College of Pharmacy, Kyungsung University, Nam-ku, Busan, Republic of Korea.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Sep 15;905:113-7. doi: 10.1016/j.jchromb.2012.08.010. Epub 2012 Aug 13.
The objective of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine the stability of collagen pentapeptide (KTTKS), which is a subfragment of collagen and has been proved to promote the extracellular release of collagen in skin fibroblast, in rat skin. The chromatographic condition was optimized on an Acclaim C-18 column (2.1 mm × 150 mm, 3 μm) under isocratic elution using a mobile phase consisting of deionized water and acetonitrile (87:13, v/v) mixture containing 5mM pentafluoropropionic acid as an ion-pairing reagent. The quantitation of KTTKS was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode. The calibration curve showed good linearity in the concentration range of 0.05-10.0 μg/mL (r(2)>0.999). The intra- and inter-day precisions were 0.8-6.5% and 2.4-5.8%, respectively, and the intra- and inter-day accuracies were 96.3-102.7% and 92.8-98.5%, respectively. The developed LC-MS/MS method was successfully applied to investigate the degradation rate and sites of KTTKS in rat skin homogenate. KTTKS was found to be very susceptible to the peptide bond cleavage by aminopeptidases present in the skin.
本研究旨在开发一种液相色谱-串联质谱（LC-MS/MS）方法来测定胶原五肽（KTTKS）的稳定性，胶原五肽是胶原的一个亚片段，已被证明可促进皮肤成纤维细胞中外源胶原的释放。在等度洗脱条件下，优化了 Acclaim C-18 柱（2.1mm×150mm，3μm）的色谱条件，流动相由去离子水和乙腈（87:13，v/v）混合物组成，其中含有 5mM 全氟丙酸作为离子对试剂。采用三重四极杆质谱仪在多重反应监测模式下对 KTTKS 进行定量分析。校准曲线在 0.05-10.0μg/mL 浓度范围内具有良好的线性（r²＞0.999）。日内和日间精密度分别为 0.8-6.5%和 2.4-5.8%，日内和日间准确度分别为 96.3-102.7%和 92.8-98.5%。所建立的 LC-MS/MS 方法成功应用于研究 KTTKS 在大鼠皮肤匀浆中的降解率和位点。研究发现，KTTKS 非常容易被皮肤中存在的氨肽酶切割肽键。
