响应面法优化浙贝母氨基酸提取工艺及含量测定

The use of response surface methodology to optimize the extraction process and determine amino acids in Fritillaria thunbergii Mig

采用响应面法优化浙贝母氨基酸提取条件,建立柱前衍生-超高效液相色谱法(ultra-performance liquid chromatography,UPLC)测定浙贝母鳞茎中14种氨基酸含量方法,比较不同产地栽前与栽后浙贝母鳞茎氨基酸含量差异性。采用6 mol/L HCl 8 mL,超声30 min,在110℃下水解20 h提取浙贝母鳞茎中氨基酸,以异硫氰酸苯酯(phenyl isothiocyanate,PITC)柱前衍生化,UPLC检测,使用ACQUITY UPLC BEH C 18色谱柱(2.1 mm×150 mm,1.7μm),流动相A为0.1 mol/L乙酸钠溶液(冰醋酸调pH=6.5)-乙腈(体积比97∶3),B为乙腈-水(体积比4∶1),梯度洗脱,检测波长为254 nm,流速0.2 mL/min。14种氨基酸线性关系良好(r≥0.9992),平均加样回收率为95.12%~105.53%,RSD<3%,表明该方法准确可靠。不同产地栽前栽后浙贝母氨基酸含量具有一定差异。该方法确定了提取浙贝母氨基酸的最优条件,可用于浙贝母鳞茎中氨基酸的含量测定。

To optimize the amino acid extraction conditions of Fritillaria thunbergii Mig by response surface methodology,a pre-column derivatization-ultra-performance liquid chromatography method was establish to determine the contents of 14 amino acids in Fritillaria thunbergii Mig bulbs before and after planting in different areas.The following conditions were used for the extraction of amino acids from bulbs in Fritillaria thunbergii Mig,which was 8 mL hydrochloric acid(6 mol/L)for ultrasonication 30 min and hydrolysis at 110℃for 20 h,and then derivatized with phenyl isothiocyanate pre-column and followed by the detection by UPLC which used ACQUITY UPLC BEH C 18 column(2.1 mm×150 mm,1.7μm).Mobile phase A was 0.1 mol/L sodium acetate solution(pH adjusted by glacial acetic acid=6.5)∶acetonitrile(97∶3),and mobile phase B was acetonitrile∶water(4∶1);followed by gradient elution and the detection wavelength was 254 nm with flow rate 0.2 mL/min.The calibration curves of 14 kinds of amino acids showed good linearity within their test ranges(r≥0.9992).The average recoveries were between 95.12%and 101.05%,and the RSD s were less than 3.00%which indicating that the method was accurate and reliable.The amino acid content of Fritillaria thunbergii Mig before and after planting in different producing areas were different.This method finds the optimal conditions for the extraction of amino acids from Fritillaria thunbergii Mig,which can be used for the determination of amino acids in bulbs of Fritillaria thunbergii Mig.