Interfacing on-line solid phase extraction with monolithic column multisyringe chromatography and chemiluminescence detection: An effective tool for fast, sensitive and selective determination of thiazide diuretics.

A new, multisyringe flow injection set-up has been developed for the completely automated determination of trace thiazide compounds with diuretic action in different types of samples. The proposed instrumental set-up exploits for the first time, a low pressure on-line solid phase extraction-liquid chromatography-chemiluminescence detection method. This novel combination of sample treatments in flow systems expands the current applicability of low pressure liquid chromatography due to the isolation/preconcentration of the target compounds, besides high selectivity and sensitivity. For the determination of three thiazide compounds named hydroflumethiazide, furosemide and bendroflumethiazide, the proposed set-up provided with the preconcentration of only 1mL of sample, limits of detection of 3, 60 and 40microgL(-1), respectively. Furthermore wide linear dynamic ranges of 6-4000, 140-20,000 and 90-40,000microgL(-1), respectively, were obtained. Besides of this, a high injection throughput of 12h(-1) was also achieved. As in sports, thiazide diuretics are prohibited substances, the proposed method has been applied to their determination in urine samples. Furthermore the potential of the proposed method as a fast-screening approach for emerging contaminants in waters has been also tested by applying it to well water and leachates from a solid waste landfill.

Stability of selected chlorinated thiazide diuretics.

In sports, diuretics are used for two main reasons: to flush previously taken prohibited substances with forced diuresis and in sports where weight classes are involved to achieve acute weight loss. A common property observed for thiazides is hydrolysis in aqueous media resulting in the formation of the degradation product aminobenzenedisulphonamide. This degradation product can be observed for several thiazides. Because there is limited information regarding the effect of pH, temperature and light on the stability of thiazides, these parameters were investigated for chlorothiaizide, hydrochlorothiazide and altizide. For all three compounds the degradation product could be detected after incubation at pH 9.5 for 48h at 60 degrees C. At lower pH and temperature the degradation product could not be detected for all compounds. When samples were exposed to UV-light altizide and hydrochlorothiazide were photodegraded to chlorothiazide. When the degradation rate between the different compounds was compared for a given temperature and pH, altizide is the most unstable compound. This study confirms that thiazide degradation products can be formed in urine during transport. Hence doping control laboratories shall include them into their routine testing methods as required by WADA.

Gas-phase behaviour of negative ions produced from thiazidic diuretics under electrospray conditions.

A systematic mass spectrometric study of 10 thiazidic diuretics and related compounds was undertaken by mass spectrometry (MS) with electrospray ionization in the negative ion mode. Collisional dissociation 'in-source' (CID-MS) and in a low-pressure collision cell (CID-MS/MS) were compared in both excitation regions. Spectra obtained by CID-MS and by CID-MS/MS were matched. Using the two methods, loss of HCl and consecutive dissociations from 2HCl losses were exhibited from compounds such as methyclothiazide and trichlormethiazide but not from other thiazidic diuretics that contain chlorine substituents in the aromatic moiety. However, deprotonated dichlorphenamide gave rise to loss of HCl by CID-MS and CID-MS/MS. For other diuretics such as hydroflumethiazide and hydrochlorothiazide, the loss of HCN and [HCN + SO(2)] was relevant. Reaction mechanisms were checked by means of deuterium-hydrogen exchange, which showed that deprotonation took place regioselectively on the heterocyclic moiety. The cleavage pathways require molecular isomerization forming ion-dipole complexes prior to decompositions, allowing long-distance proton transfer for neutral elimination. Identifications of the most specific fragmentations presented in this paper were applied to the screening and unambiguous identification of diuretics for horse doping control.

Epithelial Na channels and short-term renal response to salt deprivation.

