Abstract: The application of MS/MSin newborn screening is one of the most important branches of MS/MS's current clinical applications. In this paper, we firstly reported a new method of newborn screening for Phenylketonuria(PKU) by coupling LCwith MS/MS, which was relatively cheap, fast, sensitive, and accurate. In this method, only a few steps of sample preparation were required and no isotopically labeled internal standard was required. Phe and Tyr were extracted from dry blood spots with 0.1 mol/L HCl-methanol and reacted with 2.8 mol/L HCl-butanol at 68 ℃ for 35 min. MS/MSwas used as a detector for LCand multi reactions monitoring (MRM) scan mode was employed to determine phenylalanine and tyrosine in blood samples. Among the product ions of Phe and Tyr butyl esters, [M+H-102]⁺was the most abundant in intensity and unique to α-amino acids, therefore the precursor-daughter ion pairs of m/z 222-120 and m/z 238-136 were selected as MRMion pairs. The contents of these two amino acids in blood samples were determined by calculating the areas of their ionic flow peaks. Finally, the molar ratios of Phe to Tyr in blood samples were calculated as the basis of PKUdiagnosis. The curve for Phe showed excellent linearity over the concentration range of 24_1 152 μmol/L(r²=0.999). As to Tyr, the range of linearity was 22_1 056 μmol/L(r²=0.998). The average analytical recoveries of added Phe and Tyr from blood were 98% and 96%, respectively. The limits of detection for Phe and Tyr were respectively 0.5 pg and 2.0 pg , which were 10³times lower than that of reported MS/MSmethod. About 40 blood samples were analyzed by this method, and the results proved that this method could successfully discriminate between normal infants and PKUpositive ones. Each sample's analysis time was about 4 minutes; therefore it was capable for large-scale screening.

Key words: Phenylketonuria, Amino acids, HPLC, MS/MS, Screening