The gold standard for quantitative analysis of amino acids for inborn errors of metabolism has long been considered to be ion-exchange chromatography coupled to ninhydrin detection. This methodology is known to suffer from interferences, poorly resolved analytes, and exceptionally long sample analysis times (greater than 3 hours per sample). We have developed as isotope dilution liquid chromatography-tandem mass spectrometry method for quantitative analysis of amino acids with sample acquisition times of less than 6 minutes per sample.
Leveraging the capability of a multiplexed LC-MS/MS system (Aria TX4/API 5000), 3 discreet chromatographic systems were developed to optimize analytical requirements of a bioanalytical assay (linearity, precision, accuracy, lack of matrix effects). Isomers and closely related amino acids and/or internal standards were fully resolved to alleviate concerns of selectivity in quantitation. The chromatographic methods are truly orthogonal in design, which provided constraints in operational parameters (injection solution, sample preparation), but did allow for flexibility in development to ensure analytical accuracy. Challenges in developing this type of methodology will be discussed.
Samples are prepared by aliquoting 60 µL of urine, plasma, or cerebral spinal fluid to a well of a 96 well plate. 180 µL of precipitating solution containing internal standards is aliquoted to each well. Samples are vortexed, centrifuged, and 5-10 uL supernatant is injected onto each chromatographic system. Analytes are ionized in positive electrospray and distinct precursors and product ions are monitored for each amino acid and internal standard. Due to the disparate values of normals for the spread of amino acids analyzed, calibration curves of 0.1-100 µM, 0.5-500 µM, 1-1000 µM, and 5-5000 µM are prepared for quantitation.
Acquisition of 48 amino acids and 33 internal standards created a quandary in data reduction as each sample produces 81 distinct chromatograms that require review. To alleviate excessive resource overhead associated with this review, an automated data review and release program will be demonstrated that requires the review of only chromatograms that are unacceptable by a set of user-controlled criterion.
Additionally, application of Amino Acids by Multi-LC-MS/MS (AAMLC-MS/MS) method will be shown to be appropriate in the use of clinical decision making for inborn errors of metabolism. Reference intervals, normal populations, and correct identification of patients with disorders will be demonstrated. |
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