A rapid LC-MS/MS assay for detection and monitoring of underivatized branched-chain amino acids in maple syrup urine disease

Hamed Piri-Moghadam; Alan Miller; Debra Pronger; Faye Vicente; Joel Charrow; Shannon Haymond; David C Lin

doi:10.1016/j.jmsacl.2022.04.003

A rapid LC-MS/MS assay for detection and monitoring of underivatized branched-chain amino acids in maple syrup urine disease

J Mass Spectrom Adv Clin Lab. 2022 Apr 30:24:107-117. doi: 10.1016/j.jmsacl.2022.04.003. eCollection 2022 Apr.

Authors

Hamed Piri-Moghadam¹, Alan Miller¹, Debra Pronger¹, Faye Vicente¹, Joel Charrow^{2

3}, Shannon Haymond^{1

4}, David C Lin^{1

4}

Affiliations

¹ Clinical Mass Spectrometry Laboratory, Department of Pathology and Laboratory Medicine, Ann & Robert H. Lurie Children's Hospital of Chicago, USA.
² Biochemical Genetics Laboratory, Ann & Robert H. Lurie Children's Hospital of Chicago, USA.
³ Northwestern University, Feinberg School of Medicine, Department of Pediatrics, USA.
⁴ Northwestern University, Feinberg School of Medicine, Department of Pathology, USA.

Abstract

Introduction: Quantitation of the isomeric branched-chain amino acids (BCAA; valine, alloisoleucine, isoleucine, leucine) is a challenging task that typically requires derivatization steps or long runtimes if a traditional chromatographic method involving a ninhydrin ion pairing reagent is used.

Objectives: To develop and perform clinical validation of a rapid, LC-MS/MS-based targeted metabolomics assay for detection and monitoring of underivatized BCAA in human plasma.

Methods: Various columns and modes of chromatography were tested. The final optimized method utilized mixed mode chromatography with an Intrada column under isocratic condition. Sample preparation utilized the 96-well format. Briefly, extraction solvent containing the internal standard is added to 20 uL of sample, followed by shaking and positive pressure filtering, and the resulting extracted sample is analyzed. The assay was validated based on accepted quality standards (e.g., CLIA and CLSI) for clinical assays.

Results: The method is linear over a wide range of concentrations, 2.0-1500 µM, with LOD of 0.60 µM and LOQ of 2.0 µM. The precision of the assay was 4-10% across analytes. The method was also validated against reference laboratories via blinded split-sample analysis and demonstrated good agreement with accuracy: 89-95% relative to the external group mean.

Conclusion: We have developed a method that is accurate, rapid, and reliable for routine clinical testing of patient sample BCAA, which is used in the diagnosis and management of maple syrup urine disease (MSUD). The assay also has desirable characteristics, such as short run time, small sample volume requirement, simple sample preparation without the need for derivatization, and high throughput.

Keywords: 3NPH, 3-nitrophenylhydrazine; ACN, Acetonitrile; AMR, Analytical measurable range; BCAA, Branched-chain amino acids; BCKD, Branched-chain ketoacid dehydrogenase complex; Branched-chain amino acid; CAP, The College of American Pathologists; CLIA, The Clinical Laboratory Improvement Amendments; CLSI, The Clinical & Laboratory Standards Institute; CN, Cyano; CRR, Clinical Reportable Range; Clinical assay; ESI, Electrospray ionization; FA, Formic Acid; GC–MS, Gas chromatography-mass spectrometry; HILIC, Hydrophilic interaction liquid chromatography; HMDB, Human metabolome database; IEX, Ion exchange; LC, Liquid chromatography; LC-MS/MS, Liquid chromatography-tandem mass spectrometry; LC-UV, Liquid chromatography-ultra violet; LDT, Laboratory-developed tests; LLE, Liquid-liquid extraction; LOD, Limit of detection; LOQ, Limit of quantitation; MSUD, Maple syrup urine disease; Maple syrup urine disease; Mass spectrometry; MeOH, Methanol; NMR, Nuclear magnetic resonance; PBS-BSA, Phosphate buffered saline with bovine serum albumin; PITC, Phenylisothiocyanate; PTFE, Polytetrafluoroethylene; QC, Quality control; Quantitation; RP, Reverse phase; RPLC, Reverse phase liquid chromatography; S/N, Signal-to-noise ratio; SCX, Strong cation exchange; SPE, Solid phase extraction; SRM, Selected reaction monitoring; UHPLC, Ultra-high-performance liquid chromatography; WAX, Weak anion exchange.