|
Abstract INTRODUCTION
Quantitative plasma amino acid analysis is primarily used in clinical biochemical genetics laboratories for the diagnosis and monitoring of inborn errors of metabolism (IEM). A majority of biochemical genetics laboratories measure amino acids using chromatographic methods that utilize optical detectors. These methods are limited by long analytical run times and are prone to interferences. Our laboratory uses an Acquity™ ultra-performance liquid chromatography (UPLC) system with an integrated UV detector and the MassTrak™ AAA Solution Kit (Waters Corporation). This method utilizes pre-column derivation of amino acids with 6-aminoquinoyl-N-hydroxysuccinimidyl carbamate (AccQTag®) followed by reversed-phase UPLC on a MassTrak™ C18 column (2.1 x 150 mm, 1.7µm, Waters Corporation) followed by single wavelength UV detection at 260 nm. The challenges associated with chromatographic resolution of all plasma amino acids with this UPLC method (35 min runtime) and their potential impact on IEM patient management have been previously reported (1). Transitioning this testing to a liquid chromatography tandem mass spectrometry (LC-MS/MS) based procedure represented an opportunity to improve the efficiency, sensitivity and specificity of our existing plasma amino acid protocol.
OBJECTIVES
To develop an LC-MS/MS assay to quantify plasma amino acids and evaluate its potential use within our patient population with suspected IEMs.
METHODS
Method development and clinical feasibility studies were performed using an Acquity™ iClass UPLC coupled to Xevo TQ-S Micro tandem mass spectrometer (Waters Corporation) operated in positive electrospray ionization mode (ESI+). Sample pretreatment was performed using a Karios™ Amino Acid Kit (Waters Corporation), which utilizes protein precipitation and AccQTag® chemical derivation prior to analysis. This kit also contains calibration standards and QC for 45 different amino acids, including several isotopically labelled internal standards. Optimization of the sample preparation protocol permitted the use of 20 µL of material for testing. All amino acids and internal standards were detected as their derivatized products using their respective MS/MS transitions. Three different reverse phase C18 columns were tested to develop the final chromatographic conditions: CORTECS® C18 (2.1 x 150 mm, 1.6 µm), HHS C18 (2.1 x 150 mm, 1.8 µm), and ACCQ-TAG™ Ultra C18 (2.1 x 15 mm, 1.7 µm) columns were all obtained from Waters Corporation. Mobile phase composition, flow rate and gradient conditions were optimized for each column to provide the greatest isobaric amino acid resolution and sensitivity, within the shortest analytical runtime. Performance of each LC-MS/MS assay was preliminarily evaluated through plasma amino acid correlation studies with the predicate LC-UV method and an LC-MS/MS assay from a national reference laboratory. Method accuracy was then further assessed using archived amino acid external quality assurance (EQA) material from the European Research Network for the evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDiM).
RESULTS
The optimized sample preparation and chromatographic conditions of each C18 column yielded LC-MS/MS assays that were linear over a wide amino acid concentration range (2.5 to 4000 µM, R>0.99). LC-MS/MS derived plasma amino acid results were relatively higher than the UPLC method, with average plasma amino acid recoveries from the CORTECS®, HHS, and ACCQ-TAG™ Ultra C18 column methods being 130%, 119% and 117%, respectively. The blinded split sample analysis with a national reference laboratory demonstrated good agreement for the ACCQ-TAG™ Ultra C18 column method, with an average plasma amino acid accuracy of 102% relative to the reference laboratory mean. The average plasma amino acid accuracy from the HHS (114%) and CORTECS® (119%) C18 methods relative to the reference laboratory were respectively higher than the ACCQ-TAG™ Ultra C18 column method. The highest relative resolution of all isobaric amino acids included in the Karios™ Amino Acid kit (e.g., 1- and 3-methylhistidine; isoleucine, alloisoleucine and leucine; γ-, β-, and α-aminobutyric acid; sarcosine, β-alanine and alanine) was also achieved with the ACCQ-TAG™ Ultra C18 column method. Resolution of isoleucine and alloisoleucine ranged from 0.68 to 0.85 with the HHS and ACCQ-TAG™ Ultra C18 column methods, respectively. Based on these studies, the optimized plasma amino acid LC-MS/MS method chosen for validation will utilize the ACCQ-TAG™ Ultra C18 column with mobile phase compositions of 0.1% (v/v) formic acid in water (mobile phase A) and acetonitrile (mobile phase B). Under the devised gradient conditions of this method, which utilized a flow rate of 0.5 mL/min and column temperature of 55°C, all amino acids were eluted within a 17 min runtime. The accuracy of this protocol was further challenged by testing archived amino acid EQA material (N=4 samples) from ERNDiM. Good agreement relative to the ERDiM all method laboratory means was achieved, with amino acid (N=27) accuracies ranging from 87% (citrulline) to 111% (sarcosine) and an average accuracy of 98%.
CONCLUSION
We have developed an accurate LC-MS/MS method for plasma amino acids that utilizes the Kairos™ Amino Acid kit. This assay is desirable as a full amino acid panel may be achieved within a relatively short runtime (17 min), while also maintaining adequate resolution of clinically relevant isobaric analytes. The targeted metabolomics assay also requires a small sample volume (20 µL), which is desirable for potential use within our pediatric IEM patient population. The devised LC-MS/MS protocol must be validated as a laboratory developed test (LDT) within our institution.
REFERENCES
(1) Peake RWA, Law T, Hoover PN, Gaewsky L, Shkreta A, Kellogg MD. Clin Chim Acta 2013; 423:75-82.
|
= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
