高效液相色谱-柱前衍生化法测定饲料中的含硫氨基酸

摘要

A method of quantitative analysis of sulfur amino acids in feedstuffs by high performance liquid chromatography coupled with pre-column derivatization was developed. Before the feedstuffs were hydrolyzed under acidic condition, the cystine and methionine in the feedstuffs were oxidized to cysteic acid and methionine suffone respectively by performic acid, and then derivatized by 2,4-dinitrofiuorobenzene. The separation was performed on an Elite AAK C18 column ( 250 mm × 4. 6 mm, 5 μm ) by the gradient elution of 0.05 mol/L sodium acetate and acetonitrile-water (50: 50, v/v) as the mobile phase with a flow rate of 1.2 mL/min at 31 ℃.The detection wavelength was set at 360 nm. The linearities of cystine and methionine were good in the ranges of 0. 4 - 16.0 mg/L and 0. 7 - 29.6 mg/L with the correlation coefficients of 0. 999 9 and 0. 999 8, respectively. The quantification limits (S/N= 10) were 2.6 μg/kg, 3.1 μg/kg, and the recoveries were 100. 28％ - 102.00％ and 105.72％ - 107.89％, respectively. The method can be adapted to the accurate quantification of the suffur amino acids in feedstuffs.%发展了一种饲料中含硫氨基酸的检测方法.样品经过甲酸氧化,特其中的胱氨酸和蛋氨酸分别氧化为磺基丙氨酸和蛋氰酸砜;再经酸水解后,采用2,4-二硝基氟苯进行柱前衍生化,衍生物经高效液相色谱分析.使用EliteAAK C18色谱柱(250 mm×4.6 mm,5 μm)分离;以0.05 mol/L乙酸钠和乙腈-水(50∶50,v/v)为流动相,梯度洗脱,流速为1.2 mL/min;检测波长为360 nm;柱温为31 ℃.结果表明,胱氨酸在0.4～16.0 mg/L、蛋氨酸在0.7～29.6 mg/L范围内的线性相关系数分别为0.999 9、0.999 8;胱氨艘和蛋氨酸的定量限(信噪比(S/N)=10)分别为2.6 μg/kg和3.1 μg/kg;3次样品平行测定的胱氨酸和蛋氨酸含量的相对标准偏差(RSD)分别为0.79%和2.56%,加标回收率分别为100.28%～102.00%和105.72%～107.89%.该方法分析成本低,测定结果准确,灵敏度高,符合倒料中含硫氨基酸的检测要求.