Dariusz WIDEŁ, Jerzy OSZCZUDLOWSKI, Zygfryd WITKIEWICZ ? Institute of Chemistry Jan Kochanowski University, Kielce, Poland; Marcin MOCZULSKI ? Comesa Polska Sp. z o.o., Beckman Coulter Biotechnology Division, Warszawa, Poland
In the present work microemulsion electrokinetic chromatography (MEEKC) was applied for quantitative determination of vitamin E in ?-tocopherol acetate form. Capillary electrophoresis system with diode array detector was used to perform the analysis. Microemulsion consists of: 10 % (w/w) SDS, 6.6 % (w/w) 1-butanol, 0.8 % (w/w) n-octane, 12.5 % (w/w) 2-propanol, 40% (w/w) phosphate buffer with pH=2.5 and 30.1 % (w/w) demineralized water, was used in capillary. The quantitative determination of vitamin E was performed with the external standard method with the use of the 32 Carat ver. 8.0 software from Beckman Coulter Inc. Additionally the quantitative determination of the vitamin E by ultrafast liquid chromatography (UFLC) method was performed for the purpose of the comparison of results with the MEEKC method.
Please cite as: CHEMIK 2013, 67, 2, 111-118
Vitamins are a large group of organic compounds which constitute small, but the essential component of the food necessary to the normal growth and the kilter of the human and animal organism. The deficiency of the vitamins can cause serious health problems. Vitamins can be divided on two core groups: water-soluble and fat-soluble . It is difficult to perform the simultaneous determination of water and fat-soluble vitamins applying traditional high-performance liquid chromatography methods, but some studies described in the work  suggest that such determination is possible by capillary electrophoresis (CE). The determination of different forms of tocopherols using electrokinetic separation techniques have been demonstrated, including capillary electrochromatography and micellar electrokinetic chromatography (MEKC) . Over the recent years the popularity of CE method in pharmaceutical analyses grew up due to its good resolution abilities, the possibility of the analyses of very small amount of the samples and the small consumption of solvents .
One of the CE techniques which enable better resolution of components of some mixtures than other techniques is the microemulsion electrokinetic chromatography (MEEKC) . MEEKC is an electrodriven separation technique, it uses microemulsion buffer to separate charged or neutral analytes based on both their hydrophobicities and electrophoretic mobility . MEEKC is a variation of MEKC where a microemulsion droplet, a non-polar pseudophase, is formed by mixing an immiscible organic solvent, such as octane, with a buffered aqueous solution. The use of MEEKC has the advantage over MEKC that its microemulsion dissolves highly lipophilic compounds more efficiently, reduces the loss of solvent through evaporation, and results in a greater separation of the lipophilic compounds. The main disadvantage of MEEKC is relatively long times needed for the separation of lipophilic compounds . MEEKC is a useful technique for the separation of both charged and neutral solutes, covering a wide range of solubility. Sodium dodecyl sulfate (SDS) is the most commonly used surfactant in microemulsions; the immiscible oil is typically octane or heptane, and the most frequently employed co-surfactant is 1-butanol; microemulsion contains also a proper buffer, like boric or phosphate buffer .
Vitamin E is customarily called tocopherols. It has been shown to be the most effective lipid-soluble antioxidant in nature, interfering with one or more propagation steps of the lipid peroxidation process. Among four tocopherols (?-, ?-, ?- and ?-tocopherol), ?-tocopherol, especially RRR diastereomer, has been reported to possess the highest biological activity. In natural products vitamin E appears mainly as ?-tocopherol however in pharmaceutical products as the ?- tocopherol acetate due to its larger durability . A number of methods such as normal phase high-performance liquid chromatography, reversed-phase high performance thinlayer chromatography, reversed-phase high- performance liquid chromatography (C18RP-HPLC), gas chromatography (GC) have been developed for the separation of tocopherols. Owing to a difficulty of separating ?- and ?-tocopherol, they were sometimes measured as a combined fraction .
