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Robust and high-throughput lipidomic quantitation of human blood samples using flow injection analysis with tandem mass spectrometry for clinical use

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Abstract

Direct infusion of lipid extracts into the ion source of a mass spectrometer is a well-established method for lipid analysis. In most cases, nanofluidic devices are used for sample introduction. However, flow injection analysis (FIA) based on sample infusion from a chromatographic pump can offer a simple alternative to shotgun-based approaches. Here, we describe important modification of a method based on FIA and tandem mass spectrometry (MS/MS). We focus on minimizing contamination of the FIA/MS both to render the lipidomic platform more robust and to increase its capacity and applicability for long-sequence measurements required in clinical applications. Robust validation of the developed method confirms its suitability for lipid quantitation in human plasma analysis. Measurements of standard human plasma reference material (NIST SRM 1950) and a set of plasma samples collected from kidney cancer patients and from healthy volunteers yielded highly similar results between FIA-MS/MS and ultra-high-performance supercritical fluid chromatography (UHPSFC)/MS, thereby demonstrating that all modifications have practically no effect on the statistical output. Newly modified FIA-MS/MS allows for the quantitation of 141 lipid species in plasma (11 major lipid classes) within 5.7 min. Finally, we tested the method in a clinical laboratory of the General University Hospital in Prague. In the clinical setting, the method capacity reached 257 samples/day. We also show similar performance of the classification models trained based on the results obtained in clinical settings and the analytical laboratory at the University of Pardubice. Together, these findings demonstrate the high potential of the modified FIA-MS/MS for application in clinical laboratories to measure plasma and serum lipid profiles.

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Abbreviations

ABPR:

Automatic back pressure regulator

CAD:

Collision gas (QTRAP)

ccRCC:

Clear cell renal cell carcinoma

CE:

Cholesteryl esters

Cer:

Ceramides

CUR:

Curtain gas (QTRAP)

DG:

Diacylglycerol

DI-MS:

Direct infusion mass spectrometry

DP:

Declustering potential (QTRAP)

EMA:

European Medicines Agency

ESI:

Electrospray ionization

ESM:

Electronic supplementary materials

FDA:

Food and Drug Administration

FDR:

False discovery rate

FIA:

Flow injection analysis

FIA-MS/MS:

Flow injection analysis coupled with tandem mass spectrometry

FT-ICR:

Fourier-transform ion cyclotron resonance mass spectrometer

GlcCer:

Glucosylceramide

GS1:

Nebulizer gas (QTRAP)

GS2:

Heater gas (QTRAP)

HL:

High concentration level (30 µL spike)

HEX:

Hexane

Hex2Cer:

Di-hexosylceramides

HexCer:

Hexosylceramides

ID:

Internal diameter

IPA:

Isopropanol

IS:

Ion spray voltage (QTRAP)

IS mix:

Internal standard mixture

LL:

Low concentration level (10 µL spike)

LacCer:

Lactosylceramides

LC:

Liquid chromatography

LC/MS:

Liquid chromatography coupled with mass spectrometry

LLOQ:

Lower limit of quantitation

LPC:

Lysophosphatidylcholines

LPE:

Lysophosphatidylethanolamines

LPG:

Lysophosphatidylglycerols

LPI:

Lysophosphatidylinositols

LPS:

Lysophosphatidylserines

ML:

Medium concentration level (20 µL spike)

MeOH:

Methanol

MG:

Monoacylglycerols

MS/MS:

Tandem mass spectrometry

MS:

Mass spectrometry, mass spectrometer

N:

Healthy volunteers (controls)

NLS:

Neutral loss scan(s)

OPLS-DA:

Orthogonal projections to latent structures discriminant analysis

PA:

Glycerophosphates

PC:

Phosphatidylcholines

PC O- :

Ether phosphatidylcholines

PC P- :

Plasmenylcholines (PC plasmalogens)

PCA:

Principal component analysis

PDAC:

Pancreatic ductal adenocarcinoma

PE:

Phosphatidylethanolamines

PE O-:

Ether phosphatidylethanolamines

PE P-:

Plasmenylethanolamines

PG:

