Abstract
Objectives
Measurement of the serum levels of vitamin B12 (VB12) is key for evaluating VB12 deficiency-dependent anemia. Immunoassay, the major method for determining VB12, tends to give false-normal results because of the presence of anti-intrinsic factor (IF-Ab) or other factors such as heterophilic antibodies et al. This study aimed to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that is helpful for distinguish false normal VB12 results measured by the immunoassay.
Methods
Different forms of VB12 were derivatized into CN-B12, which was collected through solid-phase extraction and analyzed via LC-MS/MS. 236 serum samples were measured both by LC-MS/MS and immunoassay, results were compared, and the IF-Ab effect was evaluated.
Results
The LC-MS/MS assay afforded a linear slope from 20 to 4,000 pmol/L for CN-B12. OH-VB12, methyl-VB12, and CoA-VB12 showed recovery within 89.3–109.5%. The intra-assay CV of VB12 was 2.6–4.1%, whereas the total CV was 9.3–9.8%. Passing–Bablok regression between LC-MS/MS and immunoassay results showed that the slope was 1.085 and the intercept was −15.691. The Bland–Altman plot showed that the mean difference and difference% were −34.6 pmol/L and 0.3%, respectively. Inter-rater agreement analysis showed that the linear weighted kappa value was 0.885, implying good agreement between the two methods. However, two samples were falsely elevated and one sample was falsely normal in the immunoassay compared with LC-MS/MS. The LC-MS/MS method helped in the distinction of false-normal VB12 results shown by the immunoassay.
Conclusions
The VB12 LC-MS/MS method can be used as an arbiter of clinically discordant immunoassay results.
Funding source: National Key Research and Development Program of China http://dx.doi.org/10.13039/501100012166
Award Identifier / Grant number: 2021YFC2401100
Funding source: National High Level Hospital Clinical Research Funding
Award Identifier / Grant number: 2022-PUMCH-A-138
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Research funding: This work was funded by the National Key Research and Development Program of China (No. 2021YFC2401100), and National High Level Hospital Clinical Research Funding (2022-PUMCH-A-138). The funding bodies were not involved in conducting or analyzing this work or in the decision to publish. The authors have no additional financial disclosures.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: Authors state no conflict of interest.
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Informed consent: Informed consent was obtained from all individuals included in this study.
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Ethical approval: Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013), and has been approved by the authors’ Institutional Review Board (Peking Union Medical College Hospital) or equivalent committee. (ZS-2795).
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Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2022-0523).
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