Screening procedure for detection of diuretics and uricosurics and/or their metabolites in human urine using gas chromatography-mass spectrometry after extractive methylation.

A gas chromatography-mass spectrometry (GC-MS)-based screening procedure was developed for the detection of diuretics, uricosurics, and/or their metabolites in human urine after extractive methylation. Phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by GC and identified by MS in the full-scan mode. The possible presence of the following drugs and/or their metabolites could be indicated using mass chromatography with the given ions: m/z 267, 352, 353, 355, 386, and 392 for thiazide diuretics bemetizide, bendroflumethiazide, butizide, chlorothiazide, cyclopenthiazide, cyclothiazide, hydrochlorothiazide, metolazone, polythiazide, and for canrenoic acid and spironolactone; m/z 77, 81, 181, 261, 270, 295, 406, and 438 for loop diuretics bumetanide, ethacrynic acid, furosemide, piretanide, torasemide, as well as the uricosurics benzbromarone, probenecid, and sulfinpyrazone; m/z 84, 85, 111, 112, 135, 161, 249, 253, 289, and 363 for the other diuretics acetazolamide, carzenide, chlorthalidone, clopamide, diclofenamide, etozoline, indapamide, mefruside, tienilic acid, and xipamide. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with reference spectra. This method allowed the detection of the abovementioned drugs and/or their metabolites in human urine samples, except torasemide. The limits of detection ranged from 0.001 to 5 mg/L in the full-scan mode. Recoveries of selected diuretics and uricosurics, representing the different chemical classes, ranged from 46% to 99% with coefficients of variation of less than 21%. After ingestion of the lowest therapeutic doses, furosemide was detectable in urine samples for 67 hours, hydrochlorothiazide for 48 hours, and spironolactone for 52 hours (via its target analyte canrenone). The procedure described here is part of a systematic toxicological analysis procedure for acidic drugs and poisons.

Screening for 18 diuretics and probenecid in doping analysis by liquid chromatography-tandem mass spectrometry.

A fast and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the screening of 18 diuretics and probenecid in human urine is presented. Analyses were performed on a LCQ-Deca instrument equipped with ESI-interface using scan by scan polarity changing. All diuretics and probenecid were separated in less than 20 min after liquid-liquid extraction with ethyl acetate. The LOD for all substances was 100 ng/mL or better. The method was applied to detect diuretics after the oral administration of several drugs including hydrochlorothiazide, bumetanide, spironolactone, furosemide, amiloride, triamterene, chlortalidone and epithizide. All diuretics could be detected for periods up to 96 h after the intake of therapeutic amounts.

Utilidad de muestras de orina espontánea para el control del tratamiento con uricosúricos / Utility of urine spot samples for the follow-up of uricosuric therapy

Objetivo: Estudio abierto y prospectivo en 15 pacientes con gota para valorar la utilidad de las muestras de sangre y orina espontánea para el seguimiento de la eficacia y seguridad del tratamiento con uricosúricos. Métodos: Todos los pacientes eran hipoexcretores de ácido úrico, no presentaban tofos y carecían de antecedentes de litiasis renal. Se realizó un estudio basal con orina de 24 h (aclaramiento de creatinina, aclaramiento de ácido úrico, excreción fraccionada de ácido úrico y reabsorción tubular de fosfato) y otro con orina espontánea (excreción fraccionada de ácido úrico y reabsorción tubular de fosfato). Posteriormente, se monitorizaron la excreción fraccionada de ácido úrico, la reabsorción tubular de fosfato, el pH, la densidad y el sedimento urinarios tras la administración de 100 mg de benziodarona los días 2, 4, 11, 18 y 25 de tratamiento. Resultados: La reducción de ácido úrico en plasma obtuvo su máximo valor el día 11 de tratamiento y la excreción fraccionada de ácido úrico entre los días 4 y 11 de tratamiento. La reabsorción tubular de fosfato no presentó variaciones durante el seguimiento. Tres pacientes presentaron alteración del sedimento urinario sin síntomas. Este grupo tenía mayor concentración de ácido úrico y creatinina en orina, así como mayor densidad urinaria, indicando una concentración de la orina secundaria a escaso volumen de diuresis. Conclusiones: Los parámetros que aportan las muestras de orina espontánea permiten un adecuado seguimiento de la eficacia y la seguridad del tratamiento con uricosúricos, evitando el inconveniente de la recogida de muestras de orina de 24 h (AU)

Potentiation of anticoagulant effect of warfarin caused by enantioselective metabolic inhibition by the uricosuric agent benzbromarone.

