The use of a response surface methodology on HPLC analysis of methyldopa, amiloride and hydrochlorothiazide in tablets

A multifactor optimisation technique is successfully applied to develop a n ew HPLC method in which methyldopa, hydrochlorothiazide and amiloride were analysed and determined on a C18 column with detection at 286 nm. The optim al conditions of HPLC separation were determined with the aid of the respon se surface diagram - 'window diagram'. The effect of simultaneously varying the pH, proportion aqueous acetic acidum and methanol in the mobile phase were studied to optimise the separation. The mobile phase composition that provides an acceptable resolution methyldopa, hydrochlorothiazide and amilo ride in a short elution time is water-methanol (75:25) and pH 3.60. The k' values for methyldopa, hydrochlorothiazide and amiloride after optimisation were 1.40, 2.50 and 5.33, respectively. Relative retention (a) for ratio h ydrochlorothiazide/methyldopa and amiloride/hydrochlorothiazide were 1.767 and 2.159, respectively. Correlation coefficients of the calibration curves for all analytes were greater than 0.995 and the R.S.D. values for the slo pe and the intercept with respect to the linearity were less than 2%. A met hod is applied for the quantitative analysis of Aatan(R) tablets (Lek-Ljubl jana). The powdered tablets are extracted with methanol, containing caffein e as the internal standard and assayed by comparison of peak areas after li quid chromatography. The high recovery (for all analytes about 100%) and th e low R.S.D. (<2%) confirm good precision and reproducibility of the chroma tographic method. (C) 2001 Elsevier Science B.V. All rights reserved.