Analytica Chimica Acta 520 (2004) 5767

Simultaneous determination of water- and fat-soluble vitamins in pharmaceutical preparations by high-performance liquid chromatography coupled with diode array detection
Bo rivoj Klejdus, Jitka Petrlov, David Pot eil, Vojt ech Adam, Radka Mikelov, Jan Vacek, Rene Kizek, Vlastimil Kub n
Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Zem ed elsk 1, CZ-613 00 Brno, Czech Republic Received 28 November 2003; received in revised form 27 January 2004; accepted 5 February 2004 Available online 9 April 2004

Abstract Water- and fat-soluble vitamins were separated on a MetaChem Polaris C18-A (150 mm 4.6 mm, 3 m particle size) in a single run using combined isocratic and linear gradient elution with a mobile phase consisting of 0.010% triuoroacetic acid of pH 3.9 (solvent A) and methanol (solvent B) at the ow rate 0.7 ml min1 . A linear gradient prole (A:B) started at 95:5 and was constant in the rst 4 min, then linearly decreased up to 2:98 during the next 6 min, then it was constant in the next 20 min and nally linearly increased up to 95:5 ratio of water phase in the last 5 min of the separation. The most suitable detection wavelength for simultaneous vitamin determination was 280 nm. The method was applied for the solid sample of pharmaceutical preparation (B-Komplex), fortied powdered drinks (multi-vitamin) and food samples. The results were in good agreement with the declared values. 2004 Elsevier B.V. All rights reserved.
Keywords: Simultaneous determination; Vitamins; Water- and fat-soluble vitamins; High-performance liquid chromatography; Pharmaceutical preparations; Drugs; Diode array detection; Mass spectrometry

1. Introduction Vitamins represent a group of various compounds, both chemically and analytically, because they comprise a wide range of biomolecules (Fig. 1). They may be present in several chemically diverse but biologically interconvertible forms. Their common properties reside solely in fact that they are essential dietary components for animals and/or humans [14]. They are needed in relatively small amounts to sustain life and good health. Furthermore, a signicant association between plasma l-ascorbic acid and initial non-verbal intelligence quotient (IQ) has been found in the boys [2,3]. UV-Vis spectrophotometry [57], uorimetry [8,9], chemiluminiscence [10,11], capillary electrophoresis [1214], microbiology [15,16] and high-performance liquid chromatography [8,1725] have been proposed for the
Corresponding author. Tel.: +420-5-4513-3285; fax: +420-5-4521-2044. ). E-mail address: kuban@mendelu.cz (V. Kub an 0003-2670/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.02.027

determination of vitamins. Nevertheless, there is no single analytical approach to determine water- and fat-soluble vitamins within a complicated matrix in a single run. In this work, we developed and optimised a highperformance liquid chromatographic method using diode array detection for determination water- and fat-soluble vitamins at a single run. The method was successfully applied to the determination of vitamins in pharmaceutical preparations, fortied powdered drinks and food samples.

2. Experimental 2.1. Chemicals and reagents All water- and fat-soluble vitamins and triuoroacetic acid (TFA) were purchased from Sigma Aldrich (St. Louis, MO, USA). Potassium dihydrogen phosphate and potassium hydroxide of analytical grade purity chemicals were from Pliva-Lachema (Brno, Czech Republic). HPLC-grade acetonitrile and methanol were from Merck (Darmstadt,

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Fig. 1. Chemical structures, basic biological activities and recommended daily doses of water- and fat-soluble vitamins.

Germany). The vitamin stock standard solutions at 100 g ml1 were prepared in ACS water (Sigma Aldrich, St. Louis, USA) or methanol (fat-soluble vitamins) and stored in the darkness at 4 C. Working standard solutions were prepared daily by mixing of the stock standard solutions in appropriate proportions and diluting with water or methanol, if necessary. All solutions were ltered through a 0.45 m Teon membrane lters (MetaChem, Torrance, CA, USA) prior to HPLC analyses. The pH value of the

aqueous solution was controlled using a MV870 pH meter (Praecitronic, Germany). The pH meter was regularly calibrated by a set of NBS buffers. 2.2. Chromatographic apparatus An HP 1100 liquid chromatographic system (HewlettPackard, Waldbronn, Germany) was equipped with a vacuum degasser (G1322A), a binary pump (G1312A), an auto

