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Cited by 12Analytical Methods in PharmaceuticalsAnalytical Chemistry and ChromatographyMass Spectrometry Techniques and Applications

UPLC-MS/MS method for determination of retinol and α-tocopherol in serum using a simple sample pretreatment and UniSpray as ionization technique to reduce matrix effects

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Peersman, Nele, Elslande, Jan Van, Lepage, Yannick, De Amicis, Samira, Desmet, Koenraad, Vermeersch, Pieter·2020·Clinical Chemistry and Laboratory Medicine (CCLM)
DOI 10.1515/cclm-2019-1237
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Abstract

Background: Our goal was to develop a simple, rapid and precise ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of retinol and α-tocopherol in serum. Currently published LC-MS/MS methods either require complex extraction procedures (liquid-liquid or solid-phase) or do not meet desirable specifications for imprecision in serum (coefficient of variation [CV] <6.8% and 6.9%, respectively). Methods: Sample preparation consisted of a simple protein precipitation with ethanol and acetonitrile. Stable isotope-labeled internal standards (IS) and a homemade calibration curve were used for quantification. The analysis was performed using an Acquity I-class Xevo TQ XS LC-MS/MS. Chromatographic runtime was 6.0 min using a reversed phase gradient elution. UniSpray (US) as an ionization technique was compared to electrospray ionization (ESI). Analytical validation included matrix effect, recovery and trueness compared to National Institute of Standards and Technology (NIST) standards and United Kingdom National External Quality Assessment Service (UK NEQAS) samples. Results: Intra- and inter-run CVs were <4.9% for retinol and <1.7% for α-tocopherol, both complying with desirable specifications for imprecision. Bias compared to NIST standards was <3.1% for both compounds. The method was linear over the entire tested range. The lower limit of quantification (LLOQ) with US was lower than with ESI for both retinol (0.022 vs. 0.043 mg/L) and α-tocopherol (0.22 vs. 0.87 mg/L). Matrix effects were not significant (<15%) for retinol. However, for α-tocopherol matrix effects of on average 54.0% were noted using ESI, but not with US. Conclusions: We developed a fast, precise and accurate UPLC-MS/MS method for the determination of retinol and α-tocopherol in human serum using a single-step sample pretreatment. Ionization using US eliminated the matrix effects for α-tocopherol.

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  • MS/MS offers appropriate sensitivity for the detection through background reductions of 100-1000-fold over MS.

    “MS/MS offers appropriate sensitivity for the detection through background reductions of 100–1000-fold over MS.”

    From Vitamin A Update: Forms, Sources, Kinetics, Detection, Function, Deficiency, Therapeutic Use and Toxicity (2021) · cites this paper as [300]

    The Introduction describes LC-MS/MS as a sensitive technique for measuring retinol and α-tocopherol, and the Discussion reports that the authors’ method achieved a low limit of quantification. Neither section compares MS/MS with MS alone or reports a 100–1000-fold reduction in background; the stated mechanism and magnitude are unsupported. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • Triple-quadrupole LC-MS/MS facilitates the most effective qualitative-quantitative analysis of retinoic acid with sufficient sensitivity and specificity, yet no requirement for derivatization or definite mass identification.

    “Triple-quadrupole LC–MS/MS facilitates the most effective qualitative–quantitative analysis of retinoic acid with sufficient sensitivity and specificity, yet no requirement for derivatization or definite mass identification.”

    From Vitamin A Update: Forms, Sources, Kinetics, Detection, Function, Deficiency, Therapeutic Use and Toxicity (2021) · cites this paper as [300]

    The Introduction calls LC-MS/MS state of the art for measuring retinol and α-tocopherol, and the UPLC-MS/MS conditions describe a triple-quadrupole instrument with quantifier and qualifier measurements (page numbers unavailable). The paper studies retinol, not retinoic acid. Its simple protein-precipitation preparation does not establish that retinoic acid analysis needs neither derivatization nor definitive mass identification. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • MS/MS is currently the most sensitive method used for making bioanalyses of retinoic acid and is readily coupled with LC separations capable of resolving isomers of retinoic acid.

    “MS/MS is currently the most sensitive method used for making bioanalyses of retinoic acid and is readily coupled with LC separations capable of resolving isomers of retinoic acid.”

    From Vitamin A Update: Forms, Sources, Kinetics, Detection, Function, Deficiency, Therapeutic Use and Toxicity (2021) · cites this paper as [300]

    The Introduction describes LC-MS/MS as the state-of-the-art technique for measuring retinol and α-tocopherol and says diode-array detection has lower sensitivity and selectivity. It does not make that claim for retinoic acid. The Discussion reports separation of retinol-related compounds from retinol, not resolution of retinoic acid isomers (page numbers unavailable in our copy). (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • GC-MS allows for good sensitivity but requires additional derivatization steps for making retinoid acid analyses, often representing labor-intensive procedures and a potential source of errors.

    “GC–MS allows for good sensitivity but requires additional derivatization steps for making retinoid acid analyses, often representing labor-intensive procedures and a potential source of errors.”

    From Vitamin A Update: Forms, Sources, Kinetics, Detection, Function, Deficiency, Therapeutic Use and Toxicity (2021) · cites this paper as [300]

    The Introduction (page unavailable) says complex sample pretreatment for retinol and α-tocopherol is laborious and increases the risk of errors. It does not address GC–MS sensitivity or derivatization for retinoic acid; searches for GC–MS and gas-chromatography derivatization found no passage supporting those points. (LLM, conf=0.76)

    Experimental — accuracy not yet measured

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1 of the 40 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. [34]34. Rifai N, Horvath AR, Wittwer C. Tietz textbook of clinical chemistry and molecular diagnostics, 6th ed. Amsterdam, The Netherlands: Elsevier, 2018.

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Authors
Peersman, N., Elslande, J. V., Lepage, Y., De Amicis, S., Desmet, K., & Vermeersch, P.
Affiliations
  • Universitair Ziekenhuis Leuven (BE)
  • Clinical Department of Laboratory Medicine , University Hospitals Leuven , Leuven , Belgium
Corresponding author
Vermeersch, Pieter
ORCID
Venue
Clinical Chemistry and Laboratory Medicine (CCLM)
Publisher
Walter de Gruyter GmbH
Published
2020-03-02
Volume
58
Issue
5
Pages
769-779
ISSN
  • 1434-6621 (ISSN-L)
  • 1437-4331
DOI
10.1515/cclm-2019-1237
OpenAlex
W3010420188
Cited by
12
References
40
Citations by year
2026: 2 · 2025: 2 · 2023: 2 · 2022: 2 · 2021: 1 · 2020: 3
FWCI
1.04
Type
journal article
Language
en
Open access
Licence
other-oa
Locations
  • Clinical Chemistry and Laboratory Medicine (CCLM) (publishedVersion)
  • PubMed (publishedVersion)
  • Lirias (submittedVersion, other-oa)
Topics
  • Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
  • Physical Sciences › Chemistry › Spectroscopy › Analytical Chemistry and Chromatography
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
Keywords
retinol, alpha-tocopherol, serum vitamin E, matrix effects, protein precipitation, sample preparation, electrospray ionization comparison, vitamin A
MeSH terms
Chromatography, High Pressure Liquid, Humans, Isotope Labeling, Reference Standards, Vitamin A, Reproducibility of Results, alpha-Tocopherol, Solid Phase Extraction, Tandem Mass Spectrometry, Limit of Detection, Liquid-Liquid Extraction

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