Detection of urinary markers for thiazide diuretics after oral administration of hydrochlorothiazide and altizide-relevance to doping control analysis.

In sports, thiazide diuretics are used to flush out previously taken prohibited substances with forced diuresis and in sports where weight classes are involved to achieve acute weight loss. Thiazide diuretics include compounds which are very unstable and hydrolyse in aqueous media. Because information regarding the urinary detection of the hydrolysis products is limited, urinary excretion profiles for the hydrolysis product 4-amino-6-chloro-1,3-benzenedisulphonamide were established in 6 healthy volunteers after oral administration of altizide (15 mg per tablet) and hydrochlorothiazide (25mg per tablet). Additionally, the excretion profile of chlorothiazide, a metabolite of altizide and hydrochlorothiazide, was also determined. A quantitative liquid-chromatographic tandem mass spectrometric method to detect the 4 substances was developed and validated. The result of this work shows that altizide is eliminated within 48 h in urine whereas hydrochlorothiazide was detectable after 120 h. Chlorothiazide was determined to be a minor metabolite of altizide and hydrochlorothiazide and could be detected up to 120 h. The hydrolysis product, 4-amino-6-chloro-1,3-benzenedisulphonamide, was detectable 120 h after administration, with concentrations at least 10 times higher than the parent drug. Concentrations ranged between 41-239 and 60-287 ng/mL after altizide and hydrochlorothiazide administration, respectively. The study shows that 4-amino-6-chloro-1,3-benzenedisulphonamide is an important target compound for the long time detection of thiazide diuretics in urine.

Carbonic anhydrase-immobilized precolumn for selective on-line sample pretreatment in high-performance liquid chromatographic determination of certain sulphonamide drugs.

Carbonic anhydrase (CA)-immobilized aminopropyl silica precolumn was developed for direct injection determination of certain sulphonamide drugs in biological fluids by column-switching (CS) high-performance liquid chromatography. Under the optimized conditions, only the sulphonamide drugs with an unsubstituted sulphonamide group were retained on the CA precolumn and separated on a reversed-phase analytical column. Of these, the retention of hydrochlorothiazide (HCT), chlorothiazide, acetazolamide, furosemide (FS) and chlorthalidone was almost quantitative. The peak area of HCT was proportional to the concentration in the range of 1-100 nmol/mL with relative standard deviations of 3.7% (5 nmol/mL) and 0.7% (100 nmol/mL). This CS system was applied to urine and plasma samples spiked with HCT and FS. Endogenous components of these were effectively removed, and HCT and FS were selectively retained on the CA precolumn. Almost quantitative recoveries and reproducibility were obtained.

High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography.

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.

Screening for diuretics in human urine by gas chromatography-mass spectrometry with derivatisation by direct extractive alkylation.

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 phenomenon and cause of the dose-dependent oral absorption of chlorothiazide in rats: extrapolation to human data based on the body surface area concept.

The reported incomplete and dose-dependent absorption of chlorothiazide in humans was demonstrated in six rats after five oral solutions at doses of 0.93, 2.55, 9.23, 25.6, and 70.2 mg/kg. Mean 48-hr urinary recoveries of intact drug were 57.3, 50.4, 36.7, 22.8, and 15.3%, respectively. A similar degree of dose dependency in absorption was found in rat, dog, and human when the doses were related to unit body surface area (BSA) but not on unit body weight, indicating similar interspecies absorptive capacity in terms of unit BSA. This finding may be partly rationalized by marked similarities in the reported solution transit time (2-3 hr) in the small intestine as well as in the calculated gross surface area of the small intestine per unit BSA (0.163 for rat and 0.132 for human). Contrary to the previous postulation of a specific absorption site, the drug was absorbed from different regions of the GI tract with apparent 1-hr absorption rates, studied by the in situ closed-loop method, in the following rank order: jejunum (34.6%) greater than duodenum (32.7%) greater than large intestine (20.1%) greater than ileum (18.0%) greater than stomach (12.4%). Different from the commonly assumed first-order absorption process, the intestinal loop absorption was concentration-dependent, suggesting a saturable mechanism. For example, the absorption rate at 0.008 mg/mL was higher than that at 0.2 mg/mL in ileal loops (61%, p less than 0.01) and jejunal loops (22%, p less than 0.1). In addition, the absorption rates at pH 6 and 7.4 were statistically identical, indicating a lack of ionization effect that is important in the passive absorption process. The solubility-limited absorption could probably be ruled out at doses below 2.55 mg/kg for rat and 125 mg for human in view of higher aqueous solubilities at 37 degrees C (e.g., 1.3 mg/mL at pH 7) found in the present study. Contrary to the previous hypothesis of low membrane permeability as a limiting factor for absorption, the "intrinsic" partition coefficient in 1-octanol/aqueous buffer was moderate, 0.6. Furthermore, the absorption in ileal and jejunal loops was enhanced by an apparent increase in mesenteric blood flow by caffeine. The existence of prolonged oral absorption in rats and humans is discussed.

