Analysis of Water-Soluble Vitamins Using the Prominence System

Contents
1. Introduction 1 2. Simultaneous Analysis of Vitamin B Group 2 2.1 Target Components 2 2.2 Unit Configuration 2 2.3 Optimizing the Analytical Conditions 2 2.3.1 Setting the Separation Conditions 2 2.3.2 Setting the Detection Wavelength 3 2.3.3 Standard Analytical Conditions 4 2.4 Sample Preparation 5 2.5 Analysis Examples 6 3. Analysis of Other Water-Soluble Vitamins 8 3.1 Setting the Detection Wavelength 8 3.2 Analysis of Cyanocobalamin 9 3.3 Analysis of Ascorbic Acid 10 3.4 Analysis of Carnitine 11 3.5 Analysis of Hesperidin 12 4. Conclusions 13

1. Introduction
Vitamins are essential nutrients and, recently, they have been supplemented with medications or supplements. The analysis method for quantitation of vitamins is shifting from a biological way to a chemical way, and highperformance liquid chromatography (HPLC) is widely used as the analysis method. This paper reports on the analysis methods for water-soluble vitamins in medications or vitamin supplements (Figure 1) using the Prominence HPLC System.

Riboflavin (B2) Thiamin (B1)

Pantothenic Acid (B3, B5)

Riboflavin phosphate (B2) Nicotinic Acid (B3, B5) Nicotinamide (B3, B5, PP) Pyridoxine (B6)

Folic Acid (M)

Biotin (H)

Cynanocobalamine (B12) Ascorbic Acid (C) Carnitene (BT)

Hesperidin (P)

Fig. 1 Structures of Water-Soluble Vitamins

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2. Simultaneous Analysis of Vitamin B Group

2.1 Target Components The vitamin B group includes many components. We investigated a new method for simultaneous analysis of nine water-soluble vitamins (thiamin (vitamin B1), riboflavin and riboflavin phosphate (both vitamin B2), pyridoxine (vitamin B6), nicotinic acid (niacin), nicotinamide (niacinamide), pantothenic acid (vitamin B5), biotin (vitamin B7 or H), and folic acid (vitamin B9 or M)) and caffeine. 2.2 Unit Configuration The Prominence system was used for this study: LC-20AD Solvent Delivery Unit, CTO-20AC Column Oven, SPD-20AV UV-VIS Detector and SPD-M20A Photodiode Array Detector, SIL-20AC Autosampler, and LCsolution Workstation. 2.3 Optimizing the Analytical Conditions 2.3.1 Setting the Separation Conditions To investigate simultaneous analysis of this vitamin B group using reverse-phase chromatography, which is the most widely used with HPLC analysis, the separation conditions must be set to achieve good separation with an appropriate ratio of retention between thiamin and pyridoxine, which have weak retention, and riboflavin, which has strong retention. Thiamin is a strongly basic substance containing quaternary nitrogen in its molecules and carries a positive charge in the mobile phase. It often passes through the reverse-phase column, as no hydrophobic interaction occurs with a solid phase. This problem can be solved by adding an ion-pair reagent with negative charge and a hydrophobic function to the mobile phase. This reagent is hydrophobically retained by the solid phase where it causes an ionic interaction with the target components, allowing reverse-phase ion-pair chromatography to be used. Alkyl sulfonates are often used as ion-pair reagents for strongly basic substances, such as sodium octanesulfonate or sodium pentasulfonate. Generally, the retention of the target components increases as the length of the alkyl-chain in the ion-pair reagent gets longer and as the ion-pair reagent concentration rises (up to a certain concentration). Conversely, as riboflavin has strong retention, an organic solvent (acetonitrile or methanol) must be added to the mobile phase to accelerate its elution. As described above, in the case of the reverse-phase chromatography for this vitamin B group, the ion-pair reagent must be added to selectively strengthen the retention of thiamin and pyridoxine, and the ratio of organic solvent in the mobile phase must be controlled to reduce the analysis time, while ensuring the separation of all components. For this analysis, the mobile phase conditions were optimized using a Shim-pack VP-ODS (4.6 mm ID x 150 mm long) reverse-phase chromatography column according to the procedure below. 1) o strengthen the retention of pantothenic acid, riboflavin phosphate, and folic acid, the pH of mobile T phase was set to the acidic side and the organic solvent concentration was reduced. In addition, the salt concentration was increased in consideration of the salting-out effect.