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sampler (G1313A), a column thermostat (G1316A), and a UV-Vis diode array detector (model G1315A) working at 190690 nm. The ChemStation software (Rev. A 08.01) controlled the whole liquid chromatographic system. Spectra were registered in the range of 190400 nm (SBW 100 nm). Chromatograms were registered at 280 nm. 2.3. Chromatographic conditions for sample analysis Reversed-phase chromatographic columns Zorbax AAA (150 mm 4.6 mm, 3.5 m particle size, Agilent Technologies, USA), Atlantis dC18 (150 mm 2.1 mm, 3 m particle size, Waters, Milford, MA, USA) and MetaChem Polaris C18-A (150 mm 4.6 mm, 3 m particle size, Ansys Technologies, Torrance, CA, USA) were tested for the separation of vitamins. The ow rate was 0.7 ml min1 . Auto sampler injection was 3 l. Gradient of mobile phase and its composition was tested, too. For other chromatographic conditions see Section 3. 2.4. High-performance liquid chromatographymass spectrometry An HP 1100 chromatographic system (Hewlett-Packard, Waldbronn, Germany) was equipped with a vacuum degasser (G1322A), a binary pump (G1312A), an auto sampler (G1313A), a column thermostat (G1316A) and a diode array detector (model G1315A). The system was coupled on-line to a mass selective HP MSD detector (G 1946A, Hewlett-Packard, Palo Alto, CA, USA). The ChemStation software (Rev A07.01) controlled the whole liquid chromatographic system. Vitamins were separated on a reversed-phase chromatographic column MetaChem Polaris C18-A (150 mm 4.6 mm, 3 m particle size, Ansys Technologies). The ow rate was 0.7 ml min1 . The column efuent was monitored with diode array detector at 280 nm and directly introduced into the quadruple mass spectrometer operated in positive ESI mode. The nebulizer gas pressure was 50 psi, the drying gas was nitrogen at a 10 l min1 at the temperature of 350 C and capillary voltage was 4000 V. The fragmentor voltage was set to 40 eV and the gain was one. The m/z spectra and data for the full scan mode were recorded in range 501500 m/z. The mass spectrometer was regularly calibrated with an ESI tuning solution obtained from Hewlett-Packard (Palo Alto, CA, USA). 2.5. Sample preparations 2.5.1. Pharmaceutical preparation and food supplement B-Komplex tablets (L civa, Prague, Czech Republic) consisted of vitamins (thiamine, riboavin, pyridoxine, pantothenic acid, nicotinamide) and adjuvants (lactose mohydrine, may amylose, gelatine, calcium stearate, talcum, saccharose, titanium dioxide, sodium caramel, parafn, silica gel anhydride, natural identical aromas, bres and additives).

