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-Lys(6)-GnRH contained in a microemulsion-type 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 C(18) column at 40 degrees C, using a gradient of 10-35% CH(3)CN for peptide elution. The correlation of peak intensity measured at 220 nm and peptide concentration was linear over the range 2.5-60 microg/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 microg/mL. The lower limit of quantitation was calculated to be 0.38 microg/mL, and the lower limit of detection was 0.13 microg/mL. The assay was applied to samples that were stressed under physiological conditions (37 degrees 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.
Kafka
Alexandra P
AP
School of Pharmacy, University of Otago, Dunedin 9054, New Zealand.
Rades
Thomas
T
McDowell
Arlene
A
D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D004655:Emulsions; D007987:Gonadotropin-Releasing Hormone / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000032:analysis; D007202:Indicators and Reagents; D016014:Linear Models; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000737:chemistry*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004655:Emulsions; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010455:Peptides; D007987:Gonadotropin-Releasing Hormone; C035029:LHRH, Lys(6)-