Cheryl Garganta (Presenter)
University of Florida
Bio: MD, PhD (Human Genetics), Pediatrics residency at Virginia Commonwealth University. Clinical and Clinical Biochemical Genetics residency, Yale University. Director of Metabolic Lab, UF Health PathLabs. Associate Professor of Pathology and Pediatrics, University of Florida.
Authorship: Cheryl L Garganta
University of Florida, Gainesville FL
Measurement of amino acids is important for diagnosis and management of disorders such as MSUD, PKU and tyrosinemia. Newborn screening allows early detection of these disorders but is limited by lack of separation of allo-isoleucine, a specific marker of MSUD. This method allows isocratic separation of leucine isomers within 3 minutes. It is suitable for use as a second tier test in newborn screening labs to evaluate specimens with elevated "leu" on flow-injection analysis. Preparation of calibrators in blood with hematocrit of 35-40% yields results that correlate well with results from plasma amino acid analysis. Multiple disorders can be monitored in a single assay.
Newborn screening uses flow-injection tandem mass spectrometry to measure amino acids in dried blood spots. High biological variation of branched chain amino acid concentrations and lack of separation of allo-isoleucine, a specific marker for maple syrup urine disease (MSUD), results in considerable overlap between affected and unaffected babies. Second tier testing to quantify allo-isoleucine can be used to improve test sensitivity and specificity, but an ideal method should not require extra specimen processing or additional equipment. Since many newborn screening labs use only a single LC pump, an isocratic separation is preferred.
Calibrators for monitoring older patients were prepared in whole blood with hematocrit adjusted to 35-50%. Amino acids in methanolic extract of dried blood spots were separated in 3 minutes on Pentfluorophenyl column (Kinetex, 2.1x50 mm) using 30% methanol/70% water with 0.1% formic acid. Phenylalanine, tyrosine, valine, isoleucine, leucine and allo-isoleucine were detected by selected reaction monitoring on a Quantiva triple quadrupole mass spectrometer.
Leucine isomers were separated almost to baseline resolution. Results were linear to at least 2000 µmol/L for allo-isoleucine, 3500 µmol/L for valine and tyrosine and 7000 µmol/L for isoleucine, leucine and phenylalanine. CVs were <10% for all compounds across clinically relevant concentrations. Results were within 20% of those obtained on plasma amino acid analysis without significant bias.
Conclusions & Discussion
This isocratic method for separation of leucine isomers is suitable for use as a second tier test in newborn screening labs to evaluate specimens with elevated "leu" on flow-injection analysis. Preparation of calibrators in blood with hematocrit of 35-40% yields results that correlate well with results from non-neonates obtained using plasma amino acid analysis. Inclusion of phenylalanine and tyrosine allows monitoring of PKU, MSUD and tyrosinemia in a single assay.
References & Acknowledgements:
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