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Determination of Vitamin B12 in human milk


Vitamin B12, also known as cobalamin, is a coordination complex of cobalt, which occupies the center of a corrin ligand and is further bound to a benzimidazole ligand and adenosyl group.

Figure: Structure of vitamin B12               

Different cobalamins, in which the adenosyl ligand is replaced by other groups also show the vitamin activity. Vitamin B12 has many essential biological functions, it is a cofactor in DNA synthesis, in both fatty acid and amino acid metabolism.  It is important in the normal functioning of the nervous system via its role in the synthesis of myelin and plays a significant role for the neurological development of infants.

Since breast milk is the recommended source of nutrition in the first 6 month of life, its vitamin B12 content is of high importance. Therefore, a reliable method for quantification of vitamin B12 is of vital importance to assess the nutritional status of the mother-infant dyad, to support the evaluation of the efficacy of supplementation and to ensure further development of vitamin B12 research.

The main challenge for the determination of the vitamin B12 in human milk is its low concentration which is generally one order of magnitude lower than in cow's milk. Available methods such as the microbiological assay (MBA) or the competitive chemiluminescence enzyme immunoassay call for long or extensive sample preparation.

The molecular structure of cobalamins, characterized by a cobalt (Co) complex containing a corrin system, opens the door for using Co speciation analysis for selective quantification of vitamin B12. The hyphenation of Liquid Chromatography to ICP-MS (LC-ICP-MS) is the most often used technique for trace metal speciation analysis. While this technique has been used already for the determination of vitramin B12, its applicability to the analysis of human milk has not been investigated until now.

The new study:
A group of researchers from Nestlé Research Laboratories now aimed at the development and validation of LC-ICP-MS for the accurate determination of vitamin B12 in human milk. For sample preparation, a 1.0 mL portion of human milk was heated for 30 min at 120°C in presence of potassium cyanide at pH 4.0 in order to transform all cobalamins to cyanocobalamin. Cyanocobalamin is the most stable species among the cobalamins. After cooling and centrifugation to separate fat and precipitated proteins, 9 ml of sample solution was passed through an immunoaffinity cartridge. After drying the cartridge was eluted with 5 mL of methanol in four steps. The collected eluates were dried at 50°C and the final residue dissolved in 200 µL of water and directly transferred into HPLC vials for subsequent LC-ICP-MS analysis. Quantification was achieved via external calibration with cyanocobalamin standards in the range from 0.1 to 4.0 µg/L.   

Separation was based on reversed-phase HPLC using a silica-based, C18 column under isocratic conditions. The mobile phase was composed of water-methanol (3:1 v/v) containing 5 mmol/L ethylenediaminetetraacetic (EDTA) disodium salt hydrate and 10 ppb Germanium as internal standard. The chromatographic conditions provided the separation of cyanocobalamin from all other forms of vitamin B12 within 4 min.

The HPLC eluent was directly fed to the ICP-MS sample introduction system. In order to stabilize the plasma against the solvent load, the spraychamber was cooled to 4°C and a low amount of oxygen (5.5%) was added to the argon carrier gas. While a triple quadrupole ICP-MS/MS was used for quantification, the cell mode using He as collision gas proved to be pointless, since Co detection was interference free after separation.

The practical limit of quantification, defined as the minimum concentration that can be quantitated with acceptable precision, was thus established at 40 pmol/L (54 ng/L). This value was found to be appropriate to quantify all levels of vitamin B12 in human milk. Within- and between-day variability were lower than 10% and 15% respectively. The accuracy of the method was validated by recovery test from spiked human milk and cow’s milk and infant formula Certified Reference Materials (CRMs) with reference Vitamin B12 values.  All recovery values were well within acceptability limits (80–120%). Also a comparison with results obtained using a microbiological assay showed strong correlation with results obtained by the new method. Based on these results, the authors concluded, that the new method can be used to routinely monitor vitamin B12 in clinical studies or in population observational studies, to determine infants’ intake or assess efficacy of mother’s supplementation in a non-invasive manner.

The original publication

Stephane Dubascoux, Janique Richoz Payot, Paul Sylvain, Marine Nicolas, Esther Campos Gimenez, Vitamin B12 quantification in human milk – Beyond current limitations using liquid chromatography and inductively coupled plasma – Mass spectrometry, Food Chemistry, 362 (2021) 130197. DOI: 10.1016/j.foodchem.2021.130197

Used Instrumentation:

Agilent Technologies - HPLC 1200
Agilent Technologies - 8800 Triple Quad ICP-MS

Related studies (newest first):

Che-Wei Wui, Shiuh-Jen Jiang, A.C. Sahayam, Determination of cobalt compounds in dietary supplements using liquid chromatography inductively coupled plasma mass spectrometry, Spectrochim. Acta Part B., 154 (2019) 70-74. DOI: 10.1016/j.sab.2019.02.010

R. Wenzel, D. Major, K. Hesp, P. Doble, Determination of vitamin B12 in equine urine by liquid chromatography – inductively coupled plasma – mass spectrometry, J. Trace Elem. Med. Biol., 50 (2018) 634–639. DOI: 10.1016/j.jtemb.2018.05.005

J. Bosle, S. Goetz, A. Raab, E.M. Krupp, K.G. Scheckel, E. Lombi, A.A. Meharg, P.A. Fowler, J. Feldmann, Cobalamin Concentrations in Fetal Liver Show Gender Differences: A Result from Using a High-Pressure Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry as an Ultratrace Cobalt Speciation Method. Anal. Chem., 88/24 (2016) 12419–12426. DOI: 10.1021/acs.analchem.6b03730

Fang-Yu Yang, Shiuh-Jen Jiang, A.C. Sahayam, Combined use of HPLC–ICP-MS and microwave-assisted extraction for the determination of cobalt compounds in nutritive supplements, Food Chem., 147 (2014) 215–219. DOI: 10.1016/j.foodchem.2013.09.141  

B.D. Kerger, R. Gerads, H. Gurleyuk, K.A. Thuett, B.L. Finley, D.J. Paustenbach, Cobalt speciation assay for human serum, Part I. Method for measuring large and small molecular cobalt and protein-binding capacity using size exclusion chromatography with inductively coupled plasma-mass spectroscopy detection. Toxicol. Environ. Chem., 95/4 (2013) 687–708. DOI: 10.1080/02772248.2013.793444.

C.S.K. Raju,  L.L. Yu, J.E. Schiel, S.E. Long, A simple and sensitive LC-ICP-MS method for the accurate determination of vitamin B12 in fortified breakfast cereals and multivitamin tablets. J. Anal. At. Spectrom., 28/6 (2013) 901-907. DOI: 10.1039/c3ja30383g.

E.G. Yanes, N.J. Miller-Ihli, Cobalamin speciation using reversed-phase micro-high-performance liquid chromatography interfaced to inductively coupled plasma mass spectrometry. Spectrochim. Acta Part B., 59/6 (2004) 891–899. DOI: 10.1016/j.sab.2004.03.006

K. Honda, M. Imanishi, T. Takeda, M. Kimura, Determination of Vitamin B12 in Serum by HPLC/ICP-MS, Anal. Sci., 17/Sup. (2001) i983. DOI: 10.14891/ANALSCISP.17ICAS.0.I983.0

A. Makarov, J. Szpunar, Species-selective determination of cobalamin analogues by reversed-phase HPLC with ICP-MS detection. J. Anal. At. Spectrom., 14/9 (1999) 1323–1327. DOI: 10.1039/A900633H

Related EVISA News (Newest first)

last time modified: July 10, 2021


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