Development and validation of an advanced electrochemical sensor for the fast and cheap determination of hydrochlorothiazide in urine samples using the Monte-Carlo method for uncertainty evaluation.

This work describes the development, optimization, and validation of an electrochemical method for the determination of hydrochlorothiazide (HCTZ) in urine. The method allows fast, cheap and reliable determinations of recent administrations of this diuretic that can be used in doping control in sport. The response of the sensor was determined by differential pulse voltammetry (DPV). The glassy carbon electrode was modified with multiwall carbon nanotubes (MWCNT) and gold nanoparticles. The sensor is calibrated in the analysed sample matrix by the cumulative standard addition method. The method validation was based on the bottom-up evaluation of the measurement uncertainty were components were combined using the Monte Carlo Method (MCM) applicable with no restrictions regarding components uncertainty value and measurement function linearity. The developed metrological models were implemented in MS-Excel spreadsheets. The adequacy of the electrochemical measurements was assessed by comparing their relative standard uncertainty with a target value of 20% and by evaluating the compatibility of measurements with determinations performed by a reference procedure. The tools developed for the construction and optimization of working electrodes are applicable to measurements of other analytes and matrices. The used cumulative standard addition method and respective measurement uncertainty models are applicable to any kind of non-destructive chemical measurement of a solution.

Multiple incidence of the prescription diuretic hydrochlorothiazide in compounded nutritional supplements.

Diuretic agents are prohibited in sports in- and out-of-competition according to the regulations of the World Anti-Doping Agency (WADA) because of their possible masking effects on other doping agents in urine samples, and their ability to produce fast acute weight losses. Despite previous studies reported adverse analytical findings (AAFs) resulting from contaminations at ppm level (µg/g) of medicinal products, and recommended to introduce reporting limits for diuretics in doping controls, these are not adopted in analyses performed by WADA-accredited laboratories. We report the case of an athlete with two AAFs for hydrochlorothiazide (HCTZ) at low urinary concentrations (<10 ng/mL), who declared the use of nutritional supplements prepared in a compounding pharmacy. His nutritional supplements were analyzed revealing HCTZ presence in different concentrations, at the ppm level (µg/g and ng/mL). With the aim of testing the plausibility of the observed urinary HCTZ concentrations with the nutritional supplement ingestion, a urinary excretion study with three healthy volunteers was performed. HCTZ-contaminated powder (6.4 µg/g of HCTZ) was administered to each subject in different dosages, reproducing the possible ingestion pattern occurred. Urine specimens were collected before and after ingestion of the powder, up to 24 hours, and underwent liquid-liquid extraction and liquid chromatography-tandem mass spectrometry determination. Post-administration specimens were found to contain HCTZ at concentrations of 5-230 ng/mL, which supported the accidental inadvertent intake of the prohibited substance by the athlete. This study makes the argument that the introduction of reporting limits for diuretics are warranted in doping control samples, in order to protect against inadvertent AAFs due to contaminated products.

Two-phase and three-phase liquid-phase microextraction of hydrochlorothiazide and triamterene in urine samples.

This paper reports the applicability of two-phase and three-phase hollow fiber based liquid-phase microextraction (HF-LPME) for the extraction of hydrochlorothiazide (HYD) and triamterene (TRM) from human urine. The HYD in two-phase HF-LPME is extracted from 24 mL of the aqueous sample into an organic phase with microliter volume located inside the pores and lumen of a polypropylene hollow fiber as acceptor phase, but the TRM in three-phase HF-LPME is extracted from aqueous donor phase to organic phase and then back-extracted to the aqueous acceptor phase, which can be directly injected into HPLC for analysis. Under optimized conditions preconcentration factors of HYD and TRM were obtained as 128 and 239, respectively. The calibration curves were linear (R(2) ≥ 0.995) in the concentration range of 1.0-100 µg/L for HYD and 2.0-100 µg/L for TRM. The limits of detection for HYD and TRM were 0.5 µg/L. The intra-day and inter-day RSD based on four replicates were obtained as ≤5.8 and ≤9.3%, respectively. The methods were successfully applied for determining the concentration of the drugs in urine samples. Copyright © 2015 John Wiley & Sons, Ltd.

