LC Simultaneous Determination of the Free Forms of B Group Vitamins and Vitamin C in Various Fortied Food Products

2010, 71, 10691074

va Stefanovits-Ba nyai, La szlo Abranko & Rita Engel, E

nyi u t 29-31, Budapest 1118, Hungary; Department of Applied Chemistry, Faculty of Food Science, Corvinus University of Budapest, Villa E-Mail: laszlo.abranko@uni-corvinus.hu

Received: 11 September 2009 / Revised: 12 March 2010 / Accepted: 15 March 2010 Online publication: 11 April 2010

Abstract
An LCUV screening method for simultaneous determination of ascorbic acid (C), and the free forms of thiamine (B1) riboavin (B2), niacin (B3), pyridoxine (B6) in enriched food products was developed and validated. The chromatographic separation was accomplished within 18 min using a gradient of water with 0.1% formic acid (pH 2.5) and methanol with 0.1% formic acid on a C18 reverse phase column (5 lm, 150 9 3.2 mm) while detection was performed at two wavelengths (266 and 290 nm). Sample preparation was based on an extraction method originally developed for vitamin C. This procedure besides extracting vitamin C was extended to the extraction of the free forms of vitamins B1, B2, B3, B6 and B9. The developed analytical method was successfully applied for the simultaneous determination of the vitamin C content along with the free vitamin B forms of three different enriched food products.

Keywords
Column liquid chromatography Water-soluble vitamins Enriched foodstuff Cereal, cacao and fruit juice

Introduction
Vitamins are a diverse group of organic compounds essential in trace amounts for the normal growth and maintenance of life [1]. To ensure the adequate intake of vitamins the human diet can be com-

pleted with a high range of multivitamin tablets and food products enriched with vitamins. In the case of vitamin enriched food products, the analytical vitamin determination procedures should consider that apart from the added quantity of various vitamins the food may also

contain its own so-called endogenous vitamins [25]. It means that the total amounts present of the vitamins consist of the endogenous free and bound forms together with the added quantity. According to the relevant European regulation this total sum of vitamins is the one that should be given on the label of the food product [6]. This is the reason why the majority of methods are targeting the determination of the total vitamin content. Meanwhile, the same regulation species that the presence of too small and insignicant amounts of added vitamins in fortied foods would not oer any benet to consumers and would be misleading. Thus, enriched food products usually contain signicant amounts of added vitamins. Additionally, the regulation also species that in order to avoid any confusion for the consumer as to the natural nutritional value of fresh foods, the addition of vitamins and minerals thereto should not be allowed. It means that vitamin fortication can only be performed in those groups of foodstus that originally did not contain signicant amounts of endogenous vitamins, thus the added signicant quantities of vitamins dominate the total content. The above facts open the door to develop rapid and cost eective analytical monitoring methods for such special

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food products, since the determination of the bound, endogenous vitamin content (that would need enzymatic sample preparation) is not a strict requirement, because in this case the added vitamins are the major contributors to the total vitamin content. It means that for everyday screening and quality control purposes these less complex methods can be complementary to laborious reference methods. On the other hand it is advantageous if the developed method is able to quantitatively determine the free forms of vitamins (mainly coming from the fortication process). This aspect is important for the indication of any undesirable excess of vitamins in enriched food products. Conventional vitamin analytical methods including colorimetric [7], uorometric [8], spectrophotometric [9, 10], titrimetric [11], microbiological [1214] and immunoassay [14] techniques mostly require the determination of each vitamin individually. When multi-component analyses are required, liquid chromatographic separation techniques [14, 15], quite often reversed-phase liquid chromatography (RP-LC) are preferred to provide separation of the components prior to detection [5, 1619]. Majority of the available multivitamin methods using various detection techniques such as uorimetry [20, 21] UV detection [22, 23] and mass spectrometry (MS) [24, 25] are quite often targeting only the determination of the B group vitamins. Until recently, only a limited number of RP-LC methods were published dealing with the simultaneous determination of B group vitamins and vitamin C [15, 17], moreover most of these methods are mainly applied for analysing only less complex matrices such as polyvitaminated premixes or multivitamin tablets [5, 24]. The reason for the reduced number of multi-component methods equally including B group vitamins and vitamin C might reside in the diering stability of the target analytes. Stability of vitamins is not equally aected by factors such as heat, light and oxygen exposure or reactions with other food components and storage [7, 26, 27]. Most of the B group vitamins are tolerant against heat, so extraction of free B vitamins is quite