The effect of beta-adrenoceptor blockers on the absorption and excretion of chlorothiazide in man.

The effect of beta-adrenoceptor blockers on the absorption and elimination of the diuretic chlorothiazide was studied in healthy subjects. A week of pretreatment with either pindolol (10 mg twice daily) or propranolol (80 mg twice daily) resulted in significant reduction in 36 h mean cumulative urinary recovery of chlorothiazide in two groups of six subjects compared with a control (untreated) group. A week of pretreatment with atenolol (100 mg daily) did not significantly alter 36 h cumulative urinary excretion in another group of six subjects. None of the beta-blockers significantly changed chlorothiazide half-life. It is suggested that the non-selective (as opposed to the cardioselective) beta-blockers reduce chlorothiazide absorption by the mechanism(s) discussed.

Cellulose phosphate and chlorothiazide in childhood idiopathic hypercalciuria.

A calcium loading test performed on seven of eight children with idiopathic hypercalciuria identified the hyperabsorptive form of hypercalciuria in five and renal hypercalciuria in one. The type of hypercalciuria was not identified in the other patient. Three children presented with hematuria without calculus formation. Chlorothiazide reduced the urinary calcium excretion level in two of six patients to the normal range. The addition of cellulose phosphate to chlorothiazide reduced the urinary calcium excretion level to the normal range in those four patients who showed an incomplete response to chlorothiazide alone. There was clinical improvement with cellulose phosphate in another child whose symptoms did not disappear after chlorothiazide had reduced urinary calcium level to the normal range. Cellulose phosphate is effective in children with recurrent stone formation who have shown inadequate response to chlorothiazide.

Chlorothiazide absorption in humans--possible example of Michaelis-Menten kinetics.

Chlorothiazide absorption was studied in five healthy adult males at 1,000, 750, 500 and 250-mg dose levels. The 24-hour-absorbed fraction fell from a mean value of 20.15% of the orally administered dose at the 250-mg level to 8.38% at 1,000 mg. Analysis of data at the four dosage levels for each subject is suggestive of the fact that chlorothiazide absorption is possibly an example of Michaelis-Menten kinetics. Possible factors responsible for the saturable absorption are discussed.

On-line direct liquid introduction interface for micro-liquid chromatography/mass spectrometry: application to drug analysis.

We describe an integrated micro-liquid chromatograph/mass spectrometer (micro-LC/MS) system capable of performing routine determinations for 1--10 ng of drugs and their metabolites extracted from biological fluids. The micro-LC is constructed from conventional "high-performance" liquid-chromatographic instrumentation by using commercially available components. The mass spectrometer is operated in the chemical ionization mode. The direct liquid introduction micro-LC/MS interface can be constructed from commercially available materials. Chromatographic and mass spectral results demonstrate the ability of the micro-LC and micro-LC/MS system to separate and determine multiple components in standards of trace concentrations and in equine urinary extracts. The stability and sensitivity of this micro-LC/MS system are demonstrated through determinations of trichlormethiazide.

Bioavailability of chlorothiazide from 50, 100, and 250 MG solution doses.

