Folic Acid Analysis on the Lucidity LC-UV

Folic Acid, Folate, or Vitamin B9 is a key vitamin used by the human body to generate new healthy red blood cells, form DNA and RNA, and break down a certain amino acid, homocysteine, that can be harmful in high amounts.  The need for Folic Acid is especially important during pregnancy, which is why there is a lot of interest in Folic Acid as a supplement particularly for women who are planning to become pregnant, are pregnant, or are breast-feeding.  Typically, you can get Folic Acid or Folate from leafy greens, fruits, beans, eggs, and other food sources, but there are a lot of supplements now that allow people to ensure they are getting a desired amount.

Samples:

In this study we analyze the following samples:

Sample A: Folic Acid diet supplement tablet

Sample B: Folic Acid supplement tablet

Sample C: B12 lozenge with Folic Acid

Sample Prep:

Each tablet or lozenge was placed into 25mL of water and agitated at 30C using the SimplePrep for 75 min to make the stock solution.  For Sample A, 300uL of the stock solution was diluted to 1000uL using water to make the final solution.  For Sample B, 150uL of stock solution was diluted to 1000uL using water to make the final solution.  For Sample C, the final solution was the stock solution.

For our standards we started with a Folic Acid standard from Supelco (F-087-1ML), and diluted the standard to 2.50 mcg/mL, 5.00 mcg/mL, and 15.0 mcg/mL in water, which encompassed the range of concentrations in the diluted samples.

Method:

We used the HPLC method shown in Figures 1 and 2 to run the standards and samples.

Figure 1: HPLC method for Folic Acid

Figure 2: HPLC method for Folic Acid

Figures 3 and 4 show the overlaid chromatograms of the 3 concentrations of the Folic Acid standard used to create the calibration curve.  Figure 4 shows a zoomed in view.

Figure 3: The 3 concentrations of the Folic Acid standard used to make the calibration curve

Figure 4: Zoomed in view of the chromatograms from Figure 3

Figure 5 shows the calibration curve using these three standard runs.  The R2 of the calibration curve is >0.999, indicating a reasonable fit of the curve to the standard concentrations.

Figure 5: Calibration curve for Folic Acid

Figures 6,7, and 8 show the chromatograms from Samples A, B, and C, respectively, with the Folic Acid peak at around 3 minutes.  The concentrations of these peaks as determined by the calibration curve is shown in the tables to the right of the chromatograms.  These show concentrations of 7.72 mcg/mL for Sample A, 6.98 mcg / mL for Sample B, and 5.77 mcg/mL for Sample C.

Figure 6: Sample A

Figure 7: Sample B

Figure 8: Sample C

Accounting for the dilutions of each tablet / lozenge during sample prep we arrive at the numbers shown in the table below, which shows the amount of Folic Acid measured in each tablet or lozenge versus the labeled amount of Folic Acid in each tablet / lozenge.

We see recoveries of 86-97% for the 3 samples indicating a reasonable method for determination of Folic Acid in these supplements.  We could potentially improve the recoveries by further optimization of the sample prep, but for the scope of this study, we’re happy with the results.