High-Throughput Analysis of Underivatized Amino Acids and Acylcarnitines in Infant Serum: A Micromethod Based on Stable Isotope Dilution Targeted HILIC-ESI-MS/MS

Amino acids and acylcarnitines are important biomarkersof thebody's energy state and can be used as diagnostic markers ofcertain inborn errors of metabolism. Few multianalyte methods forhigh-throughput analysis in serum exist for these compounds, but micromethodssuitable for use in young children and infants are lacking. Therefore,we developed a quantitative high-throughput multianalyte hydrophilicinteraction liquid chromatography-tandem mass spectrometrymethod preceded by a derivatization-free sample preparation usingminimum amounts of serum (25 mu L). Isotopically labeled standardswere utilized for quantification. Forty amino acids and amino acidderivatives and 22 acylcarnitines were detected by applying a multiplereaction monitoring mode within a 20 min run. The method was comprehensivelyvalidated, comprising linearity, accuracy, (intraday/interday) precision,and quantitation limits, of which the latter ranged from 0.25 to 50nM for acylcarnitines and from 0.005 to 1 mu M for amino acidsand their derivatives. Application of the method to 145 serum samplesof three- to four-month-old healthy infants showed excellent reproducibilityfor multiday analyses and enabled simultaneous amino acid and acylcarnitineprofiling in this age group.

Automated summary

Amino acids and acylcarnitines are important biomarkers for the body's energy state and can be used to diagnose certain inborn errors of metabolism. This study developed a quantitative high-throughput method to analyze these compounds in serum samples, using minimal amounts of serum and isotopically labeled standards for quantification. The method was successfully validated and applied to serum samples from infants, showing excellent reproducibility and enabling simultaneous profiling of amino acids and acylcarnitines.