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目的采用柱前衍生高效液相色谱法对L-肌肽原料药中的游离氨基酸进行测定。方法采用异硫氰酸苯酯(phenyl isothiocyanate,PITC)柱前衍生高效液相色谱法,使L-肌肽中游离的β-丙氨酸和L-组氨酸衍生物达到完全分离,并测定其含量。色谱柱为DiamonsilTM C18(200 mm×4.6 mm,5μm),流动相为甲醇-0.05 mol.L-1醋酸钠溶液(体积比为13∶87),流速为1.0 mL.min-1,检测波长为254 nm,柱温为35℃。结果HPLC法测定β-丙氨酸和L-组氨酸的线性范围分别为2.01～20.10 mg.L-1(r=0.999 7)和2.00～20.00 mg.L-1(r=0.999 3);平均回收率分别为110.6%和98.2%。结论HPLC法无需专业的氨基酸分析仪,可用于L-肌肽中游离氨基酸的检查。
Objective To determine the free amino acids in L-carnosine by using pre-column derivatization high performance liquid chromatography. Methods The pre-column derivatization with phenyl isothiocyanate (PITC) was used to separate the free β-alanine and L-histidine derivatives in L-carnosine and to determine content. The column was DiamonsilTM C18 (200 mm × 4.6 mm, 5 μm). The mobile phase was methanol-0.05 mol·L-1 sodium acetate solution (volume ratio was 13:87) at a flow rate of 1.0 mL · min- 254 nm, the column temperature is 35 ℃. Results The linear range of β-alanine and L-histidine were 2.01-20.10 mg · L-1 (r = 0.999 7) and 2.00-20.00 mg · L-1 (r = 0.999 3), respectively. The average recovery was 110.6% and 98.2% respectively. Conclusion HPLC method does not require a professional amino acid analyzer, which can be used for the examination of free amino acids in L-carnosine.