液相色谱-串联质谱法测定水稻中生长素及其氨基酸结合物

Analysis of Auxins and Their Amino Acid Conjugates in Rice by Liquid Chromatography Tandem Mass Spectrometry

建立了同时测定水稻中吲哚-3-丁酸(IBA)、吲哚-3-乙酸(IAA)及其7种氨基酸结合物的液相色谱-串联质谱(HPLC-MS/MS)检测方法。样品在4℃下于80%甲醇中浸提12 h后,经混合阴离子交换反相固相萃取(MAX)净化,以5 mmol/L的甲酸铵溶液和甲醇为流动相,在C18柱上进行液相色谱分离,电喷雾正离子模式下用三重四极杆质谱仪多反应监测方式测定。对水稻根、茎、叶和籽粒4种基质分别进行加标回收实验,9种分析物的平均加标回收率为75%～106%,RSD为3.6%～15.2%,方法检出限(S/N=3)为0.01～25μg/kg。方法准确、灵敏,能满足水稻中9种生长素及其结合物在其生理水平的测定。

A liquid chromatography -tandem mass spectrometric （LC -MS/MS） method was developed for the simultaneous determination of indole-3-butyric acid （ IBA）, indole-3-acetic acid （IAA） and its 7 amino acid conjugates including IAA - Asp, IAA - Ala, IAA - Val, IAA - Trp, IAA - Leu, IAA - Ile and IAA - Phe in rice. The sample was extracted with 80% methanol for 12 h at 4 ℃. The extract was cleaned up on an Oasis MAX cartridge （60 mg × 3 mL） , and eluted with methanol contained 0.5% formic acid. 9 analytes were separated on a C18 column （1.8 μm, 100 mm × 2, 1 mm i. d. ） using 5 mmol/L ammonium formate（A） -acetonitrile（B） as mobile phase by gradient elution. The analytes were detected by positive-ion electrospray ionization - mass spectrometry under multiple reaction monitoring （MRM） mode. The recoveries of 9 analytes from the matrixes of root, sheath, leaf and seed in rice tissue ranged from 75% to 106% with relative standard deviations（RS- Ds） of 3.6% - 15.2% . The detection limits（S/N =3） ranged from 0.01 μg/kg to 25 μg/kg. The method was sensitive and reliable, and could meet the requirements for quantification of auxins and their amino acid conjugates in rice at the physiological level.