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Food Analytical Methods

, Volume 5, Issue 5, pp 1167–1176 | Cite as

Multivitamin Analysis of Fruits, Fruit–Vegetable Juices, and Diet Supplements

  • Joanna PłonkaEmail author
  • Agata Toczek
  • Violetta Tomczyk
Open Access
Article

Abstract

Vitamins are organic compounds that are required for various biological functions. In general, vitamins are not synthesized in the human body, but lack or deficiency of them may lead to certain diseases. Determinations of 11 vitamins in various products were performed, which included ascorbic acid (C), seven vitamins of the B group (thiamine B1, riboflavin B2, nicotinamide B3, pantothenic acid B5, pyridoxine B6, folic acid B9, and cyanocobalamin B12), as well as three fat-soluble vitamins (retinol A, cholecalciferol D3, and α-tocopherol E). A column with RP18 stationary phase and a diode array detector with properly selected analytical wavelengths for each compound were used. A gradient of trifluoroacetic acid in water with methanol was used as the mobile phase. Limits of quantification in the range of 0.70–2.90 μg/mL for water-soluble vitamins and 1.85–15.84 μg/mL for fat-soluble vitamins were obtained. Those values are sufficient for determinations of the aforementioned compounds in foodstuff. The developed procedure of sample preparation together with chromatographic system can be used for food quality monitoring in the food industry.

Keywords

Water-soluble vitamins Fat-soluble vitamins Fruits Juices High-performance liquid chromatography SPE 

Introduction

Vitamins are a vital group of food components that must be provided to the human body in sufficient amounts. They support metabolism processes and improve the efficiency of proteins and enzymes. Vitamins are provided in final form or as provitamins that are subsequently transformed into vitamins. The main sources of vitamins are fruits, vegetables, meats, and fish. However, when acquisition from food is insufficient, the vitamins can be acquired from pharmaceutical preparations. Currently, the increased interest in a balanced and wholesome diet has caused increased demand for multivitamin diet supplements.

Vitamins are divided into two groups based on their solubility. The fat-soluble vitamins A, D3, E, and K are non-polar, hydrophobic compounds that are isoprene derivatives. They are absorbed by the human organism also with fat and can be stored in liver, kidneys, and fat tissue. Fat-soluble vitamins perform essential roles in the regulation of height, calcium and phosphorus absorption, and the development and functionality of bones. They also act as anticoagulants. The water-soluble vitamin group contains vitamin C (ascorbic acid), B1 (thiamine), B2 (riboflavin), B3 (nicotinamide), B5 (pantothenic acid), B6 (pyridoxine), B9 (folic acid), and B12 (cyanocobalamin). They have different chemical structures because they represent acid, pyrimidine, and imidazole derivatives as well as acid amides. They are responsible for the proper functioning of the nervous and respiratory systems, synthesis of nucleic and fatty acids, and creation of red blood cells (National Academy of Sciences 1989; Sikorski 2007). Lack or deficiency of vitamins in consumed food can lead to deficiency states and diseases.

A widely used analytical method for the determination of vitamins in food and diet supplements is chromatography, especially high-performance liquid chromatography (HPLC). HPLC is described by high resolution and selectivity, short time of analysis, and ability to couple with other techniques. Literature publications describe numerous methods for vitamin determination; however, particular procedures allow only determining one, two, or several vitamins at the same time, but only from either the fat- or water-soluble group. Table 1 shows parameters of chromatographic procedures for the determination of fat- and water-soluble vitamins. Literature data allow the selection of a chromatographic procedure that enables the determination of vitamins in food samples with adequate concentration levels with the use of both gradient and isocratic systems. The disadvantage of the described procedures is the small number of analytes that can be determined at the same time. Most of the methods allow the determination of several vitamins from one group at once. Most often, it is vitamin A together with E or three to four vitamins of the B group. Only in single particular cases does a chromatographic procedure allow for the simultaneous separation of several compounds of both fat- and water-soluble groups without requirement of stationary or mobile phase changes.
Table 1

Procedures for the selected chromatographic determination methods of vitamins in foodstuff

No.

