Direct absorptiometric quantitation in high-performance liquid chromatography in the absence of concomitant reference standards

A method for absorptiometric quantification in high-performance liquid chromatography that utilizes the known molar absorption coefficients of the individual components of a mixture of analytes to enable a series of determinations to be carried out without the concomitant use of reference standards is described. An equation is derived that describes the dependence of the molar amount of a defined analyte on the parameters influencing the detector response and on the peak area of the analyte in the chromatogram. The equation can be used for the direct calculation ofthe molar amount of individual analytes in a well resolved mixture. A method for the analysis of two analytes, the peaks of which totally overlap but which differ by their molar absorption coefficients, is also described. The validity of the equations and the applicability of the proposed method was examined in the analysis of 5-(2-dimethylaminoethoxy)-7-oxo-7H-benzo c]fluorene hydrochloride (benflurone) and its metabolites. Some examples of the application of this approach are considered.