高效液相色谱法测定奶粉中羟脯氨酸含量

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(250mm×4.6mm i.d.,5μm);0.05mol/L乙酸钠,乙腈/水(50:50,V:V)为流动相梯度洗脱;流速1.2mL/min;检测波长360nm;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30mg/kg(S/N=10);加标浓度为0.05,8.0mg/L的平行5次测试结果相对标准偏差分别为9.64%和0.36%;0.05,0.1,0.25,0.5,1.0,2.0,8.0mg/L 7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。

Detection of hydroxyproline in milk powder by high performance liquid chromatography

In this paper, a method for measurement of hydroxyproline in milk powder by high performance 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 validation 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.