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ISSN: Print -2349-0977, Online - 2349-4387


 
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ORIGINAL CONTRIBUTION: CLINICS IN MOLECULAR BIOLOGY
Year : 2015  |  Volume : 2  |  Issue : 3  |  Page : 121-125

Characterization of partitioning behavior of vitamins in micellar systems using affinity capillary electrophoresis


Novel Drug Delivery Research Center, School of Pharmacy, Kermanshah University of Medical Sciences, 67149-67346 Kermanshah, Iran; Faculty I of Natural Science-Biological Science, Institute of Pharmacy, Martin Luther University, Wolfgang Langenbeck Str. 4, 06120 Halle-Wittenberg, Germany

Date of Web Publication2-May-2016

Correspondence Address:
Leila Behbood
Novel Drug Delivery Research Center, School of Pharmacy, Kermanshah University of Medical Sciences, 67149-67346 Kermanshah, Iran

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2349-0977.181507

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  Abstract 

Introduction: Micellar Electrokinetic Chromatography, sometimes referred as Affinity Capillary Electrophoresis, is a useful tool to the characterization of the drug partitioning in different vehicle systems such as micelles and microemulsions using the capacity factor as hydrophobicity parameter. Bile acids, because of their important role in biological systems, are applied widely in ACE method to characterization of partition properties of solutes. In this paper the interaction of some hydrophilic and lipophilic vitamins with micellar systems consisted of bile salts and phospholipids was considered using ACE. Material and Methods: Three mixed micelles and one microemulsion were employed. The samples were filtered through a 0.45 μm filter. 13 vitamins, consist of 4 lipophilic and 9 hydrophilic vitamins were investigated. Each vitamin was solved in different micellar systems (0.1 mg/ml). Then, using ACE, the electropherogram of samples was obtained under certain run conditions. The capacity factor for each sample was calculated according the migration times in the electropherogram. DMSO and Sudan red were used as EOF tracer and micelle tracer respectively. All experiments were carried out with certain instrumental conditions. Results and Discussion: The results show that the migration times of lipophilic vitamins are equal to migration time of micellar tracer and consequently the capacity factor of all of them tends to infinity. In fact these vitamins, concerning the hydrocarbonic chain in their chemical structure, are incorporated in the inner oil core of mixed micelle or microemulsion and moved with it. Four hydrophilic vitamins (niacinamid, pyridoxine, D-panthenol and cyanocobalamin) have different affinity to Micellar systems. But for each vitamin the affinity to different system is almost same. K′ zero means that vitamin has not any affinity to micelle, remains in aqueous phase and moves with EOF. Five hydrophilic vitamins (ascorbic acid, folic acid, riboflavin, biotin and thiamin) had charged behavior. The electrophoretic mobility of all these vitamins was negative, except thiamin which has positive mobility. Two important parameters that may influence these migration behaviors of charged solutes are pH and micelle concentration. Distribution properties of molecules mostly change depending to pH value of the media because of the effect of pH on the chemical interaction. In the other hand, it might be that the negative charge of bile salt's micelle increases by mixing with phospholipids, and causes to stronger repulsion between negative charged micelle and anion analyte. So the electrophoretic mobility of anionic vitamins in presence of micelle would be reduced. Conclusion: The MEKC technique as a method of ACE was employed to determination partitioning pattern of various kinds of vitamins in a micellar solutions. The affinity of both hydrophilic and lypophilic vitamins to micellar system was investigated by calculation the capacity factor. This value for lypophilic vitamins is infinite, because they incorporate with micelle compositions and moved with them. In contrast hydrophilic vitamins exhibited different partitioning behaviour in micellar solution. The negative value obtained for anionic hydrophilic vitamins can be corrected by change some condition like micellar concentration and the pH of media.

Keywords: Affinity capillary electrophoresis, capacity factor, micellar electrokinetic chromatograpgy, vitamins


How to cite this article:
Behbood L. Characterization of partitioning behavior of vitamins in micellar systems using affinity capillary electrophoresis. Astrocyte 2015;2:121-5

How to cite this URL:
Behbood L. Characterization of partitioning behavior of vitamins in micellar systems using affinity capillary electrophoresis. Astrocyte [serial online] 2015 [cited 2020 Jan 28];2:121-5. Available from: http://www.astrocyte.in/text.asp?2015/2/3/121/181507


  Introduction Top


A variety of experimental techniques are available for investigating the molecular interactions. Among the potential approaches is micellar electrokinetic chromatography (MEKC), which sometimes is referred as the affinity capillary electrophoresis (ACE).[1] A large number of literatures apply this technique to analyze of pharmaceutical and chemical substances.[2],[3],[4],[5],[6],[7],[8],[9],[10] In addition, this method is a useful tool to the characterization of the drug partitioning in different vehicle systems such as micelles and microemulsions using the capacity factor as hydrophobicity parameter.[11],[12],[13] The interaction between drugs and micellar systems has been investigated in different studies.[14],[15],[16] Moreover, determination and monitoring of vitamins and their derivatives using MEKC has been considered in some reports.[17],[18],[19]

Bile acids–because of their important role in biological systems under both physiological and pathophysiological conditions–are the most popular micelles systems in pharmaceutical studies, which are used as a vehicle system for drugs.[20],[21] They are applied widely in ACE method to the characterization of partition properties of solutes.[22],[23] In this paper, the interaction of some hydrophilic and lipophilic vitamins with micellar systems consisted of bile salts and phospholipids was considered using ACE.