commonly carried out by hot water [15, 17]. But in the case of vitamin C one of the least tolerant water-soluble vitaminsthe preservation of its integrity during the analytical procedures is one of the most important issues [2830]. For this reason diluted metaphosphoric acid is the most widely used extractant, as it provides ecient extraction and prevents the analyte from oxidation compared to other acids [31, 32]. Due to the abovementioned signicant dierences between the required analytical procedures for the rapid simultaneous determination of several B group vitamins and vitamin C in food samples is still rare. In this study a simultaneous LCUV method for B1, B2, B3, and B6 along with vitamin C in enriched foodstu has been developed for screening purposes.

phy system (Agilent Technologies, Waldbronn, Germany) equipped with a binary pump, an autosampler, a vacuum degasser system, a column thermostat compartment and a diode array detector were used. Prepared samples were centrifuged with a Hettich Mikro 22R centrifuge (Hettich, Tuttlingen, Germany).

Chromatographic Conditions
The chromatographic separation was performed on a Restek Ultra Aqueous reverse phase C18 column (5 lm, 150 9 3.2 mm) protected with guard column (Restek Corporation, Bellefonte, PA, USA). The ow rate was 0.5 mL min-1 and the injection volume 10 lL. Two detection wavelengths k = 266 and k = 290 nm were used. Two mobile phases were applied for the gradient elution program: 0.1% (v/v %) formic acid in water as solvent A (pH = 2.55) and 0.1% (v/v %) formic acid in methanol as solvent B.

Experimental
Reagents and Samples
All reagents were of analytical grade. All solutions were prepared in high purity water (Milli-Q 18.2 MX cm-1). Vitamin standards such as L-ascorbic acid (C), riboavin (B2), nicotinic acid (B3acid), nicotinamide (B3amid), pyridoxine hydrochloride (B6) and folic acid (B9) were obtained from Fluka (Buchs, Switzerland). Thiamine hydrochloride (B1), hippuric acid and crystalline methaphosphoric acid (HPO3) were purchased from SigmaAldrich (Budapest, Hungary), L-cysteine was from Reanal (Budapest, Hungary), concentrated ammonia and acetic acid (CH3COOH) were from Merck. (Darmstadt, Germany).To prepare mobile phases water, formic acid (HCOOH) (Spektrum-3D, Debrecen, Hungary) and methanol (MeOH) (Carlo Erba Reagents, Milan, Italy) were applied. The examined vitamin enriched cereal, instant cacao powder and fruit juice were commercially available products.

Sample Preparation
Sample preparation was based on the European Standard EN 14130 [32] with some minor modications. Briey, after homogenization, 1 g of cereal sample/ cacao powder or 5 mL of fruit juice were weighed into a 15-mL graded centrifuge tube. Internal standard (1200 lL of 1 mg mL-1 hippuric acid stock solution) was added and the tube was made up to 15 mL with 2% (w/v) metaphosphoric acid solution. After 10 min of sonication at ambient temperature the suspension was centrifuged for 15 min with 4100 g. The supernatant was ltered through a mixed cellulose ester (MCE) membrane (0.45 lm). Into amber asks 5 mL of each ltrate and 2.5 mL of 1% (w/v) L-cystein solution were pipetted and stirred magnetically. In order to adjust to pH of the solution into the range of 7.07.2 according to the preliminary studies 180 lL of 1:5 diluted ammonia solution was added to each vial. After 5 min of stirring the pH was decreased to 2.52.8 by adding 700 lL 20% (w/v) metaphosphoric acid solution. Resulting sample solution was used for chromatography Full Short Communication

Instrumentation
For chromatographic separations an Agilent 1100 Series liquid chromatogra-

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after ltering through a 0.2-lm MCE membrane.

