反相离子对色谱法测定手性生物转化中扁桃酸与苯乙酮酸

Determination of mandelic acid and phenylglyoxilic acid in chiral biotransformation by ion-pair reversed-phase high-performance liquid chromatography

建立一种同时测定生物不对称转化发酵液中扁桃酸及苯乙酮酸含量的反相离子对高效液相色谱方法.色谱柱为HypersilC18柱(4.6mm×250mm,5μm),流动相为甲醇-25mmol·L-1磷酸二氢钾缓冲液(内含6mmol·L-1四丁基溴化铵,NaOH调pH至7.0)(体积分数15∶85),流速为1.0mL·min-1,检测波长220nm,柱温为室温.扁桃酸与苯乙酮酸在0.5～20mmol·L-1内,其浓度与峰面积呈现良好的线性关系(r=0.9994,0.9997).二者的加样回收率均为96.5%～100.6%.当信噪比(S/N)为3时,二者的最低检测浓度分别为2.4和8μmol·L-1.该方法简便、准确、灵敏度高,可用于手性生物转化中扁桃酸与苯乙酮酸的定量分析.

To established a method for detecting mandelic acid and phenylglyoxilic acid in chiral biotransformation by an ion-pair reversed-phase HPLC. The optimal chromatography was performed with a Hypersil C18 column （4. 6 mm × 250 mm,5 μm）,the mobile phase consisted of methanol-25 mmol·L^-1 potassium dihydrogen phosphate （contained 6 mmol·L^-1 tetrabutylammonium bromide, pH was adjusted to 7. 0 with sodium hydroxide） （15∶ 85,V/V）,flow rate at 1. 0 mL·min^-1,the column temperature at room temperature,and UV detection at 220 nm. The peak area and concentration showed excellent correlation,when concentrations of mandelic acid （r = 0. 999 4） and phenylglyoxilic acid（r = 0. 999 7） were 0. 5 ～ 20 mmol·L^-1,the recoveries were 96. 5% ～ 100. 6% . The detection limits of both mandelic acid and phenylglyoxilic acid were 2. 4 and 8 μmol/L,if the rate of signal /noise was 3. The method proposed in this paper is suitable to analyze mandelic acid and phenylglyoxilic acid in fermentation.