Vitamins

Mobile phase

Stationary phase

Detector

LOD (ng/mL)

Measurement range (μg/mL)

Sample type

Reference

1

A

A: 0.010% TFA (pH 3.9)

C18. 150 × 4.6 mm (3 μm)

DAD

12.30–12.49

Vitamin preparations

Klejdus et al. (2004)

D

B: MeOH

MS

41.77–129.4

E

Gradient elution 35 min

34.78–127.30

K

83.10–197.40

B1

12.18

B2

188.00

B3

16.41–35.40

B6

21.17–23.08

B9

30.48

B12

97.47

C

25.55

2

B1

A: 0.4 M LiClO4 (pH 2.4)

C18, 75 × 2 mm (5 μm)

UV

Vitamin preparations

Kozhanova et al. (2002)

B2

B: CH3CN

B3

Gradient elution 25 min

B5

B6

B7

B9

B12

A

CH3CN/H2O (95:5, v/v)

D2

D3

E

K3

3

B1

A: 0.05 M CH3COONH4

C18. 150 × 3.9 mm (4 μm)

UV–Vis

3.18

4.93–39.44

Vitamin preparations

Moreno and Salvadó (2000)

B2

B: MeOH

1.84

2.18–17.43

B3

Gradient elution 17 min

9.92

17.53–140.24

B6

1.37

1.78–14.20

B12

0.04 (LOQ)

0.04–0.12

A

CH3CN/MeOH (95:5. v/v)

5.00

4.94–39.49

E

3.09

10.15–81.20

D3

0.05

0.16–1.30

4

B1

A: 5 mM HFBA

C18. 250 × 4.6 mm (5 μm)

MS

3

0.01–50

Vitamin preparations

Chen et al. (2006)

B2

B: MeOH

6

0.01–50

B3

Gradient elution 30 min

8–10

0.02–50

B5

8

0.02–50

B6

1–3

0.01–50

B7

3

0.02–50

B9

9

0.01–50

C

12

0.02–50

5

B1

A: 0.025% TFA (pH 2.6)

C18, 250 × 4.6 mm (5 μm)

UV

1.25–50

Infant foodstuff

Heudi et al. (2005)

B2

B: CH3CN

0.62–25

B3

Gradient elution 17 min

1.25–50

B5

2.5–500

B6

0.125–50

B8

0.5–50

B9

0.6–25

B12

0.25–10

C

5–200

6

B1

A: 16 mM SDS + 0.02 M phosphate buffer (pH 3.6) + 3.5% BuOH

C18. 150 × 4.6 mm (7 μm)

UV

17,000

125–400

Vitamin preparations

Ghorbani et al. (2004)

B2

B: 16 mM SDS + 0.02 M phosphate buffer (pH 3.6) + 10% BuOH

C18. 250 × 4.6 mm (10 μm)

4,000

11–68

B3

Gradient elution 75 min

20,000

95–750

B6

5,000

12–140

B9

7,100

50–175

B12

120

0.2–1.3

C

50,000

100–1500

7

B1

A: 0.1% HCOOH

C18. 220 × 2 mm (5 μm)

DAD

20

0.1–2.0

Vitamin preparations

Chen and Wolf (2007)

B2

B: 0.1% HCOOH in CH3CN

MS

20

0.1–10

B3

Gradient elution 25 min

100

1.0–100.0

B5

20

0.5–50

B6

20

0.1–10.0

B7

20

0.02–0.2

B9

50

0.2–1.0

8

B1

A: 0.0125 M CH3(CH2)5SO3Na in 0.1% H3PO4 (pH 2.4–2.5)

C18. 250 × 4.6 mm (5 μm)

DAD

100

18.4–50.4

Medicated syrup

Vidović et al. (2008)

B2

B: CH3CN

200

7.7–21.6

B3

Gradient elution 60 min

200

119.8–359.0

B5

700

50.3–165.9

B6

100

6.0–17.9

C

400

964.0–3004.0

9

B1

A: MeOH

Amid C16. 150 × 4.6 mm (5 μm)