Theory

ACE is usually performed by adding one of the interacting parameters, the ligand (e.g., micelle), to background electrolyte in the capillary, then the introduction of a small sample plug containing the analyte into the capillary and finally application of voltage.[24] The quantification of the interact strength between analyte and ligand is done by measuring capacity factor (k'). In fact this expression is a parameter which is used to characterize the partition behavior of solute between two phases and is defined as the ratio of the numbers of moles of solute in the micellar pseudostationary phase and that in the bulk aqueous phase. Capacity factor of uncharged solutes, which introduced by Trabe, is calculated using the following equation:



where t0 is the migration time of electroosmotic flow, tm is the migration time of solute, and tmc is the migration time of ligand.[25] Strasters and Khaledi et al. have developed this equation to calculate the capacity factor for charged solutes:



where µm is the electrophoretic mobility of solute in the presence of ligand, µ0 is the electrophoretic mobility of solute in the absence of ligand, and µmc is the electrophoretic mobility of ligand.[26]


  Materials and Methods Top


Chemicals

Glycocholic acid, glycine, soya lecithin, and vitamins were obtained from Midas Pharma (Ingelheim, Germany). Glycodeoxycholate Na was purchased from Sigma Aldrich Chemie GmbH (Steinheim, Germany). Deoxycholic acid, oleic acid, and Sudan red were obtained from Fluka Chemie GmbH (Steinheim, Germany). Dimethyl sulfoxide (DMSO) was purchased from Roth (Karlsruhe, Germany). Phosphatidylcholine was obtained from Phospholipid GmbH (Cologne, Germany).

Micellar solutions

Three mixed micelles and one microemulsion were employed [Table 1]. The MM1 was prepared according to composition of vehicle system of vitamins in formulation of a commercial multivitamin product obtained from Cernevit (Unterschleißheim, Germany). Each mixed micelle or microemulsion was degassed for 30 min in an ultrasonic bath and filtered through a 0.45 µm filter prior to use [Table 1].
Table 1: Development Mixed Micelles and Microemulsion Systems

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Sample preparation

Investigated vitamins comprised ascorbic acid, folic acid, riboflavin, biotin, thiamine, pyridoxine, D-panthenol, niacinamide and cyanocobalamin as hydrophilic drugs, and retinol palmitate (Vitamin A), cholecalciferol (Vitamin D), α-tocopherol (Vitamin E), and phytonadione (Vitamin K) as lipophilic vitamins. Each vitamin was solved in different micellar systems (0.1 mg/mL). The samples were filtered through a 0.45 µm filter. Then, using ACE, the electropherogram of samples was obtained under certain run conditions. The capacity factor for each sample was calculated according to the migration times in the electropherogram. DMSO and Sudan red were used as electroosmotic flow tracer and micelle tracer, respectively.

Instrument and conditions

Capillary electrophoresis (CE) experiments were performed on a Hewlett-Packard Model G1600A (Waldbronn, Germany) three-dimensional CE system with a diode-array detector from 190 to 600 nm. The CS fused capillary was obtained from Chromatographie Service GmbH (Langerwehe, Germany) and had 48.5 cm (length to detector 40 cm) ×50 µm I.D. All experiments were carried out with a voltage of 20 kV at 25°C. Samples were injected with 50 mbar for 10 s. The detection wavelengths were 200 and 250 nm.

Each new capillary was rinsed with 1 M NaOH for 15 min prior each run the capillary was flushed with 0.1 M NaOH for 1 min, water for 5 min, and background electrolyte for 5 min.


  Results and Discussion Top


The information of k' is useful for estimating the partition behavior of solutes in multiphasic environments. We calculated k' for vitamins in mentioned micellar systems.

Capacity factor of lipophilic vitamins

The results show that the migration times of lipophilic vitamins are equal to migration times of micellar tracer (Sudan red), and consequently the capacity factor of all of them tends to infinity. In fact, these vitamins–concerning the hydrocarbonic chain in their chemical structure–are incorporated in the inner oil core of mixed micelle or microemulsion and moved with it. [Figure 1] shows the electropherogram of vitamin D [Figure 1].
Figure 1: Electropherogram of vitamin D in MM1, obtained by affinity capillary electrophoresis. Run condition: Apply voltage: 20kV; Temperature: 25 °C; Injection: 50 mbar in 10 sec; detection wavelength: 250 nm.

