Self-Classified Topic Area(s): Small Molecules / Tox / TDM
Development of an LC-MS/MS Assay for Biotin Quantitation to Support Research on Biotin Interference of Immunoassays
A. IJpelaar , A. Beijers, H. van Daal, J.M.W. van den Ouweland Department of Clinical Chemistry, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands
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Antoinette IJpelaar (Presenter) CWZ Hospital
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Background: Recent studies have shown that the results of immunoassay methods, using streptavidin-biotin interaction methodology, can be affected by the presence of high biotin circulating levels due to supplemental therapy leading to a growing number of adverse events. The evidences so far reporting on biotin interference by immunoassay methods are mainly based on case reports, rather than on experimental or clinical studies.
Aim: We developed an LC-MS/MS method for measuring biotin in serum to support research on biotin interference of immunoassays.
Methods: Samples underwent trichloroacetic acid precipitation prior to chromatography using a ACQUITY BEH C18 1.7 μm, 2.1 x 50 mm Column (Waters) with gradient elution. Mobile phases A and B consisted of ammonium bicarbonate (10 mM, pH 8.75) and MeOH (100%), respectively. Purified biotin, >99% purity (Cat. No. PHR1233, Sigma-Aldrich), and its deuterated internal standard, biotin-d4 (Sigma-Aldrich) were used to establish a 5-point calibration curve (range: 0-1315 ng/mL). Fragments of biotin and biotin-d4 were detected using positive ESI (Xevo TQS, Waters) by SRM using m/z 245.0>226.9 (96.9 qual.) and 249.0>230.9, respectively. Method accuracy was verified using a biotin European Pharmacopoeia Reference Standard (Sigma-Aldrich).
Results: The limit of quantitation was 1 ng/mL (20% CV), and inter-run precision was <10% at 10 ng/mL and 100 ng/mL. On the basis of our LC-MS /MS measurements, we noticed discrepancies in the magnitude of observed immunoassay interference between patient sera following biotin supplementation and when the same amount of biotin was spiked to blank serum. This indicates that not only free biotin but also biotin metabolites (e.g. biotin sulfoxide and bisnorbiotin) are likely to contribute to immunoassay interference, an issue not always accounted for in manufacturer-established thresholds (often based on exogenous addition of pure biotin).
Conclusion: Our LC-MS/MS assay will allow for more precise data of biotin prevalence, serum biotin interference and better understanding of the systemic concentrations seen after moderate- and high-dose biotin supplementation.
= Discovery stage.
= Translation stage.
= Clinically available.