MS

1

0.005–10

Pasta

Leporatia et al. (2005)

B2

B: 20 mM HCOONH4 (pH 3.75)

3–5

0.005–12.5

B3

Gradient elution 12 min

0.5

0.01–25

B5

5

0.01–50

B6

1–3

0.005–25

B9

5

0.01–50

10

B1

A: Phosphate buffer (pH 6)

Amid C16 (5 μm)

DAD

10

0.05–1.00

Baby food

Viñas et al. (2003)

B2

B: CH3CN

3

0.02–1.0

B3

Gradient elution 45 min

19–38

0.07–1.5

B6

19–20

0.07–1.5

B9

5

0.02–1.0

B12

10

0.05–1.0

11

B1

A: 0.1 M SDS

C18. 120 × 4.6 mm (5 μm)

UV

20

0.5–25

Vitamin preparations

Monferrer-Pons et al. (2003)

B2

B: 4% PnOH in 0.1 M phosphate buffer (pH 3)

3

0.5–25

B3

Gradient elution 22 min

10

0.5–25

B6

5–12

0.5–25

12

B1

A: 0.1% HCOOH

Hydro-RP 250 × 2 mm (4 μm)

MS

Vitamin preparations

Chen et al. (2007)

B3

B: 0.1% HCOOH in CH3CN

B5

Gradient elution 25 min

B6

13

B1

Phosphate buffer/MeOH (90:10, v/v) adjusted to pH 3.55 with 0.018 M TMA + 85% H3PO4

C18. 250 × 4.6 mm (5 μm)

DAD

9.2

22–8200

Juices, sea fruit, vitamin preparations

Lebiedzińska et al. (2007; Marszałł et al. (2005)

B6

ED

0.19–0.29

9.5–2800

B12

0.0021

2–25

14

B1

A: 0.5% TEA + 15% MeOH + 2.4% CH3COOH + 5 mM OSA

C8, 250 × 4.6 mm (5 μm)

DAD

50

Meat

Riccio et al. (2006)

B6

B: CH3CN

50

B12

Gradient elution 17 min

50

15

A

A: MeOH/H2O (90:10, v/v)

C18. 250 × 2 mm (5 μm)

MS

0.0025

0.097–1.783

Human milk

Kamao et al. (2007)

D2

B: CH3CN

0.00005

0.010–1.116

D3

Gradient elution 30 min

0.00005

0–1.300

E

For D3—A :CH3CN/H2O (30:70, v/v)

0.005

0.387–35.664

K1

B: CH3CN

0.0005

0.953–12.382

K2

Gradient elution 35 min

0.0005–0.004

0.074–15.861

16

A

A: MeOH/H2O (99:1, v/v)

C18, 150 × 0,3 mm (3 μm)

UV

0.02

Milk

Gomis et al. (2000)

D2

B: MeOH/THF (70:30, v/v)

2

D3

Gradient elution 16 min

0,2

E

2

K1

0.4

17

A

3% SDS + 15% BuOH adjusted to pH 7 with 0.02 M phosphate buffer

C18. 250 × 4.6 mm (5 μm)

DAD

810

1.0–60

Food, vitamin preparations

Kienen et al. (2008)

D3

910

1.0–40

E

1120

1.0–60

K1

830

1.0–60

18

A

MeOH/H2O (99:1, v/v)

C18, 120 × 2 mm (5 μm)

UV–Vis

Vitamin preparations

Kozlov et al. (2003)

D2

D3

E

19

A

MeOH

C18, 125 × 4 mm (5 μm)

UV–Vis

15–36

1.6–7.7

Vitamin preparations

Paulo et al. (1999)

C

65

5.0–100.7

E

53

0.04–0.19

20

A

Heksan + dioksan + i-PrOH (96.7:3:0.3, v/v/v)