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Capacity factor of hydrophilic vitamins

The pH values of micellar systems were 5.9 (MM1, MM2) and 7.2 (MM3, ME). In these pH values, some hydrophilic vitamins were charged and others were uncharged. [Table 2] shows the capacity factor of hydrophilic vitamins in Micellar systems. As it shown, these vitamins (niacinamide, pyridoxine, D-panthenol, and cyanocobalamin) have different affinity to micellar systems. But for each vitamin, the affinity to different system is almost same. k' = zero means that vitamin has not any affinity to micelle, remains in aqueous phase, and moves with EOF [Figure 2].
Table 2: Capacity Factor of Hydrophilic Vitamins in Different Micellar Solutions, Obtained Using Affinity Capillary Electrophoresis (n=3)

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Figure 2: Electropherogram of pyridoxine in MM1, obtained by affinity capillary electrophoresis. For condition see the Figure 1.

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Five hydrophilic vitamins (ascorbic acid, folic acid, riboflavin, biotin, and thiamin) had charged behavior. The electrophoretic mobility of these was measured by capillary zone electrophoresis (CZE). It has indicated that glycine changes both electroosmotic and electrophoretic mobility,[27] and it is expected that the capacity factor affected by the presence of glycine. Hence, in the cases that micellar system contains glycine, it was added to background electrolyte in CZE to eliminate its effect. As is presented in [Table 3], all of these vitamins have negative electrophoretic mobility, except thiamin which has positive mobility. Other words, concerning equation 2, in these pH values (5.9 and 7.2) thiamin dissociated as cationic analyte and ascorbic acid, folic acid, and riboflavin and biotin presence as anionic analytes. According to the electrophoretic mobility of vitamins in CZE and MEKC, their capacity factor was calculated using Equation 2. The results indicate that k' values of anionic vitamins are negative [Table 2].
Table 3: Electrophoretic Mobility of Charged Hydrophilic Vitamins, Obtained by Capillary Zone Electrophoresis (n=3)

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However, a negative value of k' is impossible due to its definition, it results when the effective electrophoretic mobility of solute is higher in the MEKC than in the CZE. Two important parameters that may influence these migration behaviors of charged solutes are pH and micelle concentration [Table 2], [Table 3] and [Figure 2], [Figure 3].
Figure 3: Electropherogram of folic acid in MM1, obtained by affinity capillary electrophoresis. For condition see the Figure 1.

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Effect of micelle concentration and pH on capacity factor

k' of solutes (neutral or charged) is linearly related to the micelle concentration through Equation 3:



where CMC is critical micelle concentration, Pmw is the partition coefficient of solute in the micelle, v is the partial molar volume of the surfactant, and Ct is the total surfactant concentration.[28] It seems that capacity factor increases with an increasing in surfactant concentration, but one should note that Pmw for ionic solutes is a function of pH.[29],[30] In different pH values, concerning dissociation constant, these solutes are partly dissociated and due to the electrostatic repulsion between the dissociated solute and the surfactant with the same charge, the Pmw values will be smaller as compared to uncharged solute.

Distribution properties of molecules mostly change depending on the pH value of the media because of the effect of pH on the chemical interaction. The migration velocity of an anionic analyte is the weighted average of the migration velocity of its uncharged form, charged form, and the solute interacting with micelle. Hence, the total k' will be a function of the k' of these forms. Khaledi et al. have identified this concept with following equation:



Where Ka is dissociation constant, k'HA and k'A - are the capacity factor of solute in the associated and dissociated forms, respectively.[29] It is clear that the capacity factor of acidic analyte depends on the pH value of background electrolyte and Ka of solutes. This equation predicts a sigmoidal relationship between k' and pH of background electrolyte. Hence, appropriate k' can be obtained in a suitable pH range; however, this range will not be the same for all solutes because of the difference in Ka.

On the other hand, it might be that the negative charge of bile salt ' s micelle increases by mixing with phospholipids and causes stronger repulsion between negatively charged micelle and anion analyte. Hence, the electrophoretic mobility of anionic vitamins in the presence of micelle would be reduced. This suggestion was confirmed by the results obtained in micellar systems which contain tween (MM3 and ME). In these systems, negative surface charge of micelle decreases by adding a nonionic surfactant (tween). Consequently, the affinity of solute to micelle sharply increases and k' tends to infinite. Comparing the effective electrophoresis mobility of the micellar systems also emphasizes this claim [Table 4].
Table 4: Effective Electrophoretic Mobility of Mixed Micelles and Microemulsion, Obtained by Affinity Capillary Electrophoresis

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  Conclusion Top


The MEKC technique as a method of ACE was employed to determine partitioning pattern of various kinds of vitamins in a micellar solutions. In this method, micelle was added to the background electrolyte in capillary, and the affinity of both hydrophilic and lipophilic vitamins to micellar system was investigated by calculation the k' value. This value for lipophilic vitamins is infinite because they incorporate with micelle compositions and moved with them. In contrast hydrophilic vitamins exhibited different partitioning behavior in micellar solution. The negative value obtained for anionic hydrophilic vitamins can be corrected by varying some conditions like micellar concentration and the pH of media.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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