Abstract
An improved high-performance liquid chromatographic (HPLC) method for the separation of zwitterionic detergents is described. It is based on a reversed-phase liquid chromatography with evaporative light-scattering detection (ELSD). The method was shown to be highly specific, allowing the separation of three detergents of the alkyl sulfobetaine family: 3-( N-dodecyl- N, N-dimethyl-ammonio)-propane-1-sulfonate (SB12), 3-( N-tetradecyl- N, N-dimethyl-ammonio)-propane-1-sulfonate (SB14) and 3-( N-hexadecyl- N, N-dimethyl-ammonio)-propane-1-sulfonate (SB16). It was further used to develop a quantitation method for SB14, which was validated for linearity, precision, robustness, limits of detection and quantitation, specificity and accuracy. Linearity was found in the range of 50–500 μg/ml with a correlation coefficient of 0.9938 ± 0.0029. The mean value of slope and intercept were 1.567 ± 0.06 and 0.1541 ± 0.0271, respectively. The limits of detection (LOD) and quantitation (LOQ) were 2 and 10 μg/ml, respectively. The validated method was used to determine the concentration of SB14 in different biological samples, specially in bulks of a recombinant membrane protein, the Klebsiella pneumoniae outer membrane protein A, which is produced at the pilot scale for human clinical studies.
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