Vitamin drink (Penko, Krnov, Czech Republic) consisted of vitamins (thiamine, riboavin, pyridoxine, pantothenic acid, nicotinamide, -tocopherol, cyanocobalamin, l-ascorbic acid, folic acid) and adjuvants (saccharose, glucose, E330, maltodextrine, natural identical aroma, milk proteins, lemon pectin, soy lecithin, ZnO, MnSO4 and Na2 SeO3 ). The tablets (100 mg) were homogenised by an A 11 basic IKA analysis mill (IKA-Werke, Staufen, Germany). The homogenised samples of B-Komplex and Vitamin drink (1 g of powder) were dissolved in 10 ml of solution consisted of 0.010% TFA:methanol (50:50) and stirred on Vortex (Scientic Industries, Vortex2 Genie, USA) for 15 min. 2.5.2. Food samples Soja milk (dry soy milk, ASP CZECH, Mod rice, Czech Republic) consisted of vitamins (trans-retinol, thiamine, cyanocobalamin, pyridoxine, l-ascorbic acid, pantothenic acid and nicotinamide) and adjuvants (soy protein isolate, dry maize syrup, soy oil, inulin, fructose, oat farina, lecithin, K3 PO4 , E471 and natural identical aroma). Nestl BEBA (infant milk food, Nestl Cesko, Prague, Czech Republic) consisted of vitamins (trans-retinol, vitamin K1 , l-ascorbic acid, thiamine, cyanocobalamin, pyridoxine, nicotinamide and pantothenic acid) and adjuvants (cow milk, lactose, plant oils, maize and potatoes amyl, soy lecithin, mineral compounds, trace elements and lyophilic bacteria). Fortuna silueta (milk drink, OLMA, Olomouc, Czech Republic) consisted of vitamins (thiamine, riboavin, pyridoxine, pantothenic acid, nicotinamide, -tocopherol) and adjuvants (lactose, cow milk, l-carnitin, roughage, mineral compounds and trace elements). Nestl BEBA and Soja milk samples were weighed (1 g), placed in a centrifuge tube and dissolved in 4 ml of solution consisted of 0.010% TFA:methanol (50:50). The mixtures were stirred on Vortex (Scientic Industries, Vortex2 Genie, USA) for 15 min. The solutions were centrifuged (14 000 g, 15 min, 4 C) on Hettich-Zentrifugen (Universal 32 R, Germany). Fortuna silueta was diluted (1 ml) with 9 ml of solution consisted of 0.010% TFA:methanol (50:50) and shaken by Vortex (Scientic Industries, Vortex2 Genie, USA) for 15 min. The solution was centrifuged (14 000 g, 15 min, 4 C, Hettich-Zentrifugen, Germany) on Universal 32 R centrifuge [26]. All supernatants and homogenates were evaporated to dryness in a rotary vacuum evaporator. The residues were dissolved in 1 ml of solution consisted of 0.010% TFA:methanol (50:50) and ltered through a 0.45 m Teon membrane lters (MetaChem) prior to HPLC analyses before injection on the reverse-phase column.

3. Results and discussion A simultaneous analysis of water- and fat-soluble vitamins (Fig. 1) by chromatographic methods is very difcult

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due to their completely different physical and chemical properties [14]. In addition, the major analytical challenges they present are derived from the fact that there is a need to quantify them in a wide range of biological matrices, which include both foodstuff and body uids [11,23,24,27]. In our work we attempted to simultaneously analyse water- and fat-soluble vitamins by high-performance liquid chromatography coupled with a diode array detector (HPLCDAD). 3.1. Separation optimisation and chromatographic column selection Primarily it was necessary to select the most suitable wavelength for the simultaneous detection of water- and fat-soluble vitamins since spectral properties of individual vitamins differ very seriously [22,23]. Several wavelengths in UV spectral range (190400 nm) were tested for the detection using a mixture of water-soluble vitamins B (thiamine, riboavin and pyridoxine) and fat-soluble vitamins (retinol and -tocopherol). Based on our measurements, on theoretical values of molar absorptivity coefcients of the individual detected vitamins and on the most commonly used wavelengths for vitamin determination we selected three suitable wavelengths 230, 266 and 280 nm for the following experiments [7,13,20,21,23,25,28,29]. A type of chosen chromatographic column has a signicant inuence on separation of vitamins. Due to the serious differences in polarity of both groups of vitamins the compromise between hydrophilic and lipophilic character of sorbents has to be found. That is why three kinds of chromatographic columns: Zorbax AAA, Atlantis and MetaChem Polaris, were compared on the basis of retention factor of very neighbouring vitamin peaks (thiamine and nicotinic acid; retinol acetate and trans-retinol, respectively) to evaluate the column performance (results not shown). Results of this comparison show that the column MetaChem Polaris was the most suitable for the separation of both groups of vitamins in a single run and this column was used for all other experiments. 3.2. Separation of water-soluble vitamins Mobile phases containing potassium dihydrogen phosphate or sodium acetate as buffering compounds are obviously recommended as the most suitable to assure the very good chromatographic separation of water-soluble vitamins. The pH-values of mobile phases were adjusted by addition of sodium or potassium hydroxide and/or acetic or phosphoric acid [2022]. The baseline separation of the water-soluble vitamins was also achieved using mobile phases consisting of relatively low concentrations of organic, water miscible solvents and aqueous solution of inorganic components as buffering agents [2022,30]. During our preliminary experiments, several different mobile phases were tested (10 mM potassium dihydrogen phosphate with acetonitrile or methanol and 0.010% tri-

