A simple and rapid high-performance liquid chromatography method for determining furosemide, hydrochlorothiazide, and phenol red: applicability to intestinal permeability studies.

A simple reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection at 280 nm was developed for simultaneous quantitation of furosemide and hydrochlorothiazide along with phenol red as a nonabsorbable marker for in situ permeability studies in anaesthetized rats. A jejunal segment of approximately 10 cm was isolated and cannulated in both ends for inlet and outlet solution. The perfusate was collected every 10 min, and samples were analyzed using the developed method. The mobile phase was acetonitrile-water-triethylamine-glacial acetic acid (41.5 + 57.4 + 0.1 + 0.9, adjusted to pH 5.6) at a flow rate of 1 mL/min; the run time was 9 min. The calibration graphs were linear for all 3 compounds (r > 0.999) across the concentration range of 7.93-125 microg/mL for phenol red and 6.25-100 microg/mL for hydrochlorothiazide and furosemide. The limits of quantitation were 7.2, 8.9, and 6.8 microg/mL for furosemide, hydrochlorothiazide, and phenol red, respectively. The coefficients of variation for intraassay and interassay precision were less than or equal to 7.6%, and the accuracy was between 93.2-103.4%. Using the single pass intestinal perfusion technique and the suggested HPLC method for sample analysis, mean values of 0.25 x 10(-4) (+/-0.16) cm/s and 0.22 x 10(-4) (+/-0.13) cm/s were obtained for furosemide and hydrochlorothiazide, respectively.