离子对反相液相色谱-原子荧光光谱法测定畜禽肉中5种硒形态含量

建立了一种测定畜禽肉类中硒代胱氨酸、甲基硒代半胱氨酸、硒代蛋氨酸、硒酸根和亚硒酸根含量的离子对反相液相色谱-原子荧光光谱分析方法。样品中有机硒通过胰蛋白酶和蛋白酶（XIV，链霉蛋白酶）酶解提取，无机硒通过碘乙酰胺溶液提取，于55 ℃水浴200 r·min-1振荡提取20 h，提取液高速离心后再经超滤管离心净化，C18反相色谱柱分离，30 mmol·L-1磷酸氢二铵、0.5 mmol·L-1四丁基溴化铵和5% （V/V）甲醇为流动相，用20%（V/V）甲酸调节流动相溶液pH至6.0，离子对反相液相色谱-原子荧光光谱法测定样品溶液中5种硒形态含量。采用与标准样品对照法定性，峰面积外标法定量。保留时间定性，外标法峰面积定量。硒代胱氨酸、甲基硒代半胱氨酸、硒代蛋氨酸、硒酸根和亚硒酸根在5~200 μg·L-1范围内线性良好，相关系数均大于0.999，其检出限分别为0.89，0.78，0.55，0.94和0.70μg·L-1，加标回收率为76.8%~109%，批内精密度和批间精密度分别为2.7%~7.8%和3.5%~12.3%。本方法具有快速简便、灵敏和准确等优点，适用于畜禽肉类样品中硒形态分析测定。

Determination of 5 Kinds of Selenium Species in Livestock and Poultry Meat With Ion Pair Reversed Phase Liquid Chromatography-Atomic Fluorescence Spectrometry

A method of ion-pair reversed phased liquid chromatography-ultraviolet (IP-RP-HPLC)-hydride generation-atomic fluorescence spectrometry (AFS) was established to determine the contents of selenocystine, methylselenocysteine, selenotmethionine, selenateand selenite in livestock and poultry meat. Organic Se species contents in samples were extracted by trypsin and protease (XIV, pronase), and inorganic Se species contents in samples were extracted with iodine acetamide solution and water incubated at 55 ℃ with shaking at 200 r·min-1for 20 h. The extraction solution of samples was centrifuged at high speedand then centrifuged with microsep for purification. C18 separated the solution of samples reversed phase column by using 30 mmol·L-1diammonium hydrogen phosphate, 0.5 mmol·L-1tetrabutyl-ammonium bromide and 5% (V/V) methanol as moblie phase. The pH of the mobile phase was adjusted to 6.0 by 20% (V/V) formic acid. IP-RP-HPLC-AFS determined the contents of five Se species in samples solution. The impurities are qualitatively determined by contrasting with the standard sample and quantitatively determined by calculating the peak areas. Selenocystine, methylselenocysteine, selenotmethionine, selenate and selenite had good linearities in the range of 5~200 μg·L-1, and the correlation coefficients were all greater than 0.999, the detection limits of five Se species were 0.89, 0.78, 0.55, 0.94 and 0.70 μg·L-1, respectively. There coveries were between 76.8%~109%, the within-run precisions and between-run precisions were 2.7%~6.8% and 3.5%~12.3% respectively. The proposed method has the advantages of rapid, simple, high sensitivity and high accuracy, and it is suitable for Se species analysis in livestock and poultry meat samples.