To test the role of epithelial Na channels in the day-to-day regulation of renal Na excretion, rats were infused via osmotic minipumps with the Na channel blocker amiloride at rates that achieved drug concentrations of 2-5 microM in the lumen of the distal nephron. Daily Na excretion rates were unchanged, although amiloride-treated animals tended to excrete more Na in the afternoon and less in the late evening than controls. When the rats were given a low-Na diet, Na excretion rates were elevated in the amiloride-treated group within 4 h and remained higher than controls for at least 48 h. Adrenalectomized animals responded similarly to the low-Na diet. In contrast, rats infused with polythiazide at rates designed to inhibit NaCl transport in the distal tubule were able to conserve Na as well as did the controls. Injection of aldosterone (2 microg/100 g body wt) decreased Na excretion in control animals after a 1-h delay. This effect was largely abolished in amiloride-treated rats. On the basis of quantitative analysis of the results, we conclude that activation of amiloride-sensitive channels by mineralocorticoids accounts for 50-80% of the immediate natriuretic response of the kidney to a reduction in Na intake. Furthermore, the channels are necessary to achieve minimal rates of Na excretion during more chronic Na deprivation.

Rapid screening for diuretic doping agents in urine by C60-assisted laser-desorption-ionization-time-of-flight mass spectrometry.

This study describes a matrix-assisted laser-desorption-ionization (MALDI) mass spectrometry for rapid screening of 12 diuretics in spiked urine. C60 is used as the matrix for MALDI. Diuretics are directly analyzed by C60-MALDI without previous derivatization. The fact that most diuretic molecules contain sulfate groups accounts for why anions of the molecules can be easily desorbed and ionized in MALDI. Using C60 as the matrix is advantageous because of the low background in the low mass range on the negative MALDI mass spectrum. A clear mass window between m/z 200 and 600 in negative ion mode is also obtained. Only a minimum amount of the sample (< 1 microL) is necessary to perform the analysis. The detection limit of diuretics is approximately 0.1-1 microg/mL.

Simple method for determination of hydrochlorothiazide in human urine by high performance liquid chromatography utilizing narrowbore chromatography.

A simple high performance liquid chromatographic (HPLC) method utilizing narrowbore chromatography was developed for the determination of hydrochlorothiazide in human urine. A mobile phase of 0.1% aqueous acetic acid--acetonitrile (93:7, v/v) pH 3 was used with a C18 analytical column and ultraviolet detection (UV). The method demonstrated linearity from 2 to 50 micrograms ml-1 using 50 microliters of urine with a detection limit of 1 microgram ml-1. The method was utilized in a study evaluating if racial differences are present in the pharmacokinetic and pharmacodynamic effects of hydrochlorothiazide.

Detection of thiazide-based diuretics in equine urine by liquid chromatography/mass spectrometry.

Thiazide-based diuretics are included in the list of banned drugs in the horse-racing industry. One effect of their misuse is increased urine flow, contributing to dilution of other doping agents. Their determination is essential in ensuring compliance to horse-racing regulation. This study evaluates the feasibility of using liquid chromatography/mass spectrometry (LC/MS) with electrospray and atmospheric pressure chemical ionization interfaces to analyze thiazidic diuretics in equine urine samples. Existing LC and gas chromatography/MS methods are limited in their applicability to thiazide analysis. Sample preparation, analyte extraction, chromatographic separation, ion-source collision induced dissociation, solvent composition, ionization mode, and ion polarity are discussed. The practicality of LC/MS for this analysis is demonstrated with actual equine administration samples collected at specified time intervals. Detection limits were 270 ng/mL for chlorothiazide, 131 ng/mL for hydrochlorothiazide, and 384 ng/mL for trichlormethiazide.

3-Bromomethyl-propyphenazone as a new derivatization reagent for high performance liquid chromatography of captopril and hydrochlorothiazide with UV-detection.

