Abstract: A high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method for determination of content of collagen in snake skin was established.After the collagen in snake skin sample was hydrolyzed with hydrochloric acid,separation was performed by a Thermo Hypersil Gold C₁₈(250 mm×4.6 mm,5 μm) chromatographic column and acetonitrile (containing 0.2% formic acid,mobile phase A)-water (containing 0.2% formic acid,mobile phase B) as the mobile phase were used for gradient elution.The elution time is 15 min.The column temperature is 30 ℃;and the flow rate is 0.4 mL/min.The ELSD detector parameters are as follows:nebulization temperature,85 ℃;evaporation temperature,85 ℃;carrier gas,N₂;air flow of the evaporator,1.2 L/min.The injection volume of the sample is 10 μL.Under these chromatographic conditions,the linearity range of hydroxyproline is 10 - 100 mg/L,R² = 0.999 6;the lowest limit of detection is 5.56 mg/L,and the lowest limit of quantification is 10 mg/L.The average recovery rate of snake skin collagen is 94.53%;the RSD is 2.15%;and the average of the mass fraction of collagen in snake skin is 40.21%.

Key words: cosmetic ingredient, collagen, hydroxyproline, HPLC-ELSD, snake skin