The determination of tocopherols by GC has also been carried out together with some of its oxidation products, ?-tocopherolquinone. Melcher et al.  have quantitatively determined ?-, ?-, ?- and ?-tocopherols in human serum by HPLC with fluorescence detection and GC-MS to analyze trimethylsilyl (TMS) derivatives. Since the introduction of MEKC by Terabe et al. in 1984, it has been extensively applied to a wide variety of analytes, especially for the separation of neutral compound. Hydrophobic interaction electrokinetic chromatography was used to simultaneously separation of seven active ingredients, including ?-tocopherol acetate . Mottier et al.  for quantitative analysis of ?-tocopherol acetate and ?-tocopherolquinone in human serum compared GC-MS and HPLC-MS/MS methods. Applying GC-MS method the derivatization of tocopherols to TMS derivatives was carried out. The both methods showed the similar accuracy in tocopherols determination.
A large review of chromatographic tocopherol analysis in different matrices is shown in work . There are a number of articles about the vitamin separation by MEEKC method. Boso et al.  studied water- and fat-soluble vitamins separation using anionic surfactant (SDS) and cationic trimethyltetradecylammonium bromide. Altria  showed the application of MEEKC to analysis of a large group of pharmaceutical preparations, both soluble and insoluble in water, vitamins and different additions to medicines, in this of the vitamin A and D3 as fat-soluble. Pedersen-Bjergaard et al.  applied MEEKC for separation and determination vitamin A palmitate, vitamin D3 and vitamin E acetate in pharmaceutical preparations. Sanchez and Salvado  have separated water- and fat-soluble vitamin mixtures, using SDS as a surfactant, 1-butanol as co-surfactant, n-octane as nonpolar solvent and 2-propanol as the second co-surfactant. Aurora-Prado et al.  determined folic acid dissolved in water by MEEKC method. Chang et al.  have separated ?-, ?-, ?-tocopherol and ?-tocopherol acetate by microemulsion chromatography with cyclodextrin used as modifier . A rapid and simple voltammetric method was developed for quantitative analysis of ?-tocopherol acetate in pharmaceutical preparations .
The aim of the present work was the execution of the quantitative determination of ?-tocopherol acetate in four pharmaceutical preparations of vitamin E by MEEKC and UFLC methods. The quantitative analysis of ?-tocopherol acetate is not easy and creates large difficulties. Particularly important is the elaboration of conditions of the obtaining of repeatable retention and migration times and peak areas during the determination of the calibration curve. The repeatability of analysis in MEEKC is unusually difficult to obtain, mainly due to the volatility of some components of the microemulsion what influences to its composition at each moment. The established purpose was reached across the use of the phosphate buffer of the pH= 2.5 as the main component of the microemulsion in capillary and through the suitable selection of other organic components of the microemulsions.
2.1. Chemicals and reagents
The standard of vitamin E that is DL-?-tocopherol acetate in a form of the 100 milligram pastille was provided by the Supelco and this amount was checked on the analytical balance. SDS was from Sigma, 2-propanol, methanol and acetonitrile from Fluka, 1-buanol from Merck, n-octane and from Institute of Physical Chemistry of Polish Academy of Sciences. Phosphate buffer, pH = 2.5 for high performance capillary electrophoresis was provided by Fluka. Furthermore demineralized water and NaOH aqueous solution as the conditioner, with the concentration 0.1 M received from the Beckman Coulter Company were used. Four pharmaceutical preparations of vitamin E were analyzed. The pharmaceuticals have been in form of drops and tablets, and they contained ?-tocopherol acetate as the main component and auxiliary substances, e.g. cleaned peanut oil.
