Validation of an automated method for the liquid chromatographic determination of atenolol in plasma: application of a new validation protocol

In order to test the applicability of a new strategy by a Commission of the Societe Francaise des Sciences et Techniques Pharmaceutiques (SFSTP) for t he validation of bioanalytical methods, an automated method was developed f or the determination of atenolol in human plasma. This method was based on the use of dialysis as sample purification step, followed by enrichment of the dialysate on a precolumn and liquid chromatographic analysis. All sampl e handling operations were carried out automatically by means of an ASTED s ystem. Atenolol and its internal standard (sotalol) were separated on a C-8 column with a mixture of pH 7.0 phosphate buffer containing 1-octanesulpho nate and methanol (81/19; v:v) as mobile phase and monitored photometricall y at 225 nm. The validation strategy comprises two steps. The experiments performed duri ng the first step, the so called pre-validation step, have permitted the se lection of the most appropriate model for the calibration curve by means of a decision tree, i.e. a least squares regression model obtained after tran sformation of data (square root in this case), the estimation of the limit of quantitation at 25 ng/ml by means of an accuracy profile, the determinat ion of the calibration range (from 25 to 1000 ng/ml), the estimation of the limit of detection at 9 ng/ml and the calculation of the mean extraction e fficiency (about 65%). The second step is the validation itself, comprising the evaluation of method selectivity towards endogenous components, the co nfirmation of the limit of quantitation, the verification of linearity and the assessment of method precision (repeatability and intermediate precisio n) as well as accuracy using quality control samples at different concentra tion levels over the range investigated. The relative standard deviation va lues for repeatability and intermediate precision were between 2.7% and 9.0 %. Moreover, the method was found to be accurate. Indeed, the 95% one-sided confidence limits of the mean recovery did not exceed the acceptance limit s of 80% and 120%. (C) 1999 Elsevier Science B.V. All rights reserved.