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2) lkyl sufonates were added as an ion-pair reagent to the mobile phase to retain basic substances such A as thiamin, pyridoxine, and biotin. We investigated the retention behavior of basic substances using several types of alkyl sulfonate with different chain lengths for several combinations of the organic solvent concentration (set in 1) above and alkyl sulfonate concentration. As a result of this investigation, we adopted a mobile phase comprising 100 mmol/L phosphoric acid (sodium) buffer solution (pH 2.1) containing 0.8 mmol/L sodium octanesulfonate mixed with acetonitrile (mixing ratio 19/2). 2.3.2 Setting the Detection Wavelength Figure 2 shows the UV absorption spectra for the vitamin B group and caffeine using the mobile phase set in section 2.3.1. Table 1 shows the wavelength of maximum absorbance for each component. The pantothenic acid was detected at a short wavelength of 210 nm, while other components having enough absorption near 250 to 300 nm were detected at 270 nm where impurity compornents had less of an adverse effect. Simultaneous detection is possible using the SPD-M20A photodiode array detector or using the SPD-20A/20AV UV-VIS detectors two-wavelength simultaneous measurement function.

Nicotinamide Thiamin

Riboflavin Nicotinic Acid Folic Acid

Riboflavin Phosphate

Caffeine

Pyridoxine

Biotin Pantothenic Acid

Fig. 2 UV Absorption Spectra of the Vitamin B Group and Caffeine Table 1 Wavelength of Maximum Absorbance for the Vitamin B Group Component Thiamin Riboflavin Riboflavin Phosphate Pyridoxine Nicotinic Acid Nicotinamide Pantothenic Acid Biotin Folic Acid Wavelength of Maximum Absorbance (nm) 192, 247 190, 223, 267, 373 190, 223, 267, 373 190, 290 192, 210, 260 192, 210, 260 192 192 195, 285

 How to prepare 100 mmol/L phosphoric acid (sodium) buffer solution (pH 2.1) Sodium dihydrogenphosphate dehydrate (MW = 156.01) 50 mmol (7.8 g) Phosphoric acid (85%, 14.7 mol/L) 50 mmol (3.4 mL) Dissolve the above in water and make up to 1000 mL.

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2.3.3 Standard Analytical Conditions The standard analytical conditions shown in Table 2 were set based on the investigation results.
Table 2 Analytical Conditions Column Mobile Phase : Shim-pack VP-ODS (150mmL. x 4.6mmi.d.) : A) 100 mmol/L (Sodium) phosphate buffer (pH 2.1) containing 0.8 mmol/L sodium 1-octanesulfonate B) Acetonitrile A / B = 19 / 2 (v/v) : 1.2 mL/min : 40C : 210 nm, 270 nm

Flow Rate Column Temp. Detection

Figure 3 shows the analysis results of a standard mixture of the vitamin B group and caffeine.
Peaks 1. Niacin 2. Nicotinamide 3. Ca Pantothenate 4. Pyridoxine 5. Riboflavin Phoshate 6. Thiamin 7. Caffeine 8. Folic Acid 9. Biotin 10. Riboflavin
10 9

2 1

210nm

3 5

(20mg/L each, 10L inj.)