2.2. Preparation of microemulsions
Two microemulsions were prepared, first for the capillary filling and second for the dilution of samples. As result of the optimization of the composition of the microemulsion it has been realized that good results were obtained when the microemulsion for the capillary filling was prepared in the following way: 10% (w/w) SDS, 6.6 % (w/w) 1-butanol, 0.8 % (w/w) n-octane, 12.5 % (w/w) 2-propanol, 40 % (w/w) phosphate buffer pH= 2.5 and 30.1 % (w/w) demineralized water. The mixture was subjected to the mixing with sonification, about 45 minutes, to the moment till the microemulsion became clear. It is very important to remember that the microemulsion has to be clear, because this influences substantially to migration time of the analyte and the repeatability of the analyses. During the mixing it is important to close tightly the vials with the microemulsion to protect it before the evaporation of volatile components of the mixture, what can cause changes in the composition of solution.
The composition of the microemulsion to the preparation of samples is much alike to the microemulsion applied to the capillary filling. The only difference is that in the first one there is no phosphate buffer. The SDS concentration in both microemulsions carried out 30 mM and was selected on the basis of earlier research. It turned out that the lower concentration of SDS made difficult or impossible even to mix the microemulsion properly. Ready microemulsions were put away into the dark place on a few days to final clarifying.
2.3. Preparation of standard solutions and sample solutions
The standard solution of ?-tocopherol acetate was prepared through the quantitative dissolution of the 100 mg pastille in 10 ml of 2-propanol, receiving this way the stock solution with the concentration of 10 mg/ml. The stock solution was stored in 5°C without the light. Then through the dilution of the stock solution with the microemulsion without the phosphate buffer standard-solutions with different concentrations were prepared for the determination of the calibration curve by the external standard method. Five standard solutions of ?-tocopherol acetate were prepared with concentration of: 0.05; 0.10; 0.25; 0.50 and 1.00 mg/ml.
Samples of the ?-tocopherol acetate contained in pharmaceutical preparations were prepared through the dissolution of pharmaceuticals in 2-propanol for the level of concentration being situated within the range of calibration, established on the basis the mass given by the producer. Then propanol solutions were mixed against 100 ?l:100 ?l with the microemulsion without the phosphate buffer, what enabled the analysis with the MEEKC method.
2.4. Instrumentation and conditions
2.4.1. MEEKC method
For the investigations P/ACE MDQ Capillary Electrophoresis System from Beckman Coulter Inc. (USA) with diode array detector was used. In the present work the acidic environment was applied with the phosphate buffer about the pH = 2.5. At that pH level the electroosmotic flow is suppressed enough, to provide the maximum migration velocity to negatively charged droplets of the microemulsion . Different times of capillary rinsing with methanol, 0.1M NaOH aqueous solution, demineralized water and microemulsion filling the capillary were applied to obtain the fastest stabilization of the capillary inside. The temperatures of the analyses from 20 to 27?C and also values of the current voltage from 12 to 30 kV were changed to obtain optimum conditions. The influence of capillary length on migration time of the analyte was also tested, by applying 37 and 57 cm capillaries. It turned out that the reduction of the capillary length effected in shorter migration times but with good results.
Before the introducing to the capillary every series of standardsolutions and pharmaceutical preparations capillary was conditioned through the rinsing: 4 min. with the methanol, 0.1 M NaOH, and then with demineralized water and 5 min. with the microemulsion. Received electropherograms were saved and elaborated in the 32 Karat ver. 8.0 software. At the beginning standard solution of ?-tocopherol acetate with the concentration of 1 mg/ml was introduced to the capillary and analyzed, to made some modification of the method described in the work  through the selection of the suitable temperature of the analysis, the value current voltage and the use of suitable aqueous solutions and solvents for the rinsing of the capillary.
Traditionally, MEEKC is carried out with an alkaline separation medium providing a substantial electroosmotic flow in the direction of the detector. In this type of separation system, where the negative electrode is placed at the capillary outlet, the electroosmotic flow towards the detector exceeds the electrophoretic mobility of the negatively charged microemulsion droplets in direction of the capillary inlet. Thus, compounds of low affinity towards the oil droplet phase rapidly pass through the effective length of the capillary to the detector, whereas compounds with strongly partitioning into the oil droplet phase are significantly retained .
