小儿尿液中的新蝶呤及生物蝶呤检测方法优化

Optimization of Detection Methods for Neopterin and Biopterin in Children’s Urine

目的基于高效液相色谱法(HPLC)同时检测小儿尿液中新蝶呤(N,CAS No:2009-64-5)和生物蝶呤(B,CAS:22150-76-1)的含量并探究KI-I氧化的条件。方法采用KI-I2法氧化原型新蝶呤及生物蝶呤,在激发波长为350 nm,发射波长460 nm下使用HPLC进行检测,其中色谱柱选用Kromasil EternityXT C18(4.6*250mm,5μm),流动相为甲醇:水(10:90)。结果新蝶呤及生物蝶呤的线性均在0.9999以上,检测范围为新蝶呤:0.00625mg/mL~1.0mg/mL,生物蝶呤:0.00675 mg/mL~1.08 mg/mL,加标回收率在92.18%~98.24%。氧化过程中,普通实验室的光照对实验无明显的影响,氧化前不酸化尿液会造成测量的N、B值减小。结论使用优化方法检测小儿尿液中的新蝶呤、生物蝶呤与尿肌酐的含量比值,并通过计算B%(百分比),有助于高苯丙氨酸的鉴别分型,对患儿的治疗及预防发病具有重要意义。

Objective The content of neopterin(N)and biopterin(B)in children’s urine was simultaneously detected by High Performance Liquid Chromatography,and the conditions of KI-I oxidation were investigated at the same time.Methods The prototype neopterin and biopterin were oxidized by KI-I2 method and detected at excitation wavelength 350nm and emission wavelength 460nm.The column was selected Kromasil EternityXT C18(4.6*250mm,5μm),and the mobile phase was methanol:water(10:90).Results The linearity of neopterin and biopterin was above 0.9999.The detection range was 0.00625mg/mL~1.0mg/m L for neopterin and 0.00675mg/mL~1.08mg/m L for biopterin.The recovery rate was 92.18%~98.24%.The light of the general laboratory has no obvious effect on the experiment during the oxidation process,and the measured N and B values will be reduced if the urine is not acidified before oxidation.Conclusion Using this method to detect the content ratio of neopterin,biopterin and urinary creatinine in children,and by calculating B%.It is helpful to identify the type of hyperphenylalanine.This method is accurate and feasible,which is of great significance for differentiating the classification of hyperphenylalaninemia and for the treatment and prevention of disease onset in children.