色谱条件对HPLC检测大豆中赤霉酸含量的影响

Effect of chromatography condition on determing gibberellic acid content in soybean by HPLC

以赤霉酸的保留时间、谱图峰形、标准偏差、分离度、拖尾因子、线性相关系数等为指标,考察流动相的甲醇体积分数及其pH和检测波长对其检测结果的影响。结果表明,HPLC法检测大豆中赤霉酸含量的适宜条件为:色谱柱为C18(250 mm×4.6 mm,5.0μm),进样量为20μL,流动相为pH 4.0的35%甲醇溶液,流速为1.0 mL/min,柱温为25℃,紫外检测器波长为210 nm。评价指标显示,采用该色谱条件,不同质量浓度赤霉酸(0.2μg/mL和1.0μg/mL)的加标回收率分别为99.52%和99.44%,RSD分别为1.05%和0.90%;不同质量浓度赤霉酸(10μg/mL和50μg/mL)对照品5次重复检测结果的RSD分别为0.37%和0.13%,表明其精密度和准确度均较高。

The influences of three factors on determination of gibberellic acid by HPLC, such as volume fraction of methanol, pH of mobile phase and wavelength, were compared by investigating the retention time, peak shape, standard deviation, resolution, tailing factor and linear related coefficient. The data indicated that the appropriate HPLC condition for gibberellic acid was as follows： C18 column （250 mm ×4.6 mm, 5.0 um）, sample load 20 uL, mobile phase 35% methanol （pH 4.0）, flow rate 1.0 mL/ min, column temperature 25 ℃ and UV wavelength 210 nm. The evaluation results showed that the mean recovery was 99.52% and 99.44% for 0. 2 ug/mL and 1.0 ug/mL gibberellic acid, and the RSD was 1.05% and 0.90% ,respectively under the HPLC condition. Different mass concentrations of gibberellic acid standard solution were tested 5 times repeatedly, the RSD of peak area was 0.37% and 0.13% for 10 ug/mL and 50 ug/mL gibberellic acid. The HPLC method for determining gibberellic acid in soybean was proved to be sensitive and accurate.