6. AOACSPIFANMethods-2018Awards

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L in et al .: J ournal of AOAC I nternational V ol . 100, N o . 1, 2017  145

INFANT FORMULA AND ADULT NUTRITIONALS

Determination of Biotin in Infant, Pediatric, and Adult Nutritionals by High-Performance Liquid Chromatography and Fluorescence Detection: Single-Laboratory Validation, First Action 2016.11 Q i L in , Y i D ing , F iona P oh , C hunyan Z hang , S hang -J ing P an , and K aren J. S chimpf Abbott Nutrition Research and Development, 20 Biopolis Way #09-01/02, Singapore 138668

A reversed-phase HPLC method with postcolumn protein conjugation and fluorescence detection for the quantitative determination of biotin in infant, pediatric, and adult nutritionals was developed and evaluated in a single-laboratory validation (SLV). Sample of appropriate size is mixed with 2% metaphosphoric acid to precipitate out the protein. The filtrate is injected onto a C18 HPLC column in which biotin and riboflavin are separated with an appropriate mobile phase. The biotin, after eluting from the column, binds with the streptavidin fluorescein to become a fluorescent conjugate. The conjugate is then detected by fluorescence at λ ex = 495 nm and λ em = 518 nm. A column switch is used in the method as an option to shorten the run time from 30 to 15 min, by eluting out riboflavin at a higher flow rate. In this SLV, a total of 19 AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals matrixes representing a range of infant, pediatric, and adult formulas were evaluated for their biotin content. The analytical range was 1.66–142 μg/100 g reconstituted final product. The repeatability and intermediate precision ranged from 0.5 to 3.0% RSD r and from 1.3 to 4.5% RSD iR , respectively. Recovery from spiked matrixes varied from 95 to 111%, and accuracy of quantification using Standard Reference Material 1849a ranged from 99 to 105%. The LOQ in reconstituted product was estimated to be 0.8 μg/100 g. The method was approved by the Expert Review Panel as First Action at the 2016 AOAC INTERNATIONAL Mid-Year Meeting. T heAOAC Stakeholder Panel on Infant Formula andAdult Nutritionals (SPIFAN) developed Standard Method Performance Requirements (SMPRs ® ) for Biotin in Infant Formula and Adult/Pediatric Nutritional Formula and Received August 11, 2016. Accepted by SG September 09, 2016. Corresponding author’s e-mail: qi.lin@abbott.com This method was approved by the AOAC Expert Review Panel for SPIFAN Nutrient Methods as First Action. The Expert Review Panel for SPIFAN Nutrient Methods invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit-for-purpose and are critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. DOI: 10.5740/jaoacint.16-0257

called for reference methods to determine total biotin in all forms (powders, ready-to-feed liquids, and liquid concentrates) of infant, adult, and/or pediatric formula for dispute resolution (1). Biotin is a water-soluble vitamin also known as vitamin B 7 or vitamin H. It functions in important metabolic processes of carbohydrates, fats, and amino acids. Biotin is a monocarboxylic acid containing a cyclic urea structure with the sulfur atom in a thioether linkage; biocytin, the intermediate metabolite of biotin, is an amide formed from biotin and lysine (Figure 1). This method is revised from a method published in 2006, in which SPE was used for sample preparation to remove riboflavin and HPLC with a fluorescence detector (FD) was used for the detection of the biotin/streptavidin/fluorescein conjugate formed during a postcolumn derivatization (2). In this new method, the SPE clean-up step is omitted from the sample preparation, and the revised sample preparation involves simple reconstitution, dilution with methanol/water, and protein precipitation with metaphosphoric acid. A column switch is used as an option to elute out riboflavin in a shorter run time. Biocytin was subjected to the sample preparation condition and verified to be stable. It eluted out as a well-resolved peak from the biotin peak using this HPLC method. All of the 19 tested SPIFAN samples were found to be free of biocytin. To find out the biotin and biocytin content from the potential inherent biotin conjugates and biocytin conjugates in the SPIFAN placebo formula, three ways of hydrolysis were attempted during sample preparation, adapting the protocols reported by Lahély et al. (3) and Höller et al. (4): ( 1 ) acidic hydrolysis with 2 N sulfuric acid in an autoclave at 120°C for 30 min; ( 2 ) enzymatic digestion in citric buffer with papain at 37°C for 16 h; and ( 3 ) acidic hydrolysis with 2 N sulfuric acid in an autoclave at 120°C for 30 min, followed by pH adjustment to 5.7, and enzymatic digestion in citric buffer with papain at 37°C for 16 h. All of the three tested SPIFAN placebo samples were found to be free of biotin conjugates or biocytin conjugates. AOAC Official Method 2016.11 Biotin in Infant, Pediatric, and Adult Nutritionals High-Performance Liquid Chromatography and Fluorescence Detection First Action 2016

A. Principle

The basis of this method is the strong affinity between biotin and streptavidin. This method is applicable to infant, pediatric, and adult nutritional products. Samples of appropriate size are mixed with 2% metaphosphoric acid to precipitate out the