12

270nm
7

10

4 8

Fig. 3 Chromatograms of a Standard Mixture of the Vitamin B Group and Caffeine

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2.4 Sample Preparation Table 3 shows the solubility and stability of the vitamin B group and caffeine. Considering that thiamin and pyridoxine have strong adsorption to the glass surface and sample matrix, an acid solution is effective for sample preparation. However, riboflavin, riboflavin phosphate, biotin, and folic acid have poor solubility below neutral pH. Therefore, they were first dissolved in an aqueous sodium hydroxide solution and then diluted with an acidic solution. The sample must be prepared in the dark as many components are unstable to light.

Table 3 Solubility and Stability of Vitamin B Group and Caffeine Compound Name Thiamin Riboflavin Riboflavin Phosphate Pyridoxine Nicotinic Acid Nicotinamide Pantothenic Acid Biotin Folic Acid Caffeine Solubility Acidic Weakly Basic Acidic Stability Weakly Basic Light

( : good : bad : unknown)

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2.5 Analysis Examples Figure 4 shows an example of the analysis of a commercial vitamin tablet (dietary supplement). 100 mg of the pulverized tablet was added to 20 mL 100 mmol/L sodium hydroxide solution and subjected to ultrasonic extraction for 10 minutes. After centrifuging, 1 mL supernatant was made up to 50 mL with mobile phase, and filtered through a 0.45 m membrane filter. 10 L of this sample was injected for analysis.

210nm

Peaks 1. Nicotinamide 2. Ca Pantothenate 3. Pyridoxine 4. Thiamin 5. Riboflavin

5 4

270nm

Fig. 4 Chromatograms of a Vitamin Tablet

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Figure 5 shows an example of the analysis of a commercial drink (nonmedicinal product). 1 mL of the sample was made up to 10 mL with mobile phase, and filtered through a 0.45 m membrane filter. 10 L of this sample was injected for analysis.

210nm

Peaks 1. Nicotinamide 2. Pyridoxine 3. Riboflavin Phoshate 4. Thiamin 5. Caffeine

2 3 4

270nm

2 3 4

Fig. 5 Chromatograms of a Vitamin Drink

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3. Analysis of Other Water-Soluble Vitamins

This section introduces some examples of the analysis of other major water-soluble vitamins: cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), carnitine (vitamin BT), and hesperidin (vitamin P). 3.1 Setting the Detection Wavelength Figure 6 shows the UV absorbance spectrum for each component. Table 4 shows the wavelength of maximum absorbance for each component.

Carnitine

Ascorbic Acid

Hesperidin

Cyanocobalamin

Fig. 6 UV Absorption Spectra of Water-Soluble Vitamins

Table 4 Wavelength of Maximum Absorbance for Water-Soluble Vitamins Component Cyanocobalamin Ascorbic Acid Carnitine Hesperidin Wavelength of Maximum Absorbance (nm) 190, 278, 362, 550 243 190, 204 199, 283

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3.2 Analysis of Cyanocobalamin As cyanocobalamin is strongly retained in a reverse-phase chromatography column, acetonitrile was added to the mobile phase to reduce the elution time. As the content of cyanocobalamin in vitamin tablets is only 1/1000 to 1/3000 of the other component, it was necessary to set the detection wavelength to 550 nm to inhibit the effects of the interfering components, and to increase the absolute amount of cyanocobalamin for analysis. Figure 7 shows the chromatogram of a cyanocobalamin standard solution (10 mg/L, 10 L injected volume) under the analytical conditions shown in Table 5. Figure 8 shows the chromatogram of a commercial vitamin tablet (supplement). 9.6 g of the pulverized tablet were added to 10 mL 1 mmol/L sodium hydroxide solution and subjected to ultrasonic extraction for 10 minutes. This was made up to 100 mL with mobile phase A, subjected to ultrasonic extraction for 10 minutes, and filtered through a 0.45 m membrane filter. 10 L of this sample was injected for analysis.
Table 5 Analytical Conditions Column Mobile Phase : Shim-pack VP-ODS (150mmL.x 4.6mmi.d.) : A) 100 mmol/L (Sodium) phosphate buffer (pH 2.1) B) Acetonitrile A / B = 8 / 1 (v/v) : 1.2 mL/min : 40C : 550 nm