Electrochemical investigation of the voltammetric determination of hydrochlorothiazide using a nickel hydroxide modified nickel electrode.

The preparation and electrochemical characterization of a nickel hydroxide modified nickel electrode as well as its behavior as electrocatalyst toward the oxidation of hydrochlorothiazide (HCTZ) were investigated. The electrochemical behavior of the modified electrode and the electrooxidation of HCTZ were explored using cyclic voltammetry. The voltammetric response of the modified electrode in the detection of HCTZ is based on the electrochemical oxidation of the Ni(II)/Ni(III) and a chemical redox process. The analytical parameters for the electrooxidation of HCTZ by the nickel hydroxide modified nickel electrode were obtained in NaOH solution, in which the linear voltammetric response was in the concentration range from 1.39×10(-5) to 1.67×10(-4)mol L(-1) with a limit of detection of 7.92×10(-6)mol L(-1) and a sensitivity of 0.138 µA Lmmol(-1). Tafel analysis was used to elucidate the kinetics and mechanism of HCTZ oxidation by the modified electrode.

Cyclodextrin Micellar LC for Direct Selective Analysis of Combined Dosage Drugs in Urine.

Two cyclodextrin micellar liquid chromatographic methods were developed and applied to the simultaneous determination of bisoprolol/hydrochlorothiazide and atenolol/chlorthalidone combinations in urine matrices without sample pretreatment. These combined ß-blockers and diuretics chemotherapies are commonly used in the treatment of hypertension and cardiovascular diseases. Hybrid isocratic mobile phases containing hydroxypropyl-ß-cyclodextrin, sodium dodecyl sulfate, phosphate buffer and methanol on a Luna C18 column with 0.5 mL min(-1) flow rate and 25.0°C column temperature were used. The methods were sensitive enough for the determination of analytes at the therapeutic urine levels with limits of detections down to 1.0 µg mL(-1); relative standard deviations and recoveries were ranged between 1.5-4.4% and 98.00-109.52%, respectively. Urinary excretion studies showed that the detection of drugs is possible up to 24 h after their ingestion. The selective proposed separations with less consumption of organic solvents over the hitherto ones could be attributed to the four point competitive interactions among analysts, pseudostationary phases and a real stationary phase.

A novel dilute and shoot HPLC assay method for quantification of irbesartan and hydrochlorothiazide in combination tablets and urine using second generation C18-bonded monolithic silica column with double gradient elution.

Irbesartan (IRB) and hydrochlorothiazide (HCT) are angiotensin-II receptor antagonist and thiazide-class diuretic compounds, respectively, which are in use in the treatment of hypertension. A novel dilute-and-shoot HPLC assay method for simultaneous quantification of IRB and HCT in fixed-dose combination tablets and urine samples was described. The separation of IRB, HCT and agomelatine (internal standard) was carried out using a second generation C18-bonded monolithic silica column (Chromolith(®) High Resolution RP-18e, 100×4.6mm, Merck KGaA), utilizing both mobile phase and flow rate gradient elution programs. The analytes were detected at 230 nm wavelength using photodiode array detector within 24 minutes with high resolution, observing about 50 percent more peak capacity when using second generation C18-bonded monolithic silica column. Urine samples were introduced into the system effortlessly, with only filtration and subsequent dilution. Validation studies were performed according to the official recommendations of USP and ICH, and the developed method was successfully applied to pharmaceutical tablets and urine samples.

Population-based meta-analysis of hydrochlorothiazide pharmacokinetics.

