AOAC Final Action Methods in 2017

J OSEPH ET AL . : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . 3, 2018 831

INFANT FORMULA AND ADULT NUTRITIONALS

Determination of Total Biotin by Liquid Chromatography Coupled with Immunoaffinity Column Cleanup Extraction: Multilaboratory Testing, Final Action 2016.02

G EORGE J OSEPH and R ANJANI D EVI AsureQuality Ltd, PO Box 41, Shortland St, Auckland 1140, New Zealand E LAINE C. M ARLEY and D AVID L EEMAN R-Biopharm Rh ˆ one Ltd, West of Scotland Science Park, Glasgow G20 0XA, United Kingdom

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Collaborators: S. Bhandari; L. Butler Thompson; E. Campos Giminez; H. Chen; N. Dekker; R. Devi; M. Dole; T. Gallegos-Peretz; B. Gill; T. Gill; Y. Green; M. Hodgson; X. Huo; H. Indyk; G. Joseph; L. Kennedy; D. Leeman; F. Lim; C. Macfadzean; A. McMahon; I. Malaviole; E. Marley; M. Seegers; K. Schimpf; J. Wang; D. Wollard; S. Yadlapalli; C. Yin

Single- and multilaboratory testing data have provided systematic scientific evidence that a simple, selective, accurate, and precise method can be used as a potential candidate reference method for dispute resolution in determining total biotin in all forms of infant, adult, and/or pediatric formula. Using LC coupled with immunoaffinity column cleanup extraction, the method fully meets the intended purpose and applicability statement in AOAC Standard Method Performance Requirement 2014.005. The method was applied to a cross-section of infant formula and adult nutritional matrixes, and acceptable precision and accuracy were established. The analytical platform is inexpensive, and the method can be used in almost any laboratory worldwide with basic facilities. The immunoaffinity column cleanup extraction is the key step to successful analysis. A collaborative study was carried out on AOAC First Action Official Method SM 2016.02 Determination of Total Biotin by Liquid Chromatography Coupled with Immunoaffinity Column Cleanup Extraction . Subsequent to the successful method optimization and the analysis of practice samples by 12 laboratories from 10 different countries, nine laboratories completed the analysis of 12 pairs of blind duplicates before the due date of the study. Carefully selected AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) matrixes were used for the multilaboratory testing (MLT). Received June 12, 2017. Accepted by SG September 13, 2017. The method was approved by the AOAC Official Methods Board as Final Action. See “ Standards News, ” (2017) Inside Laboratory Management , July/August issue. The AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) invites method users to provide feedback on the Final Action methods. Feedback from method users will help verify that the methods are fit for purpose and are critical to gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author. Corresponding author ’ s e-mail: george.joseph@asurequality.com DOI: https://doi.org/10.5740/jaoacint.17-0242

The sample is dispersed in phosphate-buffered saline (PBS) and autoclaved at 121 ± 2°C for 25 min. The sample is cooled to room temperature and then diluted to 100 mL in a volumetric flask. The extract is centrifuged and filtered usingWhatman glass microfiber filter paper (GE Healthcare Life Sciences, Amersham, Buckinghamshire). Clear filtrate is collected for cleanup and extraction. A biotin immunoaffinity column (IAC) is mounted onto an SPE manifold. A disposable syringe barrel is connected to the IAC as a reservoir. The buffer in the affinity column is drained, and the sample filtrate is loaded through the reservoir and allowed to flow through by gravity. The column is washed with PBS followed by water. Air is passed through the column to remove residual liquid. Biotin and biocytin from the column are eluted with methanol and collected in a Reacti-Vial ™ (Cat. No. 13223; Thermo Scientific). The eluent is evaporated to dryness using a heating block set at 85 ± 5°C under a gentle stream of nitrogen, and the sample is reconstituted in 1 mL water. The biotin and biocytin in the reconstituted sample are analyzed simultaneously by HPLC using a photodiode-array (PDA) detector set at 200 nm. Identification of the peaks is based on absolute retention time. Quantification is by multipoint external calibration using peak area responses of the analytes. Spectrum scan (200 – 350 nm) can be used for purity and peak identity confirmation as required. Biotin (also called vitamin H or vitamin B 7 ) is a B-group vitamin involved in the production of energy and functions as a coenzyme in bicarbonate-dependent carboxylation reactions. Biotin exists as free biotin and in protein-bound forms in foods. Signs of biotin deficiency are evident in humans who consume raw egg white over long periods. Raw egg white contains avidin, a protein that has shown to bind biotin in the small intestine and prevent its absorption. Clinical findings of biotin deficiency include dermatitis, conjunctivitis, alopecia, and central nervous system abnormalities. Biotin (hexahydro-2-oxo- 1 H-thieno[3,4-d]imidazol-4-pentanoic acid; CAS 58-85-5) is the fusion of an imidazolidone ring with a tetrahydrothiophene group linked to a valeric acid side chain. The chemical formula of biotin is C 10 H 16 N 2 O 3 S, with a MW of 244.31. Biocytin (N6-[5-[(3aS,4S,6aR)-hexahydro-2-oxo- 1 H-thieno[3,4- d]imidazol-4-yl]-1-oxopentyl]- L -lysine; CAS 576-19-2) is a bound form of biotin (the linked molecule is lysine) and is also biologically