C18 250 × 4.6 mm

MS

0.0014

0.15–12

Infant nutrition

Heudi et al. (2004)

D3

0.000008

0.005–0.4

E

0.00025

0.25–20

21

A

MeOH/H2O (50:50, v/v)

C18. Monolith 270 × 10 mm

UV–Vis

3.9

0.2–2

Cereals

Xu and Jia (2009)

E

173

1–10

K1

16.4

0.2–2

22

D3

CH3CN with Chl + H2TPP

C18. 300 × 3.9 mm (5 μm)

UV

3.5–2300

Vitamin preparations

Lazareva et al. (2002)

E

120

23

A

MeOH/H2O (96:4, v/v)

C18, 250 × 4 mm (5 μm)

UV

Meat, milk, powdered milk

Berg et al. (2000)

E

RP-60 select B 250 × 4 mm (5 μm)

FD

24

A

A: CH3CN/0.04% TFA (1:1, v/v)

C18, 120 × 2 mm (5 μm)

UV

Vitamin preparations

Kozlov et al. (2004)

E

B: CH3CN/MeOH (3:7, v/v)

Gadient elution 15 min

25

A

A: MeOH/H2O (96:4, v/v)

C30. 250 × 4.6 mm (5 μm)

DAD

100–1,100 (LOQ)

10–400

Vitamin preparations

Breithaupt and Kraut (2006)

E

B: TBME/MeOH/H2O (90:6:4, v/v/v)

MS

100–10,000 (LOQ)

10–400

Gradient elution 30 min

26

A

MeOH/H2O (96:4, v/v)

C18, 250 × 4 mm (5 μm)

UV

 

Turner and Mathiasson (2000)

E

27

A

MeOH

C18, 250 × 4.6 mm (5 μm)

DAD

0.33

5–100

Milk

Rodas Mendoza et al. (2003)

E

3.2–32.9

5–100

28

A

0.5% CH3COOC2H5/hexane (1:1, v/v)

50 × 2.1 mm (3 μm)

DAD

0.2–0.3

0.2–5

Baby food

Chávez-Servín et al. (2006)

E

4.9–8.8

1–100

29

A

MeOH/H2O (96:4, v/v)

C18, 250 × 4.6 mm (5 μm)

UV

189/100 g

0.04–3.73

Cooked meals, milk and milk products

Escrivá et al. (2002)

E

8,333/100 g

5.5–550

30

A

76.9 mM SDS/1-BuOH (88.3:11.7, v/v) adjusted to pH 6.37 with 0.02 M phosphate buffer

C18, 100 × 3.9 mm (5 μm)

UV

1,710

150–1,500

Medicated syrup

Momenbeik et al. (2005)

E

4,520

2,000–8,000

31

A

A: CH3CN

C8. 250 × 4.6 mm (5 μm)

DAD

0.58

0.86–17.16

Polymeric diets for enteral nutrition

Kuhn et al. (2008)

E

B: H2O C/MeOH

10

0.86–32.21

Gradient elution 24 min

BuOH butyl alcohol, Chl chlorophyll, EtOH ethyl alcohol, H2TPP tetraphenylporphyrin, HFBA heptafluorobutyric acid, i-PrOH isopropyl alcohol, MeOH methyl alcohol, OSA 1-octanesulfonic acid, PnOH amyl alcohol, SDS sodium lauryl sulfate, TBME methyl tert-butyl ether, TEA 2,2′,2″-trihydroxytriethylamine, TFA trifluoroacetic acid, THF tetrahydrofuran, TMA trimethylamine

Another issue is proper food sample preparation, which would allow the simultaneous extraction of all analytes from a matrix. Solid–liquid and solid phase extraction are the most often used extraction techniques. Most of the procedures described in the literature concern single vitamins of water-soluble vitamins (Hussein et al. 2000; Kurilich et al. 1999; Wang et al. 1996; Franke et al. 2004; Frenich et al. 2005; Gil et al. 2006; Mahattanatawee et al. 2006; Wall 2006; Singh et al. 2007; Lopez-Berenguer et al. 2009) and fat-soluble vitamins (Kurilich et al. 1999; Mahattanatawee et al. 2006; Singh et al. 2007; Ching and Mohamed 2001). Only two papers (Ndaw et al. 2000; Lebiedzińska and Szefer 2006) describe procedures that allow the simultaneous extraction of three to four vitamins from one group. Other studies (Moreno and Salvadó 2000; Munzuroglu et al. 2003) show that it is possible to extract vitamins from both groups; however, this requires a few additional changes in the procedures, like adding a secondary solvent.