Phosphatidylglycerols

PI:

Phosphatidylinositols

PIS:

Precursor ion scan(s)

PS:

Phosphatidylserines

QC:

Quality control sample(s)

QTOF:

Quadrupole time-of-flight mass spectrometer

QTRAP:

Quadrupole ion trap mass spectrometer

RSD:

Relative standard deviation

SFC:

Supercritical fluid chromatography

SM:

Sphingomyelins

T:

Cancer patients, cancer cases

TEM:

Source temperature (QTRAP)

TG:

Triacylglycerols

TIC:

Total ion current

UHPSFC:

Ultra-high-performance supercritical fluid chromatography

UHPSFC/MS:

Ultra-high-performance supercritical fluid chromatography coupled with mass spectrometry

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Acknowledgements

The authors would like to acknowledge Ali Talebi, Vincent de Laat, and Shuncong Wang from KU Leuven, Belgium, for their help in the preparation of the manuscript.

Funding

This work was supported by the grant project NU21-03–00499 funded by Czech Health Research Council. J.B. and K.P. thank the support of the Ministry of Health of the Czech Republic – conceptual development of research organization: RVOVFN 64165/2012 (a program of the General University Hospital in Prague). The obtaining of serum samples was supported by BBMRI-CZ no. LM2018125.

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Authors and Affiliations

  1. Faculty of Chemical Technology, Department of Analytical Chemistry, University of Pardubice, Studentská 573, 532 10, Pardubice, Czech Republic

    Jakub Idkowiak, Robert Jirásko, Denisa Kolářová, Tomáš Hájek, Michela Antonelli, Zuzana Vaňková, Denise Wolrab & Michal Holčapek

  2. Department of Pediatrics and Inherited Metabolic Disorders, Charles University, First Faculty of Medicine and General University Hospital in Prague, Ke Karlovu 2, 12808 2, Prague, Czech Republic

    Josef Bártl & Karolína Pešková

  3. Research Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Žlutý kopec 7, 656 53, Brno, Czech Republic

    Roman Hrstka

  4. Medical School and Teaching Hospital, Department of Oncology, Palacký University, I.P. Pavlova 6, 775 20, Olomouc, Czech Republic

    Hana Študentová & Bohuslav Melichar

Authors
  1. Jakub Idkowiak
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  2. Robert Jirásko
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  8. Denise Wolrab
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  13. Michal Holčapek
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Contributions

Jakub Idkowiak: Methodology, Formal analysis, Investigation, Writing – original draft, Visualization; Robert Jirásko: Methodology, Formal analysis, Investigation, Visualization Writing – review & editing; Denisa Kolářová: Formal analysis, Investigation, Visualization, Writing – review & editing; Josef Bártl: Methodology, Formal analysis, Writing – review & editing; Tomáš Hájek: Methodology, Writing – review & editing; Michela Antonelli: Formal analysis, Investigation, Writing – review & editing; Zuzana Vaňková: Methodology, Writing – review & editing; Denise Wolrab: Methodology, Writing – review & editing; Roman Hrstka: Resources, Writing – review & editing; Hana Študentová: Blood collection, Resources, Writing – review & editing; Bohuslav Melichar: Resources, Writing – review & editing; Karolína Pešková: Methodology, Writing – review & editing; Michal Holčapek: Resources, Project administration, Supervision, Writing-review & editing.

Corresponding author

Correspondence to Robert Jirásko.

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Ethics approval

All human plasma samples were obtained from University Hospital in Olomouc and the kidney cancer study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University Hospital Olomouc (No. 46/17, approval date 30 March 2017). All subjects gave informed consent. All human serum samples together with clinical data were obtained from the Bank of Biological Material in Masaryk Memorial Cancer Institute in Brno, approved by the institutional ethical committee, and all blood donors gave informed consent.

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The authors declare no competing interests.

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Idkowiak, J., Jirásko, R., Kolářová, D. et al. Robust and high-throughput lipidomic quantitation of human blood samples using flow injection analysis with tandem mass spectrometry for clinical use. Anal Bioanal Chem 415, 935–951 (2023). https://doi.org/10.1007/s00216-022-04490-w

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