Quantitation of non-derivatized free amino acids for detecting inborn errors of metabolism by incorporating mixed-mode chromatography with tandem mass spectrometry

Patrick D DeArmond; Dustin R Bunch

doi:10.1016/j.jmsacl.2022.05.002

Quantitation of non-derivatized free amino acids for detecting inborn errors of metabolism by incorporating mixed-mode chromatography with tandem mass spectrometry

J Mass Spectrom Adv Clin Lab. 2022 May 21:25:1-11. doi: 10.1016/j.jmsacl.2022.05.002. eCollection 2022 Aug.

Authors

Patrick D DeArmond¹, Dustin R Bunch^{1

2}

Affiliations

¹ Department of Pathology and Laboratory Medicine, Nationwide Children's Hospital, Columbus, OH 43205, USA.
² Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.

Abstract

Introduction: Amino acids are critical biomarkers for many inborn errors of metabolism, but amino acid analysis is challenging due to the range of chemical properties inherent in these small molecules. Techniques are available for amino acid analysis, but they can suffer from long run times, laborious derivatization, and/or poor resolution of isobaric compounds.

Objective: To develop and validate a method for the quantitation of a non-derivatized free amino acid profile in both plasma and urine samples using mixed-mode chromatography and tandem mass spectrometry.

Methods: Chromatographic conditions were optimized to separate leucine, isoleucine, and allo-isoleucine and maintain analytical runtime at less than 15 min. Sample preparation included a quick protein precipitation followed by LC-MS/MS analysis. Matrix effects, interferences, linearity, carryover, acceptable dilution limits, precision, accuracy, and stability were evaluated in both plasma and urine specimen types.

Results: A total of 38 amino acids and related compounds were successfully quantitated with this method. In addition, argininosuccinic acid was qualitatively analyzed. A full clinical validation was performed that included method comparison to a reference laboratory for plasma and urine with Deming regression slopes ranging from 0.38 to 1.26.

Conclusion: This method represents an alternative to derivatization-based methods, especially in urine samples where interference from metabolites and medications is prevalent.

Keywords: AMR, analytical measurement range; AQC, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; ASA, argininosuccinic acid; Allo-isoleucine; Amino acid; CEX, cation exchange; CS, Fcerebrospinal fluid; CV, coefficient of variation; GABA, gamma-aminobutyric acid; GC/MS, gas chromatography-mass spectrometry; HCl, hydrochloric acid; HILIC, hydrophilic interaction liquid chromatography; IS, internal standard; Inborn errors of metabolism; LC-MS/MS; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LLOQ, lower limit of quantitation; MM, mixed-mode; MS/MS, tandem mass spectrometry; MSU, Dmaple syrup urine disease; Maple syrup urine disease; QC, quality control; RPL, Creversed phase liquid chromatography; ULO, Qupper limit of quantitation.