HPLC法测定L-肌肽中的游离氨基酸的含量

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。

Determination of contents of the free amino acids in L-carnosine by HPLC

Objective To establish an HPLC method with precolumn derivatization for determining the contents of two free amino acids in L-carnosine. Methods Using phenyl isothiocyanate (PITC) as the derivating reagent, the contents of β-alanine and histidine in L-carnosine were determined, which were completely separated. The column was DiamonslTM C18 (200 mm×4.6 mm,5 μm); the mobile phase was methanol-0.05 mol·L ^-1 sodium acetate solution (V:V=13:87); the flow rate was 1.0 mL·min-¹; the detection wavelength was set at 254 nm and the column temperature was 35 ℃. Results The liner ranges of β-alanine and 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; their average recoveries were 110.6 % and 98.2 %. Conclusion The method does not need the special amino acid analyzer, and it is efficient, sensitive and accurate for determination of the free amino acids in L-carnosine.