Vitamin B12 - A review of analytical

methods for use in food

Government Chemist Programme Report
March 2015

Vitamin B12 - A review

of analytical methods
for use in food

Author: Paul Lawrance

Contact point: Michael Walker

[email protected]
07738 179 985

Report no. LGC/R/2014/378

Approved by:

___________________________
Date: March 2015

Preparation of this report was funded by the UK

National Measurement System.
Project code: CB1114-4
LGC Limited 2015

Abstract
Vitamin B12 - a water-soluble vitamin that is found naturally in some foods, added to others,
and is available as a dietary supplement - plays an important role in the functioning of the
brain and nervous system, and in the formation of red blood cells. The requirement for
vitamin B12 is low and the storage capacity in humans is high, therefore deficiency is rare in
healthy populations. However certain population groups, such as infants, the elderly,
vegetarians or vegans, can be prone to deficiency. This can be controlled in vegetarian
groups by supplementation. Deficiency, particularly in the elderly, is often a result of
inadequate metabolism and is not related to dietary intake; the need for analysis of foods is
therefore low. In specific population groups, such as vegetarians, intake may be reliant on
supplementation and in others, such as infants, fortification of infant formula and follow-on
foods is important. Analysis may be required in fortified foods or supplements, primarily to
confirm label declarations. Methods for the analysis of vitamin B12 include microbiological
assay, polarographic, spectrophotometric, radio-ligand binding and various chromatographic
techniques. Microbiological assay has been the most commonly used assay technique for
foods and has been used for the majority of available food nutrition datasets. LGC has
extensive experience of the determination of vitamin B12 using an in-house microbiological
assay procedure although this technique is no longer supported. In this report, we review
methods for the analysis of vitamin B12 and describe methods that may be applied in the
absence of a supported microbiological assay.

Contents
............................................................................................................................................. 1
Abstract................................................................................................................................. 3
Introduction ........................................................................................................................... 2
Dietary sources of vitamin ..................................................................................................... 2
Stability ................................................................................................................................. 3
Analytical requirements ......................................................................................................... 3
Extraction .............................................................................................................................. 3
Analytical methods ................................................................................................................ 4
Official methods .................................................................................................................... 4
Commercial methods: ........................................................................................................... 5
Methods used at LGC ........................................................................................................... 6
Conclusion ............................................................................................................................ 7
References ........................................................................................................................... 7

Introduction
Vitamin B12 is a water-soluble vitamin that is found naturally in some foods, is added to
others, and is also available as a dietary supplement. In fact, vitamin B12 exists in several
forms, all of which contain cobalt and are called cobalamins. Vitamin B12 is commonly
called cyanocobalamin; however the name can refer to any of the cobalamins that exhibit
similar biological activity. Methylcobalamin and 5-deoxyadenosylcobalamin are the forms of
vitamin B12 that are active in human metabolism. Cyanocabalamin and hydroxycobalamin
are used pharmacologically with cyanocobalamin primarily being used for fortification.

Vitamin B12 plays an important role in the functioning of the brain and nervous system, and in
the formation of red blood cells. The requirement for vitamin B12 is low and the storage
capacity in humans is high therefore deficiency is rare in healthy populations. Certain
population groups such as infants, the elderly, vegetarians or vegans can be prone to
deficiency. Severe deficiency, primarily caused by malabsorption or metabolic failure,
causes pernicious anemia, a failure to produce sufficient red blood cells or haemoglobin.
This disease, primarily of the elderly, can be treated by vitamin B12 injections but not cured.
Vitamin B12 is very important for the development and growth of children and infant formula
is an important vehicle for fortification.

This report summarises a review of analytical methods for the determination of vitamin B12 in
food. Mention of a commercial analytical product in this report does not imply endorsement
by the Government Chemist.

Dietary sources of vitamin

Vitamin B12, occurring naturally in food, derives from bacterial synthesis and therefore occurs
almost exclusively in foods of animal origin. Small amounts have been determined in certain
plant sources such as seaweeds and algae but are not all available for human consumption
and digestion. Cobalamins are usually bound to other food components, especially proteins,
and must undergo enzymatic and acid hydrolysis in the gut to be released. Similar
processes are required for in-vitro analysis.
Dairy foods, milk and milk products, eggs, meat and seafoods are the main natural sources
in the diet. Vitamin preparations or supplements containing vitamin B12 are widely available
and foods, such as breakfast cereals, are often fortified with vitamin B12. The capacity to
utilise vitamin B12 however, depends upon the availability of intrinsic factor, a glycoprotein

required for its absorption. Intake cannot therefore be used as a sole measure of nutritional
status. Infant formula and follow-on foods are an important area for fortification.