(a)
1

4
4

Standard Solutions
Three standard stock solutions were prepared. 10 mg of vitamin B1, B3amid, B3acid and B6 were weighed into a 10-mL volumetric ask and dissolved in water. After adding 12 lL concentrated acetic acid the ask was made up to the mark with water (solution 1). 10 mg of B2 and 6 mg of B9 were weighed into a 100-mL volumetric ask. Than 50 mL of water and 6.25 mL of ammonia (25% w/v) were added. After the compounds had dissolved completely, the pH of the solution was adjusted to 7 with formic acid. Then the ask was made up to the mark with water (solution 2) [24]. In a 10-mL volumetric ask 24 mg vitamin C was dissolved and made up to the mark with 2% (w/v) metaphosphoric acid solution (solution 3). Multicomponent standard solution was prepared daily. 100 lL of solution 1, 1 mL of solution 2 and 1 mL of solution 3 were pipetted into a 10-mL volumetric ask and made up to the mark with 2% (w/v) metaphosphoric acid. This multicomponent standard mix was the stock solution of calibration. Hippuric acid was used as internal standard. 100 mg of hippuric acid was dissolved in hot water. After cooling, the solution was transferred to a 100-mL volumetric ask and made up to the mark with water [24].

12 10

8
3 2 1

Absorbance (mAU)

8
0

290 nm
5 6 7 8

6 4
2

2
266 nm

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(b)
12 10
1

4
1

Absorbance (mAU)

8 6 4 2 0 0 1 2 3 4 5 6 7 8
2 3
0 5

290 nm
6 7 8

6 8 7 266 nm

9 10 11 12 13 14 15 16 17 18 19

Retention time (min)

Fig. 1. Chromatograms of multicomponent standard solution of water-soluble vitamins (ascorbic acid = 120 lg mL-1, thiamine, nicotinic acid, nicotinamide, pyridoxine, riboavine = 5 lg mL-1, folic acid = 3 lg mL-1) (a) and cereal sample (b) at 266 and 290 nm (1 = ascorbic acid, 2 = thiamine, 3 = nicotinic acid, 4 = pyridoxine, 5 = nicotinamide, 6 = hippuric acid, 7 = folic acid, 8 = riboavine) (stationary phase: Ultra Aqueous C18 5 lm, 150 9 3.2 mm, mobile phases: 0.1% HCOOH/water, 0.1% HCOOH/MeOH, injected volume: 10 lL, ow rate: 0.5 mL min-1)

Results and Discussion

Chromatographic Conditions
After several modications an appropriate chromatographic separation of the examined vitamins were performed using the following gradient program. The eluent composition was initially for 4 min 99.9% A and 0.1% B. In the next 14 min a linear gradient was increased to 60% B. The separation was achieved within 17.8 min. After the acquisition a 7-min-long regeneration step using 90% solvent B was inserted followed by

5-min-long equilibration with the initial mobile phase prior to the next injection. Based on the UV spectra of each vitamin two wavelengths were used for detection. To observe vitamin C, B1, B2, B3acid, B3amid and B9 266 nm were appropriate. To improve the selectivity 290 nm were chosen for vitamin B6 (Fig. 1). Under the described chromatographic condition the void volume well separated from the rst compound of

interest. Retention times of the vitamins with their relative standard deviation (RDS%) values are shown in Table 1.

