Application of Computer-Aided Spectral Deconvolution Technique in Validation of High Performance Liquid Chromatographic Peaks

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Abstract
Reliable analysis with high performance liquid chromatography (HPLC) requires purity of the eluting peak. The present work has combined the advantages of the availability of full spectral data from HPLC photodiode array UV detector and computer algorithms to perform chromatographic peak purity check. A deconvolution technique based on multicomponent analysis has been applied to the UV spectra of co-eluting components. This method employs residual error (Relative-fit-error, RFE) between predicted spectrum and analyte's spectrum to detect presence of other component or contaminant. Typical RFE values for uncontaminated chromatographic peaks of norethisterone and ethynyloestradiol range between 1 and 3, while contaminated peaks have RFE values as large as 145. A systematic increase in ‘relative-fit error’ from 1.10 to 145 was observed for peaks of norethisterone when contaminated to varied extent with ethynyloestradiol. Extent of peak overlap in chromatogram was also mapped out with this technique. The co-prescribed oral contraceptive, norethisterone and ethynyloestradiol were used as model in this work. An advantage of the method is its applicability when the contaminant's spectrum is unavailable. The method, unlike several earlier techniques, is also applicable to chromatograms with concidental elution time for the components.