Stability
Cyanocobalamin is the most stable form of vitamin B12 and is normally used for fortification.
It is most stable at pH 4 - 4.5 and to heat, including autoclaving between pH 4 and 7.
Crystalline forms are stable when protected from light. Severe acid or alkali, strong light or
the presence of oxidising agents will however destroy the vitamin. Excess cyanide will
displace other moieties from the cobalt beta position to form cyanocobalamin.

Analytical requirements
Nutritional status is determined by analysis of plasma or serum concentrations of
cyanocobalamin or related forms and metabolites. Since deficiency is rare and often not
related to dietary intake, the need for analysis of foods is low. The exception is in specific
population groups where intake may be reliant on supplementation or fortification. Since the
requirement for vitamin B12 is small and there is no risk associated with excessive intake, the
need for regulation in this area is limited. Analysis may be required in fortified foods or
supplements, primarily to confirm label declarations.

Extraction
When synthetic vitamin B12 is added to fortified foods and dietary supplements, it is already
in free form and does not require vigorous extraction. Simple aqueous extraction can be
carried out, usually at a pH of around 4. Heating may be used and clean-up may be
required to remove other analytical components.
For natural foods, vitamin B12 is bound to protein and must be released before analysis.
This is often accomplished simply by denaturing the protein by heating during the extraction,
although pepsin or other protease enzymes may also be used. For starchy foods, amylase
may also be added to help break down the food matrix. Excess cyanide is added when
determining natural vitamin B12 to convert all of the cobalamins to the cyanocobalamin form.
Analysis of vitamin B12 at natural levels in foods is difficult as it is normally present at very
low concentration and may be accompanied by much higher levels of other food

components which may interfere with subsequent analysis. These need to be removed
before quantitation of the vitamin B12.

Analytical methods
Methods for the analysis of vitamin B12 include microbiological assay, polarographic,
spectrophotometric, radio-ligand binding and various chromatographic techniques. For
clinical use, radio-ligand binding assays are commonly used but these are not normally
suitable for use in foods.
Microbiological assay has been the most commonly used assay technique for foods and has
been used for the majority of available food table data. It relies on the specific requirement
for vitamin B12 by certain bacterial organisms (e.g. Lactobacillus delbreueckii) to enable their
growth in a supporting medium. Under appropriate conditions, the amount of growth
obtained is proportional to the amount of vitamin B12 in the test extract. Such assays can be
considered routine for vitamin B12 but require overnight incubation, can be subject to
contamination and require considerable laboratory set-up and maintenance time. The
sensitivity is high enabling the detection of low concentrations of vitamin B12 but other food
components may interfere with the determination.
The development of commercial test kits for the analysis of water-soluble vitamins in foods
using microbiological assay (e.g. VitaFast kits) has simplified these procedures. However
careful use is still required and the extraction procedures given in the kit may need further
validation depending on the samples to be analysed.

Official methods
There are few official methods for vitamin B12 in food. The following methods are available:
USP/BP:
Determination of cyanocobalamin and hydroxocobalamin in pure substance, injectable
solutions and oil or water soluble vitamins tablets and capsules. Spectrophotometric and
HPLC methods with specific scope as above.
AOAC:
There are a number of approved procedures within AOAC although almost all of these are
for the analysis of infant formula and related nutritional products as below. The more recent
methods were developed under the AOAC Stakeholder Panel on Infant Formula and Adult
Nutritionals (SPIFAN) project.

AOAC 952.20 Vitamin B12 in vitamin preparations - Microbiological assay

AOAC 986.23 Vitamin B12 in milk-based infant formula - Microbiological assay
AOAC 2011.08 Vitamin B12 in infant formula and adult nutritionals - HPLC-UV with
immunoaffinity extraction (1st action)
AOAC 2011.09 Determination of vitamin B12 in infant formula and adult nutritionals using
HPLC after purification on an immunoaffinity column (1st Action) HPLC with immunoaffinity
extraction
Note: For final action status, AOAC recommended combining 2011.08 with 2011.09 as they
are very similar. SPIFAN, however, rejected both of these as they use a proprietary (singlesource) IAC cartridge and are proceeding with AOAC 2011.10.

AOAC 2011.10 Determination of vitamin B12 in infant formula and adult nutritionals by HPLC:
(1st Action) HPLC with SPE clean-up (column switching)
AOAC 2011.16 Determination of vitamin B12 in infant formula and adult nutritionals by
surface plasmon resonance: (test kit method) Biacore technology (1st action)
ISO: There are no specific methods for vitamin B12 but the recommended CODEX
procedures refer to the AOAC microbiological methods 952.20 and 986.23.

