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Experimental conditions have been studied in order to improve the sensitivity for the analysis of diuretics and probenecid in urine samples by high-performance liquid chromatography with ultraviolet detection. Sample clean-up and chromatographic parameters have been optimized to obtain a suitable sensitivity for the detection or quantification of each diuretic using an HP-Hypersil ODS-C18 column (5 microns, 250 mm x 4 mm I.D.), taking into account the pharmacological properties of each compound. The reliability of this method was tested by analysing urine samples after a minimum single-dose administration of chlorthalidone and probenecid.

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS-methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS-5% methanol (v/v) at 50 degrees C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.

A simple and efficient procedure has been developed for the derivatization of diuretic agents in human urine by direct extractive alkylation and their detection by gas chromatography-mass spectrometry. The procedure is an improvement over previous extractive alkylation methods because of the development of a simple clean-up step using a macroreticular acrylic copolymer (SM-7 resin) to remove the coextracted phase-transfer reagent from the organic phase after derivatization. With 1 ml of sample the method gives detection limits in the range 10-50 ng/ml for acetazolamide, probenecid, dichlorphenamide, hydroflumethiazide, furosemide, chlorthalidone, bumetanide, hydrochlorothiazide, quinethazone, bendroflumethiazide, metolazone and cyclopenthiazide.

A quantitative method for the simultaneous high-performance liquid chromatographic (HPLC) resolution and determination of the enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2,3-dihydrobenzofuran-2-carboxyl ic acid, a new diuretic, and its N-monodemethylated metabolite in monkey plasma and urine is described. The method includes diethyl ether extraction of the samples and S-(-)-alpha-methylbenzylamide derivatization of the extract, followed by reversed-phase solid-phase extraction and injection of the resulting diastereoisomers onto a reversed-phase HPLC column. Baseline separation was obtained. The assay showed linearity over the range 0.1-50 micrograms/ml of plasma and 0.25-500 microliters of urine, with a lower limit of detection of ca. 0.01 micrograms/ml for each of the enantiomers. The method is adequate for pharmacokinetic and enantioselective disposition studies of both the diuretic and its metabolite.

A rapid, sensitive and reliable gas chromatographic-mass spectrometric (GC-MS) screening procedure for diuretics in human urine has been developed. The procedure uses derivatisation by extractive methylation directly from the urine. The suitability of a number of phase transfer reagents and solvents were studied for the detection of sixteen diuretics. The results obtained indicate that the screening procedure employing tetrahexylammonium hydrogensulphate at pH 12 with methyl iodide in toluene at room temperature was the most effective. This method gives selectivity and sensitivities down to 0.03-0.1 microgram/ml and provides a substrate suitable for GC-MS confirmation without further manipulation. The application of the method is demonstrated by the screening of urine for bumetanide, ethacrynic acid, acetazolamide, chlorothiazide and hydrochlorothiazide.

The application of thermospray and plasmaspray high-performance liquid chromatography-mass spectrometry to the analysis of diuretics and probenecid has been investigated. The latter method gave better ionization efficiency than the former, and its response was optimized by altering the solvent composition: best results were obtained with water-methanol-acetonitrile-trifluoroacetic acid. Using different proportions of these solvents, three isocratic systems were developed to separate the compounds under study. The principal characteristic of plasmaspray positive-ion mass spectra was a protonated molecular ion and very little fragmentation was evident. In the negative ionization mode, the plasmaspray method gave mass spectra showing more fragmentation, which resulted in additional structural information. The ability of trifluoroacetic acid to form negative cluster ions precluded its use as a mobile phase component. The minimum detectable amounts determined by the analysis in the positive-ion mode was compound-dependent, but generally ca. 10-150 ng. In many cases the compounds could be detected in urine extracts.

A sensitive and specific thin-layer densitometric method was developed for the determination of muzolimine (BAY g 2821), a structurally new diuretic drug of the pyrazolinone type. For detection of the drug on thin-layer chromatographic plates, a colour reaction with 4-dimethylaminocinnamaldehyde is carried out. The detection limit was 50 pg per spot on commercially available silica gel 60 plates. A nearly linear relationship between integrated peak area and amount per spot was obtained in the range from 100 pg to 1 mug per spot. For quantitative determination in biological fluids, a recovery of about 100% is achieved by a single extraction with dichloromethane, and the extract is spotted directly on to the plate. The detection limit of muzolimine in plasma and urine was 1 ng/ml, and inaccuracy in the nanogram range was found to be 5-8%. The assay was applied to pharmacokinetic studies and can be recommended for monitoring plasma levels of muzolimine in patients.

