Portable collection of breath phenylpyruvic acid for noninvasive assessment of pediatric hyperphenylalaninemia by mass spectrometry

Despite the efforts of Hyperphenylalaninemia (HPA) prevention in prenatal testing, many infants with HPA are still found in new-born screening. Because there is no definitively effective cure for HPA, routine examination and monitoring is of paramount importance to effective management of HPA conditions through dietary restriction. Blood phenylalanine (Phe) has been one of the metabolic biomarkers of the HPA and thus is considered as indicators for HPA management but poses a challenge in invasive sampling. Phenylpyruvic acid (PPA) is also a biomarker of HPA with greatly increased levels in blood and urine of the patients. In the present study, a portable breath sampler was developed for in situ extraction and storage of PPA from exhaled breath metabolites of HPA patients and healthy volunteers, and breath PPA was then identified and quantified by mass spectrometry. The analytical parameters of PPA detection were optimized, showing the good sensitivity (LLOQ: 0.04 ng/mL, S/N = 10), reproducibility (intraday RSDs: 6.18 % for 0.2 ng/mL, 7.32 % for 1.0 ng/mL, n = 6; inter-day RSDs: 6.08 % for 0.2 ng/mL, 11.29 % for 1.0 ng/mL, n = 6), and recoveries (intraday: 83.34 % for 0.2 ng/mL, 81.73 % for 1.0 ng/mL, n = 6; inter-day: 88.44 % for 0.2 ng/mL, 85.11 % for 1.0 ng/mL, n = 6), good quantitation capacity (linear range: 0.05-1.0 ng/mL, R² = 0.9968), and low matrix effect (1.0 ng/mL, 98.25 %). It was observed that there is a significant difference (p < 0.01) of breath PPA between HPA patients and healthy volunteers. Furthermore, the detection results of breath PPA and blood Phe were compared, showing a linear correlation (R² = 0.6262) of breath PPA and blood Phe. Overall, this work provided valuable insights into breath PPA in HPA patients and showed a potential tool for noninvasive investigating of HPA.