Simultaneous determination of candesartan and hydrochlorothiazide in human plasma by LC-MS/MS

Abstract A simple, sensitive, rapid and highly efficient LC-MS/MS method was developed for the determination of Candesartan and Hydrochlorothiazide simultaneously in human plasma. The method employed Zorbax eclipse C18 (150 X 4.6 mm, 5µ) column using acetate buffer: acetonitrile (25:75%, v/v) as the mobile phase. The mobile phase flow rate is 1 mL/min which was delivered into the mass spectrometer electron spray ionization chamber. The Liquid/liquid extraction procedure was used in the method for the extraction of analytes. The chromatograph was attached to a negative ion mode tandem mass spectrometer and the method was validated for all the parameters as per the guidelines of US-FDA. The ions were detected in multiple reaction monitoring mode and the transitions are m/z 439.00®309.10 and 295.80®268.80 for candesartan and hydrochlorothiazide respectively. Isotopic standards were used as internal standards for effective recovery of the analytes. The drugs were analyzed over a calibration range of 1.027-302.047 ng/mL for candesartan and 1.044-306.945 ng/mL for hydrochlorothiazide respectively with regression coefficient greater than 0.99. The mean extraction recoveries are 96.95±5.61 and 100.55±4.82 for candesartan and hydrochlorothiazide respectively. The precision and accuracy values for all the studies were within the range of ≤15% and 85-115%. The performed stability studies indicate that the developed method is stable in plasma for 15 h at room temperature (bench top); 52 h (in injector); for 112 days at -70 ºC for long term stability; five successive freeze and thaw cycles. The developed method could be successfully employed for the determination of selected drugs in biological samples.

Kinetics study of hydrochlorothiazide lactose liquid state interaction using conventional isothermal arrhenius method under basic and neutral conditions

ABSTRACT The Maillard reaction of hydrochlorothiazide (HCTZ) and lactose has been previously demonstrated in pharmaceutical formulations. In this study, the activation energy of - hydrohlorothiazide and lactose interaction in the liquid state was ascertained under basic and neutral conditions. Conventional isothermal High Performance Liquid Chromatography (HPLC) technique was employed to ascertain the kinetic parameters using Arrhenius method. Results: The activation energy obtained was 82.43 and 100.28 kJ/mol under basic and neutral conditions, respectively. Consequently, it can be inferred that Maillard reaction is significantly affected by pH, which can be used as a control factor whenever the reaction potentially occurs.

Bioanalytical method development and validation for determination of metoprolol tartarate and hydrochlorothiazide using HPTLC in human plasma

A simple, sensitive, rapid and economic chromatographic method has been developed for determination of metoprolol tartarate and hydrochlorothiazide in human plasma using paracetamol as an internal standard. The analytical technique used for method development was high-performance thin-layer chromatography. HPTLC Camag with precoated silica gel Plate 60F254 (20 cm×10 cm) at 250 µm thicknesses (E. Merck, Darmstadt, Germany) was used as the stationary phase. The mobile phase used consisted of chloroform: methanol: ammonia (9:1:0.5v/v/v). Densitometric analysis was carried out at a wavelength of 239 nm. The rf values for hydrochlorothiazide, paracetamol and metoprolol tartarate were 0.13±0.04, 0.28±0.05, 0.48±0.04, respectively. Plasma samples were extracted by protein precipitation with methanol. Concentration ranges of 200, 400, 600, 800, 1000, 1200 ng/mL and 2000, 4000, 6000, 8000, 10000, 12000 ng/mL of hydrochlorothiazide and metoprolol tartarate, respectively, were used with plasma for the calibration curves. The percent recovery of metoprolol tartarate and hydrochlorothiazide was found to be 77.30 and 77.02 %, respectively. The stability of metoprolol tartarate and hydrochlorothiazide in plasma were confirmed during three freeze-thaw cycles (-20 ºC) on a bench for 24 hours and post-preparatively for 48 hours. The proposed method was validated statistically and proved suitable for determination of metoprolol tartarate and hydrochlorothiazide in human plasma.