Flow Rate Column Temp. Detection

Peaks 1. Cyanocobalamimn

Peaks 1. Cyanocobalamimn

Fig. 7 Chromatogram of Cyanocobalamin

Fig. 8 Chromatogram of Vitamin Tablet

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3.3 Analysis of Ascorbic Acid Ascorbic acid can be analyzed by reversed-phase chromatography with tetrabutylammonium added as an ion-pair reagent. Generally, however, ascorbic acid is analyzed by normal-phase chromatography using an NH2 column, which provides good separation from other vitamins. Figure 9 shows the chromatogram of an ascorbic acid standard solution (100 mg/L, 10 L injected volume) under the analytical conditions shown in Table 6. Figure 10 shows the chromatogram for a commercial soft drink. The sample was diluted 100 times in 1% (w/v) metaphosphoric acid and filtered through a 0.45 m membrane filter. 10 L of this sample was injected for analysis.

Table 6 Analytical Conditions Column Mobile Phase : NH2P-50 4E (250mmL. 4.6mmi.d.) : A) 100 mmol/L (Triethanolamine) phosphate buffer (pH 2.2) B) Acetonitrile A / B = 1 / 4 (v/v) : 1.0 mL/min : 40C : 240 nm

Flow Rate Column Temp. Detection

* phosphoric acid 6.8 mL + triethanolamine 7.46 g + water total 1000 mL

Peaks 1. Ascorbic Acid

Peaks 1. Ascorbic Acid

Fig. 9 Chromatogram of Ascorbic Acid

Fig. 10 Chromatogram of Soft Drink

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3.4 Analysis of Carnitine As carnitine is weakly retained in a reverse-phase chromatography column and tailing occurs readily, sodium perchlorate was added to the mobile phase under an acidic condition to increase the retention effect and restrict tailing. Figure 11 shows the chromatogram of a carnitine standard solution (1000 mg/L, 10 L injected volume) under the analytical conditions shown in Table 7.

Table 7 Analytical Conditions Column Mobile Phase Flow Rate Column Temp. Detection : Shim-pack VP-ODS (150mmL. x 4.6mmi.d.) : A) 10 mmol/L (Sodium) phosphate buffer (pH 2.6) containing 0.2 mol/L sodium perchlorate : 1.0 mL/min : 40C : 210 nm

Peaks 1. Carnitine

Fig. 11 Chromatogram of Carnitine

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3.5 Analysis of Hesperidin As hesperidin is strongly retained in a reverse-phase chromatography column, like cyanocobalamin, acetonitrile was added to the mobile phase for the analysis. Figure 12 shows the chromatogram of a hesperidin standard solution (100 mg/L, 10 L injected volume) under the analytical conditions shown in Table 8.

Table 8 Analytical Conditions Column Mobile Phase : Shim-pack VP-ODS (150mmL. x 4.6mmi.d.) : A) 100 mmol/L (Sodium) phosphate buffer (pH 2.1) B) Acetonitrile A / B = 3 / 1 (v/v) : 1.5 mL/min : 40C : 265 nm

Flow Rate Column Temp. Detection

Peaks 1. Hesperidin

Fig. 12 Chromatogram of Hesperidin

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4. Conclusions
Analysis examples of the vitamin B group and other water-soluble vitamins using the Prominence system were introduced. These medications or vitamin supplements can be analyzed after simple pretreatment, as they contain relatively low levels of impurity components and high levels of the target components. However, some skill is required with the detection method and pretreatment method for the analysis of natural samples. In some cases, post-column derivatization is used to enhance sensitivity and selectivity for the analysis of water-soluble vitamins. These analyses were previously reported in Shimadzu Application News, and we intend to keep studying and introducing them in Application Reports in the future.

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