目的: 建立基于酶解法采用超高效液相色谱-串联质谱法(UPLC-MS/MS法)检测阿胶、黄明胶和鹿角胶中猪皮特征肽。方法:采用胰蛋白酶进行酶解处理;使用Xbridge BEH-C18色谱柱(100 mm×2.1 mm,2.5 μm),柱温30 ℃,以0.1%甲酸水溶液(A)-0.1%甲酸乙腈溶液(B)为流动相,梯度洗脱(0～1 min,95%A;1～5 min,95%A→70%A;5～5.1 min,70%A→10%A;5.1～7 min,10%A;7～7.1 min,10%A→95%A;7.1～10 min,95%A),流速0.4 mL·min-1,进样量为2 μL;电喷雾离子源(ESI+),采用多重反应监测模式(MRM),正离子模式,对猪皮特征肽目标离子m/z 406.5(双电荷)→657.3和m/z 406.5(双电荷)→556.3进行检测。结果:阿胶、黄明胶、鹿角胶中分别掺入猪皮特征肽,质量浓度范围为5～500 ng·mL-1时,测得浓度与色谱峰面积成正比,线性方程分别为Y=3.020 4X+8.998(r2=0.999 7),Y=3.178 6X+11.906 9(r2=0.999 7),Y=3.046 5X+6.879(r2=0.998 6)。检测限为11 mg·kg-1,阿胶样品中猪皮杂质残留限值为76.7 mg·kg-1。28批阿胶样品中,有2批样品中检出猪皮特征肽。6批黄明胶、6批鹿角胶中均未检出猪皮特征肽。结论:所建立方法灵敏度高,重复性好,操作简便快捷,专属性强,可用于阿胶、黄明胶和鹿角胶中掺假猪皮的检测。
Objective: To establish a ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (tandem mass spectrometry) method for the detection of ingredients from pig skin in donkey-hide gelatin, ox-hide gelatin and deer-horn glue. Methods: Trypsin was used in the enzymatic analysis procedure. Detection was carried out on a Xbridge BEH-C18(2.1 mm×100 mm,2.5 μm) column. Tandem mass spectrometry was used to detect ingredient from pig hide adulteration in donkey-hide gelatins, ox-hide gelatin and deer-horn glue. The mobile phase was composed of water solvent containing 0.1% formic acid (A) and 0.1% formic acid acetonitrile (B) with gradient elution (0-1 min, 95%A; 1-5 min, 95%A→70%A; 5-5.1 min, 70%A→10%A; 5.1-7 min, 10%A; 7-7.1 min, 10%A→95%A; 7.1-10 min, 95%A) at a flow rate of 0.4 mL·min-1. The injection volume was 2 μL. The electrospray ionization (ESI+) source was performed in multiple reaction monitoring (MRM) mode with the transitions of m/z 406.5(double-charge)→657.3 and m/z 406.5(double-charge)→556.3. Results: The method showed good linearity within the range of 5-500 ng·mL-1 in different matrix. The linear equations were Y=3.020 4X+8.998 (r2=0.999 7), Y=3.178 6X+11.906 9(r2=0.999 7), and Y=3.046 5 X+6.879(r2=0.998 6), respectively. The lower limit of detection concentration of pig-hide gelatin characteristic peptide was 11 mg·kg-1 in matrix. The residue limit of the characteristic peptide from pig hide in donkey hide gelatin samples was 76.7 mg·kg-1. The characteristic peptide from pig skin could be detected in two from 28 batches of donkey-hide gelatin. It was not detected in six batches of ox-hide gelatin and six batches of deer-horn gelatin. Conclusion: The established method is simple, specific, sensitive and repetitive. It can be used for detection of ingredients from pig skin in donkey-hide gelatin, ox-hide gelatin and deer-horn glue.
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