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

Abstract

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.

Separation and quantitative analysis of alkyl sulfobetaine-type detergents by high-performance liquid chromatography and light-scattering detection

“…To determine the linear range of the method, various quantities of SB14 ranging from 0.04 to 200 g were injected three times and peak areas were determined for different detector sensitivities ranging from gain 3 to 9. As observed previously for other quantitative analyses with ELSD [13,22], there was no linear relationship between the peak area and the injected detergent mass. This problem, which was shown to be related to the droplet size inhomogeneity and to depend mainly on the nebulizer geometry and gas flow rate [23,24], can be overcome by transforming the mean peak areas and standard SB14 concentrations to logarithms and drawing a log-log plot.…”

“…Indeed, it is especially attractive for analyzing compounds which do not absorb in the near UV (200-400 nm) or which have weak chromophores in this UV range, such as carbohydrates, steroids or surfactants. In the recent past, ELSD has become more popular as a universal detector, and applications are now widely reported for the determination of detergents, such as polysorbate 80 [12] and octylglucoside [13], and for the separation of surfactant mixtures [14,15].…”

Tandem mass spectrometry with online high‐flow reversed‐phase extraction and normal‐phase chromatography on silica columns with aqueous‐organic mobile phase for quantitation of polar compounds in biological fluids

“…The major advantage of this method is that it embeds the sample preparation step into the chromatographic separation step and thus minimizes the offline sample preparation step. It has been widely used by other bioanalytical groups in the pharmaceutical industry 6–10. Generally, a reversed‐phase extraction column was used and coupled with a reversed‐phase separation column.…”