CEN / BS: There are no approved methods for vitamin B12

Commercial methods:
R-Biopharm test kits: VitaFast test kit: 96 well microtiter plate-based microbiological
assay. These are produced by R-Biopharm and provide a relatively simple means of
applying the microbiological assay without the need for in-house maintenance of organisms
etc. The B12 test kit is described for the determination of vitamin B12 at natural or added
levels in food, feed and pharmaceutical products.
EASI-EXTRACT VITAMIN B12: Monoclonal antibody based immunoaffinity columns for use
in conjunction with an HPLC or LC-MS/MS system for detection of vitamin B12 in foods and
similar matrices. The columns (available in two sizes) are described for the selective
extraction of vitamin B12 before analysis using HPLC or LC-MS. The conditions required for
HPLC with UV detection are given.

Note: As used in the AOAC methods above. The extraction is mainly validated for infant
formulae and some additional validation may be required for other foods.
RidascreenFast Vitamin B12: Competitive enzyme immunoassay for the quantitative
analysis of vitamin B12 in fortified food, feed and vitamin products.
HPLC or LC-MS methods
There are few published procedures for vitamin B12 in food apart from those discussed
above. Published procedures are similar to the AOAC methods and are mainly for fortified
products. HPLC with UV detection is difficult because of the low level of vitamin B12 in
unfortified foods and relatively high detection limits with UV detectors. LC-MS procedures
are rare at present although some conditions and multivitamin methods including vitamin B12
have been published as below. These provide a useful starting point if mass spectrometry is
desired but further validation would be necessary.
Table 1: Mass spectrophotometric methods for Vitamin B12
Analyte

Technique

Reference

Vitamin B12 standard

LC-MS

UPLC / Triple Quad MS

LC/ESI-MS\MS

UPLC/MS/MS

Vitamins B5,B8,B9 & B12 in

fortified infant formula
Water soluble vitamins in
various foods
Water soluble vitamins in
fortified beverages &
supplements

Methods used at LGC

LGC has long experience of the determination of vitamin B12 using an in-house
microbiological assay procedure, although this technique is no longer supported. The
VitaFast test kit is not used routinely although similar kits are used successfully for other
water soluble vitamins.
Recent analysis has focussed on the HPLC-UV procedure with immunoaffinity cartridge
extraction. The R-Biopharm test kit is used for this purpose. It has been successfully used
for infant formulae and baby foods (fruit, pasta and cereal). The method performs well
although chromatographic problems can be encountered since the cartridges have a low
maximum retention capacity which limits the extent to which sample extracts can be

concentrated. Additional validation is required for foods other than those described in the
test kit instructions (mainly infant formula).
A method using aqueous extraction followed by ion-pair HPLC with UV detection has also
been used for analysis of vitamin premixes but is only suitable for this purpose.

Conclusion
Vitamin B12 - a water-soluble vitamin that is found naturally in some foods, added to others,
and is available as a dietary supplement - plays an important role in the functioning of the
brain and nervous system, and in the formation of the red blood cells. Vitamin B12 deficiency
is rare in healthy populations. However certain population groups, such as infants, the
elderly, vegetarians or vegans, can be prone to deficiency. This can be controlled in
vegetarian groups by supplementation. Deficiency, particularly in the elderly, is often a
result of inadequate metabolism and is not related to dietary intake; the need for analysis of
foods is therefore low. However analytical methods are required for fortified foods or
supplements, primarily to confirm label declarations. Methods for the analysis of vitamin B12
include microbiological assay, polarographic, spectrophotometric, radio-ligand binding and
various chromatographic techniques. Microbiological assay has been the most commonly
used assay technique for foods and has been used for the majority of available food nutrition
datasets. However such techniques are expensive to support in the absence of a minimum
level of use. There are other methods for the analysis for vitamin B12 reviewed above that
may be applied in the absence of a supported microbiological assay.

References
1. LC/MS Analysis of Vitamin B12: Waters application note 7250000758EN;
Waters.com/library (used for vitamin B12 standard only)
http://www.waters.com/waters/library.htm?locale=e&lid=1517173
2. Simultaneous Determination of Four Water-Soluble Vitamins in Fortified Infant Foods by
Ultra-Performance Liquid Chromatography Coupled with Triple Quadrupole Mass
Spectrometry; Baiyi et al.; J Chromatogr Sci (2008) 46 (3):225- 232.
3. Simultaneous determination of water-soluble vitamins in selected food matrices by liquid
chromatography/electrospray ionization tandem mass spectrometry; Gentili et al., Rapid
Communications in Mass Spectrometry; 22 (13) 20292043, 15 July 2008

4. Simultaneous Analysis of Water-Soluble Vitamins in Vitamin-Enriched Beverages and

Multivitamin Dietary Supplements by UHPLC-MS/MS; Wang et al.
http://www.dionex.com/en-us/webdocs/110909-PO-LC-MSMS-WSVitamins-BevSupplements-18Oct2011-LPN2906-01.pdf