Method Validation
Standard mixtures were prepared to study the linearity, the limit of detection (LOD) and limit of quantitation (LOQ) of the method. LODs were calculated as

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Table 1. Linear dynamic range, correlation coecients (R2), limits of detection (LOD), limits of quantication (LOQ), retention times (tR) of the LC method
Compound Range (mg L-1) 0.002200 0.0026 0.00910 0.0026 0.0026 0.0023 0.0026 R2 LOQa (ng mL-1) 60 140 10 80 20 8 4 (9 (3 (3 (2 (5 (2 (2 RSD%) RSD%) RSD%) RSD%) RSD%) RSD%) RSD%) LOQb (mg 100 g-1) 0.180 0.420 0.030 0.240 0.060 0.024 0.012 LODc (ng mL-1) 6 9 5 4 5 4 2 td R (min) 2.88 3.27 5.19 9.14 6.16 16.3 17.6 (0.22RSD%) (0.51RSD%) (0.23RSD%) (0.17RSD%) (0.35RSD%) (0.04RSD%) (0.02RSD%)

C B1 B3acid B3amid B6 B9 B2
a, b c

1.000 0.988 0.999 0.999 0.999 0.995 1.000

The lowest concentrations of vitamins where RSD% of precision (n = 3) <10 Three times the standard deviation of the blank sample (n = 3) divided by the slope of the calibration curve d Average retention time of analytes (in standards and samples, n = 12)

Table 2. Results of the extraction eciency gained from comparison of a once and twice extracted samples (n = 3)
Compound C B1 B3acid B3amid B6 B9 B2 Cereal (%) 104 162 88 107 108 80 97 Fruit juice (%) 98 102 98 109 97 107 Cacao (%) 98 105 79 103 100 87

The results were calculated according to the formula: Eciency (%) = [(Areav1/AreaH1)/(AreaV2/AreaH2)] 9 100 AreaV1 peak area of vitamin obtained from the single extraction, AreaH1 peak area of hippuric acid obtained from the single extraction, AreaV2 peak area of vitamin obtained from the double extraction, AreaH2 peak area of hippuric acid obtained from the double extraction

three times the standard deviation of background noise in the retention window of the given compound in a blank sample divided by the slopes of the calibration curves [23]. The LOQ was dened as the lowest concentration of the compounds where the RSD% of the precision was below 10 [33]. The values of LODs, LOQs, and the results of linearity study are summarised in Table 1. The repeatability of the LC method was assessed by injecting three times a multicomponent standard mixture (2 lg mL-1). The RSD% values were in the range of 0.21.9%.

Sample Preparation
Dierent chemical groups of vitamins require specic sample preparation methods. To release the endogenous, bound forms of B group vitamins from the samples enzymatic treatment and

acidic hydrolysis are required. However these steps can cause the degradation of vitamin C present in the sample. Thus, simultaneous determination of watersoluble vitamins can be achieved only by applying a compromised sample preparation technique. Since vitamin C is the least tolerant water-soluble vitamin a sample preparation method originally developed for vitamin C can t for this purpose. However extraction procedures, which can preserve vitamin C from decomposition, may not release the bound forms of the B group vitamins from the samples. In spite of this fact such a compromised extraction technique without further sample clean-up steps can serve screening purposes for compliance monitoring of fortied foodstus, since in these products the free vitamin forms are dominated due to vitamin enrichment. In the light of the aforementioned aims in this study an extraction proce-

dure developed for vitamin C based on EN 14130 European Standard [32] was tested for screening purposes. This sample preparation procedure contains an extraction and a reduction step. Metaphosphoric acid solution (2 w/v %) was used for the extraction to prevent the oxidation of L-ascorbic acid. To ensure the reduction of L-dehydroascorbic acid to L-ascorbic acid L-cysteine as a reducing agent was added to the sample extract solution. The required value of pH = 7 for this step was adjusted by ammonia solution instead of trisodium phosphate solution written in the European Standard. After the reduction step the total amount of vitamin C can be monitored as L-ascorbic acid. Extraction eciencies were examined using a pair of replicate samples from each type of samples being investigated. Both replicates were spiked with hippuric acid as an internal standard. Hippuric acid was chosen because of its structural similarity with the examined vitamins and its absence in foodstu. After spiking both replicates one of them was extracted once, while the other was extracted two times and in the latter case the equal volumes of supernatants were pooled. In order to conrm or reject the necessity of the second extraction step the absolute analyte amounts in the supernatants were compared. The analyte concentrations were deduced from their response relative to that produced by the internal standard. In such cases this procedure is requisite to obtain appropriate comparison, which can be explained with the following: After the Full Short Communication

