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Rapid and specific high‐performance liquid chromatography for the in vitro quantification of d ‐Lys 6 –GnRH in a microemulsion‐type formulation in the presence of peptide oxidation products
Author(s) -
Kafka Alexandra P.,
Rades Thomas,
McDowell Arlene
Publication year - 2010
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1261
Subject(s) - chemistry , chromatography , peptide , detection limit , microemulsion , high performance liquid chromatography , mass spectrometry , quantitative analysis (chemistry) , matrix (chemical analysis) , analytical chemistry (journal) , biochemistry , pulmonary surfactant
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°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 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. Copyright © 2009 John Wiley & Sons, Ltd.