Um método simples, sensível, rápido e econômico empregando a cromatografia em camada delgada de alta eficiência (HPTLC) foi desenvolvido para determinação do tartarato de metoprolol e hidroclorotiazida em plasma humano, usando paracetamol como padrão interno. Placas prontas de sílica-gel 60F254 (20 cm×10 cm), 250 µm de espessura, para HPTLC Camag (E. A Merck, Darmstadt, Alemanha) foramutilizadas como fase estacionária. A fase móvel utilizada consistiu de clorofórmio: metanol: amônia (9:1:0,5 v/v/v). A análise densitométrica foi realizada no comprimento de onda 239 nm. Os valores de Rf de hidroclorotiazida, paracetamol e tartarato de metoprolol foram 0.13±0.04, 0.28±0.05, 0.48±0.04 respectivamente. As proteínas do plasma foram extraídas por precipitação com metanol. Para construção das curvas de calibração, empregaram-se intervalos de concentração de 200, 400, 600, 800, 1000, 1200 ng/mL e 2000, 4000, 6000, 8000, 10000, 12000 ng/mL de hidroclorotiazida e tartarato de metoprolol, respectivamente. Os percentuais de recuperação do tartarato de metoprolol e de hidroclorotiazida foram de 77,30 e 77,02, respectivamente. A estabilidade do tartarato de metoprolol e de hidroclorotiazida no plasma foi confirmada durante três ciclos de congelamento e descongelamento (-20 ºC), durante 24 horas e póspreparação durante 48 horas. O método proposto foi validado estatisticamente, sendo adequado para determinação do tartarato de metoprolol e hidroclorotiazida em plasma humano.

HPLC method for simultaneous determination of amiloride hydrochloride and hydrochlorothiazide in human plasma

A simple, sensitive and rapid chromatographic method was developed and validated for the quantification of a binary mixture namely; [amiloride hydrochloride and hydrochlorothiazide] in human plasma using chlorthalidone as internal standard [IS]. The method utilized proteins precipitation with acetonitril as the only sample preparation involved prior to reverse phase-HPLC. The analytes were chromatographed on Shim-pack cyanopropyI column with isocratic elution with 10 mM KH[2]PO[4] [pH 4.50]-methanol [70:30 v/v] at ambient temperature with flow rate 1 ml/min and UV detection. The chromatographic run time was less than 10 min for the mixture. The calibration curves were linear over the range 0.1-10 micro g ml[-1]. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The within-and between-day accuracy and precision were found to be within acceptable limits <15%. The analytes were stable after three freeze-thaw cycles [deviation <15%]. The proposed method is specific for determination of the mixture in human plasma where there is no interference from endogenous biological substances

Simultaneous spectrophotometric estimation of bisoprolol fumarate and hydrochlorothiazide in tablet dosage form

Two analytical methods have been developed for simultaneous quantification of bisoprolol fumarate and hydrochlorothiazide in combined pharmaceutical dosage form using spectrophotometer. Excellent simplicity, accuracy, precision and economy were achieved by the assay. The 'method I' was based upon 'simultaneous equations' whereas the 'method II' was based upon 'multicomponent mode of analysis' of the instrument. For both these methods, 0.1 N NaOH was used as solvent. In this solvent, bisoprolol fumarate showed absorbance at 224 nm and hydrochlorothiazide at 273 nm. Linearity lies in the concentration range of 3 - 21 microg/ml for bisoprolol fumarate and 3 - 18 microg/ml for hydrochlorothiazide. The methods were validated statistically and by recovery studies

Pharmacological study of a new synthetic potential diuretic

A new synthetic disulfamyl compound with pyrimidine nucleus was studied for its oral diuretic action in both rats and dogs and it was found to have directic activity comparable to that of hydrochlorothiazide, but with the advantage of less augmentation of potassium excretion. Acute toxicity studies revealed that this compound has exceedingly high oral and intraperitoneal LD[50] [>8000 mg/ Kg and 325.6 mg/ Kgm] respectively. Again the subacute toxicity studies confirmed the very low toxicity of this compound as is induced no significant changes in all the parameters studied and was devoided of the hypokalaemic effect produced by hydrochlorothiazide. Pharmacological in vivo and in vitro studies pointed to the potential parasympathomimetic activity of the test compound