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Table 3. Concentrations and recovery values of each vitamin in cereal, fruit juice and cacao powder
C Cereal Ccalculated Cindicated Recovery (%) Fruit juice Ccalculated Cindicated Recovery (%) Cacao powder Ccalculated Cindicated Recovery (%) B1 2.3 0.18 2.4 133 6.4 0.4 0.02 0.35 132 5.2 0.85 0.04 0.8 117 0.8 B3acid 2.4 0.05 93 2.6 nd 99 1.2 nd 97 1.7 B3amid 32 0.97 31 104 3.1 4.5 0.30 4.5 100 0.7 17 0.10 14.1 123 17.2 B6 3.4 1.40 3.4 100 1.0 0.42 0.02 0.5 99 2.7 1.85 0.07 1.2 97 2.2 B9 0.52 0.015 0.34 91 13 0.12 0.007 0.05 116 1.4 1.72 0.26 0.18 114 2.1 B2 2.5 0.08 2.7 100 0.5 0.49 0.007 0.4 100 0.6 nd 103 2.4

134 3.22 102 98 3.8 54 2.06 30 100 4.1 74 1.40 43 90 4.0

Ccalculated concentration obtained by the standard addition calibration technique (mg 100 g-1), Cindicated concentration indicated on the package (mg 100 g-1), Recovery (%) mean of the recovery results, nd not detected

removal of the supernatant of the rst extraction the sediment still contains residual amounts of extraction solution (i.e., the sediment is not dry). Consequently the pure solvent of the next extraction besides re-extracting the sediment might liberate additional analytes and also diluting the residual extractant, thus simply dissolving portions of previously extracted analytes into the pure solvent. It results in the enrichment of the amount of analyte in the nal pooled supernatant, however this increment is only due to the aforementioned dilution, i.e., it is not a consequence of additionally extracted compounds. This obtained enrichment might improperly suggest the necessity of the second extraction step. However, if the above procedure is used this enrichment phenomenon analogously occurs in the case of hippuric acid, hence the internal standard corrected concentrations provide the correct comparison results. Extraction eciencies are summarised in Table 2. Based on the results it can be concluded that there is no dierence between the once and twice-extracted samples so the sample preparation procedure described in Experimental is proved suitable to extract water-soluble vitamins from the studied enriched foodstu.

enriched cereal product (Fig. 1), an instant cacao powder and a fruit juice sample. Recovery study was performed by six addition levels for each vitamin in the case of all investigated samples. The results are summarised in Table 3. In most of the cases the recovery varied between 90 and 110%. The reason for the higher dierences observed in the cases of vitamin B1 and B9 can be attributed to chromatographic interferences that especially in the case when the analyte is present in low concentrations caused severe misestimating of the concentrations. Since vitamin B9 is quite often present in much lower concentrations in enriched food products compared to the other investigated B group vitamins in the case of vitamin B9 this problem might occur regularly and cannot be avoided without applying any further sample clean-up. Regarding the other investigated B group vitamins the measured concentrations of the analytes determined by the standard addition technique and the vitamin contents indicated on the packages of the products were in good agreement meaning that the dominant portion of the vitamin content could be extracted with this rapid screening method. These data are also listed in Table 3.

ion pairing reagent. A sample preparation method basically developed for vitamin C was extended for the extraction of the free forms of the given B group vitamins. This chromatographic method combined with the optimised sample preparation methodin contrast to the unspecic detection principle and insucient sample clean-updemonstrates a simple and low-cost solution of screening several B group vitamins on the cost of a single vitamin C measurement in enriched food products. However due to the lack of any sample clean-up the quantitative values should individually be revised for every particular matrix.

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Recovery Study and Real Sample Analysis

The described extraction procedure and the chromatographic method was applied for a commercially available Full Short Communication

Conclusions
The developed and validated RP-LC UV method is proved to be suitable for the simultaneous monitoring of vitamin C, B1, B2, B3, B6 and B9 without using

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