Reliability and uncertainty in the estimation of pKa by least squares nonlinear regression analysis of multiwavelength spectrophotometric pH titration data

When drugs are poorly soluble then, instead of the potentiometric determination of dissociation constants, pH-spectrophotometric titration can be used along with nonlinear regression of the absorbance response surface data. Generally, regression models are extremely useful for extracting the essential features from a multiwavelength set of data. Regression diagnostics represent procedures for examining the regression triplet (data, model, method) in order to check (a) the data quality for a proposed model; (b) the model quality for a given set of data; and (c) that all of the assumptions used for least squares hold. In the interactive, PC-assisted diagnosis of data, models and estimation methods, the examination of data quality involves the detection of influential points, outliers and high leverages, that cause many problems when regression fitting the absorbance response hyperplane. All graphically oriented techniques are suitable for the rapid estimation of influential points. The reliability of the dissociation constants for the acid drug silybin may be proven with goodness-of-fit tests of the multiwavelength spectrophotometric pH-titration data. The uncertainty in the measurement of the pK _a of a weak acid obtained by the least squares nonlinear regression analysis of absorption spectra is calculated. The procedure takes into account the drift in pH measurement, the drift in spectral measurement, and all of the drifts in analytical operations, as well as the relative importance of each source of uncertainty. The most important source of uncertainty in the experimental set-up for the example is the uncertainty in the pH measurement. The influences of various sources of uncertainty on the accuracy and precision are discussed using the example of the mixed dissociation constants of silybin, obtained using the SQUAD(84) and SPECFIT/32 regression programs.