The investigations were performed in fused-silica capillary with internal diameter 50 ?m and effective length 30 cm (total length 37 cm). Reverse polarity of the electrodes with 15 kV in 25 °C temperature was applied. The reverse polarity of the electrodes decreases the migration time of the analyte. In order to prevent the samples evaporation, they have been stored in autosampler in 20°C. The samples were injected by hydrodynamic technique with pressure of 0.5 psi during 5 sec.
2.4.2. UFLC method
The results of quantitative determination of the ?-tocopherol acetate by MEEKC method and isocratic UFLC method were compared, using the same standard solutions and pharmaceutical preparations. The use of microemulsion in UFLC method is unnecessary, so the solutions of ?-tocopherol acetate in 2-propanol were analyzed. The external standard calibration curve was determined using the same concentration as in the MEEKC method. The UFLC system from Shimadzu with the UV-VIS detector and the rheodyne valve was applied for the determinations. The capillary column from Phenomenex Co., Kinetex 100 x 4.6 mm, 2.6 u C 18 type, with 2.6 ?m octadecyl phase. The mobile phase was 95 % acetonitrile and 5 % methanol with the constant flow of 1.5 ml/min .
The detection was made at the wavelength 280 nm, despite that the maximum of the absorbance of ?-tocopherol acetate is observed at 200 nm. Because of the detector overload in examined range of concentrations at 200 nm wavelength, 280 nm was applied. After 100 times dilution of standard solutions about concentrations: 0.05; 0.10; 0.25; 0.50 and 1.00 mg/ml, chromatograms at the wavelength 200 nm with sharp and symmetrical shapes were received. It is possible to determine ?-tocopherol acetate by UFLC method on the lower level of concentrations than with the MEEKC method. To compare the both methods with the detection at 280 nm, this wavelength in the UFLC method was applied. The absorbance is then smaller and the large dilution of pharmaceuticals is not necessary, and peaks on chromatograms are equally sharp as at 200 nm.
3. Results and discussions
After the conditions were settled, standard solutions of ?-tocopherol acetate were introduced into the capillary to determine the calibration curve. Every sample was introduced three times. Next the pharmaceutical preparations were introduced. The ?-tocopherol acetate was detected at the 200 nm wavelength of the UV light. The 3D UV spectrum of ?-tocopherol acetate is shown in Figure 1.
The example of the overlaid peaks is shown in Figure 2. Every peak shows a different concentration of ?-tocopherol acetate standard solution used to determination of calibration curve.
For the determined calibration curve the linear regression coefficient carried out 0.996. After the calibration curve designation, ?-tocopherol acetate was determined in pharmaceutical preparations. The UV spectra of standard were compared with spectra of ?- tocopherol acetate in pharmaceutical preparations and on that base its identity of was confirmed.
For the determined calibration curve by UFLC method, the linear regression coefficient carried out 0.999. The results of quantitative determination of ?-tocopherol acetate obtained for four vitamin E pharmaceutical preparations by MEEKC and UFLC methods are shown in Table 1.
The received results were given with uncertainties calculated on the basis of relative standard deviations (RSD). The RSD of retention times in UFLC method, for n = 10 carried out 1.74 %, and for peak areas 2.4 %. The RSD of the migration times in MEEKC method, for n = 10 carried out 1.65 % and for peak areas 6.5 %. In quantitative analyses by MEEKC method, the limit of detection of ?-tocopherol acetate, LOD = 0.030 mg/ml, while limit of quantification LOQ = 0.090 mg/ml. In quantitative analyses by UFLC method, the limit of detection of ?-tocopherol acetate, LOD = 0.005 mg/ml, while limit of quantification LOQ =0.015 mg/ml.
For example the quantitative determination of ?-tocopherol acetate by voltammetric method  gave the approx. results of LOD = 0.010 mg/ml.
In the present work the application of MEEKC method was described for the quantitative determination of the ?-tocopherol acetate in vitamin E pharmaceutical preparations. Additionally the quantitative determination of the ?-tocopherol acetate in pharmaceutical preparations was performed with the UFLC method, for the purpose of the comparison of results received with the MEEKC method. It was found that the UFLC method allows to determine the analyte at much lower level of concentration than the MEEKC method, what is explained in 2.4.2 capture of the article.