OBJECTIVE: To clarify the mechanism(s) for the interaction between warfarin and benzbromarone, a uricosuric agent, and to predict changes in the in vivo pharmacokinetics of (S)-warfarin from in vitro data. METHODS: Warfarin enantiomers and benzbromarone in serum, 7-hydroxywarfarin in urine, and serum unbound fractions of warfarin enantiomers were measured in patients with heart disease given warfarin with (n = 13) or without (n = 18) oral benzbromarone (50 mg/d). In vitro inhibition constants (K(i)) of benzbromarone for (S)-warfarin 7-hydroxylation were determined with use of human CYP2C9 and liver microsomes. The magnitude of changes in the formation clearance for 7-hydroxylation (CLf), the unbound oral clearance (CL(oral,u)), and the oral clearance (CL(oral)) for (S)-warfarin were predicted by equations incorporating the in vitro Ki, the theoretical maximum unbound hepatic benzbromarone concentration, and the fractions of warfarin eliminated through metabolism and of CYP2C9-mediated metabolic reaction susceptible to inhibition by benzbromarone. RESULTS: The patients given warfarin with benzbromarone required a 36% less (P < .01) warfarin dose than those given warfarin alone (2.5 versus 3.9 mg/d) to attain similar international normalized ratios (2.1 and 2.2, respectively), and the former had 65%, 53%, and 54% lower (P < .05 or P < .01) CLf, CL(oral),u, and CL(oral) for (S)-warfarin than the latter, respectively. In contrast, no significant differences were observed for (R)-warfarin kinetics between the groups. Benzbromarone was found to be a potent competitive inhibitor (Ki < 0.01 micromol/L) for (S)-warfarin 7-hydroxylation mediated by CYP2C9. The average changes in the in vivo CLf, CL(oral),u, and CL(oral)values for (S)-warfarin induced by benzbromarone were largely predictable by the proposed equations. CONCLUSION: Benzbromarone would intensify anticoagulant response of warfarin through an enantioselective inhibition of CYP2C9-mediated metabolism of pharmacologically more potent (S)-warfarin. The magnitude of changes in the in vivo warfarin kinetics may be predicted by in vitro data.

Stereoselective renal tubular secretion of a new uricosuric diuretic, 6,7-dichloro-5-(N,N-dimethylsulfamoyl)-2,3-dihydro-2-benzofurancarboxyl ic acid (S-8666), in cynomolgus monkeys.

Plasma concentration-time curves and urinary excretion of individual enantiomers of unchanged S-8666 and its N-monodemethylated metabolite, M-1, in male cynomolgus monkeys were measured after oral administration of racemic S-8666 at doses of 5, 10, and 50 mg/kg and also after intravenous injection at doses of 1, 5, and 10 mg/kg. The Tmax values for individual enantiomers of S-8666 in fasted male monkeys were 30 min after oral administration. The AUC values for S(-)-S-8666 were greater than those for R(+)-S-8666 and oral dose-AUC relationships of both enantiomers showed a linearity over the dose range used. Most of the S-8666 and a trace of M-1 were excreted in the first 24-hr urine, with no evidence of stereoselectivity from the amounts excreted. Since large portions were recovered from the urine as unchanged S-8666 after intravenous injection, most excretion occurs via the kidney. The t1/2 beta values and the Vdss for S(-)-S-8666 after intravenous injection were smaller than those for R(+)-S-8666. The CLt and the CLr values decreased with increasing intravenous doses, indicating saturation at a renal excretion process at high plasma concentrations of S-8666. The CLt and CLr values for R(+)-S-8666 were greater than those for the S(-)-enantiomer. The unbound fraction of R(+)-S-8666 in plasma was significantly greater than that of S(-)-S-8666 [21.6% for R(+), 12.0% for S(-)]. Renal clearance for the unbound fraction of S(-)-S-8666 was greater than that of R(+)-S-8666, suggesting stereoselective renal tubular secretion.

