Rapid and specific high-performance liquid chromatography for the in vitro quantification of D-Lys⁶-GnRH in a microemulsion-type formulation in the presence of peptide oxidation products

A high-performance liquid chromatography (HPLC) method for assay of D-Lys⁶-GnRH contained in a microemulsiontype formulation is described. The peptide is extracted from the microemulsion matrix and quantified using a two-step gradient method. Separation from microemulsion compounds and potential peptide oxidation products was achieved on a Jupiter C₁₈ column at 40°C, using a gradient of 10-35% CH₃CN for peptide elution. The correlation of peak intensity measured at 220 nm and peptide concentration was linear over the range 2.5-60 μg/mL with a correlation coefficient of 0.9997 and a y-intercept not significantly different from zero (p > 0.05). Intraday and interday variability of the assay was less than 5% for multiple injections of samples containing 7.5, 30 and 60 μg/mL. The lower limit of quantitation was calculated to be 0.38 μg/mL, and the lower limit of detection was 0.13 μg/mL. The assay was applied to samples that were stressed under physiological conditions (37° C, pH 7.4) over 4 days. Three degradation peaks were well resolved from the parent peptide, demonstrating the selectivity of the assay. Off-line MALDI TOF mass spectrometry was applied to identify these degradation species as oxidation products of the peptide.