One of the advantage in the MEEKC method is that the volume of solutions for the capillary inside rinsing and the microemulsion which fills the capillary is at the level of 1.4 ml each. This quantity is enough to the execution of 20 analyses. For the single sample analysis with the UFLC method approx. 15 ml of mobile phase consisting of the mixture of the acetonitrile and the methanol the purity HPLC grade is needed. On the basis of all received results of ?-tocopherol acetate quantitative analyses, it was found that the relative error for the UFLC method carried out in the range 2.3 % ? 2.9 %, and relative error for MEEKC method 6.5 % ? 8.6 %. Proposed methods can be used as the base of the creation of the other methods to the separations of fat-soluble vitamins mixtures and their qualitative and quantitative determination e.g. for vitamin A and D.
This work was supported by the ESF Human Capital Operational Programme grant 6/1/8.2.1./POKL/2009.
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Dariusz WIDEL ? a graduate in 2009, chemistry with the environment protection on the Mathematics and Sciences Faculty at Jan Kochanowski University of Humanities and Sciences in Kielce. He works as an assistant in Physical Chemistry Department of the Institute of Chemistry, UJK. He is accomplishing the doctoral thesis ,, Application of electromigration and chromatographic method in waters and drinks analysis? under the direction of Prof. Zygfryd Witkiewicz.
e-mail: email@example.com, phone: +48 41 349 70 54
Jerzy OSZCZUDŁOWSKI ? Ph.D., a graduate of Jagiellonian University. He is the author of 30 articles in international and Polish journals, several dozen lectures and communications on domestic and foreign scientific conferences . The Council Member of Editorial of the Aparatura Badawcza i Dydaktyczna journal. The member of Editorial in the Polish Normalization Committee. The reviewer applications in Operational Programme Innowacyjna Gospodarka. Currently, he holds the position of the vicedirector of Institute of Chemistry at Jan Kochanowski University in Kielce. The scientific practice: Sweden, University of Umea ? Department of Public Health and Environmental Studies, Environmental Chemistry and National Defense Research The Establishment (1993)
e-mail: Jerzy.Oszczudlowski@ujk.edu.pl, phone: +48 41 349 70 60
Zygfryd WITKIEWICZ ? Professor, Director of the Institute of Chemistry at Jan Kochanowski University in Kielce, as well as he is working at Military Technical Academy in Warsaw. Professor is the chief editor of Aparatura Badawcza i Dydaktyczna journal, edited by COBRABiD. He is the president of Technical Committee responsible for Air Quality in Polish Normalization Committee. He is also a member of The Analytical Chemistry Committee in Polish Academy of Science and a member of the Chromatographic Analysis commission, a member of PTChem, IUPAC and The Chromatographic Society. He published over 250 papers and 7 books, he is also co-author of 21 patents. Professor received the Cwiet Medal granted by the Russian Chromatographic Association.
e-mail: firstname.lastname@example.org, phone: +48 41 349 70 76
Marcin MOCZULSKI ? M.Sc., graduated from the University of Nature in Poznan (since 2008 University of Life Sciences), in the Department of Biotechnology (1999) In the years 1999-2000 he worked for Pofam-Poznan and 2000-2005 for Institute of Plant Genetics of the Polish Academy of Sciences (PAN). Since 2006, he has been working as Product Manager Electrophoresis Products at Comesa Polska Sp.z o.o. Beckman Coulter Biotechnology Division.
e-mail: email@example.com, phone: +48 668 341 900
12. Michałkiewicz S., Pryciak M., Małyszko J., Oszczudłowski J.: Voltammetric
Determination of ?-Tocopheryl Acetate in Pharmaceutical Dosage Forms.
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13. www.phenomenex.com; Application Detail (App ID: 18696) 2012.
Translation into English by the Author