Simultaneous Determination of Hydrochlorothiazide and Reserpine in Human Urine by LC with a Simple Pre-Treatment

Abstract

A simple, selective and sensitive reversed-phase high performance liquid chromatography method for simultaneous analysis of hydrochlorothiazide and reserpine in human urine was developed and subjected to primary pharmacokinetic study. After a simple protein precipitation using methanol and extraction with ethyl acetate, the analytes were separated on an Elite C 18 column at a flow rate of 0.8 mL min −1 . The mobile phase was composed of acetonitrile (A) and 0.2% ammonium chloride solution (B) for a gradient elution starting at A:B at 30:70, v/v for 0~6 min, linearly raising the percent of A from 30% to 50% (6~9 min) and ending at 50:50, v/v (9~25 min). The standard curves were linear over the range of 0.05-20 µg mL −1 for hydrochlorothiazide and 0.02-5.0 µg mL −1 for reserpine, respectively (r > 0.999). The limit of detection (LOD) and the limit of quantification (LOQ) were 5.5 ng mL −1 and 18.2 ng mL −1 for hydrochlorothiazide, and 7.1 ng mL −1 and 23.6 ng mL −1 for reserpine, respectively. The recoveries for both analytes were above 89.0±1.35%. The intra-day and inter-day precision for hydrochlorothiazide were less than 1.91% and 1.38%, and those for reserpine were below 1.61% and 2.64%, respectively. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy, and it was employed successfully for the simultaneous determination of hydrochlorothiazide and reserpine in human urine samples.

Improved simultaneous quantitation of candesartan and hydrochlorthiazide in human plasma by UPLC–MS/MS and its application in bioequivalence studies

“…The available strengths for CNC+HCT are 16+12.5 mg, 32+12.5 mg and 32+25 mg. Although some literatures for the quantitation of CN and HCT alone or in combination of other drugs are available [7] , [8] , [9] , [10] , [11] , [12] , no simultaneous sensitive bioanalytical method has been published that could be applied for bioequivalence studies of human subject samples for all available dose strengths of CN+HCT combination. A recent report for the simultaneous quantification of CN and HCT by using liquid chromatography mass spectrometry (LC–MS/MS) could only be applied for the bioequivalence of 16+12.5 mg dose strength due to its very low validated calibration curve range [13] .…”

Fluorimetric detection of reserpine in mouse serum through online post-column electrochemical derivatization

“…To date, several works have focused on the analysis of reserpine in tablets [ 4 , 5 ], plants [ 6 – 8 ], plasma [ 9 ] and urine [ 10 ]. Various methods, such as adsorptive stripping voltammetry [ 11 , 12 ], high-performance thin layer chromatography [ 13 ], spectrofluorimetry [ 14 – 16 ], chemiluminescence [ 17 ] and capillary electrophoresis [ 18 , 19 ], have been used for reserpine detection.…”

“…These methods, however, are unsuitable for the online separation and direct detection of reserpine. Thus, high-performance liquid chromatography (HPLC) equipped with UV detector [ 10 , 20 ], photochemical fluorimetry [ 21 ] and mass spectroscopy [ 22 , 23 ] have been used for direct reserpine detection. However, the accuracy of UV detection is easily influenced by solvent effect, interferents and matrix complexity; the effects of these factors are particularly robust in biological samples.…”

Simultaneous analysis of aliskiren and hydrochlorothiazide in pharmaceutical preparations and spiked human plasma by HPTLC

“…These methods have been based on spectrophotometry [6][7][8], spectrofluorometry [9,10], electrophoresis [11,12], liquid chromatography (LC) [8,[13][14][15][16][17][18][19][20][21], LC-MS/MS [22][23][24][25] and UPLC-MS/MS [26]. Similarly, a number of analytical methods have been presented for the estimation of HCTZ as a single analyte [27] and in the presence of different antihypertensive agents [8,12,14,16,18,19,[28][29][30][31][32][33][34][35][36][37][38][39] in pharmaceutical preparations and biological matrices using spectrophotometry [29,32], capillary electrophoresis [28], HPTLC [30,31], LC [33][34]…”