高效液相色谱-串联质谱法测定水果中叶酸及其稳定性

Determination of Folic Acid and Its Stability in Fruits by HPLC-MS/MS

建立了高效液相色谱-串联质谱(HPLC-MS/MS)法测定水果中叶酸(PteGlu)、四氢叶酸(H4PteGlu)、5-甲基一四氢叶酸(5-CH3-H4PteGlu)的方法,同时研究了不同温度、处理时间、pH对3种叶酸稳定性的影响。以0.1%甲酸乙腈溶液-0.1%甲酸水溶液为流动相进行梯度洗脱,在电喷雾正离子模式下,以多反应监测(MRM)模式进行检测。结果表明,3种叶酸在10-100 ng/mL质量浓度范围内线性关系良好,线性相关系数(R^2)均大于0.99,5-甲基-四氢叶酸的检出限为1 ng/mL,叶酸、四氢叶酸的检出限均为2 ng/mL,平均回收率为59.34%~65.67%。不同水果3种叶酸的含量差异较大,叶酸的含量范围在3.11~27.33μg/100 g之间,四氢叶酸的含量范围在10.65~26.87μg/100 g之间,5-甲基-四氢叶酸的含量范围在8.33~32.72μg/100 g之间。pH对3种叶酸稳定性的影响较大,温度和时间影响相对较小,叶酸在pH 7较稳定,四氢叶酸和5-甲基-四氢叶酸在pH 6较稳定。该方法简单、快速,灵敏度高,可为水果中叶酸的检测提供参考依据。

A high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method was developed for the detection of folic acid(PteGlu),tetrahydrofolate(H4 PteGlu)and 5-methyltetrahydrofolate(5-CH3-H4 PteGlu)in fruits.The influences of different temperature,treatment time and pH on stability of 3 kinds of folic acid were studied.The separation was performed using acetonitrile containing 0.1%formic acid and water containing 0.1%formic acid as mobile phase with gradient elution at a flow rate of 0.2 detected by multiple reaction monitoring(MRM)with an electrospray ionization source in the positive ion mode.The results show that the linear range of the method is from 10 ng/mL to 100 ng/mL with R^2>0.99,which means the method has a good linearity.The limit of detection of 5-CH3-H4 PteGlu is 1 ng/mL.The limits of detection of PteGlu and H4 PteGlu are 2 ng/mL.The recoveries for spiked samples are 59.34%-65.67%.The content of folic acid in different fruits varies greatly.The content of PteGlu ranges from 3.11μg/100 g to 27.33μg/100 g.The content of H4 PteGlu ranges from 10.65μg/100 g to 26.87μg/100 g.The content of 5-CH3-H4 PteGlu ranges from 8.33μg/100 g to32.72μg/100 g.pH has a great influence on the stability of three kinds of folic acid.The temperature and time have relatively small effect.PteGlu is stable at pH 7.H4 PteGlu and 5-CH3-H4 PteGlu are stable at pH 6.The method is simple,rapid and sensitive,and it can provide a reference for the detection of folic acid in fruit.