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
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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. / Kafka, Alexandra P.; Rades, Thomas; McDowell, Arlene.
In: Biomedical Chromatography, Vol. 24, No. 2, 02.2010, p. 132-139.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - 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
AU - Kafka, Alexandra P.
AU - Rades, Thomas
AU - McDowell, Arlene
PY - 2010/2
Y1 - 2010/2
N2 - 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.
AB - 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.
KW - MALDI TOF
KW - Microemulsion
KW - Oxidation
KW - Proteins and peptides
KW - Tryptophan
UR - http://www.scopus.com/inward/record.url?scp=74849096858&partnerID=8YFLogxK
U2 - 10.1002/bmc.1261
DO - 10.1002/bmc.1261
M3 - Journal article
C2 - 19517451
AN - SCOPUS:74849096858
VL - 24
SP - 132
EP - 139
JO - Biomedical Chromatography
JF - Biomedical Chromatography
SN - 0269-3879
IS - 2
ER -
ID: 299427713