Abstract

Amino acid analysis is central to newborn screening and the investigation of inborn errors of metabolism. Ion‐exchange chromatography with ninhydrin derivatization remains the reference method for quantitative amino acid analysis but offers slow chromatography and is susceptible to interference from other co‐eluting compounds. Liquid‐chromatography tandem mass spectrometry (LC‐MS/MS) provides a rapid and highly specific alternative, but sample preparation is frequently laborious and sometimes cost prohibitive. To address these limitations, we validated an LC‐MS/MS method using the aTRAQ Reagents Application Kit with a modified protocol consuming only half reagents. Adequate performance for clinical specimen measurement of 26 amino acids with high clinical relevance was achieved. An automated liquid handler and modified calibration and normalization approaches were used to ensure reproducible assay performance. Linear measurement between 5 and 2000 μM was achieved for most analytes despite use of a small, 10 μl sample size. Overall the method achieved near substantially improved throughput and enabled use of smaller samples volumes for batched analyses of clinical samples.

Highlights

  • Quantitative amino acid analysis is frequently required for the diagnosis and monitoring of inherited metabolic disorders

  • We present a method for quantitative analysis of 26 clinically relevant amino acids using derivatization via aTRAQ reagents, using a modified protocol to achieve near cost equivalence with our current ion-exchange chromatography (IEX) and ultra-performance liquid chromatography (UPLC) methods

  • In the modified procedure we report here, all reagent volumes are halved per sample

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Summary

| INTRODUCTION

Quantitative amino acid analysis is frequently required for the diagnosis and monitoring of inherited metabolic disorders. Low cost amine derivatization using butylation has longstanding use in amino acid analysis, but performance is variable.[13,14,15] The application of light and heavy isotope-coded derivatization (ICD) reagents is one alternative that is gaining more wide-spread adoption in clinical laboratories.[12,16,17,18] A key advantage is broad internal standard coverage. Amino acids in the patient samples are derivatized with a mass tag, and combined at the end of sample processing with an internal standard amino acid mix pre-derivatized with a mass tag of identical structure but alternative isotopic makeup. We present a method for quantitative analysis of 26 clinically relevant amino acids using derivatization via aTRAQ reagents, using a modified protocol to achieve near cost equivalence with our current IEX and ultra-performance liquid chromatography (UPLC) methods. A liquid handler semi-automates the extraction and derivatization process cutting down on the manual sample preparation time and ensuring consistency of handling between samples

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