超声酶水解提取/高效液相色谱-原子荧光光谱联用法测定动物源性中药中的砷形态

Determination of Arsenic Speciation by HPLC Combined with Atomic Fluorescence Spectrometry(HPLC-AFS) Using Ultrasound and Enzymatic Hydrolysis in Animal Derived Traditional Chinese Medicines

目的采用液相色谱-原子荧光光谱(HPLC-AFS)联用技术对动物源性中药中一甲基砷酸(monomethylarsine,MMA)、二甲基砷酸(dimethyarsine,DMA)、三价砷[As(Ⅲ)]、五价砷[As(V)]的形态进行研究。方法样品中加入pH 2.5的胃蛋白酶液,于55℃超声20 min,6000 r·min^-1离心3 min,取其上清液,过滤进样。采用Hamilton PPRP-X100阴离子交换色谱柱(150 mm×4.6mm,5?m)分离,1 mmol·L^-1磷酸二氢铵溶液(pH 8.5),20 mmol·L^-1磷酸二氢铵溶液(pH 7.0)体系组成的流动相按一定比例进行梯度洗脱,流速为1.0 mL·min^-1,进样量为100μL。结果4种砷形态在13 min内完成分离。MMA、DMA、As(Ⅲ)、As(V)的检出限分别为0.01,0.01,0.01和0.02 mg·kg^-1,样品中加标量为0.03~0.1 mg·kg^-1时,4种砷化合物的回收率为93.4%~105.4%,精密度RSD为0.8%~4.4%。结论本研究建立的超声酶水解提取/HPLC-AFS测定动物源性中药中砷形态的分析方法样品提取简便,提取率高,测定方法精密度好,准确度高。

OBJECTIVE To develop a method to determine four arsenic(As)speciation including arsenite[As(Ⅲ)],arsenate[As(V)],monomethylarsine(MMA),dimethyarsine(DMA)by HPLC-AFS using ultrasound and enzymatic hydrolysis in animal derived traditional Chinese medicines.METHODS Add pepsin to the samples after ultrasound and enzymatichydrolysis(pH 2.5)with 55℃for 20 min,centrifuged at 6000 r·min^-1 for 3 min.Took the supernatant,filter the sample.The sample was separated on an Hamilton PPRP-X100 anion exchange column(150 mm×4.6 mm,5μm)with the mobile phase of 1 mmol·L^-1 NH4 H2 PO4(p H 8.5)and 20 mmol·L^-1 NH4 H2 PO4(p H 7.0)by the gradient elution.The flow rate was 1.0 mL·min^-1 and the injection volume was 100μL.RESULTS The separation was achieved within 13 min for four arsenic speciations.The detect limits were 0.01,0.01,0.01 and 0.02 mg·kg^-1 for MMA,DMA,As(Ⅲ)and As(V)respectively.The recoveries of four arsenic speciations ranged from 93.4%to 105.4%,the RSDs of precision were among 0.8%to 4.4%with the spiked amounts of 0.03?0.1 mg·kg^-1 in the samples.CONCLUSION The developed HPLC-AFS method using ultrasound and enzymatic hydrolysis for determination of As speciation has the advantages of simplicity,high extraction rate,good precision and high accuracy.