Our study presents results of the development of an analytical procedure for the simultaneous extraction as well as simultaneous chromatographic determination of fat- and water-soluble vitamins. After an examination of standard samples, application to real samples was performed. The developed method allows reduction in time for the analysis of fruits, vegetables, and juices, as well as multivitamin diet supplements.

Materials and Methods

Reagents

Standard solutions (10 mg/mL) of retinol (A), cholecalciferol (D3), α-tocopherol (E), thiamine (B1), riboflavin (B2), nicotinamide (B3), pantothenic acid (B5), pyridoxine (B6), folic acid (B9), cyanocobalamin (B12), and ascorbic acid (C) (all from Sigma, St. Louis, MO, USA) were prepared in methanol. Methanol, trifluoroacetic acid (TFA), and water of HPLC grade used for the mobile phase were purchased from Merck (Darmstadt, Germany). Acetic acid 99%, zinc acetate, and potassium ferricyanide (all from POCH, Gliwice, Poland) of analytical grade were used. Standard solutions were prepared directly before use.

High-Performance Liquid Chromatography Conditions

HPLC analysis was performed using a Merck-Hitachi chromatograph equipped with an L6200A pump, L-7360 thermostat, and L4500A diode array detector. Chromatographic separation was carried out on a TSKGel ODS-100V, 150 × 4.6 mm (5 μm) column (Tosoh Bioscience). Examination was performed at a temperature of 30 °C. A gradient elution with 0.01% TFA in water (A) and methanol (B) was applied. The gradient elution parameters are shown in Table 2.
Table 2

Gradient elution parameters

Time (min)

A (%)

B (%)

Flow rate (mL/min)

0

95

5

0.6

4

95

5

0.7

10

2

98

0.7

13

2

98

0.7

15

0

100

1.3

25

0

100

1.3

A—0.01% TFA in water; B—methanol

Detection of vitamins was performed using a diode array detector (DAD) with analytical wavelengths typical for each analyte: retinol, 320 nm; cholecalciferol, 275 nm; thiamine, 253 nm; riboflavin, α-tocopherol, and cyanocobalamin, 290 nm; nicotinamide, 258 nm; pantothenic acid, 218 nm; pyridoxine, 289 nm; folic acid, 360 nm; and ascorbic acid, 262 nm.

Calibration Curves

The calibration curves for the examined compounds were prepared in a matrix of methanolic food extract. Matrix of food samples was purified by solid phase extraction with the same parameters as the extraction of analytes (see “Sample Preparation”). The effluent formed during sample passing through solid phase was collected. Then, appropriate amounts of standard solutions were added.

Curves were determined in a concentration range appropriate for the expected content of analytes in real food samples. The number of experimental points taken for regression was n = 6. Every analyte was injected three times. The volume of each injection was 20 μL. Values of standard deviation of slope and intercept for calibration curves were calculated.

Carrez I and Carrez II Solution Preparation

Carrez I solution was prepared by dissolving 10.6 g of potassium ferricyanide (K4Fe(CN)6⋅3H2O) in 100 mL of distilled water. Carrez II solution was prepared by dissolving 21.95 g of zinc acetate (Zn(CH3COO)2⋅2H2O) and 30 mL of 99% acetic acid in 100 mL of distilled water.

