HPLC法在小儿尿液新蝶呤和生物蝶呤检测中的应用

Determination of neopterin and biopterin in pedo-urine by HPLC

目的建立 HPLC 法测定小儿高苯丙氨酸血症(HPA)患者尿液中新蝶呤(N)和生物蝶呤(B)含量的方法,以帮助临床对经典型苯丙酮尿症(PKU)和四氢生物蝶呤(BH_4)缺乏症的诊断和鉴别诊断。方法选用 Cosmosil 5C_(18)-AR-Ⅱ色谱柱(4.6 I.D.×250 mm),甲醇-水(8:92)为流动相,用三维荧光检测器检测。结果新蝶呤线性范围为1.60～40.0 ng/μl,生物蝶呤线性范围为1.73～43.2 ng/μl;最小检测限为0.16 ng/μl和0.1728 ng/μl。样品测定的变异系数(CV)值分别为2.18%和1.70%;原尿样品前处理时氧化的最佳时间为30 min;原尿样品不便于保存,氧化后可于室温或4℃冰箱保存3 d。结论本方法准确、可靠、简便易行,灵敏度高,方法学实验结果满意,适用于临床 HPA 患儿的鉴别诊断。

Objective To establish the method for determination of neopterin and biopterin in pedourine by HPLC for differential diagnosis of the classic phenylketonuria and tetrahydrobiopterin deficiency. Methods The COSMOSIL 5C18-AR-Ⅱ column was selected; the mobile phase was methanol： water （8： 92） and the fluorescence detector was used. Results The linear range for neopterin was 1.60-40. 0 ng/μl and for biopterin was 1.73-43.2 ng/μl; The lowest limit were 0. 16 ng/μl for neopterin and 0. 1728 ng/μl for biopterin, the coefficient of variation of the sample were 2. 18% for neopterin and 1.70% for biopterin; The optimizing pre-processing oxidation time of urine was 30 minutes. The conservation of urine sample was difficult and the product of oxidation can be conserved for 3days at room temperature or at freezer 4℃ o Conclusions This method is accurate, simple, sensitive and easy for operation. The results from the experiments of methodology is satisfied . This method is suitable for differential diagnosis of the classic phenylketonuria and tetrahydrobiopterin deficiency.