Hydrochlorothiazide (HCTZ) is a thiazide diuretic used for the treatment of hypertension and edema associated with fluid overload conditions such as congestive heart failure (CHF). A population-based meta-analysis approach in NONMEM® was used to develop a PK model to characterize the time-course of HCTZ concentrations in plasma and excretion into the urine for healthy subjects and CHF patients. Data from healthy subjects receiving 100 mg of oral HCTZ were supplemented with additional plasma concentration and urinary excretion versus time data published in the literature following administration of oral HCTZ doses ranging from 10 to 500 mg to healthy subjects or patients with renal failure, CHF or hypertension. A two-compartment model with first-order oral absorption, using a Weibull function, and first-order elimination best described HCTZ PK. Creatinine clearance (CLCR ) was a statistically significant predictor of renal clearance (CLR ). Non-renal clearance was estimated to be 2.44 l/h, CLR was 18.3 l/h and T1/2,α was 1.6 h and T1/2,ß was 14.8 h for a typical individual with normal renal function (CLCR = 120 ml/min). However, CLR was reduced to 10.5, 5.47 and 2.70 l/h in mild (CLCR = 80 ml/min), moderate (CLCR = 50 ml/min) and severe (CLCR = 30 ml/min) renal impairment, respectively. Model diagnostics helped to demonstrate that the population PK model reasonably predicts the rate of urinary HCTZ excretion over time using dosing history and estimated CLCR , allowing for the convenient assessment of PK-PD relationships for HCTZ when given alone or in combination with other agents used to treat fluid overload conditions.

Simultaneous determination of aliskiren and hydrochlorothiazide in tablets and spiked human urine by ion-pair liquid chromatography.

An alternative method for analysis of aliskiren (ALI) and hydrochlorothiazde (HCT) in combined dosage forms by ion-pair reversed phase high performance liquid chromatography was developed and validated. The pharmaceutical preparations were analyzed using a C18 column (250 mm x 4.6 mm, 3 microm) with a mobile phase consisting of 25% methanol, 50% sodium monobasic phosphate aqueous solution containing 6 mM tetrabutylammonium bromide and 25% water at pH 7.2. Isocratic analysis was performed at a flow rate of 1 mL/min and a column temperature of 30 degrees C under direct UV detection at 210 nm. Paracetamol was used as internal standard. The validation was performed according to the ICH guidelines. The proposed method was linear over the concentration range of 0.250 to 60 and 0.1 to 10 microg/mL for ALI and HCT, respectively. The limits of detection and quantitation (LOD and LOQ) were 0.075 and 0.198 microg/mL, respectively, for ALI and 0.04 and 0.062 microg/mL, respectively, for HCT. The method proved to be specific, sensitive, precise and accurate with mean recovery values of 101.1 +/- 0.32% and 100.9 +/- 0.41% for ALI and HCT, respectively. The method robustness was evaluated by means of an experimental design. The proposed method was applied successfully to spiked human urine samples with mean recoveries of 98.8 +/- 0.36% and 98.1 +/- 0.21% for ALI and HCT, respectively.

Assessment of the drug-drug interactions between fimasartan and hydrochlorothiazide in healthy volunteers.

AIM: Fimasartan is a selective angiotensin II receptor blocker. Hydrochlorothiazide (HCTZ), which is used to treat hypertension and edematous conditions, is coadministered with many antihypertensive agents. METHODS: An open-label, randomized, multiple-dosing, 2-arm, 1-sequence, 2-period study was conducted to assess the effects of fimasartan (240 mg) on HCTZ (25 mg) or vice versa in 18 and 14 healthy male volunteers, respectively. During each drug administration period, drugs were given once daily for 7 days, with a 7-day washout period between the 2 administration periods. RESULTS: The respective geometric mean ratios of fimasartan for AUC τ,ss and C max,ss with HCTZ were 1.30 [90% confidence interval (CI), 0.84-2.01] and 1.17 (90% CI, 0.93-1.47) compared with fimasartan alone. The respective geometric mean ratios of HCTZ for AUC τ,ss and C max,ss with fimasartan were 0.94 (90% CI, 0.84-1.04) and 0.88 (90% CI, 0.80-0.97) compared with HCTZ alone. Plasma renin activity indicated no significant differences between fimasartan monotherapy and coadministered treatment. CONCLUSIONS: Fimasartan administered alone or in combination with HCTZ was well tolerated at the described dosages. Coadministration of fimasartan increased the urinary excretion of HCTZ and urine volume, but these observations are unlikely to have any clinical relevance.

