QuEChERS净化-超高效液相色谱-串联质谱法测定番茄中5种链格孢霉毒素的含量

Determination of Five Alternaria Toxins in Tomatoes by UHPLC-MS/MS with Purification by QuEChERS

取经粉碎匀浆的番茄样品(5.0g)于50mL聚丙烯离心管中,加入酸化乙腈(每升中加入甲酸4.0mL)10mL,超声提取30min,使得测定的5种链格孢霉毒素[链格孢酚(AOH)、交链格孢酚单甲醚(AME)、交链孢烯(ALT)、腾毒素(TEN)及细交链格孢菌酮酸(TeA)]溶入乙腈中,加入氯化钠0.5g,无水硫酸镁5g脱水后高速离心5min,取提取液1.5mL,加入C1825mg作为净化剂,涡旋1min除去其中色素等共提取物,取经净化的提取液0.5 mL,置于离心管中,加入1mmol·L-1碳酸氢铵溶液0.5mL,再次高速离心5min,取上清液供超高效液相色谱-串联质谱分析。选择ACQUITY UPLC BEH C18色谱柱作为固定相,以不同比例的1mmol·L-1碳酸氢铵溶液(A)和甲醇(B)的混合液作为流动相进行梯度洗脱。串联质谱测定中采用电喷雾离子源正负离子切换扫描和多反应监测模式。用基质匹配法绘制标准曲线,所测定的5种链格孢霉毒素的线性范围均为0.5~100μg·L-1,其检出限(3S/N)在0.6~3.0μg·kg-1之间。以空白样品为基体,加入5种链格孢霉毒素的标准溶液按所述方法测定后计算其回收率,所得结果在81.6%~115%之间,测定值的相对标准偏差(n=6)在2.1%~11%之间。

A portion of homogenized pulp of tomato sample(5.0 g)was place in 50 mL polypropyrene centrifugation tube and extracted ultrasonically with 10 mL of acidified acetonitrile(4.0 mL of formic acid was added to 996 mL of acetonitrile)for 30 min,to dissolute the 5 alternaric toxins(i.e.AOH,AME,ALT,TEN and TeA)into acetonitrile.Sodium chloride(0.5 g)and anhydrous magnesium sulfate(5 g)were added and centrifuged at high speed for 5 min to have the extract dehydrated.An aliquot of 1.5 mL was taken from the extract,and purified by swirling with 25 mg of C18 for 1 min to remove interfering co-extracted substances,e.g.some pigments and etc.An aliquot of 0.5 mL of the purified extract was transferred to a centrifugation tube,and after adding 0.5 mL of 1 mmol·L-1(NH4)HCO3 solution,centrifuged at high speed for 5 min.The supernatant was used for UHPLCMS/MS analysis.ACQUITY UPLC BEH C18 chromatographic column was chosen as stationary phase,and mixtures of 1 mmol·L-1(NH4)HCO3 solution(A)and methanol(B)in various proportions were used as mobile phases in gradient elution.In MS/MS determination,electrospray ionization with switching scanning of ESI+and ESI-,and the mode of MRM were adopted.Standard curves for the 5 alternaric toxins were prepared by the matrix matching method,and linear relationships were found in the same range of 0.5 to 100μg·L-1,with values of detection limits(3 S/N)in the range of 0.6 to 3.0μg·kg-1.Using blank sample as matrix,tests for recovery were made by standard addition method,giving values of recovery in the range of 81.6%to 115%,and values of RSDs(n=6)were in the range of 2.1%to 11%.