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.

A rapid and reliable screening procedure using high-performance liquid chromatography for the detection of 23 diuretics (belonging to five different pharmacological groups) in urine has been developed. Two aliquots of 2-ml urine samples were extracted separately under acidic and basic conditions. The acidic and basic extracts were pooled, evaporated to dryness and reconstituted in methanol. The methanolic extract was injected onto a Hewlett-Packard Hypersil ODS C18 (5 microns) column (column I) and a Hewlett-Packard LiChrosorb RP-18 (5 microns) column (column II; an alternative column). The same gradient mobile phase was used for both columns. A diode array ultraviolet detector was set to monitor the signal to the integrator (Chem Station) at 230 and 275 nm. Recovery studies of the 23 diuretics were performed under acidic and basic conditions. The overall lower limits for detection on column I using both extraction procedures ranged from 0.5 to 1.5 micrograms/ml of urine (average 1.0 micrograms/ml). Amiloride, ethacrynic acid and probenecid could not be detected below 5 micrograms/ml of urine. No interference from the biological matrix was apparent. Amiloride could be detected in urine 4 h after oral administration of 15 mg of amiloride to a healthy volunteer, when the sample was extracted under alkaline conditions. The suitability of the screening method for the analysis of urine samples was tested by studying the variation with time of chlorthalidone, furosemide, probenecid, acetazolamide, quinethazone, spironolactone, bendroflumethiazide, bumetanide, triameterene and hydrochlorothiazide concentrations in the urine of normal human volunteers after minimum single or multiple (probenecid) doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitive and specific thin-layer (TLC) and high-performance liquid chromatographic (HPLC) methods were developed for the determination of the diuretic agent 2-chloro-5-[4-hydroxy-3-methyl-2-(methylimino)-4-thiazolidinyl]benzenesulphonamide hydrochloride (HOE 740). HOE 740 can be determined in serum by HPLC. The detection is performed at a very short wavelength (202 nm), resulting in a detection limit of 10 ng/ml. By TLC only urine levels that are normally high can be determined directly (the detection limit is 70 ng/ml). For the determination of the lower serum levels it is necessary to convert the drug into its dehydration product, which has a higher absorbance and gives sufficient sensitivity (the detection limit is 10 ng/ml). Serum levels determined by the two methods correlate well. Some pharmacokinetic and excretion-kinetic data were computed using two-compartment open models.

We describe a liquid chromatographic screening procedure for the detection, in urine, of twelve of the fifteen potassium-depleting diuretics available in Australia. A 2-ml urine sample was acidified with NaH2PO4 (pH 4.1) and extracted with 4 ml ethyl acetate. The sample was cleaned up further by washing with 5 ml Na2HPO4 (pH 7.5). The ethyl acetate was then evaporated to dryness, the residue reconstituted in 100 microliters mobile phase and 5 microliter were injected onto a Merck LiChrosorb RP-18 (5 microns) column. The ultraviolet absorbance of the eluent was monitored at 271 nm for 10 min. The screen was evaluated by giving each of thirty volunteers the lowest recommended dose of one of the diuretics in the study and obtaining urine samples 4, 8 and 24 h after having taken the dose. Twelve diuretics, chlorothiazide, hydrochlorothiazide, quinethazone, chlorthalidone, methyclothiazide, clopamide, frusemide, metolazone, mefruside, bendrofluazide, cyclopenthiazide and bumetanide, were all detectable up to 24 h after a dose. We therefore conclude that the screen would be reliable for the detection of these diuretics in urine.

A sensitive, selective and easy to use high-performance liquid chromatographic method for the determination of cicletanide, a new diuretic, in plasma, red blood cells, urine and saliva is described. After extraction of cicletanide together with an internal standard with diethyl ether, or diethyl ether-n-hexane (20:80) for urine, the sample extracts are chromatographed with water-methanol-acetic acid (50:50:0.3) as eluent on to a Nucleosil C18 column. Both compounds are detected by their ultraviolet absorption at 280 nm. The calibration graph was linear between 0.2 and 20 micrograms/ml for plasma and between 0.2 and 5 micrograms/ml for the other biological fluids. The sensitivity limit was 20 ng/ml for plasma, red blood cells and saliva and 30 ng/ml for urine. The coefficients of variation of the between-day assays did not exceed 4.6% in plasma, 8.3% in red blood cells, 7.8% in urine and 4.2% in saliva for the lowest concentrations studied. The application of the method to a pharmacokinetic study of cicletanide after a single oral therapeutic dose in humans is reported.