Metabolomics

, Volume 10, Issue 2, pp 312–323 | Cite as

Validation of a dual LC–HRMS platform for clinical metabolic diagnosis in serum, bridging quantitative analysis and untargeted metabolomics

  • Ilya Gertsman
  • Jon A. Gangoiti
  • Bruce A. Barshop
Original Article

Abstract

Mass spectrometry-based metabolomics is a rapidly growing field in both research and diagnosis. Generally, the methodologies and types of instruments used for clinical and other absolute quantification experiments are different from those used for biomarkers discovery and untargeted analysis, as the former requires optimal sensitivity and dynamic range, while the latter requires high resolution and high mass accuracy. We used a Q-TOF mass spectrometer with two different types of pentafluorophenyl (PFP) stationary phases, employing both positive and negative ionization, to develop and validate a hybrid quantification and discovery platform using LC–HRMS. This dual-PFP LC–MS platform quantifies over 50 clinically relevant metabolites in serum (using both MS and MS/MS acquisitions) while simultaneously collecting high resolution and high mass accuracy full scans to monitor all other co-eluting non-targeted analytes. We demonstrate that the linearity, accuracy, and precision results for the quantification of a number of metabolites, including amino acids, organic acids, acylcarnitines and purines/pyrimidines, meets or exceeds normal bioanalytical standards over their respective physiological ranges. The chromatography resolved highly polar as well as hydrophobic analytes under reverse-phase conditions, enabling analysis of a wide range of chemicals, necessary for untargeted metabolomics experiments. Though previous LC–HRMS methods have demonstrated quantification capabilities for various drug and small molecule compounds, the present study provides an HRMS quant/qual platform tailored to metabolic disease; and covers a multitude of different metabolites including compounds normally quantified by a combination of separate instrumentation.

Keywords

Untargeted metabolomics Targeted metabolomics Bioanalytical validation Mass spectrometry Q-TOF LC–HRMS Comprehensive metabolite profiling 

Abbreviations

CLIA

Clinical laboratory improvement act

CAP

College for American pathologists

GLP

Good laboratory practices

HCC

Hepatocellular carcinoma

Cirr

Cirrhotic patient

ND

Not determined

TOF

Time of flight

LOQ

(Lower) limit of quantification

QC

Quality control

CV

Coefficient of variation

HRMS

High resolution mass spectrometry

MSUD

Maple syrup urine disease

MCADD

Medium chain acyl-CoA dehydrogenase deficiency

PKU

Phenylketonuria

Supplementary material

11306_2013_582_MOESM1_ESM.docx (120 kb)
Supplementary material 1 (DOCX 119 kb)
11306_2013_582_MOESM2_ESM.jpg (92 kb)
Supplementary material 2 (JPEG 92 kb)
11306_2013_582_MOESM3_ESM.pdf (2.9 mb)
Supplementary material 3 (PDF 3002 kb)

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Ilya Gertsman
    • 1
  • Jon A. Gangoiti
    • 1
  • Bruce A. Barshop
    • 1
  1. 1.Biochemical Genetics LabUniversity of California, San DiegoLa JollaUSA

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