Analytica Chimica Acta 396 (1999) 5560

Micellar electrokinetic capillary chromatography analysis of water-soluble vitamins

Domingo Blanco Gomis , Luis Laviana Gonzlez, Dolores Gutirrez lvarez
Departamento de Qu mica F sica y Anal tica, Universidad de Oviedo, 33006, Oviedo, Spain Received 8 December 1998; received in revised form 23 March 1999; accepted 26 April 1999

Abstract This paper describes the development of an electrophoretic method used to analyse seven water-soluble vitamins, and its use in the analysis of pharmaceutical formulations. Thiamine, nicotinamide, nicotinic acid, pyridoxine, ascorbic acid, folic acid and riboavin were separated from one another in 7 min by micellar electrokinetic capillary chromatography using a carrier containing sodium dodecyl sulphate as surfactant, and were subsequently detected spectrophotometrically using a diode-array detector. The limits of detection (S/N = 3) ranged between 0.08 g ml1 for folic acid and 0.25 g ml1 for nicotinamide and thiamine. The relative standard deviation of the proposed method was less than 6% for all formulation samples. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Water-soluble vitamins; MEKC

1. Introduction It is well known that vitamins are a group of indispensable compounds for the development and normal growth of living beings, their absence being the cause of serious physiological problems. There is no doubt that vitamin analysis is becoming more and more widespread, and hence a rapid, easy and reliable method for vitamin analysis is required by the food and pharmaceutical industries. In recent years, liquid chromatography (LC) techniques have been used for this purpose. In order to analyse vitamins by LC, reversed-phase (RP) LC is required (by using an ion-pair technique [1,2] or gradient
Corresponding author. Tel.: +34-9-8510-3490; fax: +34-9-85103125 E-mail address: dbg@sauron.quimica.uniovi.es (D.B. Gomis)

elution [3,4]) with the resulting increased costs and problems of reproducibility. Furthermore, the efciency of this methodology is poor and peak tailing occurs. A simple, cheap and efcient alternative approach to the chromatographic devices employed for these analyses has been the use of capillary electrophoresis (CE). In fact, of the several operational modes existing in CE, capillary zone electrophoresis (CZE) has been used by different authors to separate mixtures of water-soluble vitamins such as total ascorbic acid [5], B group vitamins [6,7] and also mixtures containing other water-soluble vitamins [8]. In these studies, phosphate, borate or phosphate-borate mixtures (pH 79) were used as running buffers. However, neutral species cannot be separated by CZE. In order to extend the advantages of CE to neu-

0003-2670/99/$ see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 9 9 ) 0 0 3 6 3 - 3

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tral compounds, micellar electrokinetic capillary chromatography (MEKC), rst introduced by Terabe and co-workers [9], was developed. The separation and analysis of charged and uncharged analytes [10,11] is made possible by the inclusion of micelle-forming surfactants in the electrophoretic medium. The most commonly used surfactants for analysis of water-soluble vitamins are alkyl sulphates such as sodium dodecyl sulphate (SDS). The papers published in this eld have demonstrated the applicability of MEKC in the separation of mixtures of vitamins of the B group [1214], as well as other water-soluble vitamins such as ascorbic acid, folic acid, cyanocobalamin and calcium pantothenate [1518]. From the optimisation studies carried out in these reports, it could be concluded that the best separation of vitamins was obtained at an SDS concentration between 25 and 50 mM [12,14,15,17,18], although the use of SDS concentrations from 10 to 100 mM have been reported [13,19]. Some authors [19,20] have reported the use of CZE to separate mixtures of water-soluble vitamins as it is the simplest operational mode in CE; but as is known, they could not resolve uncharged vitamins. The addition of surfactants to the running buffer is employed to overcome this problem. Thus, Fotsing et al.[20] reported the separation of ten water-soluble vitamins by CZE in about 10 min by means of a 50 mM borax buffer at pH 8.5, the anionic surfactant SDS being added to the running buffer at 25 mM to separate cyanocobalamin from nicotinamide. Recently, bile salts such as sodium cholate have been proposed as the surfactant for determining 14 water-soluble vitamins and vitamin cofactors. This alternative to anionic surfactants avoids the concurrence of two interactions, hydrophobic and ionic/ion pairing, between the micelles and positively charged vitamins, which complicate the predictions of migration times [16]. This paper reports the MEKC separation of seven water-soluble vitamins. The vitamins were separated and quantitated in a pharmaceutical formulation using UV detection. The inuence of the nature of the buffer on migration time and separation of the seven vitamins was evaluated. Limits of detection, linearity, reproducibility and recoveries are presented for these analytes.

