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Folic acid content in fortified cereals
Online Cleanup of Sample or Matrix Components
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printable Application Sheet
Notes: Folic acid (pteroyl-L-monoglutamic acid) is a member of a biologically important class of compounds known as folates and is added to
many foods and beverages by what is referred to as fortification. To verify that the correct amount of folic acid has been added to fortified
products, reliable analytical methods are needed for its quantitation in situ. Folate metabolites play a vital role in nutrition. Deficiency of
folates in the diet leads to an accumulation of homocysteine [1]. Elevated levels of homocysteine, in turn, have been shown to inhibit purine
biosynthesis [2].
1. C. M. Ulrich, M. C. Reed, H. F. Nijhout, Nutr. Rev. 66 (2008) S27–S30.
2. Y. Fujita, E. Ukena, H. Iefuji, Y. Giga-Hama, K. Takegawa, Microbiology. 152 (2006) 397.
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Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| Catalog No. |
70000-7.5P |
| Dimensions |
4.6 x 75 mm |
| Solvents |
A: DI water + 10 mM ammonium formate
B: 90% acetonitrile/10% DI water/10 mM ammonium formate
Both solutions were vacuum filtered through a 0.45 µm nylon filter (MicroSolv Technology Corp). |
| Gradient |
| Time (min) | %B | Time (min) | %B |
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| 0 | 100 | 19 | 50 |
| 10 | 90 | 20 | 100 |
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| Post time |
5 min. |
| Flow rate |
0.5 mL/min. |
| Samples |
Fortified whole wheat flour-based cereals.
Ground, dispensed in DI H2O + 10 mM ammonium formate + 0.05% (w/v) sodium L-ascorbate + 12 mM NH3. Next sample was centrifuged at
10,000 g. The supernatant was then collected and filtered through a 0.45 µm nylon membrane HPLC filter prior to HPLC-UV injections. (MicroSolv Technology Corp.) |
| Peaks |
1. Folic acid
2. Methotrexate: internal standard |
| Detection |
UV 284 nm |
Discussion
This application note demonstrates an effective means of separating and resolving folic acid from undesired matrix components in cereal extracts
without the use of SPE or other sample cleanup techniques. SPE is normally performed to remove compounds from the matrix which would co-elute with
folic acid before injection but by performing the separation shown above, this step can be virtually omitted. Many of the matrix components in this
cereal are less polar than folic acid and therefore elute earlier in the HPLC run. In this manner, the separation can act as an online sample cleanup
and eliminate chances of losing any folic acid during this step. This helps to provide better reproducibility.
The precision of this method is clearly demonstrated by the low %RSD (0.1% and below) of the folic acid retention times. The calibration curve showed
good linearity (R2 = 0.9997).
Results obtained by this method agree with the amount of the folic acid reported by the manufacturer. Methotrexate was used as an internal standard
for the developed method.
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