uoroacetic acid with methanol or acetonitrile (ACN) of different composition. We obtained well-separated symmetric peaks of individual B vitamins with exception of co-eluting thiamine and nicotinic acid when we applied the mixture of 10 mM potassium dihydrogen phosphate at pH 6.0 and 08% ACN as a mobile phase [2022]. The best chromatographic separation of the water-soluble vitamins was obtained at the highest content of inorganic part (99.5% phosphate of pH 6; 0.5% acetonitrile) in the mobile phase (not shown). This mobile phase was suitable for the very effective separation of the water-soluble vitamins but unfortunately serious silting of column was observed due to relatively high contents of inorganic salts. This fact signicantly limited applicability of the method for routine analysis. Thus we tried to nd a different mobile phase. It is well known that triuoroacetic acid (TFA) is a very strong acid and the very suitable buffering agent. It is also a very strong ion-pairing agent that can react with individual vitamins and may assign them charge. Thus, we selected a mixture of 0.010% triuoroacetic acid with methanol at different ratios as another type of the mobile phase. 3.3. Effect of pH The pH value of aqueous phase of the 0.010% triuoroacetic acid/methanol mobile phase dramatically inuenced chromatographic separation of water-soluble vitamins (l-ascorbic acid, thiamine, nicotinic acid, pyridoxal, pyridoxine, nicotinamide, pantothenic acid, cyanocobalamin, riboavin and folic acid) and p-aminobenzoic acid and thioctic acid. Three basic chromatographic parameters (the retention factor, the symmetry factor and the peak area) were evaluated in our experiments. The retention factor was calculated as k = (tR tM )/tM ; where tR is the retention time of a studied compound and tM is the dead retention time. The changes in retention factor, which are dependent on pH value, of the water-soluble vitamins are well detectable in Fig. 2A. A very good and even relatively very fast (less than 15 min) separation of individual water-soluble vitamins was obtained using the 0.010% triuoroacetic acid/methanol mobile phase at the pH value 3.9 of the TFA aqueous solution. We dened the symmetry of peaks by a symmetry factor equal to one for absolutely symmetrical chromatographic peak. The chromatographic peaks with symmetry factors equal to 0.81.2 were considered as highly symmetric (graph is shown in Fig. 2B). Majority of studied compounds pertained to interim of good symmetric chromatographic peak (symmetry factor, 1.0 0.2) at pH 3.9 (see Fig. 2B). Beyond good chromatographic separation, it was necessary to ensure good sensitivity of the developed method. The evaluation was based on the peak area of the water-soluble vitamins measured in the pH-range from 3.2 to 5.1. The maximum values were observed either at upper or at lower pH-values with the local maximum at pH value 3.9 of aqueous phase (Fig. 3A). At the lowest pH value (pH < 3.2),

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Fig. 2. Dependences of retention (A and C) and symmetry (B and D) factors of water- and fat-soluble vitamins (100 g ml1 of each) on pH values, respectively. MetaChem Polaris C18-A column, mobile phase consisted of 0.010% TFA and methanol; gradient prole started at 92:8 (TFA:methanol; (v/v)) and was constant in the rst 2 min, then decreased to 2:98 during the next 10 min and nally linearly increased up to 92:8 from 32 to 35 min. Vitamin concentrations were 100 g ml1 . Autosampler injection was 3 l. HPLCUV parameters: MetaChem Polaris C18-A (150 mm 4.6 mm, 3 m particle size, Ansys Technologies, Torrance, CA, USA; isocratic ow rate of 0.7 ml min1 ; column temperature 20 C; detection wavelength of DAD is 280 nm. Symbols of individual vitamins in gures are given in Table 1.

