A quantitative method for acylcarnitines and amino acids using high resolution chromatography and tandem mass spectrometry in newborn screening dried blood spot analysis

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2012-08, Vol.903, p.142-149
• Main Authors: Miller, John H., Poston, Philip A., Karnes, H. Thomas
• Format: Article
• Language: English
• Subjects: acetonitrile; Acylcarnitines; Amino acids; Amino Acids - blood; Amino acids UHPLC; ammonium acetate; Blood; Carnitine - analogs & derivatives; Carnitine - blood; Chromatography; Chromatography, High Pressure Liquid - methods; Disorders; Dried Blood Spot Testing - methods; Formic acid; heparin; Humans; Infant, Newborn - blood; ionization; isotope labeling; Linear Models; Mass spectrometry; metabolic diseases; methanol; monitoring; Neonatal Screening - methods; neonates; Newborn screening; quantitative analysis; Reproducibility of Results; Screening; Second-tier test; Sensitivity and Specificity; spectrometers; tandem mass spectrometry; Tandem Mass Spectrometry - methods; ultra-performance liquid chromatography
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2012.07.008

► We developed a rapid second-tier test for acylcarnitines and amino acids. ► We evaluated response factors and matrix effects not previously report in NBS. ► Fast chromatographic separation of acylcarnitines and amino acids in 3.1min. ► Our method allows for differential diagnosis of MSUD and other disorders. ► We provide chromatographic separation of C4 and C5 isomers. We have developed a high resolution liquid chromatographic separation with electrospray ionization (ESI) mass spectrometry detection for the combined analysis of twelve acylcarnitines and seven amino acids commonly measured in newborn screening heritable metabolic disorders. Samples were prepared by punching 3.2mm disks out of dried blood spots and extracting with a mixture of methanol and 0.1% formic acid containing stable isotopically labeled internal standards. Analysis was performed on an UHPLC system using a HILIC amide, 2.1mm×50mm, 1.7μm column. A normal phase gradient, employing 10mM ammonium acetate in 90:10 acetonitrile/water for mobile phase B and 0.1% formic acid in water for mobile phase A, was used. Optimized multiple reaction monitoring (MRM) was used for detection of amino acids and acylcarnitines on a Waters Premier mass spectrometer. Quantification of analytes was performed using internal calibration by fortification of sodium heparin whole blood with analytes at appropriate levels to encompass the range around the reported cut-off values. The method was fully validated with respect to precision, accuracy, recovery, linearity, matrix suppression and extraction robustness. Precision and accuracy were evaluated over 3 days and determined to be acceptable with an overall precision within 10% and accuracy within 15% of theoretical for all analytes except for acetylcarnitne at one fortified level, which quantitated 21.8% lower than the expected value. This method is suitable as a second-tier test for newborn screening of specific disorders associated with abnormal levels of acylcarnitines and amino acids, potentially reducing false positive cases and shortening the time to diagnosis.