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A reversed-phase high-performance liquid chromatography method using evaporative light-scattering detection is developed for the determination of residual octylglucoside (OG) levels after a detergent exchange step for in-process samples of a vaccine antigen. The reversed-phase column not only provides separation of the OG but also functions as an extraction column to remove the vaccine antigen from the sample, thereby eliminating off-line sample manipulations. In addition to column selection, the mobile phase is optimized to enhance extraction and separation. The vaccine antigen is irreversibly bound to the column, allowing nonprotein components to interact with the column for separation and elution. The assay is linear over the range of 0.00050-0.050% OG. Precision tested at 0.0010% and 0.0050% OG is 2.9% and 7.2% relative standard deviation, respectively. The limits of quantitation and detection are determined to be 0.00050 and 0.000125% OG, respectively. Accuracy is determined to be 103 and 98%, based on spike recoveries of 0.0010% and 0.0050% OG, respectively.

journal of chromatographic science

Online Extraction and Determination of Octylglucoside by Reversed-Phase High-Performance Liquid Chromatography with Evaporative Light-Scattering Detection