3-Bromomethyl-propyphenazone (BMP) was used as a derivatization reagent for the detection and quantification of captopril (CAP) and hydrochlorothiazide (HCT) by high performance liquid chromatography using Zorbax C8 column, and 0.05M sodium acetate, acetonitrile, methanol (14:17:4; pH 6.5) as mobile phase system with UV-detection at 254 nm. The cited reagent reacts with the mercapto and amino groups of CAP and HCT in acetone using anhydrous potassium carbonate as hydrobromide acceptor. The reaction was completed within 30 min for CAP and 60 min for HCT with heating at 105 +/- 5 degrees C in mini-reaction vial. The linear concentration ranges for both CAP and HCT were 8 to 160 and 6 to 140 ng per injection, respectively. The derivatized captopril was synthesized and confirmed with spectral analysis. This method was applied for determination of spiked captopril in human urine after extraction with Extrelut-20 column using ethyl acetate:isopropanol (85:15 v/v) as eluant.

[The phenomenon of resistance to a thiazide diuretic in acute kidney failure]. / Izuchenie fenomena rezistentnosti k tiazidovomu diuretiku pri ostroi pochechnoi nedostatochnosti.

The resistance to hypothiazid (hydrochlorothiazide) during the development of acute renal insufficiency (ARI) induced by glycerin injection was studied in rat experiments. Natriuretic, kaliuretic, and hydrouretic activity of the diuretic decreased two days after injection of a glycerin solution. In this period hypothiazid excretion with the urine was least. Ten days later, despite the restoration of the water- and electrolyte-excretion function of the kidneys and normalization of hypothiazid excretion with the urine, the natriuretic effect of the drug was still very poor. This is indirect evidence of the long-term affection of the receptors for the thiazid diuretics during the development of ARI. The affection remains even after the excretory function of the kidneys is restored. The high correlation between the cumulative excretion of hypothiazid with the urine and natriuresis encountered in intact animals was weaker in rats with acute renal insufficiency.

Effect of hydrochlorothiazide on the pharmacokinetics of enalapril in hypertensive patients with varying renal function.

An open, randomised, cross-over study was performed to investigate the pharmacokinetics of enalaprilat, administered as 20 mg enalapril both as monotherapy and in combination with hydrochlorothiazide (HCTZ 12.5 mg). Three groups of 6 hypertensive patients were enrolled [untreated diastolic blood pressure (DBP) 90-115 mm Hg]; normal renal function [glomerular filtration rate (GFR) > 81 ml min-1 1.73 m-2], mild renal impairment (GFR 51-80 ml min-1 1.73 m-2), and moderate renal impairment (GFR 31-50 ml min-1 1.73 m-2). The pharmacokinetics of enalaprilat and enalaprilat plus HCTZ correlated predictably with renal impairment with increased plasma concentrations and decreased urinary elimination at lower values of GFR. The coadministration of HCTZ had no significant effect on the pharmacokinetics of enalaprilat in any group. We conclude that although the pharmacokinetics of both enalaprilat and HCTZ are related to renal function, HCTZ has no significant effect on the pharmacokinetics of enalaprilat and that dosage adjustment for both regimens should be based on renal function.

Interaction of bemetizide and indomethacin in the kidney.

The effect of a single oral dose of 25 mg bemetizide on renal function without and with concomitant administration of the prostaglandin synthesis inhibitor indomethacin was investigated in ten healthy volunteers during sustained water diuresis. Bemetizide induced a significant increase in urinary sodium and chloride excretion from 196 +/- 30 and 163 +/- 28 mumol/min to 690 +/- 54 and 537 +/- 51 mumol/min (P less than 0.01). This effect occurred in the absence of changes in glomerular filtration rate, urinary excretion of phosphate or the delivery of chloride beyond the proximal nephron to the distal tubules (distal delivery) [(CH2O + CCl)/GFR . 100], but was associated with a significant decrease in distal fractional chloride absorption (DFACl) [CH2O/(CH2O + CCl)] from 0.84 +/- 0.02 to 0.63 +/- 0.02 (P less than 0.01). Bemetizide also increased urinary excretion of prostaglandin (PG) E2. Concomitant indomethacin administration significantly suppressed urinary excretion of PGE2 and markedly decreased urinary excretion of sodium and chloride during control and following bemetizide administration. Indomethacin had no effect on glomerular filtration rate, urinary excretion of phosphate, distal delivery or the urinary excretion of bemetizide but significantly increased DFACl both during control and after bemetizide administration. Our results show that bemetizide as a thiazide-diuretic acts in the diluting segments of the nephron. Indomethacin administration induces retention of sodium and chloride and blunts the renal effects of bemetizide via increased absorption in the diluting segments. The interaction of both drugs most likely represents a pharmacodynamic interaction.