the results were irreproducible: the peaks were broad and poorly resolved. Primarily, for comparison of peak areas of individual vitamins at pH = 3.6, 3.7, 3.9, 4.3 and 5.1 we calculated relative sensitivities (as a maximal peak factorMPf ) according to the formula: MPf = PA /PA,max 100 (%); PA is the peak area of individual vitamin at the given pH value; PA,max is the highest peak area of an individual vitamin in the whole range of pH values. Furthermore, for complete set of fatand water-soluble vitamins we compared the total number of MPf values exceeding value of MPf = 80, 90 and 95% (Fig. 3C). We found the highest frequency of MPf exceeding the critical values at pH 3.9 (Fig. 3C) for all vitamins thus the pH (3.9) value was selected as the most advisable for separation of water-soluble vitamins. 3.4. Inuence of triuoroacetic acid on vitamin separation We assumed that the content of TFA in the mobile phase seriously inuenced chromatographic separation of individual compounds. The inuence of different concentration of

triuoroacetic acid (0.005, 0.010, 0.015, 0.020 and 0.030%) on separation of water-soluble vitamins was studied. On the basis of evaluation of retention factors, symmetry factors and peak areas of individual analytical signals we concluded that the TFA concentration equal to 0.010% was the most suitable for the baseline separation. TFA probably inuenced acid-base equilibrium of studied vitamins due to its acid-base properties (very strong acid, pKa1 0) and markedly improved efciency of chromatographic separation of the vitamins. 3.5. Effect of temperature on vitamin separation The column temperature affects efciency of chromatographic separation too. We tested the impact of four different temperatures (15, 20, 25 and 30 C) using the retention factor, the symmetry factor and the peak area as the basic criteria. A less efcient and signicantly slower separation (about 15% longer than at 20 C) was observed at 15 C. The separation was markedly faster at higher temperatures (25 and 30 C) but co-elution appeared gradually. Thus the temperature of 20 C we selected as the optimal temperature

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Fig. 3. Dependence of peak areas of water- (A) and fat-soluble (B) vitamins on pH and the total number of MPf values exceeding MPf = 80, 90 and 95%, respectively. (C) Other conditions are the same as in Fig. 2.

for the very effective and fast separation of water-soluble vitamins and used in all further experiments. 3.6. Gradient elution Gradient and isocratic elution proles were used for the water-soluble vitamins separation [2022,31]. Mixtures of inorganic salts with organic solvents (acetonitrile) were most frequently applied as the mobile phase [20,21]. In our experiments, we tested the applicability of combined isocratic and linear gradient prole of the mobile phase on the separation of water-soluble vitamins (B group). The mobile phase consisted of 0.010% aqueous TFA at pH 3.9 and methanol (in ratio 92:8). Primarily we selected prole that started at 92:8 and was constant in the rst 2 min, then decreased to 2:98 during the next 6 min, was kept constant for the next 20 min and nally linearly increased up to 92:8 in the last 5 min. The baseline separation of B vitamins was achieved but the conditions were not very suitable for separation of other water-soluble vitamins (pantothenic acid, cyanocobalamin, riboavin and folic acid) and thioctic acid

and p-aminobenzoic acid. Thus different linear gradient proles were tested. The other water-soluble vitamins and p-aminobenzoic acid and thioctic acid were added to group of vitamins B in the next experiments. Different ratios of 0.010% aqueous TFA at pH 3.9 and methanol (88:12, 92:8, 95:5 and 97:3, respectively) were tested for the initial isocratic elution. The retention factors and the symmetry factors decreased with the decreasing TFA ratio. The symmetry factors, the peak areas and the retention factors for chromatographic separation of the vitamins were the best for the 92:8 and 95:5 TFA/methanol ratios. Increasing dispersion of the chromatographic peaks was observed at the higher ratios of water phase (95:5). Next we tested different time intervals in which the composition of mobile phase was kept constant (isocratic elution). The time of the initial interval of isocratic ow varied from 2 to 8 min. After that period the optimised linear gradient prole consisting of decrease from 95:5 TFA:methanol in the rst 6 min up to 2:98, isocratic elution at 2:98 in the next 20 min and nal linear increase up to