2. Experimental 2.1. Apparatus A Hewlett-Packard (Waldbronn, Germany) threedimensional capillary electrophoresis system was used in all measurements. The fused silica capillary (48.5 cm 50 m i.d., effective length 40.0 cm) was supplied by Hewlett-Packard. 2.2. Chemicals Thiamine hydrochloride, pyridoxine, nicotinamide, nicotinic acid, folic acid, and riboavin were obtained from Sigma (St. Louis, MO), L-ascorbic acid, disodium hydrogenphosphate 2-hydrate, Tris (hydroxymethyl)aminomethane (Tris) and [2-(N-Morpholino) ethane sulfonic acid] (MES) were purchased from Merck (Darmstadt, Germany). Phosphoric acid was obtained from Probus (Badalona, Spain). All were of analytical-reagent grade. Milli-Q water (Millipore, Milford, MA) was used throughout. 2.3. Sample and buffer preparation The standard vitamins thiamine, nicotinamide, nicotinic acid, pyridoxine and ascorbic acid were dissolved in water. Both folic acid and riboavin needed a basic medium for dissolution (0.8 and 4% (1 M) NaOH, respectively). The buffers were prepared by dissolving the required amount of the salt in water, and subsequently adding 1 M NaOH (for pH 9). SDS-containing carriers were prepared by mixing the required volume of 100 mM SDS with the buffer. Samples and buffers were ltered through a 0.22 m syringe lter prior to use. The commercial multivitamin formulation analysed was diluted with water containing 1 M NaOH (to a nal concentration of 0.6%), the mixture was homogenised with a Bunser homogeniser, ltered through a 0.22 m syringe lter and injected into the system. 2.4. Analytical conditions The capillary was preconditioned for 10 min with 1 M NaOH before the rst run and then for 3 min with

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Fig. 1. Separation of thiamine (1), nicotinamide (2), riboavin (3), pyridoxine (4), ascorbic acid (5), nicotinic acid (6) and folic acid (7). Phosphate buffer (20 mM, pH 9.0), 35 C. Separation voltage 20 kV; absorbance detection at 215 nm (mAU = 103 ).

Fig. 2. Variation in migration times with surfactant concentration. Phosphate-borate (20 mM, pH 9.0) containing different concentrations of SDS [0, 25, 50 and 75 mM]. Separation voltage 20 kV. Capillary temperature 35 C. ( )EOF, ()nicotinamide, () pyridoxine, ( ) ascorbic acid, () nicotinic acid, ( ) riboavin, () folic acid, ( ) thiamine.

0.1 M NaOH and for 5 min with the run buffer prior to each subsequent run, since both reproducibility and peak shape in CE are sensitive to the state of the inner wall of the capillary. The injection was carried out under pressure (hydrodynamic) at 50 mbar for 3.7 s (approx. 5.9 nl). In order to ensure area reproducibility and a quantitative injection of the sample, subsequent injection under the same conditions of the running buffer was necessary. The standard separation conditions were voltage 20 kV (positive polarity), capillary temperature 35 C, and a 20 mM phosphate-borate (50 mM SDS) run buffer at pH 9.0.

