Figure 1. Folic acid (pteroylglutamic acid), C19H19N7O6, Mw = 441,4 g/mol.
Different forms of folate in foods include 5,6,7,8-tetrahydrofolate and several 5- or 10-substituted forms of folates. Folate can occur in the polyglutamate or monoglutamate forms. Foods do not naturally contain folic acid (oxidised form of folate), but it can be found in fortified foods and food supplements or if the reduced forms of folate in food are oxidised.
Synonyms for folate:
- folic acid, folacin, pteroylglutamic acid, vitamin M, vitamin B9
- 1 Golden standard
- 2 Method indicator
- 3 Scope
- 4 Principle
- 5 Key steps
- 6 Remarks
- 7 Criteria for analytical performance and analytical quality control
- 8 Certified Reference Materials/Standard Reference Material
- 9 Proficiency testing schemes
- 10 Other methods available
- 11 Literature
- 12 EuroFIR assistance to this method/guidelines
EN 14131:2003 Foodstuffs - Determination of folate by microbiological assay
The European Standard EN 14131 specifies a method for the determination of the total folate content of foodstuffs by microbiological assay (MBA). The growth of the test organism Lactobacillus casei sp. rhamnosus (ATCC 7469) is measured by turbidimetric detection. Both naturally occurring folate and added folic acid are determined with this method.
Phosphate buffer is added to the sample, and the sample suspension is heated to extract folate. The food matrix may be further digested by adding protease and α-amylase. Polyglutamate forms of folate are hydrolysed enzymatically to mono- or diglutamate forms. The sample is diluted with medium, which has all the nutrients needed for the growth of the test organism (Lactobacillus casei sp. rhamnosus (ATCC 7469)), except folate. The growth response of the test organism is measured turbidimetrically and is compared to the growth responses obtained with calibrant solutions with known concentration.
- Sample must be properly homogenised.
- Avoid high temperatures during homogenisation.
- Extraction is performed by adding phosphate buffer (including ascorbate; pH 6,1) and heating the sample solution.
- heat treatment 100-121 °C, 15 min
- heat treatment 100-121 °C, 15 min
- Protease and/or α-amylase treatments may be included depending on the sample type.
- γ-glutamyl hydrolase (e.g. from hog kidney) is added, and the sample solution is incubated (37 °C, 3 h).
- Optimal pH, incubation temperature and incubation time must be checked for every enzyme used.
- The sample solution is centrifuged to remove precipitated proteins.
- The folic acid content of the enzyme preparations should be determined, and the results of the samples should be compensated accordingly.
- Calibration solutions and sample dilutions are prepared.
- The pH of the solutions should be 6,1 during incubation.
- Test tubes or 96-well microplates may be used for the microbiological assay.
- Test tube format:
- Test tubes for blanks, calibration solutions and samples are prepared in triplicate.
- In addition to the calibration solution or sample solution, water and basal medium solution are added to each test tube.
- The total volume of the solution in every test tube is 10 ml.
- Test tubes, except uninoculated blanks, are inoculated after heat treatment (121-123 °C, 5 min).
- Tubes are incubated at 37 °C for 15-24 h.
- Spectrometer is set to 550-650 nm and adjusted to 100 % transmission for uninoculated blanks.
- After achieving a steady state, absorbance of test tubes are read.
- Microplate format:
- The sample solutions are pipetted in duplicates.
- In addition to the calibration solution or sample solution, phosphate buffer and basal medium are added to the wells.
- Plates are put to a plastic container.
- To inhibit the evaporation of water from the wells, adequate humidity must be maintained during incubation.
- Microplate is incubated at 37 °C for 15-24 h.
- Absorbance of test tubes is read.
- Inoculated blanks are used as reference.
- Calibration graph is formed according to the absorbance data of each calibration point and the corresponding mass of folic acid.
- The mass of folic acid is determined in nanograms for each sample dilution.
- The mass fraction of folate in µg/100g is calculated.
- Folate is sensitive to UV light and oxidation.
- Samples should be protected from natural light during sample treatments.
- There are many alternative sources of γ-glutamyl hydrolase (e.g. hog kidney), and some of these are described in EN 14131.
- The activity and appropriateness of the enzyme preparation should be checked.
- The concentration of folic acid stock solution must be checked spectrophotometrically.
- The checked concentration must be taken into consideration when preparing the standard solution.
- Minimum criteria for acceptance of data:
- inoculated blank solutions should not exceed the maximum turbidity set
- the calibration solution with the highest concentration level must exceed the minimum turbidity set
- variation between replicates and different dilution levels should not exceed the amount of variation accepted
- suitable reference control samples should be analysed
- Old data:
- Radio-immune assay
- AOAC using Streptococcus faecalis
- Latest review
- Arcot J, Shrestha A. 2005. Folate: methods of analysis. Trends Food Sci Technol. 16:253.266.
- Quinlivan EP, Handon AD, Gregory JF. 2006. The analysis of folate and its metabolic precursors in biological samples. Anal Biochem, 348:163-184.
Criteria for analytical performance and analytical quality control
- Greenfield and Southgate discuss the criteria for analytical performance and quality of analytical data in their book.
Certified Reference Materials/Standard Reference Material
Proficiency testing schemes
Here are listed some completed, on-going and/or upcoming proficiency testing schemes concerning folate:
Some upcoming proficiency testing schemes can be found in the EPTIS database.
Other methods available
- AACC 86-47.01 Total Folate in Cereal Products---Microbiological Assay Using Trienzyme Extraction
- AOAC 944.12 Folic Acid (Pteroylglutamic Acid) in Vitamin Preparations
- AOAC 992.05 Folic Acid in Infant Formula
- AOAC 2004.05A Total Folates in Cereals and Cereal Foods
- microbiological, spectroscopy
- see separate child page below