提出了反相高效液相色谱同时测定新生儿尿液中4-羟基苯乙酸、2-羟基苯乙酸、苯乙酸和苯丙酮酸等4种苯丙酮尿症特征酸的方法。取尿液样品5.00 mL，乙腈5 mL，混合振荡1 min，离心10 min，取上清液进行液相色谱分离。用Waters Symmetry C₁₈色谱柱作为固定相，用不同比例的乙腈和0.15%（体积分数）磷酸溶液进行梯度洗脱，检测波长为210，289 nm。在上述条件下，4种特征酸分离完全，并且不受尿液中内源性物质的干扰。4种特征酸的质量浓度在一定范围内呈线性，检出限（3S/N）分别为0.43，0.22，0.62，0.42 mg·L^-1。按标准加入法在3个浓度水平上进行加标回收试验，测得回收率在89.1%~108%之间，测定值的相对标准偏差（n=6）小于4%。

Reversed-phase high performance liquid chromatography (RP-HPLC) was applied to the determination of 4 phenylketonuric characteristic acids, including 4-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, phenylacetic acid and phenylpyruvic acid in newborn urine. The urine sample (5.00 mL) was shaked with acetonitrile (5.0 mL) by swirling for 1min and centrifuged for 10 min. 4 analytes in the supernatant were separated by LC using Waters Symmetry C₁₈ column as stationary phase, and mixtures of acetonitrile and 0.15% (φ) phosphoric acid solution in various ratios as mobile phase in gradient elution. Detections were taken at the wavelengths of 210, 289 nm. Under the above conditions, the 4 characteristic acids were separated completely, and no interference by the endogenous substances in the urine was observed. Linearity for the 4 characteristic acids were kept between definite mass concentration ranges. Values of detection limits (3S/N) for the 4 characteristic acids were 0.43, 0.22, 0.62, 0.42 mg·L^-1 respectively. Tests for recovery were made by standard addition method at 3 concentration levels, giving results in the range of 89.1%-108%, with RSDs (n=6) less than 4%.