Fast gradient high performance liquid chromatography method with UV detection for simultaneous determination of seven angiotensin converting enzyme inhibitors together with hydrochlorothiazide in pharmaceutical dosage forms and spiked human plasma and urine.

The development of a reversed phase liquid chromatographic method for the simultaneous determination of seven angiotensin converting enzyme (ACE) inhibitors; five drugs namely benazepril HCl (BZL), enalapril maleate (ENL), fosinopril sodium (FSP), lisinopril (LSP) and ramipril (RMP) and two metabolites captopril disulfide (CPD) and enalaprilat (ENT) together with hydrochlorothiazide (HCT) is described. The method can serve as a substitute for many published papers for the analysis of the targeted compounds with or without hydrochloothiazide in pharmaceutical formulations as well as in spiked human plasma and urine samples. The method utilizes a simple gradient procedure for the separation in a 11 min run time using acetonitrile aqueous ammonia buffer (pH 9) solution and an Extend RP-C18 (25 µm particle size, 4.6 mm×250 mm, Agilent) HPLC column. The effluent was monitored on a UV detector at 215 nm. The effect of pH, solvent strength and analysis time on the peak shape and quantification were carefully studied in order to optimize the method. Adopting the proposed procedure, the analytes produce well-shaped peaks with good linear relationship over the investigated concentration ranges. The limits of detection (LOD) and limits of quantification (LOQ) from standard drug solutions lie in the range of 17-64 and 56-212 ng mL(-1), respectively. Correlation coefficient values (r) higher than 0.997 were obtained for all the studied drugs in spiked human plasma and urine samples. The intra-day and inter-day precision of the method was evaluated with relative standard deviation values being satisfactory for their purposed analysis. The method was validated with respect to specificity, recovery, accuracy, precision and linearity.

Simultaneous quantitative determination of olmesartan and hydrochlorothiazide in human plasma and urine by liquid chromatography coupled to tandem mass spectrometry.

A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the simultaneous determination of olmesartan (OLM) and hydrochlorothiazide (HCTZ) in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the analytes from biological matrices followed by injection of the extracts onto a C(18) column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using negative electrospray ionization (ESI). The method was validated over the concentration range of 1.00-1000 ng/mL and 5.00-5000 ng/mL for OLM in human plasma and urine as well as 0.500-200 ng/mL and 25.0-25,000 ng/mL for HCTZ in human plasma and urine, respectively. Inter- and intra-run precision of OLM and HCTZ were less than 15% and the accuracy was within 85-115% for both plasma and urine. The average extraction recoveries were 96.6% and 92.7% for OLM, and 87.2% and 72.1% for HCTZ in human plasma and urine, respectively. The linearity, recovery, matrix effect and stability were validated for OLM/HCTZ in human plasma and urine.

Development and validation of an LC method for simultaneous determination of amiloride hydrochloride and hydrochlorothiazide in human urine.

An HPLC method with photometric detection has been developed for determination of a binary mixture of amiloride hydrochloride and hydrochlorothiazide in human urine using chlorthalidone as the internal standard. Reversed-phase chromatography was performed at room temperature on a cyanopropyl column with the mobile phase consisting of a 10 mM KH2PO4 solution (pH 4.5)-methanol (70 + 30, v/v) at a flow rate of 1 ml/min. The detector was set at 214 nm. The total analysis time was 10 min. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability, and re-injection reproducibility. The procedure shows good accuracy, repeatability, and selectivity. Moreover, the method was applied directly to urine that had not undergone prior treatment. The intra- and interday coefficients of variation for all compounds were below 4%, and the method was highly accurate, with a relative error for all compounds that was below 8%. No interference from endogenous compounds in urine samples was found. The proposed method, which is rapid, simple, and does not require any separation steps, has been successfully applied to the assay of human urine containing amiloride hydrochloride and hydrochlorothiazide.

