Determination of B-vitamin concentrations in supplements

1st April 2013

The rapidly increasing usage of vitamins as supplements in foods has created a need to minimise the turnaround time of testing. Lennart Wahlström reports.

Surface plasmon resonance (SPR) technology offers an automated platform with a high degree of accuracy and reproducibility for B-vitamin concentration determination in a variety of food matrices, with minimal sample preparation, and low requirements for re-testing (Fig.1).

Compared with more traditional methods of testing, eg microbiological analysis (MBA), Biacore's SPR technology provides a highly accurate alternative method for the rapid and sensitive analysis of vitamins. Same-day analysis and generation of results using SPR technology enables food manufacturers to maintain closer control of the production process allowing early release of final product, in some cases as much as several days earlier.

In addition, single sample analysis can be achieved in around 30 minutes, providing the capability for introducing real-time process control of vitamin concentration. This well-established technology provides a rapid automated system for the concentration analysis of additives, residues and contaminants in a variety of food products. Because of the increased speed, sensitivity and reliability of SPR analysis, food testing laboratories worldwide are already benefiting from faster results, higher productivity and subsequent cost reduction.

Qflex Kits provide the flexibility required during method development, together with the reliability essential for routine assays. When used with BiacoreQ (Fig.2.), Biacore's specialised food analysis instrument, Qflex Kits facilitate the performance of automated, label free assays for concentration analysis, offering significant savings in time and reducing the risks of experimental errors.

Biacore has five kits for determination of water-soluble vitamins: Qflex Kit Biotin, Qflex Kit Folic Acid, Qflex Kit VitaminB12, Qflex Kit VitaminB2 and Qflex Kit Pantothenic Acid.

In the following sections some of the important B-vitamins and the Biacore products available for their analysis will be presented.

Folic acid

Folic acid is a water-soluble B-vitamin that takes its name from the Latin word for leaf, folium, because it was first isolated from spinach leaves. Biochemically, folic acid (or folate) functions as a methyl donor after being enzymatically reduced to tetrahydrofolate by the enzyme dihydrofolate reductase.

Biacore's SPR technology provides a rapid method for measuring folic acid in hours rather than days, and is usually unaffected by matrix effects which interfere with many conventional detection methods used in food analysis.

Qflex Kit Folic Acid offers a detection limit for folic acid of 1.0ng/ml in the prepared samples. The quantification range is 2.0­70ng/ml in the prepared samples, corresponding to „2.0ng/ml in liquid matrices and „4ng/100g in solid matrices.

Collaborative studies performed on a broad range of different sample matrices have shown of repeatability values in the range of 3­5% CV and reproducibility values in the range of 5­10% CV.

Biacore Qflex Kit Folic Acid is internationally validated and certified by the AOAC (Association of Analytical Communities) Research Institute as a Performance Tested MethodSM. As the foremost independent body determining food testing standards worldwide, AOAC Performance Tested Method status provides complete assurance that a totally independent third-party review has been undertaken and the test kit fully meets all performance claims.

Vitamin B12

Naturally occurring vitamin B12 originates solely from synthesis by bacteria and other micro-organisms growing in soil or water, in sewage, and in the rumen and intestinal tract of animals. This vitamin is added as a supplement to a variety of foodstuffs including infant formula, cereals, vitamin pre-mixtures, juice, flour, jam and pet foods.

Quantitative determination of vitamin B12 content is often complicated by the nature of the sample matrices and the low concentrations normally present in typical samples. The combination of Qflex Kit Vitamin B12 with Biacore Q offers a rapid and reliable quantification technique. The vitamin B12 derivative has been optimised for easy sensor surface preparation. The vitamin B12 binding protein has been specifically adapted for Biacore's SPR technology to ensure a high sensitivity response to cyanocobalamin (a synthetic and stable form of vitamin B12).

The day to day reproducibility for a homogeneous sample is typically < 10% CV and the awithin assay' reproducibility is typically <5%CV. The detection limit is 0.06ng/ml and the quantification limit is 0.2ng/ml.

Pantothenic acid

Pantothenic acid, also known as Vitamin B5, is a water-soluble vitamin and a member of the B-group of vitamins. It is found in high levels in many food types and indeed takes its name from the Greek aPantos' meaning aeverywhere'. The vitamin is integral to many functions in the human body such as the synthesis of essential molecules, eg acetylcholine, and for the proper functioning of the adrenal glands and nervous system.

Pantothenic acid is routinely added to foods as a supplement during processing in its more stable form, calcium pantothenate. Manufacturers and processors of food products have a constant and increasing demand to demonstrate the quality of their products to customers and to minimise wastage of resources. The quick and reliable determination of levels is rapidly becoming more important.

The most commonly used method for the determination of pantothenic acid is a microbiological assay. Ligand binding techniques have been developed but are not commercially available. Chromatographic methods have been used for research purposes but are not widely available. Each of these techniques is hampered by low sample throughput, extensive sample preparation or limitation to simple matrices. In comparison with these methods, Biacore's SPR technology, in combination with Qflex Kit Pantothenic Acid, provides a highly accurate alternative method for the rapid and sensitive analysis of pantothenic acid.

Performing an automated analysis on Biacore Q is straightforward. The calibration curve and up to 86 samples are analysed in a single run. The results from 20 samples can be obtained in less than 12 hours, including sample preparation.


As consumer pressure increases, both regulatory bodies and food producers need rapid, robust and accurate methods for determination of vitamins in food products. SPR technology offers a means of achieving this, with results that are comparable to microbiological assays in terms of accuracy, but that additionally offer the time saving that the industry needs.

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Lennart Wahlström is with Biacore AB, Uppsala, Sweden.





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