Rapid quantification of underivatized alloisoleucine and argininosuccinate using mixed-mode chromatography with tandem mass spectrometry

Plasma elevations of the amino acids alloisoleucine and argininosuccinic acid (ASA) are pathognomonic for maple syrup urine disease and argininosuccinate lyase deficiency, respectively. Reliable detection of these biomarkers is typically achieved using methods with tedious sample preparations or long chromatographic separations, and many published amino acid assays report poor specificity and/or sensitivity for one or both of these compounds. This report describes a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method that provides rapid quantification of alloisoleucine and ASA in human plasma. The basis of this method is a mixed-mode solid phase separation that achieves baseline resolution of alloisoleucine from isobaric interferents without the use of derivatization or ion pairing agents. The inject-to-inject time is 6 min including elution, column washing and re-equilibration. Validation studies demonstrate excellent limits of quantification (1 μmol/L), linearity (r = 0.999 from 1 to 250 μmol/L), accuracy (bias = −3.8% and −10.1%), and inter-assay imprecision (CV < 8.06%) for plasma analyses. Data from long-term clinical application confirms chromatographic consistency equivalent to more traditional reversed-phase or HILIC based columns. Additional matrix studies indicate low suppression (