Determination of Ascorbic Acid and Dehydroascorbic Acid in Different Food Products and Supplements - A Simple HPTLC Based Approach

Markus Burholt, Scientist Instrumental Analytics R&D, Monika Bäumle, Global Product Manager Thin-Layer Chromatography

Merck

Article from Analytix Reporter - Issue 14

Introduction

Vitamin C or ascorbic acid is a vitamin found naturally in many fruits and some vegetables or is added to certain processed foods or dietary supplements. It is water-soluble and has antioxidative capacities by degrading to dehydroascorbic acid. The human organism cannot produce ascorbic acid and it must be ingested through food or supplements. It has essential functions in human body and maintains numerous vital processes. When vitamin C is deficient (scurvy), symptoms can occur such as fatigue, tiredness, and inflammations. The recommended daily dose of ascorbic acid is about 100 mg per day and can easily be reached with a healthy, balanced diet. Typically, ascorbic acid is quantified by iodometric titration according USP method.¹ An additional substance identification is required and performed by e.g. infrared analysis.¹

In the following application, we show an easy and fast screening approach for the simultaneous quantitative analysis of ascorbic acid and dehydroascorbic acid by High Performance Thin Layer Chromatography (HPTLC). Thin layer chromatography (TLC) and HPTLC are convenient, fast, and efficient separation techniques that enable the development of analytical methods without the need for extensive sample preparation or high investments.² When combined with MS, a subsequent substance identification is possible. Low cost and short analysis time per sample are given by the parallel analysis of ascorbic acid in food products on one plate. Furthermore, the high matrix tolerance of TLC offers additional opportunities to existing routine methods. The high viscosity and high sugar content of many ascorbic acid products (e.g. fruit juice) makes them very complex and matrix-rich samples to analyze.

Experimental Conditions

Five different commercially available ascorbic acid containing products, like juice concentrate, fruit gums, vitamin C effervescent tablet, multi vitamin effervescent tablet, and a tablet with cranberry extract were analyzed using conditions in Table 1.

Table 1. Experimental conditions for determination of ascorbic acid and dehydroascorbic acid

HPTLC Conditions
Plate:	HPTLC Silica gel 60 F254s, 20x10cm (1.15696)
Application volume:	0.3 – 5.0 µL (as indicated in Tables 2 &4a), bandwise application, 6 mm bands
Detection:	366 nm
Chamber:	20x10 Chamber without filter paper
Mobile phase:	Acetone/toluene/formic acid 60:30:10 (v/v/v)
Migration distance:	6 cm
hRf:	Ascorbic acid= 45; Dehydroascorbic acid= 58
Drying:	50 °C
Samples
Solvent mixture:	Ethanol/water 1:1 (v/v)
Standard preparation:	Ascorbic acid: about 10 mg of ascorbic acid was weighed in a 10.00 mL volumetric flask and filled up with a solution of solvent mixture Dehydroascorbic acid: about 10 mg of dehydroascorbic acid was weighed in a 10.00 mL volumetric flask and filled up with a solution of solvent mixture
Samples:	Juice concentrate: 1.0 mL of the juice was transferred to a volumetric flask and filled up with the solvent mixture Fruit gums: the fruit gums were stored in a freezer for several hours. Afterwards they were grounded with a mixer (blender) for a few seconds. About 2.0 g of the chunks were weighed into a flask, filled up with 10.0 mL solvent mixture and centrifuged. The supernatant was used for the analysis Vitamin C effervescent tablet: the tablet was pulverized with a mortar. About 250 mg was weighed into a flask and filled up with 10.0 mL solvent mixture Multi vitamin effervescent tablet: the tablet was pulverized with a mortar. About 250 mg was weighed in a flask and filled up with 10.0 mL solvent mixture Tablet with cranberry extract: the tablet was pulverized with a mortar. About 100 mg was weighed in a flask and filled up with 10.0 mL solvent mixture
HTPLC-MS
MS Measurement:	Extraction solvent: Acetonitrile/water 95:5 (v/v) + 0.1% formic acid The samples were extracted with the Plate Express and measured with the single-quadrupole mass spectrometer expression CMS from Advion MS Mode: ESI Mode-, Spectrum 1, ascorbic acid, Measured molecule mass 175.0 (M-H). Spectrum 2, dehydroascorbic acid, measured molecule mass 173.1 (M-H)

Due to its oxidative capacity, ascorbic acid gets rapidly decomposed and dehydroascorbic acid is formed. A reliable quantification of ascorbic acid can be challenging and requires a gentle but rapid analysis of the samples. In practice, ascorbic acid might be quantified together with a low amount of its dehydration product dehydroascorbic acid. (Figure 1). To simulate this effect, in this experiment, standards of ascorbic acid were over-spotted with dihydroxyascorbic acid.

Figure 1. Chemical structure of ascorbic acid and dehydroascorbic acid.

Calibration curves of ascorbic acid and dehydroascorbic acid were established based on 3 different applied standard volumes (Table 2). After separation, a fast and simple substance confirmation by MS was performed.³

Table 2. Calibration solutions applied

Spots	Application volume (µL)	Description
1, 9, 17	1.5	Ascorbic acid standard solution, applied first to standard tracks
2, 10, 18	3.0
3, 11, 19	5.0
1, 9, 17	0.3	Dehydroxyascorbic acid standard solution, applied over ascorbic acid spots.
2, 10, 18	1.0
3, 11, 19	2.0

The samples and standards were applied bandwise (6.0 mm). At first, the concentration series of the ascorbic acid standards were applied and afterwards over-spotted with the dehydroascorbic acid standards. Due to expected lower concentration of dehydroascorbic acid, the sample volume was lower than for ascorbic acid.

