In this paper, a method for measurement of hydroxyproline in milk powder by high perform ance liquid chromatography(HPLC)with pre-column derivation was established. 2, 4-dinitrofluorobenzene (DNFB) was used as derivative reagent, and the separation was performed on Elite AAK C18 column (250 mm×4. 6 mm I. D. , 5 μm) with gradient elution of 0. 05mol/L sodium acetate and acetonitrile/water (50: 50, V:V) at 27 ℃. The flow rate was 1. 2 mL/min and the detection wavelength was 360 nm. Method val idation was performed. The results showed that the linear range was from 0. 04 to 10 mg/L with a linear correlation coefficient of 0. 9999. The limit of quantification was 30 mg/kg in milk powder sample (S/N = 10). The relative standard deviations of parallel determination (n=5) were 9. 64% and 0. 36% with spiked of 0. 05 mg/L and 8. 0 mg/L respectively. The recoveries were from 95. 94% to 114. 87% in the spiked range of 0. 05~8. 0 mg/L in milk powder. It was indicated that the method was accurate and repeatable for the determination of hydroxyproline in milk powder.%建立了柱前衍生化高效液相色谱法(HPLC)测定奶粉中羟脯氨酸含量的方法.以2,4-二硝基氟苯为衍生试剂,采用Elite AAK C18色谱柱(250 mm×4.6 mm i.d.,5μm)；0.05 mol/L乙酸钠,乙腈/水(50∶50,V∶V)为流动相梯度洗脱；流速1.2 mL/min;检测波长360 nm；柱温27℃.结果表明:羟脯氨酸标准品在0.04～10.00mg/L范围内线性相关系数为0.9999;方法检出限为30 mg/kg(S/N=10)；加标浓度为0.05,8.0 mg/L的平行5次测试结果相对标准偏差分别为9.64％和0.36％；0.05,0.1,0.25,0.5,1.0,2.0,8.0 mg/L7个加标水平测试,加标回收率在95.94％～114.87％之间,加标线性曲线的线性相关系数为0.9998.该方法定量结果准确,满足奶粉中羟脯氨酸含量测定的要求,可以作为判断奶粉中是否违禁添加皮革水解蛋白的依据.
展开▼
