ABSTRACT
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.
Subject(s)
Chemistry Techniques, Analytical/methods , Chemistry, Pharmaceutical/methods , Chromatography, High Pressure Liquid/methods , Furosemide/analysis , Hydrochlorothiazide/analysis , Intestines/drug effects , Animals , Calibration , Chromatography , Dose-Response Relationship, Drug , Furosemide/chemistry , Hydrochlorothiazide/chemistry , Permeability , Phenolsulfonphthalein/chemistry , Phenolsulfonphthalein/pharmacology , Rats , Reproducibility of Results , Sensitivity and Specificity , Sodium Potassium Chloride Symporter Inhibitors/pharmacology , Time FactorsABSTRACT
A simple reversed-phase high performance liquid chromatographic method with UV detection at 270 nm was developed for simultaneous quantitation of ketoprofen and naproxen sodium along with phenol red as a non-absorbable marker for in situ permeability studies. The mobile phase was a mixture of 20% methanol, 28% of acetonitrile, 52% water and 0.4 ml triethylamine (adjusted to pH 3.2 using orthophosphoric acid). Analysis was run at a flow of 1.5 ml/min with a 20 min run time. The calibration curves were linear for all three compounds (r>0.999) across the concentration range of 15.6-250 microg/ml with a limit of quantitation of 0.3, 0.25 and 0.2 ng/ml for naproxen, ketoprofen and phenol red, respectively. The coefficient of variation for intra-assay and inter-assay precision was less than or equal to 5.3% and the accuracy was between 95.36 and 101.6%. Using the SPIP technique and the suggested HPLC method for sample analysis, the mean values of 1.17e(-4) (+/-0.28) cm/s and 0.97e(-4) (+/-0.2) cm/s were obtained for naproxen and ketoprofen, respectively.