3. Results and discussion Initially, the separation of the seven water-soluble vitamins was carried out using the CZE technique. When CZE is employed, it is necessary to optimise the pH because it is important that the analytes are in their ionic forms. The best separation is obtained at pH 9, as can be seen in Fig. 1, since only one of the seven vitamins remains uncharged (nicotinamide). Furthermore, the pH inuences the magnitude of the electroosmotic ow (EOF), which is directed towards the negative end of the fused-silica capillary. In order to carry out simultaneous determination of cations and anions in CZE in a reasonable time, it was necessary to force an EOF towards the detector at a velocity which exceeds the migration velocity of the anions. We hence selected a basic medium (pH 9), with which the separation run time did not exceed 7 min, and an optimum separation could be achieved.

The main drawback of this method was the quantication of the nicotinamide now that it remained uncharged and it migrated with the rest of the uncharged species. Separation of uncharged molecules may, in rare cases, be done by CZE due to differences in the viscous drag of different sized molecules. These differences are small, and CZE is not usually a good technique for separating uncharged solutes. Thus, in this paper we studied the separation of water-soluble vitamins using MEKC. In MEKC, a buffer solution that contains micelles is used as the run buffer. We selected pH 9 buffers for all the assays, as this provides a good separation in CZE and, in general, high pH buffers are used in MEKC to maintain reasonable EOF and ensure the migration direction. The surfactant selected for this study was SDS. With this surfactant, negatively charged solutes are not strongly attracted to the micelles, and they are separated primarily as a result of differences in their electrophoretic mobilities, just as in CZE. Moreover, the analysis time is not very large. 3.1. Effect of SDS concentration Fig. 2 shows the effect of adding various concentrations of SDS to the buffer. At an SDS concentration of zero, nicotinamide was not separated and eluted with methanol, which was used as a tracer for EOF. As the SDS concentration was increased, the retention time of nicotinamide became greater than that of methanol, and it was separated from the rest of the uncharged species. The increase in its retention is due to its sol-

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ubilization by the micelles. The retention times of the rest of the solutes increased at about the same rate with increasing SDS concentration, due to their solubilization by the micelles. However, the retention time of thiamine increased more dramatically with an increase in SDS concentration because of the formation of ion pairs between this positively charged vitamin and the negatively charged groups on the micelles [15,17,20]. In order to separate all the analytes in the least time possible, 50 mM SDS was selected as optimum, since lower concentrations led to incorrect separations, and increasing the concentration above 50 mM involved an increase in the time of analysis. 3.2. Separation conditions Four buffers, namely phosphate, phosphate-borate, Tris and Tris-MES were tested at different separation voltages. The resulting plots of observed current versus applied voltage for these buffers showed a deviation from linearity above 15 kV for phosphate and above 20 kV for the phosphate-borate buffer. Ohms law plots for the Tris and Tris-MES buffers were linear up to 25 kV. In order to obtain short analysis times and narrow peaks, the phosphate buffer was discarded. The electropherograms obtained with the phosphate-borate, Tris and Tris-MES buffers at 20 kV were subsequently examined. Phosphate-borate gave the best separation, as is shown in Fig. 3. With this buffer, the analysis of the seven water-soluble vitamins was carried out in seven minutes, all of them being perfectly separated from one another. Using the Tris buffer, it was impossible to separate nicotinamide since it migrates at the same rate as the EOF. The Tris-MES buffer gave an incomplete separation. The suggested analytical conditions were therefore: analysis temperature 35 C, voltage 20 kV and a 20 mM phosphate-borate pH 9.0 (50 mM SDS) run buffer. 3.3. Analytical performance Table 1 summarises the quantitative analytical parameters of MEKC separation of the water-soluble vitamins. As can be seen, satisfactory reproducibility was obtained for 5 injections. These range from 0.7 to 3.1% relative standard deviation (RSD) at

