Validation of a rapid, comprehensive and clinically relevant amino acid profile by underivatised liquid chromatography tandem mass spectrometry
-
Rachel S. Carling
,
Kate John
Abstract
Background
Quantification of plasma amino acids is key to the diagnosis of inherited defects of amino acid synthesis, catabolism and transport, many of which present as clinical emergencies. The utility of this test is limited by the long analysis time and subsequent inability of laboratories to provide results in real-time. Traditionally, analysis has been performed by ion exchange chromatography (IEC) but recently there has been a move towards liquid chromatography tandem mass spectrometry (LC-MS/MS) which provides the potential for faster analysis. However, the necessity to derivatise the sample and/or utilise an ion-pair reagent, combined with lack of commercially available stable isotope internal standards (IS) has prevented laboratories fully exploiting the benefits of this methodology. We describe an underivatised LC-MS/MS method enabling patient results to be reported with an improved turnaround time (<1 h).
Methods
Methanolic IS was added to plasma (10 μL) to precipitate protein. Following centrifugation amino acids were analysed by LC-MS/MS using selected reaction monitoring (SRM) for each analyte and corresponding IS.
Results
Patient samples (n = 57) and external quality assessment (EQA) material (n = 11) were analysed and results compared with IEC. Comparable accuracy and precision were obtained with 15-min analysis time.
Conclusions
This method enables the analysis of a clinically comprehensive amino acid profile without the need for derivatisation/ion-pair reagents and benefitting from improved analytical quantitation through multipoint calibration and use of stable isotope IS. The analysis time is fast in comparison to IEC, improves efficiency of laboratory workflow and enables stat analysis of clinically urgent samples.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: CT and RND are minority shareholders in SpOtOn Clinical Diagnostics Ltd.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2019-0604).
©2020 Walter de Gruyter GmbH, Berlin/Boston
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- Method development for quantitative determination of seven statins including four active metabolites by means of high-resolution tandem mass spectrometry applicable for adherence testing and therapeutic drug monitoring
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- Validation according to European and American regulatory agencies guidelines of an LC-MS/MS method for the quantification of free and total ropivacaine in human plasma
- Enhanced specificity due to method specific limits for relative ion intensities in a high-performance liquid chromatography – tandem mass spectrometry method for iohexol in human serum
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- Applying mass spectrometry-based assays to explore gut microbial metabolism and associations with disease
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- Development of a total serum testosterone, androstenedione, 17-hydroxyprogesterone, 11β-hydroxyandrostenedione and 11-ketotestosterone LC-MS/MS assay and its application to evaluate pre-analytical sample stability
- Short-term stability of free metanephrines in plasma and whole blood
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- LC-MS/MS analysis of plasma glucosylsphingosine as a biomarker for diagnosis and follow-up monitoring in Gaucher disease in the Spanish population
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