Use of addition calibration technique for determination of acetaminophen and hydrochlorothiazide in human urine by high-performance liquid chromatography.

The quantitation of target analytes in complex matrices such as biological samples requires special calibration approaches to compensate for additional capacity or activity in the matrix samples. A conventional calibration curve, obtained with standard solutions, is one of the most important calibration procedures for quantitation of target analytes in such matrices. However, these technique require a great number of reagents and material, and consume a considerable amount of time throughout the analysis. In this work, a new calibration procedure to analyze urine samples is proposed for the first time in chromatography procedures. The proposed calibration, called the addition calibration technique, was used for the determination of acetaminophen and hydrochlorothiazide in urine samples. The results obtained for the proposed calibration mode were compared to those obtained using standard addition and standard calibration techniques. The proposed addition calibration was validated by statistical studies between results obtained by the addition technique and conventional techniques, using the ANOVA test and linear regression. The results demonstrated good agreement among them. The performance of the analytical method was evaluated. Relative standard deviation, limit of detection, and limit of quantification are respectively 0.5-0.6%, 0.169-0.75 microg/mL, and 0.565-2.5 microg/mL. Linear range falls within the range of 0.3 to 63.8 microg/mL for both compounds. Accuracy ranged between 94% and 101%.

Measurement of glomerular filtration rate in the conscious rat.

INTRODUCTION: Glomerular filtration rate (GFR) is an important parameter for studying drug-induced impairments on renal function in rats. The GFR is calculated from the concentration of creatinine and blood urea nitrogen (BUN) in serum and in urine, respectively. Following current protocols serum and urine samples must be taken from the same animal. Thus, in order to determine time-dependent effects it is necessary to use for each time point one separated group of animals. We developed a statistical test which allows analyzing the GFR from two different groups of animals: one used for repeated serum and the other one used for repeated urine analysis. METHODS: Serum and urine samples were taken from two different sets of rats which were otherwise treated identically, i.e. drug doses, routes of administration (per os or per inhalation) and tap water loading. For each dose group GFR mean, standard deviation and statistical analysis to identify differences between the dose groups were determined. RESULTS: After determination of the optimal time points for measurements, the effect on GFR of the three reference compounds, furosemide, hydrochlorothiazide and formoterol, was calculated. The results showed that the diuretic drugs furosemide and hydrochlorothiazide decreased the GFR and the antidiuretic drug formoterol increased the GFR, as counter regulation on urine loss or urine retention, respectively. DISCUSSION: A mathematical model and the corresponding algorithm were developed, which can be used to calculate the GFR, and to test for differences between groups from two separated sets of rats, one used for urine, and the other one for serum analysis. This new method has the potential to reduce the number of animals needed and to improve the quality of data generated from various groups of animals in renal function studies.

Multidrug resistance-associated protein 4 is involved in the urinary excretion of hydrochlorothiazide and furosemide.

The role of ATP-binding cassette transporters in the urinary excretion of diuretics was investigated. Significant ATP-dependent uptake of hydrochlorothiazide (HCT) and furosemide was observed in membrane vesicles that expressed multidrug resistance-associated protein 4 (MRP4) and breast cancer resistance protein (BCRP). Unlike taurocholate uptake, S-methylglutathione had no effect on the ATP-dependent uptake of both compounds by MRP4. The functional importance of MRP4 and BCRP in the urinary excretion of HCT and furosemide was investigated using gene knockout mice. The renal clearance of HCT and furosemide was reduced significantly but not abolished in Mrp4 knockout mice compared with wild-type mice (9.0 +/- 0.9 versus 15 +/- 2 ml/min per kg for HCT and 1.9 +/- 0.3 versus 2.7 +/- 0.1 ml/min per kg for furosemide), and the amount of HCT that was associated with the kidney specimens was greater in Mrp4 knockout mice (21 +/- 3 versus 13 +/- 1 nmol/g kidney). In contrast, Bcrp makes only a negligible contribution because the urinary excretion was unchanged in Bcrp knockout mice. Our results suggest that Mrp4, together with other unknown transporters, accounts for the luminal efflux of HCT and furosemide from proximal tubular epithelial cells.