The bioavailability of chlorothiazide was examined following single oral solution doses to eight healthy male volunteers. Drug was administered in 250 ml of water after overnight fast. Bioavailability was determined by measuring 72 h urinary recovery of unchanged drug. Mean urinary recovery from 50, 100, and 250 mg doses was, respectively, 28.3, 47.0 and 83.3 mg, representing 56.4, 47.0, and 33.3 per cent of the administered dose. The correlation coefficient between dose size and percentage recovery was -0.662. These results add support to previous suggestions that the absorption of chlorothiazide from the gastrointestinal tract is saturable, and that the availability of chlorothiazide may be similar to that of hydrochlorothiazide when these compounds are administered in the same dosage range.

High-pressure liquid chromatographic determination of chlorothiazide and hydrochlorothiazide in plasma and urine: preliminary results of clinical studies.

High-pressure liquid chromatographic procedures were developed for the determination of chlorothiazide and hydrochlorothiazide in plasma and urine. The plasma assay incorporates a preextraction procedure that eliminates interference by endogenous substances. Chromatography is carried out on an octadecyl reversed-phase column. Mobile phases are 15% methanol in 0.01 M acetic acid for plasma and 4% acetonitrile in 0.01 M sodium perchlorate, adjusted to pH 4.6, for urine. At a flow rate of 2.5 ml/min, the retention times for chlorothiazide and hydrochlorothiazide are 3.5 and 4.6 min for plasma and 10.5 and 13.5 min for urine, respectively. Preliminary results of a clinical study in fasting male volunteers showed that the plasma levels and urinary excretion rate of chlorothiazide peaked at 1-2 hr following a 500-mg oral dose and subsequently declined irregularly. On the other hand, the plasma levels and urinary excretion rate of hydrochlorothiazide peaked at 2-3 hr following a 50-mg oral dose and subsequently declined in biphasic fashion. Urinary excretion rates of both chlorothiazide and hydrochlorothiazide closely resemble their concentration profiles in plasma.

Apparent dose-dependent absorption of chlorothiazide in dogs.

The purpose of this study was to determine the effect of oral dose on the absorption of chlorothiazide in the dog. Chlorothiazide was quantitatively excreted in the urine after administration of 50-mg and 250-mg intravenous doses. In contrast, the urinary recovery of chlorothiazide after oral administration showed appreciable interanimal variation and decreased from 70.4% to 26.7% on the average as the oral dose was increased from 125 mg to 750 mg. Oral administration of a single 15-mg dose of propantheline bromide (a potent inhibitor of gastric emptying and intestinal motility) at--1 hr increased the absorption of a 250 mg oral dose of chlorothiazide in three out of four dogs. These results suggest that chlorothiazide absorption is dose dependent and apparently site specific.

Quantitative determination of thiazides in urine by a sensitive colorimetric method.

Chloriothiazide and hydrochlorothiazide may be determined in urine by the Bratton-Marshall reaction after extracting with ethyl acetate and treating the extract with Florisil. Such treatment reduces the background color of the urine sample and eliminates interferences. The modified procedure is thus sensitive enough to determine both drugs in 24 hour urine specimens from patients receiving therapeutic doses orally.

Effect of chlorothiazide on the pharmacokinetics of lithium in plasma and erythrocytes.

The effect of chlorothiazide on the pharmacokinetics of lithium in both plasma and RBCs was studied in normal adult males. This was accomplished by administering single, 300 mg. doses of lithium carbonate alone and concurrently with chlorothiazide (0.5 grams/day for one week). Thiazide administration resulted in increases in plasma and RBC concentrations of 26.2 and 25.4%, respectively, as well as a 26.5% decrease in renal lithium clearance. The data were analyzed in terms of a two compartment pharmacokinetic model as previously reported (8). The results of this analysis showed that the change in renal lithium clearance could be accounted for by a 24.1% reduction in the value of ke, the excretion rate constant. It was also shown that changes in plasma lithium concentration during chronic lithium therapy would be expected to increase by 25-30% when chlorothiazide therapy is employed. The model also predicts that changes in RBC concentrations would parallel those occurring in plasma and thus no change in the RBC/plasma lithium ratio would be expected.