Sample Preparation

Samples were taken from fruits (apricot, avocado), fruit–vegetable juices, and multivitamin diet supplements (powders and tablets). Apricots and avocados were peeled and cut into small pieces. Two grams for each product was weighed and then intermixed with 4 mL of methanol. Subsequently, samples were centrifuged for 5 min (2,000 rpm). The examined material was filtered using a nylon filter with a pore size of 0.45 μm.

One hundred milliliters of fruit–vegetable juice was taken and then 10 mL of Carrez I and 10 mL of Carrez II solutions were added for sedimentation and subsequently centrifuged for 10 min (7,000 rpm). The examined material was filtered using a nylon filter with a pore size of 0.45 μm, and then solid phase extraction (SPE) was performed. A RP18 Bakerbond column (500 mg, 3 mL) was used. The column was conditioned with methanol (1 × 3 mL) and water (1 × 3 mL). Analytes were eluted with portions of the solvents: 3 mL of 60% methanol in water, 3 mL of methanol, and 3 mL of chloroform. The SPE procedure was based on vitamins from multivitamin preparations extraction method (Kozhanova et al. 2002).

Two grams of diet supplements (tablet or powder) were ground, diluted in methanol, and filtered using a nylon filter with a pore size of 0.2 μm. Volumes of methanol used for dilution were matched with declared contents of analytes in the supplement. The volume of the filtered solution for every injection was 20 μL. This volume was taken three consecutive times from each analyzed sample. Matrices for standards which were used for calibration curves were prepared in the same manner as the samples.

Results and Discussion

The developed chromatographic system allows the simultaneous determination of 11 vitamins (both water- and fat-soluble). Dissociation of analytes was determined by a pK a value for each compound and mobile phase pH value. Thus, a solution of TFA with the proper pH value was used. The pH value of the mobile phase was chosen with pK a values of particular analytes taken into consideration to allow analyte separation. The pH value for trifluoroacetic acid with concentration of 0.01% is equal to 3. At the start of the mobile phase, gradient proportion of this acid to methanol is 95:5 (v/v).

With regard to the non-polar stationary phase, B1 and C vitamins eluted first from the chromatographic column. The pK a values of those compounds are 3.8 and 4.2, respectively. Applying a 0.01% solution of TFA allowed increasing the affinity of the compounds to the stationary phase in the initial part of the analysis. It allowed one to determine and elute outside of the dwell time of vitamin B1 and C. Both flow rate of the mobile phase and temperature of the stationary phase column were also subject to examinations. Optimal analytical conditions were obtained at a temperature of 30 °C. The flow rate gradient shown in Table 2 was applied. Detection was performed with the use of a diode array detector. Thanks to this, it was possible to optimize the parameters of analytical wavelengths, which allowed decreasing interferences from the matrix. Calibration curve parameters are shown in Table 3.
Table 3

Retention times and calibration curve parameters for vitamins (n = 6)

Compound

Retention times (min)

Slope

Intercept

Sa

Sb

Measurement range (μg/mL)

R 2

B1

3.36

5,632

5,407

34

1,060

11–86

0.997

C

4.51

2,259

−28,444

68

2,119

80–480

0.996

B6

8.51

7,729

235,405

282

37,424

34–204

0.995

B3

8.99

2,293

−691,107

89

66,487

432–936

0.994

B5

12.48

1,572

13,685

37

1,193

204–408

0.998

B12

13.20

20,253

−324,681

899

34,693

27–48

0.994

B9

13.41

34,595

−163,720

1,082

28,866

17–34

0.996

B2

14.00

15,516

−94,937

636

34,597

35–70

0.993

A

19.12

261

−5,492

3

306

27–108

0.999

D3

22.96

13,556

533,484

476

51,764

70–141

0.995

E

24.05

1,549

−16,579

66

1,072

105–210

0.993

The retention parameters obtained for particular analytes, capacity factor (k′), selectivity (α), and limits of detection (LOD) and quantification (LOQ) values, are shown in Table 4. Limits of detection and quantification values were determined from the signal-to-noise ratio on the assumption that the signal should be three times more intensive than the noise and the LOQ is three times higher than the LOD.
Table 4

