AOACSPIFANMethods-2017Awards

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Lin et al.: Journal of AOAC International V ol. 100, No. 1, 2017 145
Determination of Biotin in Infant, Pediatric, and Adult
Nutritionals by High-Performance Liquid Chromatography
and Fluorescence Detection: Single-Laboratory Validation,
First Action 2016.11
Qi Lin, Yi Ding, Fiona Poh, Chunyan Zhang, Shang-Jing Pan, and K aren J. Schimpf
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 λem = 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% RSDr and from 1.3 to 4.5% RSDiR,
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.
TheAOAC 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
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 B7
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
INFANT FORMULA AND ADULT NUTRITIONALS
Received August 11, 2016. Accepted by SG September 09, 2016.
Corresponding author’s e-mail:
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 .
DOI: 10.5740/jaoacint.16-0257
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