Development of an Analytical Method for the Rapid Quantitation of Peptides Used in Microbicide Formulations

Chromatographia. 2014;77(23-24):1713-1720. doi: 10.1007/s10337-014-2777-7. Epub 2014 Oct 17.


Recently, a growing number of macromolecules such as peptides and proteins have been formulated into various microbicide formulations for the prevention of sexually transmitted infections. However, a fast and reliable high-throughput method for quantitating peptide/protein in polymer-based microbicide formulations is still lacking. As a result, we developed and validated a reversed-phase high-performance liquid chromatography method for the quantitation of gp120 fragment and LL-37 simultaneously in various microbicide gel formulations. This method was capable of detecting a limit of linearity (regression coefficient of 0.999) for gp120 fragment and LL-37 within a range of 0.625-80 and 1.25-80 µg mL-1, respectively. The lower limit of quantification for gp120 fragment and LL-37 was 1.14 and 0.31 µg mL-1, respectively. Method validation demonstrated acceptable intra- and inter-day RSD % (<5 %) and accuracy (95.67-100.5 %). Formulating both peptides into polymeric pharmaceutical gel formulations showed high extraction efficiency (in the range of 95.90 ± 3.03 to 111.45 ± 2.51 %). Using this method, we were able to separate and identify the forced degraded products from both peptides simultaneously without affecting the quantitation of both peptides in the polymeric dosage forms. Furthermore, this method was able to detect and separate degradants that were unable to be revealed using gel eletrophoresis.

Keywords: Intravaginal gel; Protein degradant; Reversed-phase HPLC; Separation; Tris-tricine SDS-PAGE.