A simple, selective and accurate reverse phase HPLC method was developed for detection and quantitation of γ-conglutin from lupin seed extract. A linear gradient of water and acetonitrile containing trifluoroacetic acid (TFA) on a reverse phase column (Agilent Zorbax 300SB C-18), with a flow rate of 0.8 ml/min was able to produce a sharp and symmetric peak of γ-conglutin with a retention time at 29.16 min. The identity of γ-conglutin in the peak was confirmed by mass spectrometry (MS/MS identification) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The data obtained from MS/MS analysis was matched against the specified database to obtain the exact match for the protein of interest. The proposed method was validated in terms of specificity, linearity, sensitivity, precision, recovery and accuracy. The analytical parameters revealed that the validated method was capable of selectively performing a good chromatographic separation of γ-conglutin from the lupin seed extract with no interference of the matrix. The detection and quantitation limit of γ-conglutin were found to be 2.68 μg/ml and 8.12 μg/ml respectively. The accuracy (precision and recovery) analysis of the method was conducted under repeatable conditions on different days. Intra-day and inter-day precision values less than 0.5% and recovery greater than 97% indicated high precision and accuracy of the method for analysis of γ-conglutin. The method validation findings were reproducible and can be successfully applied for routine analysis of γ-conglutin from lupin seed extract.

建立了一种简单，选择性和准确的反相HPLC方法，用于检测和定量羽扇豆种子提取物中的γ-凝集素。在反相色谱柱（Agilent Zorbax 300SB C-18）上，水和乙腈中含有三氟乙酸（TFA）的线性梯度，流速为0.8 ml / min时，可以产生一个尖锐且对称的γ-凝集素峰，保留时间为29.16分钟。质谱（MS / MS鉴定）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析确认了峰中γ-凝集素的身份。从MS / MS分析获得的数据与指定的数据库进行匹配，以获得目标蛋白质的精确匹配。该方法在特异性，线性，灵敏度，精密度，回收率和准确度方面均得到了验证。分析参数表明，经验证的方法能够从羽扇豆种子提取物中选择性地进行γ-凝集素的良好色谱分离，而不会受到基质的干扰。γ-凝集素的检出限和定量限分别为2.68μg/ ml和8.12μg/ ml。该方法的准确性（精密度和回收率）分析是在不同日期的可重复条件下进行的。日内和日间精度值小于0.5％，回收率大于97％表明γ-凝集素分析方法具有很高的精度和准确性。该方法的验证结果是可重复的，可成功地用于羽扇豆种子提取物中γ-凝集素的常规分析。