The plate was developed with the mobile phase and afterwards dried at 50 °C until completely dry. To quantify, the plate was heated at 110 °C for 10 minutes. Examination of the plate was done at 366 nm.

Results and Discussion

At 366 nm illumination, ascorbic acid appears at hR_f 45 and dehydroascorbic acid at hR_f 58 (Figure 2). MS measurement of the spots (before heating) were carried out to confirm substance identification.³

Figure 2. Visualization of the plate under UV 366 nm. Ascorbic acid appears at hR_f 45 and dehydroascorbic acid at hR_f 58.

The calibration solution profiles (Table 2) at 366 nm were used for establishing the calibration curves and quantification (Table 3, Figure 3 & 4) related to amount applied to the plates.

Table 3. Results of 3 calibration solutions for ascorbic acid and dehydroascorbic acid

Compound	Amount (µg)	Mean Area (AU)
Ascorbic acid	1.54	0.00647
	3.08	0.01144
	5.15	0.01867
Dehydroascorbic acid	0.30	0.00461
	1.00	0.01286
	1.99	0.02822

Figure 3. Calibration plot with corresponding calibration function of ascorbic acid.

Figure 4. Calibration plot with corresponding calibration function of dehydroxyascorbic acid.

The calibration data was used to quantify the vitamin C content of the five applied samples. In all 5 samples ascorbic acid and also dehydroascorbic acid could be determined. The results are displayed in Table 4 a & b.

Table 4a. Quantitative Results of measured samples

Sample	Application position	Conc. Sample (mg/ mL)	Application- volume (µL)	Substance	Mean Area (AU)	Mean Amount (µg)	RSD %	Vitamin Content in the Sample Solution (mg/mL)
Juice concentrate	4, 12, 20	100.0	4.0	Ascorbic acid	0.01046	2.790	2.01	0.70
				Dehydroascorbic acid	0.01113	0.862	3.38	0.22
Fruit gums	5, 13, 21	200.0	3.0	Ascorbic acid	0.01076	2.881	2.08	0.96
				Dehydroascorbic acid	0.00749	0.562	3.74	0.19
Vitamin C effervescent tablet	6, 14, 22	25.0	2.5	Ascorbic acid	0.01026	2.731	3.63	1.09
				Dehydroascorbic acid	0.01014	0.783	4.78	0.31
Multi Vitamin effervescent tablet	7, 15, 23	24.9	3.5	Ascorbic acid	0.01254	3.412	2.25	0.97
				Dehydroascorbic acid	0.01604	1.225	1.87	0.35
Tablet with Cranberry	8, 16, 24	9.9	5.0	Ascorbic acid	0.01368	3.744	0.90	0.75
				Dehydroascorbic acid	0.00676	0.498	0.95	0.10

Table 4b. Calculated recovery rates (expected values are data listed on the packages of the tested products)

Sample	Conc. Sample (mg/ mL)	Substance	Vitamin content in the Sample Solution (mmol/L)	Combined Content (mmol/L)	Expected Content (mmol/L)	Recovery Rate %
Juice concentrate	100.0	Ascorbic acid Dehydroascorbic acid	3.960 1.238	5.197	3.407	153
Fruit gums	200.0	Ascorbic acid Dehydroascorbic acid	5.452 1.075	6.527	17.016	38
Vitamin C effervescent tablet	25.0	Ascorbic acid Dehydroascorbic acid	6.202 1.799	8.001	5.683	141
Multi Vitamin effervescent tablet	24.9	Ascorbic acid Dehydroascorbic acid	5.535 2.010	7.545	4.480	168
Tablet with Cranberry	9.9	Ascorbic acid Dehydroascorbic acid	4.252 0.571	4.823	4.480	108

Conclusion

The developed application procedure provides a simple method based dehydroascorbic acid and ascorbic acid analysis in different kind of samples and matrices by HPTLC. This easy and straightforward approach represents an alternative method for a reliable screening of vitamin C in food & beverage samples.

It provides a fast, economic, and simple semi-quantification of ascorbic acid and dehydroxyascorbic acid, and also demonstrates the main advantages of the TLC approach, such as quick sample preparation, high matrix tolerance, and high-throughput.

To find more on applications for food testing see SigmaAldrich.com/food.

References

1. United States Pharmacopeia (2022). USP Monographs Dietary Supplement Monographs NF Monographs, Ascorbic Acid. USP-NF. Rockville, MD: United States Pharmacopeia. https://doi.org/10.31003/uspnf_m6030_04_01

2. Schulz M, Oberle M, Burholt M, Baeumle M. 50 years of TLC-MS Thin-Layer Chromatography coupled to Mass Spectrometry and new perspectives by complementary use to HPLC as demonstrated in testing of honey Analytix Reporter. 2019;5: 3-7. [Internet]. Available from: https://www.sigmaaldrich.com/analytix

3. Unpublished results. Contact the author to get more details.