Hydrochlorothiazide efficacy and polymorphisms in ACE, ADD1 and GNB3 in healthy, male volunteers.

OBJECTIVE: The antihypertensive effect of thiazide diuretics has recently been associated with genetic variation in the angiotensin I-converting enzyme (ACE), alpha-adducin (ADD1) and the G protein subunit beta3 (GNB3). Analysis of short-term diuretic effects may provide insight into the mechanisms behind these findings. METHODS: A total of 103 male volunteers took 25 and 100 mg hydrochlorothiazide (HCT) after a placebo day, each. We measured volume, sodium, chloride, potassium, calcium excretion, blood pressure and heart rate. RESULTS: Excretion and cardiovascular parameters were highly constant between the 2 placebo days. The resting heart rate was 2-3 beats/minute (bpm) higher per ACE insertion allele on all 4 study days. The HCT-induced excretion of sodium, chloride and volume was independent of the genotypes. The additional potassium excretion induced by 100 mg HCT was 44+/-21, 33+/-27 and 16+/-26 mmol (mean+/-SD, p<0.001) in ACE II, ID and DD carriers and the same trend was observed after 25 mg HCT. As a second finding, the 100 mg HCT-induced calcium retention was 0.2+/-1.2, 0.7+/-0.8 and 1.7+/-2.1 mmol in ADD1 Gly/Gly, Gly/Trp and Trp/Trp carriers (p=0.002) and the same trend existed after 25 mg HCT. CONCLUSION: The effects of genetic polymorphisms were stronger with the higher diuretic dose. ACE insertion allele carriers appeared to be more prone to hypokalaemia than deletion allele carriers. ADD1 Trp460 carriers may especially benefit from the calcium-sparing effect of thiazides. Both associations should be further studied in long-term treatment with thiazide diuretics.

Electrochemical study of hydrochlorothiazide and its determination in urine and tablets.

A voltammetric study of hydrochlorothiazide (HCTZ) at glassy carbon electrode was carried out. The drug in Britton-Robinson buffer (pH 3.3) is oxidized at +1040mV, giving rise to a well-defined peak. Cyclic voltammetric study indicates that the oxidation process is irreversible and diffusion-controlled. A sensitive, simple and time-saving differential pulse anodic voltammetric procedure has been developed. The procedure has been applied for the drug determination in human urine with no prior extraction and in commercial tablets. The proposed method has been validated. The limit of detection for the standard solution was 5.0ngml(-1) and for the drug in urine was 14.0ngml(-1). The results obtained from the analysis of commercial tablets were compared statistically with those obtained from the USP 26 high-performance liquid chromatographic (HPLC) method.

Determination of hydrochlorothiazide and rutin in Chinese herb medicines and human urine by capillary zone electrophoresis with amperometric detection.

In this paper, capillary zone electrophoresis with amperometric detection (CZE-AD) was firstly applied to the simultaneous determination of rutin (RT) and hydrochlorothiazide (HCT) in compound Chinese herb medicines and human urine samples. The two analytes could be perfectly analyzed within 12 min and showed significant current responses at carbon electrode under the optimum conditions. It was found that the linear range of HCT was from 2.0 x 10(-6) to 1.0 x 10(-4) mol l(-1) and that of RT was from 1.0 x 10(-6) to 1.0 x 10(-4) mol l(-1). Their sensitivity was determined by linear regression and calculated as 7.02 x 10(4) and 2.17 x 10(5) nA l mol(-1), respectively, and their detection limits were 5.0 x 10(-7) and 2.0 x 10(-7) mol l(-1), respectively (S/N=3). Above results demonstrated that this method was of high sensitivity, good repeatability, high selectivity and could be used in metabolic kinetics studies of medicines. Satisfactory results were obtained when this method was used to simultaneously analyze the amounts of RT and HCT in one general compound Chinese herb medicine-Zhen Ju jiang Ya Pian and human urine samples.

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.

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.