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

关键词:  砷形态  高效液相色谱-原子荧光联用  动物源性中药

Key words:  arsenic speciation  HPLC-AFS  animal derived traditional Chinese medicines

摘要:

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，6 000 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 μm）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100 μL。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。

Abstract:

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 6 000 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, 5 μm) with the mobile phase of 1 mmol·L^-1 NH₄H₂PO₄(pH 8.5) and 20 mmol·L^-1 NH₄H₂PO₄(pH 7.0) by the gradient elution. The flow rate was 1.0 mL·min^-1 and the injection volume was 100 μL. 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.