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Analytica Chimica Acta 520 (2004) 57–67

Simultaneous determination of water- and fat-soluble vitamins in pharmaceutical preparations by high-performance liquid chromatography coupled with diode array detection
Boˇ rivoj Klejdus, Jitka Petrlová, David Potˇ ešil, Vojtˇ ech Adam, Radka Mikelová, Jan Vacek, Rene Kizek, Vlastimil Kubᡠn∗
Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Zemˇ edˇ elská 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% trifluoroacetic acid of pH 3.9 (solvent A) and methanol (solvent B) at the flow rate 0.7 ml min−1 . A linear gradient profile (A:B) started at 95:5 and was constant in the first 4 min, then linearly decreased up to 2:98 during the next 6 min, then it was constant in the next 20 min and finally 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), fortified 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 [1–4]. They are needed in relatively small amounts to sustain life and good health. Furthermore, a significant association between plasma l-ascorbic acid and initial non-verbal intelligence quotient (IQ) has been found in the boys [2,3]. UV-Vis spectrophotometry [5–7], fluorimetry [8,9], chemiluminiscence [10,11], capillary electrophoresis [12–14], microbiology [15,16] and high-performance liquid chromatography [8,17–25] have been proposed for the
Corresponding author. Tel.: +420-5-4513-3285; fax: +420-5-4521-2044. ˇ ). 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, fortified powdered drinks and food samples.

2. Experimental 2.1. Chemicals and reagents All water- and fat-soluble vitamins and trifluoroacetic 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,

an auto . 1. basic biological activities and recommended daily doses of water. St. All solutions were filtered through a 0. / Analytica Chimica Acta 520 (2004) 57–67 Fig. Klejdus et al. The pH meter was regularly calibrated by a set of NBS buffers. 2. USA) prior to HPLC analyses. The vitamin stock standard solutions at 100 ␮g ml−1 were prepared in ACS water (Sigma Aldrich. Chemical structures. Germany) was equipped with a vacuum degasser (G1322A). Louis. Waldbronn. a binary pump (G1312A). CA.58 B. Chromatographic apparatus An HP 1100 liquid chromatographic system (HewlettPackard. Torrance. if necessary.45 ␮m Teflon membrane filters (MetaChem. Working standard solutions were prepared daily by mixing of the stock standard solutions in appropriate proportions and diluting with water or methanol.2. USA) or methanol (fat-soluble vitamins) and stored in the darkness at 4 ◦ C. Germany). Germany).and fat-soluble vitamins. The pH value of the aqueous solution was controlled using a MV870 pH meter (Praecitronic.