Chronopharmacology of the new uricosuric diuretic S-8666 in rats.

A new loop diuretic with uricosuric activity, 6,7-dichloro-5-(N,N-dimethylsulfamoyl)-2,3-dihydro-2-benzofuran carboxylic acid (S-8666), was given orally at 12:00 a.m. or 12:00 p.m. in rats. The diuretic of S-8666 and the urinary excretions of the drug and its active metabolite S-8680 (N-demethyl S-8666) were greater at 12:00 a.m. than at 12:00 p.m. Thus, the present study indicates that the diuretic effects of S-8666 varies with its administration time. Time-dependent variations in the amount of urinary excretions of S-8666 and S-8680 might be involved in the mechanisms for this phenomenon.

Chronopharmacology of the new uricosuric diuretic S-8666 in rats: (II). Examination in aged rats.

We have previously demonstrated a time-dependent variability in the diuretic effects of S-8666, a new loop diuretic with uricosuric activity, in young rats. The present study was undertaken to determine whether such a daily variation in the effects of the agent exists in aged rats. S-8666 (30 and 90 mg/kg) was orally given at 12:00 a.m. (day trial) or at 12:00 p.m. (night trial) in young (10-11 week old) and aged (23-24 month old) Wistar rats. Urine was collected for 8 hours after the agent; and urinary excretions of sodium, S-8666 and its active metabolite S-8680 were determined. Urinary excretions of volume and sodium following S-8666 at 12:00 a.m. were greater than those at 12:00 p.m. in the young and aged animals. Urinary excretions of S-8666 and S-8680 were also greater in the day trial compared to those in the night trial in both groups of rats. In the day and night trials, there were significant correlations between urinary S-8666 + S-8680 and the diuretic effects in both groups of rats. These findings indicate that the diuretic effects of S-8666 also vary with its time of dosing in aged rats. The time-dependent variations in urinary S-8666 and S-8680 might be involved in this phenomenon.

Renal excretion of a slauretic-uricosuric agent (MK-196) and interaction with a urate-retaining drug, pyrazinoate, in the chimpanzee.

The excretory pattern for MK-196 is ocmpatible with that of other weak organic acids such as salicylate and probenecid. Tubular secretion of MK-196 is strongly inhibitied by probenecid and high loads of p-aminohippurate. Urinary excretion of MK-196 is increased 10-fold when the urine is alkaline. Clearances of MK-196 were not corrected for plasma protein binding of the drug which is very high (greater than 99%). Bidirectional transport processes are operative in that MK-196 is secreted by the renal tubule and passively back diffuses across the tubular epithelium by a pH-dependent process. MK-196 is able to overcome pyrazinoate-induced urate retention, whereas probenecid is not when studied by conventional clearance techniques. The uricosuric activity of MK-196 appears to be somewhat less with pyrazinoate than in its absence. When MK-196 is administered prior to pyrazinoate an attenuated uricosuric response was observed. This finding cannot be ascribed to a temporal decline in uricosuric action. Diuresis and saluresis produced by MK-196 are not influenced by pyrazinoate. The interaction of MK=196 and pyrazinoate on urate excretion is in direct contrast to results obtained with probenecid and pyrazinoate. A model has been proposed to explain this unique finding.

Gas-liquid chromatographic determination of tienilic acid (SKF 62.698) in human plasma and urine.

A gas-liquid chromatographic method is described for the determination of tienilic acid (SKF 62.698), a diuretic with uricosuric properties, in human plasma and urine. The method, which is based on the methylation of the compound, is rapid, specific and sensitive. The lowest level accurately determined is about 50 ng/ml in plasma and 1 mug/ml in urine. The first results from a human volunteer are given.