高效液相色谱-二极管阵列检测器法测定茶叶中10种有机酸含量

Determination of 10 organic acid contents in tea using high performance liquid chromatography-diode array detector

从流动相配比、柱温、流速、检测波长等方面优化色谱条件,并进行方法学考察及验证性试验,从而建立了一种同时分离和定量测定茶叶中10种有机酸(草酸、D/L-酒石酸、甲酸、L-苹果酸、抗坏血酸、乳酸、乙酸、柠檬酸、琥珀酸和富马酸)的高效液相色谱法。优化后的色谱条件为:采用TSKgel ODS-100V(4.6 mm×250 mm,5μm)色谱柱,流动相为0.10%磷酸水溶液(A相)和50%乙腈水溶液(B相),洗脱方式为100%A(0 min)→100%A(12 min)→0%A(17 min)→0%A(25 min)→100%A(30 min)→100%A(33 min),后运行2 min,柱温为40℃,流速为1.0 mL/min,二极管阵列检测器同时在210和245 nm波长下检测抗坏血酸,在210 nm波长下检测其他9种有机酸。结果显示,各有机酸组分回归方程的相关系数均在0.999 5以上,其精密度和重复性的相对标准偏差(relative standard deviation, RSD)均小于1%,稳定性的RSD小于4%(除抗坏血酸外),回收率为97%～116%。说明该方法的线性关系、精密度和重复性良好,对组分分离效果较好,可用于茶叶中10种有机酸含量的测定。

A high performance liquid chromatography(HPLC)was developed to simultaneously determine the content of 10 organic acids(oxalic acid,D/L-tartaric acid,formic acid,L-malic acid,ascorbic acid,lactic acid,acetic acid,citric acid,succinic acid and fumaric acid)in tea.The chromatographic conditions were optimized in the aspects of mobile phase composition,column temperature,flow rate and detection wavelength.The established HPLC method was methodologically assessed and applied to teas,which included maintaining TSKgel ODS-100V column(4.6 mm×250 mm,5μm)at 40℃,using 0.10%phosphoric acid-water solution(mobile phase A)and 50%acetonitrile-water solution(mobile phase B)with the gradient elution mode of 100%A(0 min)→100%A(12 min)→0%A(17 min)→0%A(25 min)→100%A(30 min)→100%A(33 min)with 2 min post-run at the flow rate of 1.0 mL/min,and detecting at the wavelengths of 210 and 245 nm for ascorbic acid,and 210 nm for other organic acids by diode array detector(DAD).The correlation coefficients of regression equations for all of the components were greater than 0.999 5,and the relative standard deviations(RSDs)of accuracy and repeatability were lower than 1%,and their RSDs of stability were lower than 4%(except ascorbic acid),as well as the recovery rate with the method of 97%-116%.It is appeared that the newly developed method can yield fine linear relationship,desirable precision,repeatability and separability for determinations of 10 organic acids in tea.