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92:8 ratio in 5 min was applied in each run. The symmetry factors and the retention time differences between the last peak of B-group vitamins and the peaks of p-aminobenzoic acid, thioctic acid and water-soluble vitamins were used as a criterion of separation efciency. Both groups of vitamins were linearly drifting (2.3tR per min) with increasing time of isocratic ow of mobile phase. The symmetry factors were changing, too. The symmetry factor was nearly one when period of isocratic elution lasted 4 min (not shown). When the initial interval of isocratic elution was excessively prolonged, the retention times of some vitamins (pantothenic acid, cyanocobalamin, riboavin and folic acid) and thioctic acid and p-aminobenzoic acid increased. Based on the chromatographic behaviour the group of water-soluble substances could be divided into two distinct sub-groupslow-polar compounds (pantothenic acid, p-aminobenzoic acid, cyanocobalamin, riboavin, thioctic acid and folic acid) and polar ones (l-ascorbic acid, thiamine, nicotinic acid, pyridoxal, pyridoxine and nicotinamide). The chromatographic behaviour of both sub-groups differed seriously. Resulted chromatographic gradient prole started at 95:5 (0.010% TFA:methanol) and

was constant in the rst 4 min, then decreased to 2:98 during the next 6 min, was constant for next 20 min and nally linearly increased up to 98:2 in the last 5 min (Fig. 5A). The optimised conditions of chromatographic separation of water-soluble substances allowed the very efcient and highly sensitive separation of l-ascorbic acid, thiamine, nicotinic acid, pyridoxal, pyridoxine, nicotinamide, pantothenic acid, cyanocobalamin, riboavin, folic acid, thioctic acid and p-aminobenzoic acid (100 g ml1 ) in the rst 15 min. The well-separated chromatographic signals at 280 nm are shown in Fig. 4A. 3.7. Separation of fat-soluble vitamins Separation of some fat-soluble vitamins was described in literature [24,28,32]. Chromatographic behaviour of fat-soluble vitamins (trans-retinol, retinol-acetate, D2, D3, K2-isomer, -tocopherol, tocopherol-acetate, K1, K1-isomer and K2) using 0.010% TFA (pH 3.9): methanol was studied in the next part of our experiment. Optimised linear gradient prole was used because of the very good solubility of the water-soluble vitamins in water and methanol and the very good solubility of fat-soluble

Fig. 4. HPLCUV chromatograms of water- (A) and fat-soluble (B) vitamins at 280 nm. Vitamin concentrations were 100 g ml1 . Other conditions are the same as in Fig. 2.

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vitamins in methanol [13]. Less pronounced changes in the chromatographic parameters (the retention factor, the symmetry factor and the peak area) with changes in separation conditions (pH, column temperature, TFA concentration, gradient prole) were observed for fat-soluble vitamins in contrast to the water-soluble substances. Variations in pH, column temperature and TFA concentration inuenced the retention factor less then 30% (not shown). The maximum of the peak area was obtained again at pH value about 3.9. The symmetry factor was approximating the criterion at pH value 3.9 and the negative values of the symmetry factors at other pH values conrmed peak tailing Fig. 2C and D). Peak areas increased at pH 3.9 and then they were constant or they slightly increased (Fig. 3B). The well-separated chromatographic peaks of fat-soluble vitamins at 280 nm are shown in Fig. 4B. 3.8. Simultaneous determination of water-soluble and fat-soluble vitamins A standard mixture of the vitamins and other compounds (100 g ml1 of each) was prepared before analyses. The baselines of the full scan separations follow the gradient prole (Fig. 5) of the simultaneous separations of waterand fat-soluble vitamins at shorter wavelengths (230 and 266 nm) and the optimised chromatographic conditions. The adverse effects of gradient elution prole disappear (see Fig. 5D) at longer wavelengths (280 nm). The wavelength at 280 nm was selected as the most suitable for detection and quantication of vitamins in the further experiments. The identity (retention time) of all individual detectable vitamins (water-soluble and fat-soluble) was conrmed by standard addition and HPLCMS detection methods [33]. The mixture was used for construction of calibration curves (peak areas versus compound concentration) by stepwise dilution with ACS water. Equations of bisectors, R2 , detection limits, quantication limits (for 3.S/N and 10.S/N criterion, respectively) and concentration range (101000 g ml1 ) are given in Table 1. Typical HPLCUV chromatogram of the mixture of water- and fat-soluble vitamins and other substances (100 g ml1 ) using the combined isocratic and linear gradient prole is presented in Fig. 6. 3.9. Identication and determination of vitamins in real samples Furthermore we tested the elaborated analytical chromatographic method for the determination of water- and fat-soluble vitamins and thioctic and p-aminobenzoic acids in a more complicated matrix represented by foods, food supplements, pharmaceutical preparation and fortied drinks. There are a lot of additives of different origin and characteristics (see lists above) in such samples that make vitamin determination more difcult.

