Abstract:

Objective To establish a method for the determination of 6 markers in neonatal phenylketonuria（PKU），congenital adrenal hyperplasia（CAH） and congenital hypothyroidism（CH） by ultra-performance liquid chromatography tandem mass spectrometry（UPLC-MS/MS）. Methods The processing conditions of dried blood spots were optimized to establish a UPLC-MS/MS that can simultaneously determine 17-hydroxyprogesterone（17-OHP），androstenedione（ASD），hydrocortisone（COR），phenylalanine（PHE），tyrosine（TYR） and thyroxine（T₄） and carry out methodological validation（linear range，accuracy，sensitivity，precision，reference material determination）. Totally，1 000 normal neonatal dried blood samples，30 CH dried blood samples，30 PKU dried blood samples and 30 CAH dried blood samples for clinical validation were collected，and a reference interval for 6 analytes was preliminarily established. Results The linear ranges for determining PHE，TYR，17-OHP，ASD，COR and T₄ by UPLC-MS/MS were 12.1-980.7 μmol/L，3.7-298.0 μmol/L，6.1-490.2 nmol/L，0.7-56.6 nmol/L，13.8-1 117.3 nmol/L and 32.2-2 606.6 nmol/L，respectively. The minimum determination limits were 0.5 μmol/L，0.7 μmol/L，1.6 nmol/L，0.2 nmol/L，2.6 nmol/L and 3.2 nmol/L，respectively. The intraday precision [coefficient of variation（CV）] of the quality control samples at 3 concentration levels was 2.2%-7.5%，the interday precision（CV） was 2.3%-7.3%，and the spiked recovery rate was 92.5%-115.4%. The relative deviations between the determined values and standard values of the 5 concentrations of the International Society for Neonatal Screening（ISNS） PHE and 17-OHP reference materials were 1.78%-7.01% and 0.70%-10.78%，respectively. Compared to normal newborns，the 17-OHP and（17-OHP+ASD）/COR ratio in CAH newborns were increased（P<0.05）. The PHE level and PHE/TYR ratio of PKU newborns were increased（P<0.05）. The T₄ level in CH newborns was decreased（P<0.05）. The positive rates of 17-OHP and 17-OHP combined with（17-OHP+ASD）/COR screening for CAH were 100% and 100%，respectively. The positive rates of PHE and PHE combined with PHE/TYR screening for PKU were 100% and 100%，respectively. The positive rate of T₄ screening for CH was 40%. Conclusions The UPLC-MS/MS has been established that can simultaneously determine 6 analytes in PKU，CAH and CH. This method has high specificity，speed and sensitivity，and it shortens sample analysis time. The method is expected to provide a new possible option for screening neonatal diseases.

Key words: Phenylketonuria, Congenital adrenal hyperplasia, Congenital hypothyroidism, Ultra-performance liquid chromatography tandem mass spectrometry, Newborn screening, Dried blood spot