Surreptitious diuretic ingestion and pseudo-Bartter's syndrome.

A patient with profound hypokalemia satisfied the criteria for Bartter's syndrome, including hyperreninemia, aldosteronism, normal blood pressure, and hyperplasia of the juxtaglomerular apparatus. Two screening tests of urine and one of plasma for diuretic agents gave negative results. A third urinary sample gave negative results for thiazide but positive for furosemide; the fourth and fifth samples gave negative results for furosemide but positive for thiazide. Urinary prostaglandin excretion was normal. We conclude that this apparent case of Bartter's syndrome was caused by long term surreptitious diuretic ingestion and suggest this may occur more frequently than is generally appreciated.

Liquid-chromatographic detection of thiazide diuretics in urine.

We describe a liquid-chromatographic procedure for detection in urine of all thiazide drugs currently used clinically. Urine is treated initially with NaBH4 to convert any chlorothiazide present to hydrochlorothiazide. The urine is acidified with NaH2PO4 (1.0 mol/L, pH 5), and thiazides are extracted with ethyl acetate. Interfering substances are then removed in two washes with 0.1 mol/L Na2HPO4 at pH 8. The ethyl acetate is evaporated and the residue redissolved in mobile phase. Thiazides are assayed by reversed-phase chromatography, with detection by ultraviolet absorption. Analytical recovery of thiazides ranged from 53 to 93%. Urines from 48 patients were so studied, and the results were compared with results by the currently used spectrophotometric method. The two methods agreed for 56% of samples. Evaluation of the discrepancies by review of patients' histories clearly showed liquid chromatography to have correctly identified seven of eight positive urines that the spectrophotometric method failed to detect. Ultraviolet scanning incorrectly identified as positive two samples, whereas liquid chromatography did not falsely identify any urines as positive. Our method was more sensitive and more specific than the spectrophotometric method.

Factitious Bartter's syndrome.

A 29-year-old man had a six-month history of fatigue and hypokalemia. Gastrointestinal losses of potassium were not judged significant. The patient denied ingestion of licorice, large quantities of laxatives, or diuretics. Clinical and laboratory findings were consistent with Bartter's syndrome in the adult. Normal blood pressure, hypokalemic alkalosis, and hyperaldosteronism, with insensitivity to the pressor effect of angiotensin infusion, were present. Another major finding in Bartter's syndrome, juxtaglomerular hyperplasia, was not demonstrated because plans for renal biopsy were cancelled when thiazide was detected in the urine, utilizing chemical extraction and spectrophotometry. Surreptitious ingestion of diuretics must be excluded in any adult patient in whom a diagnosis of Bartter's syndrome is considered.

Identification of selected antihypertensive drugs by thin-layer chromatography.

A thin-layer chromatographic procedure is described for the qualitative identification of several antihypertensive drugs including certain thiazide diuretics spironolactone, triamterene, methyldopa and their metabolites. Utilization of new solvent developing systems and spray detecting reagents provides a method useful for the identification of these compounds in biologic fluids at low therapeutic concentrations. Sensitivity limits for these antihypertensive drugs are given, and alternate techniques to provide confirmatory analyses are also presented.