Fig. 5. Gradient prole (A, mobile phase methanol) for simultaneous vitamin separation that started at 95:5 (0.010% TFA:methanol) and was constant in the rst 4 min, then decreased to 2:98 during the next 10 min and nally linearly increased up to 95:5 from 32 to 35 min is shown on (A); pH value was 3.9. Simultaneous HPLCUV chromatograms of water- and fat-soluble vitamins at three detection wavelengths: 230 nm (B), 266 nm (C) and 280 nm (D). Vitamin concentrations were 100 g ml1 . Other conditions are the same as in Fig. 2.

Vitamins in homogenised sample of pharmaceutical preparation (B-Komplex) and food supplement (multivitamin drink) were separated and determined by HPLCUV (Fig. 7A and B, respectively). An average recovery of 100 and 99% were determined, respectively. Declared and found concentrations are compared in Table 2. Vitamins in homogenised samples (Soja milk, Nestl BEBA and Fortuna silueta) were separated and determined by the same procedure. Chromatogram of vitamin separation and determination in Fortuna milk is shown in Fig. 7C. Average recoveries for individual food samples (Soja milk, Nestl BEBA and Fortuna silueta) of 103, 101 and 95% were determined, respectively. Declared and found concentrations are compared in Table 3.

B. Klejdus et al. / Analytica Chimica Acta 520 (2004) 5767 Table 1 Validation data for the determination of water- and fat-soluble vitamins (n = 5) at 280 nm Vitamin l-Ascorbic acid Cyanocobalamin D2 D3 Folic acid K1 K1 -isomer K2 K2 -isomer Nicotinamide Nicotinic acid p-Aminobenzoic acidf Pantothenic acid Pyridoxal Pyridoxine Retinol-acetate Riboavin Thiamine Thioctic acidf -Tocopherol Tocopherol acetate Trans-retinol
a b c d e f

65

tR (min)a 2.83 8.56 21.43 22.07 14.00 25.98 28.24 30.69 22.98 5.67 3.59 8.37 7.83 3.93 4.59 17.37 9.03 3.19 13.47 23.57 25.31 15.99

Regression equation y = 2.4x 4.1 y = 0.88x 1.7 y = 1.8x 21 y = 9.8x 67 y = 2.2x + 291 y = 2.5x 11 y = 2.0x 8.8 y = 7.0x 30 y = 0.68x + 15 y = 2.6x 16 y = 3.3x 40 y = 13x 28 y = 0.0097x + 0.13 y = 4.6x 80 y = 4.7x 30 y = 10x 68 y = 2.6x + 3.3 y = 10x 188 y = 0.46x 1.2 y = 7.4x 57 y = 1.4x 26 y = 7.7x 58

R2 , b 0.9986 0.9995 0.9990 0.9990 0.9887 0.9974 0.9977 0.9988 0.9975 0.9979 0.9952 0.9980 0.9958 0.9880 0.9939 0.9936 0.9992 0.9991 0.9983 0.9988 0.9998 0.9941

LOD (ng ml1 )c 25.55 97.47 129.4 41.77 30.48 157.9 171.6 83.10 197.4 35.40 16.41 16.52 865.4 23.08 21.17 12.49 188.0 12.18 394.7 34.78 127.3 12.30

LOQ (ng ml1 )d 85.16 324.9 431.1 139.4 101.1 526.2 572.1 277.0 657.9 118.1 54.71 55.05 2884 76.95 70.55 41.64 626.6 40.61 1316 115.9 424.5 40.99

R.S.D. (%)e 2.1 3.2 2.9 2.3 2.0 2.5 3.2 2.6 3.5 1.9 2.1 3.6 8.2 2.2 2.7 2.3 2.0 1.8 2.1 2.8 2.6 2.1

Symbols in gures + |

Retention times in min. Regression coefcients. Limits of detection (3.S/N). Limits of quantitation (10.S/N). Relative standard deviations. Non-vitaminous substances.

