and 2.18 +/- 0.96 before and after withdrawal, respectively. In volunteers who were smokers the molar urinary concentration ratio was 5.02 +/- 1.51, whereas in volunteers who were nonsmokers it was 3.22 +/- 1.46. Our results confirm the well-known induction of CYP1A2 activity by tobacco smoking and show that this induction is masked by long-term ethanol consumption.

Comparisons of oxidative metabolism and reductive capacity in sulfonamide-tolerant and -intolerant patients with human immunodeficiency virus.

Hypersensitivity reactions to trimethoprim/sulfamethoxazole occur with a high frequency in human immunodeficiency virus (HIV)-infected patients. This study tested whether differences in oxidative metabolism and plasma reductive capacity correlate with sulfonamide intolerance in patients with HIV. Eighteen stable outpatients with HIV were prospectively studied. Nine patients had documented histories of hypersensitivity reactions to trimethoprim/sulfamethoxazole and nine did not. Urinary caffeine metabolite ratios assessed the activity of two oxidative enzymatic pathways: cytochrome P-450 1A2 (demethylation) and 8-hydroxylation. Plasma cyst(e)ine was used as a measure of reductive capacity. The trimethoprim/sulfamethoxazole-intolerant group showed greater rates of 8-hydroxylation, lower rates of demethylation, and lower cyst(e)ine levels. The results of this pilot study extend previous observations of differences in oxidative metabolism and reductive capacity that exist within the population of HIV-infected individuals. In addition, these findings lay the groundwork for future interventional studies that could use agents to inhibit sulfonamide oxidation and increase reductive capacity in sulfonamide-intolerant patients with HIV when rechallenged with trimethoprim/sulfamethoxazole.

Sensitive liquid chromatography assay with ultraviolet detection for a new phosphodiesterase V inhibitor, DA-8159, in human plasma and urine.

A sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet absorption detection (292 nm) was developed and validated for the determination of the new phosphodiesterase V inhibitor, DA-8159 (DA), in human plasma and urine. A single step liquid-liquid extraction procedure using ethyl ether was performed to recover DA and the internal standard (sildenafil citrate) from 1.0 ml of biological matrices combined with 200 microl of 0.1M sodium carbonate buffer. A Capcell Pak C18 UG120 column (150 mm x 4.6 mm I.D., 5 microm) was used as a stationary phase and the mobile phase consisted of 30% acetonitrile and 70% 20mM potassium phosphate buffer (pH 4.5) at a flow rate of 1.0 ml/min. The lower limit for quantification was 5 ng/ml for plasma and 10 ng/ml for urine samples. Within- and between-run accuracy and precision were < or =15 and < or =10%, respectively, in both plasma and urine samples. The recovery of DA from human plasma and urine was greater than 70%. Separate stability studies showed that DA is stable under the conditions of analysis. This validated assay was used for the pharmacokinetic analysis of DA during a phase I, rising dose study.

An isocratic high-performance liquid chromatographic system for simultaneous determination of theophylline and its major metabolites in human urine.

A simple and highly selective isocratic high-performance liquid chromatography method is presented for the simultaneous determination of theophylline and its major metabolites in human urine using beta-hydroxyethyl theophylline as an internal standard. The method utilizes direct injection of diluted urine samples followed by separation and quantitation by reversed-phase isocratic elution and ultraviolet detection. The assay is accurate and reproducible with a sensitivity of 1 microgram ml-1 for theophylline and 0.5 micrograms ml-1 for its metabolites. The assay was employed for the analysis of theophylline and its major metabolites in urine following the oral administration of theophylline to four healthy volunteers.

Determination of a new phosphodiesterase V inhibitor, DA-8159, in plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method using liquid-liquid extraction for sample preparation was developed for the determination of a new phosphodiesterase V inhibitor, DA-8159, in rat plasma and urine using sildenafil citrate as an internal standard. A 100 microl aliquot of 0.1 M Na(2)CO(3) (containing sildenafil citrate, 3 microg/ml as free sildenafil) and a 1 ml aliquot of ether were added to a 100 microl aliquot of biological samples (urine samples were diluted 20 times with distilled water). After vortex centrifugation at 9000 x g for 3 min, the ether layer was collected and dried under nitrogen gas. The residue was reconstituted with a 150 microl aliquot of the mobile phase, centrifuged, and a 100 microl aliquot of the supernatant was injected onto a reversed-phase column. The mobile phases, 20 mM KH(2)PO(4) (pH 4.7):acetonitrile (70:30, v/v for plasma and tissue samples, and 75:25, v/v for urine samples), were run at a flow rate of 1.0 ml/min. The column effluent was monitored by an ultraviolet detector set at 292 nm. The retention times for DA-8159 and the internal standard were approximately 10.7 and 9.1 min, respectively, in plasma and tissue samples and the corresponding values in urine samples were 47 and 33 min. The detection limits for DA-8159 in rat plasma and urine were 20 and 100 ng/ml, respectively. The coefficients of variation of the assay were generally low: below 10% for plasma and 9.9% for urine. No interferences from endogenous substances were found.

Identification of lorazepam and sildenafil as examples for the application of LC/ionspray-MS and MS-MS with mass spectra library searching in forensic toxicology.

