Abstract: In this study, recombinant type Ⅲ collagen was used as the subject of this study. The marker peptide was digested by trypsin and identified with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This peptide was then used as a quantitative reference standard to establish the quantification method. Subsequently, the method was applied to quantify the residual amounts of recombinant type Ⅲ collagen (rhCOL-Ⅲ) following intradermal injection in rats. The results indicated that the marker peptide of rhCOL-Ⅲ was GESGPAGPAGAPGPAGSK. The peptide showed good linearity in the mass concentration range of 0.1－200 μg/mL, with a correlation coefficient over 0.99. The relative standard deviation (RSD) of reproducibility and precision were 2.65% and 4.89%, respectively. The spiked recoveries were all within the range of 96%－118%. The limit of quantification (LOQ) and the limit of detection (LOD) for the marker peptide were 0.5 ng/mL and 0.05 ng/mL, respectively. After 5 hours of intradermal injection, the concentration of rhCOL-Ⅲ remaining in the skin ranged from 0.79 μg/mg to 3.94 μg/mg. The method had high precision, good reproducibility and high sensitivity. Furthermore, it is unaffected by the animal’s endogenous collagen. This method can be used for the quantitative detection of rhCOL-Ⅲ remaining afterin vivoinjection in rats.

Key words: HPLC-MS/MS, recombinant collagen, internal injection, marker peptide