Fig. 3. Electropherograms of a mixture of nicotinamide (1), pyridoxine (2), ascorbic acid (3), riboavin (4), nicotinic acid (5), folic acid (6) and thiamine (7) with three carrier electrolytes, A: phosphate-borate, B: Tris-MES, C: Tris (all 20 mM, pH 9.0) containing 50 mM SDS. Capillary temperature 35 C. Separation voltage 20 kV. Detection at 265 nm. Table 1 Analytical characteristics of the proposed method Migration time Peak areaa LOD (g ml1 )b RSD% (n = 15) RSD% (n = 5) Thiamine Nicotinamide Nicotinic acid Folic acid Riboavin Pyridoxine Ascorbic acid
a b

0.94 0.59 0.99 1.20 1.82 0.60 0.83

3.09 0.74 1.26 1.44 1.03 0.76 0.88

0.25 0.25 0.18 0.08 0.11 0.11 0.10

RSD: relative standard deviation.(at mid-calibration range). LOD: limit of detection based on a signal to noise ratio of 3.

mid-calibration range. The calibration graphs for all the water-soluble vitamins showed good correlation between the peak areas and vitamin concentrations, with the regression coefcients >0.999 in all cases, for the range up to 100 LOD (limit of detection). The

D.B. Gomis et al. / Analytica Chimica Acta 396 (1999) 5560 Table 2 Results of the pharmaceutical formulation analysis Concentration found (g ml1 ) Nicotinamide Pyridoxine Riboavin Ascorbic acid Thiamine Fig. 4. Electropherogram of a pharmaceutical formulation. Nicotinamide (1), pyridoxine (2), ascorbic acid (3), riboavin (4), thiamine (5), retynol (6) and DL--tocopheryl (7). Conditions as in Fig. 3. 93 11 8 486 19

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Declared concentration (g ml1 ) 100 10 10 500 20

linearity of the calibration graphs was also checked with two different statistical tests (the Fisher and the lack-of-t tests). For the Fisher test, the values obtained were always higher than the tabulated values ( = 0.05), linearity thus being demonstrated. In the lack-of-t test, the calculated values were lower than the tabulated ones ( = 0.01), and the hypothesis was hence also demonstrated. The limits of detection were below the g ml1 level (between 0.08 g ml1 for folic acid and 0.25 g ml1 for nicotinamide and thiamine). These were determined by injecting serial dilutions of a concentrated standard mixture, followed by the preparation of calibration plots (peak height versus concentration injected), which were extrapolated to a signal-to-noise ratio (S/N) of 3 so as to assign the detection limit. In order to demonstrate the applicability of the MEKC method for the determination of water-soluble vitamins in pharmaceutical formulations, this was applied to a multi-vitamin formulation. Fig. 4 shows the results of this analysis. Each vitamin in the sample was identied by matching the migration time with the standard and by using a spectral library search. Quantication was carried out by calibration with standard vitamin solutions. The peak areas were corrected for the retention time. We were able to identify two additional peaks as retynol and DL- tocopheryl by comparing the migration times and the UV spectra with those of standard substances. Recovery experiments were performed in order to study the accuracy of the method. Known amounts of each vitamin were added to a variety of samples and the resulting spiked samples were subjected to

the entire analytical sequence. Each solute was spiked at three different concentrations and recoveries were calculated on the basis of the difference between the total amount determined in the spiked samples and the amount observed in the non-spiked samples. All analyses were carried out in triplicate. The average recoveries obtained, which ranged between 95 and 103%, testify to the accuracy of the proposed method, and reproducibility (between days) was <6% for all samples. Table 2 shows the results of the analysis. We found that the vitamin content declared by the producer was usually higher than the one we found. This is probably due to the low chemical stability of the analytes, since the recoveries obtained demonstrate the accuracy of the proposed method.

4. Conclusions As a result of the advantages of MEKC with regards high separation efciency, selectivity, short analysis time, ease of instrumentation and sample preconditioning and compatibility with charged and neutral solutes when compared with existing LC methods, we were able to employ this method for determining water-soluble vitamins. The method was applied to the analysis of a pharmaceutical formulation and reasonable results were obtained. The limits of detection of the method were in the sub-micrograms per milliliter range; the method had good reproducibility, as well as a very short analysis time (7 min). References
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