Retention parameters for water- and fat-soluble vitamins

Compound

k

R

α

LOD (μg/mL)

LOQ (μg/mL)

B1

3.35

0.93

1.01

0.96

2.90

C

4.50

2.05

1.44

0.90

2.70

B6

8.49

16.73

1.67

0.24

0.72

B3

8.98

4.05

1.36

0.23

0.70

B5

12.47

8.08

1.32

0.30

0.90

B12

13.19

1.91

1.07

0.41

1.22

B9

13.39

1.40

1.05

0.85

2.55

B2

13.99

3.49

1.03

0.83

2.51

A

19.10

41.23

1.35

1.66

4.98

D3

22.95

51.65

1.73

5.28

15.84

E

24.04

5.26

1.81

0.61

1.85

Comparing the obtained LOD and LOQ values with the literature, it can be noticed that in the works which present the application of the same detector, the LOD and LOQ values are slightly lower or comparable (Ghorbani et al. 2004; Vidović et al. 2008; Leporatia et al. 2005). However, these chromatographic systems do not allow the simultaneous determination of such number of analytes. It should also be noted that the obtained LOD and LOQ values are sufficient for the determination of the examined compounds in fruits and juices as well as in diet supplements.

The obtained selectivity factor (α) values above 1 (Table 4) mean total separation of signals for particular vitamins. The obtained LOD and LOQ values were in the range from 0.7 to 2.9 μg in 1 mL of sample for water-soluble vitamins and from 1.85 to 15.84 μg in 1 mL of sample for fat-soluble vitamins. When comparing the obtained LOD values with those from the literature for DAD, one can see that the developed chromatographic system has suitable sensitivity and can be used for the determination of all examined compounds in the chosen food and multivitamin diet supplement samples. Figure 1a shows a chromatogram example of methanol solution of a diet supplement.
Fig. 1

a Chromatogram of multivitamin diet supplement solution. b Chromatogram of apricot extract. c Chromatogram of fruit–vegetable juice extract

Procedures for the simultaneous extraction of both fat-soluble and water-soluble vitamins from fruit and juice samples were developed. Fresh fruits were purchased in stores in Poland, multivitamin juice came from the domestic market, and diet supplements were purchased from pharmacies. For analytes, the extraction from solid matrices, solid–liquid extraction was used. Parameters of this extraction are given above (“Sample Preparation”). Examinations included apricot and avocado because they are rich in fat-soluble and water-soluble vitamins. Avocado samples contained nine vitamins: vitamin C, five vitamins of group B (B1, B3, B6, B9, B2), and A, D3, and E vitamins. Apricot samples additionally contained vitamin B12.

Samples of fruit–vegetable juices were prepared according to the procedure specified above (“Sample Preparation”). Solid phase extraction procedure allowed extracting all eight vitamins (water-soluble C, B1, B3, B6, B9, and B2 as well as fat-soluble A and E), which were present in the juices. Figure 1b, c shows a chromatogram example of apricot extract and a chromatogram of juice extract, respectively. The presence of analytes, in particular with real samples, was confirmed by comparing the absorption spectra in the range of 200–600 nm and adding standard solutions. Signals without labels are derived from the matrix.

Method precision was examined by analysis of recoveries from apricot, avocado, and juice matrices. The procedure for determining the value of recoveries from the solid matrix consisted of the addition of all the vitamin standards to samples of fresh fruit and then extraction with methanol. For juice samples, standard solutions were added to portions of juices, thoroughly mixed, and then representative samples were collected. Afterwards, Carrez solutions were added; the procedure was the same as for real samples. Simultaneously, whole analytical procedure for “sample without added standards” was conducted. A “sample without added standards” was a real sample of the product for which no standards solutions were added as well as no purification from the analytes was made.