Fig. 6. Simultaneous HPLCUV determination of water- and fat-soluble vitamins at 280 nm. Inset shows the gradient prole (A, mobile phase methanol). Other conditions are the same as in Fig. 5.

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Fig. 7. Simultaneous HPLCUV determination of water- and fat-soluble vitamins at 280 nm in pharmaceutical preparation, B-Komplex (A); food supplement, Vitamin drink (B) and food sample, Fortuna silueta (C). Other conditions are the same as in Fig. 5.

Table 2 Declared and found vitamin concentrations in B-Komplex and in vitamin drink powder (n = 5) Vitamins B-Komplex (mg per tablet) Declared Thiamine Riboavin Pyridoxine Pantothenic acid Nicotinamide -Tocopherol Cyanocobalamin l-Ascorbic acid Folic acid nd: not declared, ND: not found. 15 15 10 25 50 nd nd nd nd Found 14. 8 0.7 15.7 0.7 9.1 1.0 25.1 0.9 53.3 1.3 ND ND ND ND Vitamin drink (mg per 100 g powder) Declared 7.0 8.5 10.0 36.0 90.0 60.0 0.025 575.0 1.0 Found 6.3 0.8 81 9.3 0.7 36 2 89 2 59 1 0.030 0.002 (5.2 0.2) 102 1.1 0.4

Table 3 Declared and found concentrations of vitamins in food samples (n = 5) Vitamins Fortuna silueta (mg per 100 ml) Declared Thiamine Riboavin Pyridoxine Pantothenic acid Nicotinamide Tocopherol Cyanocobalamin l-Ascorbic acid Folic acid Trans-retinol Vitamin K1 0.28 0.32 0.40 2.0 3.60 2.00 nd nd 0.04 nd nd Found 0.26 0.01 0.29 0.03 0.39 0.02 1.9 0.4 3.5 0.2 1.87 0.09 ND ND 0.07 0.03 ND ND Nestl e BEBA (mg per 100 g powder) Declared 1.40 nd 2.00 6.00 18 nd 1 103 60 nd 0.8 nd Found 1.2 0.2 ND 2.2 0.5 7.8 2.2 16.9 0.1 ND (8 2) 104 58 3 ND 0.9 0.2 ND Soja milk (mg per 100 g powder) Declared 0.72 1.1 0.95 3.3 13 4.0 103 9.5 104 48 0.14 0.57 21 Found 0.68 0.08 1.4 0.4 1.0 0.3 41 12.8 0.2 (2.8 0.5) 103 (9.0 0.7) 104 46 3 0.16 0.05 0.59 0.03 22 1

nd: not declared, ND: not found.

B. Klejdus et al. / Analytica Chimica Acta 520 (2004) 5767

67

4. Conclusion The conditions for the simultaneous separation and determination of fat- and water-soluble vitamins and thioctic and p-aminobenzoic acids by high-performance liquid chromatography coupled to a diode array detector were optimised and applied for the analyses of samples of pharmaceutical preparations, foods, food supplement and fortied drinks. The combined isocratic and linear gradient prole of the mobile phase consisting of 0.010% TFA of pH 3.9 and methanol allowed determination of the vitamins in three distinct groupspolar (isocratic elution at 95:5 in 6 min), low-polar (positive linear gradient elution, tR between 6th and 14th min) and non-polar (negative linear gradient elution, tR longer then 15 min). A baseline separation with good resolution of all vitamins in each group was achieved in a single run. Our results were in the very good agreement with the declared values. Nevertheless vitaminvitamin, vitaminmobile phase and vitamincolumn adsorbent interactions [13] could cause unknown and unexpected signals (see Figs. 46). Our chromatographic method allowed the very easy and fast vitamin separation of both groups of vitamins and thioctic and p-aminobenzoic acids in complicated matrices. This unique methodology can be applied for the determination of vitamins in pharmaceutical preparations, food supplement, fortied drinks and foods with relatively complicated matrices.

[3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]

Acknowledgements The nancial support from the Grant Agency of the Ministry of Education, Youth and Sports of the Czech Republic Grant Reg. No. MSM 432100001), is grate(MMT CR, fully acknowledged.

[26] [27] [28] [29] [30]

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