A mass spectra (MS) library using in-source collision induced dissociation (ESI-CID) as well as a tandem-mass spectra (MS-MS) library with product ion spectra of drugs has recently been developed with a triple-quadrupole ionspray mass spectrometer [1,2]. For the ESI-CID MS library, single-quadrupole mode and for the MS-MS library triple-quadrupole mode have been used. These mass spectra libraries were applied successfully for the general-unknown screening for drugs and metabolites in serum and urine with liquid-chromatography-mass spectrometry (LC-MS) using a PE/SCIEX API 365 with a turboionspray source. As examples, the identification of lorazepam and lorazepam-glucuronide in a serum extract and the identification of sildenafil and alkyloxidated sildenafil in urine are presented here.

Genotype and phenotype of N-acetyltransferase 2 (NAT2) polymorphism in patients with contact allergy.

We investigated whether patients with contact allergy differed from non-contact-allergic, non-atopic controls with regard to genotype and phenotype of the polymorphic enzyme N-acetyltransferase 2 (NAT2). 55 contact-allergic patients recruited from the Information Network of Departments of Dermatology (IVDK) were compared to 85 controls from among local health care personnel. NAT2 activity was calculated from HPLC analysis of the ratio of the caffeine metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 1-methylxanthine (1MX) in the urine. NAT2 genotype was determined by polymerase chain reaction (PCR). A statistically significantly increased proportion of rapid acetylators was found in contact-allergic patients. This may have 2 possible implications: acetylation may enhance contact sensitization; or NAT2 status may be a genetic marker for contact sensitizability.

Theophylline has no advantages over caffeine as a putative model drug for assessing CYPIA2 activity in humans.

AIMS: The cytochrome P4501A2 (CYP1A2) catalyses the metabolism of a number of clinically used drugs, and thus there is an interest in determining the activity of CYP1A2 in patients before treatment with CYP1A2 substrates. Caffeine is the most commonly used model drug to assess CYP1A2 function, but due to the complex metabolism of caffeine, there is a need for an alternative drug to use as an index of CYP1A2 activity. In this study the CYP1A2 substrate theophylline was tested as a possible alternative to caffeine as a model drug for CYP1A2. METHODS: Twelve healthy volunteers ingested 200 mg of caffeine, and the caffeine metabolic ratios (CMR), CMRurine = (AFMU + 1MX + 1MU)/17DMU and CMRplasma = 17DMX/137TMX were determined 6 h after drug intake. After a period of about 2 months the volunteers ingested 257 mg theophylline and blood samples were drawn and urine was collected during the following 48 h. The oral and partial clearance of theophylline were calculated via N-demethylation and 8-hydroxylation. The theophylline metabolic ratios, 1MU/13DMX and 3MX/13DMX being evaluated as indices of CYP1A2 catalysed N-demethylation and 13DMU/13DMX as an index of partly CYP1A2 catalysed 8-hydroxylation, were estimated in 0-12 h, 0-24 h and 0-48 h urine samples, and in plasma and spot urine samples 6 h after the intake of theophylline. RESULTS: The theophylline plasma ratios for the N-demethylation pathways correlated with the oral clearance of theophylline (rs = 0.881-0.934, P < 0.001) and with the respective formation clearances of the metabolites (rs = 0.712-0.925, P < 0.05). Furthermore, all of the theophylline plasma ratios correlated with the caffeine plasma ratio (rs = 0.645-0.663, P < 0.05). None of the caffeine metabolic ratios and none of the 6 h urinary theophylline ratios correlated with the oral or the partial clearances of theophylline (rs = 0.042-0.556, P < 0.05). The theophylline 0-12 h urine ratios correlated with the oral clearance of theophylline (rs = 0.677-0.757, P < 0.05) and with the respective formation clearances of the metabolites (rs = 0.705-0.750, P < 0.05). However, none of the theophylline urine ratios correlated with any of the caffeine metabolic ratios. CONCLUSIONS: In summary the theophylline 6 h plasma and 0-12 h urine ratios 1MU/13DMX and 3MX/13DMX, both reflecting N-demethylation seem to be predictors of the CYP1A2 mediated metabolism of theophylline, whereas only the plasma ratio correlated with the caffeine plasma 17DMX/13TMX ratio. Thus, it would appear that the plasma theophylline N-demethylation ratios are superior to the urine ratios as indices of CYP1A2 activity. However, because in some individuals the concentrations of theophylline metabolites in plasma were close to the limit of detection, it is concluded that theophylline does not have marked advantages over caffeine as a model drug for assessing CYP1A2 activity.

Caffeine based measures of CYP1A2 activity correlate with oral clearance of tacrine in patients with Alzheimer's disease.

AIMS: To study the potential utility of caffeine based probes of CYP1A2 enzyme activity in predicting the pharmokinetics of tacrine in patients with Alzheimer's disease. METHODS: The pharmokinetics of a single 40 mg oral dose of tacrine were measured in 19 patients with Alzheimer's disease. Each patient also received 2 mg kg(-1) [13C-3-methyl] caffeine orally and had breath and urine samples collected. RESULTS: Tacrine oral clearance (CL F(-1) kg(-1)), which varied 15-fold among the patients, correlated significantly with the 2 h total production of 13CO2 in breath (r=0.56, P=0.01), and with each of two commonly used urinary caffeine metabolite ratios: the 'paraxanthine/caffeine ratio' (1,7X + 1, 7U)/1,3,7X) (r=0.76, P=0.0002) and the 'caffeine metabolic ratio' (AFMU + 1X + 1U)/1, 7U)(r=0.76, P=0.0001). CONCLUSIONS: These observations support a central role for CYP1A2 in the in vivo disposition of tacrine and the potential for drug interactions when tacrine treated patients receive known inducers or inhibitors of this enzyme. The magnitude of the correlations we observed, however, are probably not sufficient to be clinically useful in individualizing tacrine therapy.