The applied solid–liquid extraction (SLE) procedure achieved recoveries of water-soluble vitamins from 78% to 93% for fruit matrices. The SPE procedure achieved recoveries of water-soluble vitamins from 83% to 94% for juices matrices. For fat-soluble vitamins, recoveries were from 80% to 90% for fruits (SLE) and from 86% to 95% for multivitamin juices (SPE). Recoveries are shown in Tables 5 and 6.
Table 5

Precision for vitamins from fruit matrix using SLE

Compound

Input μg/100 g

Avocado

Apricot

Measured μg/100 g

SD μg/100 g

CV (%)

Recovery (%)

Measured μg/100 g

SD μg/100 g

CV (%)

Recovery (%)

B1

85

79

1.44

2

93

68

1.48

2

80

C

355

305

6.15

3

86

311

6.30

3

87

B6

61

51

2.20

1

84

54

2.05

1

88

B3

432

382

4.30

1

88

404

4.45

1

93

B12

28

22

0.64

2

78

B9

20

17

0.86

2

83

18

0.80

2

90

B2

41

37

2.28

3

91

37

2.60

3

90

A

58

48

2.70

3

83

50

2.90

3

86

D3

127

114

1.55

1

90

102

1.21

1

80

E

110

91

3.79

3

83

88

3.81

3

80

Table 6

Precision for vitamins from juice matrix using SPE

Compound

Input μg/100 g

Multivitamins juice

Measured μg/100 g

SD μg/100 g

CV (%)

Recovery (%)

B1

80

75

1.42

3

94

C

355

299

6.56

4

84

B6

61

56

3.20

4

92

B3

432

360

5.10

3

83

B9

18

17

0.91

2

94

B2

41

37

2.66

2

91

A

58

50

2.05

3

86

E

110

104

3.70

2

95

Analyte contents determined in real samples of fruit, juice, and multivitamin preparation are presented in Table 7. Additionally, for the multivitamin preparation, this table contains the manufacturer’s declared values. The obtained results show that the developed chromatographic procedure can be used for the simultaneous separation and determination of water- and fat-soluble vitamins in fruits, juices, and diet supplements. Problems with the determination of B12 and D3 vitamins on the levels of concentration that are present in the juices and diet supplements can be eliminated by concentrating the sample before chromatographic analysis.
Table 7

Contents of water- and fat-soluble vitamins in real samples of fruits, juice, and multivitamin preparation

Compound

Avocado (μg/100 g)

Apricot (μg/100 g)

Multivitamin juice (μg/100 mL)

Multivitamin preparation

SD (μg)

Measured (μg/tab)

Declared (μg/tab)

B1

82.1

40.5

206.5

61.9

60

2.9

C

1,028.6

439.6

8,460.7

2,498.2

2,500

3.1

B6

340.1

12.5

285.1

58.3

60

2.5

B3

1,366.7

843.7

2,698.2

699.1

700

2.7

B5

nf

nf

nf

149.5

150

2.8

B12

nf

58.5

<LOQ

<LOD

0.05

3.0

B9

71.5

16.6

28.3

1.8

B2

186.3

62.9

256.9

68.0

70

2.6

A

187.4

227.9

246.1

31.0

30

3.1

D3

2.8

7.4

<LOQ

<LOD

0.25

0.1

E

838.9

184.6

1,480.3

351.0

350

2.1

nf not found, <LOD/LOQ below LOD/LOQ

Conclusion

The developed chromatographic system allows the simultaneous analysis of 11 vitamins from both groups (water- and fat-soluble) in various food samples. The procedures of sample preparation are rapid and easy to perform. They allow the simultaneous extraction of all analytes and show high recovery values. Altogether, the developed chromatographic system along with the sample preparation procedures can be routinely used in food analysis. It should be emphasized that a chromatographic system together with a sample preparation procedure allows the simultaneous determination of all examined compounds in a very short time, which has never been described before.

Notes

Open Access

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

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Copyright information

© The Author(s) 2012

Authors and Affiliations

  • Joanna Płonka
    • 1
    Email author
  • Agata Toczek
    • 1
  • Violetta Tomczyk
    • 1
  1. 1.Department of Analytical ChemistrySilesian University of TechnologyGliwicePoland

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