B. ␣-tocopherol) and adjuvants (lactose. dry maize syrup. a column thermostat (G1316A). 3.01) controlled the whole liquid chromatographic system.010% TFA:methanol (50:50). Czech Republic) consisted of vitamins (trans-retinol. Results and discussion A simultaneous analysis of water. inulin. Germany). ZnO. Spectra were registered in the range of 190–400 nm (SBW 100 nm).7 ml min−1 . pyridoxine. K3 PO4 . glucose. pyridoxine. MnSO4 and Na2 SeO3 ). a binary pump (G1312A). Auto sampler injection was 3 ␮l. Prague. 4 ◦ C) on Hettich-Zentrifugen (Universal 32 R. cyanocobalamin. and a UV-Vis diode array detector (model G1315A) working at 190–690 nm. 2. Nestlé Cesko. too.6 mm. folic acid) and adjuvants (saccharose. 15 min. nicotinamide) and adjuvants (lactose mohydrine.45 ␮m Teflon membrane filters (MetaChem) prior to HPLC analyses before injection on the reverse-phase column. Waters.5. Nestlé BEBA ˇ (infant milk food. Hewlett-Packard. riboflavin. cyanocobalamin. pantothenic acid. Waldbronn. Olomouc. The mixtures were stirred on Vortex (Scientific Industries. Ansys Technologies). 2. nicotinamide and pantothenic acid) and adjuvants (cow milk.4. trace elements and lyophilic bacteria). Germany). Nestlé BEBA and Soja milk samples were weighed (1 g). pyridoxine.5. pyridoxine.3. maize and potatoes amyl. Vortex–2 Genie. cow milk. Ansys Technologies. natural identical aromas. The tablets (100 mg) were homogenised by an A 11 basic IKA analysis mill (IKA-Werke. roughage. E471 and natural identical aroma).6 mm. milk proteins. Modˇ rice. Czech Republic) consisted of vitamins (thiamine. vitamin K1 . Sample preparations 2. pantothenic acid and nicotinamide) and adjuvants (soy protein isolate. The solutions were centrifuged (14 000 × g. Staufen. Czech Republic) consisted of vitamins (thiamine. Vortex–2 Genie. Germany) was equipped with a vacuum degasser (G1322A).7 ml min−1 . ASP CZECH. The residues were dissolved in 1 ml of solution consisted of 0. Agilent Technologies. USA) for 15 min.010% TFA:methanol (50:50) and filtered through a 0. USA) and MetaChem Polaris C18-A (150 mm × 4. pyridoxine. The fragmentor voltage was set to 40 eV and the gain was one. talcum.and fat-soluble vitamins (Fig. Chromatograms were registered at 280 nm. 3 ␮m particle size. pantothenic acid. The mass spectrometer was regularly calibrated with an ESI tuning solution obtained from Hewlett-Packard (Palo Alto. The flow rate was 0. Vitamins were separated on a reversed-phase chromatographic column MetaChem Polaris C18-A (150 mm × 4. oat farina. USA). nicotinamide. silica gel anhydride. 1) by chromatographic methods is very difficult . Vitamin drink (Penko. The m/z spectra and data for the full scan mode were recorded in range 50–1500 m/z. mineral compounds. natural identical aroma. Hettich-Zentrifugen. OLMA. a column thermostat (G1316A) and a diode array detector (model G1315A). The solution was centrifuged (14 000 × g. maltodextrine.6 mm. CA. CA. lactose. Vortex–2 Genie.5 ␮m particle size. E330. fructose. The flow rate was 0. soy lecithin. 3. riboflavin. The homogenised samples of B-Komplex and Vitamin drink (1 g of powder) were dissolved in 10 ml of solution consisted of 0. The nebulizer gas pressure was 50 psi. ␣-tocopherol. placed in a centrifuge tube and dissolved in 4 ml of solution consisted of 0. A 08. nicotinamide. USA) for 15 min.2. soy oil. For other chromatographic conditions see Section 3. fibres and additives). Milford.010% TFA:methanol (50:50) and stirred on Vortex (Scientific Industries. Czech Republic) consisted of vitamins (trans-retinol. Germany) on Universal 32 R centrifuge [26]. Fortuna silueta was diluted (1 ml) with 9 ml of solution consisted of 0. / Analytica Chimica Acta 520 (2004) 57–67 59 sampler (G1313A). cyanocobalamin. l-ascorbic acid. MA. gelatine. 2. USA). USA). l-ascorbic acid. Gradient of mobile phase and its composition was tested.5. riboflavin. USA) for 15 min. 3 ␮m particle size. Czech Republic) consisted of vitamins (thiamine. The column effluent was monitored with diode array detector at 280 nm and directly introduced into the quadruple mass spectrometer operated in positive ESI mode.010% TFA:methanol (50:50) and shaken by Vortex (Scientific Industries. USA) were tested for the separation of vitamins. lemon pectin.1 mm. mineral compounds and trace elements). pantothenic acid. 4 ◦ C. titanium dioxide. thiamine. may amylose. the drying gas was nitrogen at a 10 l min−1 at the temperature of 350 ◦ C and capillary voltage was 4000 V. 2. Fortuna silueta (milk drink. saccharose. Palo Alto. thiamine. soy lecithin. Food samples Soja milk (dry soy milk. 3 ␮m particle size. 15 min. Torrance. High-performance liquid chromatography–mass spectrometry An HP 1100 chromatographic system (Hewlett-Packard. paraffin. The ChemStation software (Rev. Prague.01) controlled the whole liquid chromatographic system. The system was coupled on-line to a mass selective HP MSD detector (G 1946A. lecithin. Chromatographic conditions for sample analysis Reversed-phase chromatographic columns Zorbax AAA (150 mm × 4. Pharmaceutical preparation and food supplement B-Komplex tablets (Léˇ civa. an auto sampler (G1313A). All supernatants and homogenates were evaporated to dryness in a rotary vacuum evaporator. Atlantis dC18 (150 mm × 2. calcium stearate. l-carnitin. l-ascorbic acid. sodium caramel. plant oils. CA. Klejdus et al. The ChemStation software (Rev A07. Krnov.1.

respectively) to evaluate the column performance (results not shown).30].and fat-soluble vitamins since spectral properties of individual vitamins differ very seriously [22. riboflavin and folic acid) and p-aminobenzoic acid and thioctic acid.23.23. 2B). riboflavin and pyridoxine) and fat-soluble vitamins (retinol and ␣-tocopherol). The maximum values were observed either at upper or at lower pH-values with the local maximum at pH value 3. it was necessary to ensure good sensitivity of the developed method.and fat-soluble vitamins by high-performance liquid chromatography coupled with a diode array detector (HPLC–DAD). which include both foodstuff and body fluids [11. The chromatographic peaks with symmetry factors equal to 0.20.5% phosphate of pH 6. thiamine. nicotinamide. which are dependent on pH value. Effect of pH The pH value of aqueous phase of the 0.1. 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. pyridoxal. 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. The pH-values of mobile phases were adjusted by addition of sodium or potassium hydroxide and/or acetic or phosphoric acid [20–22]. That is why three kinds of chromatographic columns: Zorbax AAA. cyanocobalamin.010% tri- fluoroacetic acid with methanol or acetonitrile (ACN) of different composition. Based on our measurements. During our preliminary experiments.23]. where tR is the retention time of a studied compound and tM is the dead retention time.5% acetonitrile) in the mobile phase (not shown). several different mobile phases were tested (10 mM potassium dihydrogen phosphate with acetonitrile or methanol and 0.3. on theoretical values of molar absorptivity coefficients of the individual detected vitamins and on the most commonly used wavelengths for vitamin determination we selected three suitable wavelengths 230. At the lowest pH value (pH < 3.21.13.8–1. / Analytica Chimica Acta 520 (2004) 57–67 due to their completely different physical and chemical properties [14]. A very good and even relatively very fast (less than 15 min) separation of individual water-soluble vitamins was obtained using the 0. Thus we tried to find a different mobile phase. 3. 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. Atlantis and MetaChem Polaris. The changes in retention factor. 3.010% trifluoroacetic acid/methanol mobile phase at the pH value 3. In addition.0 and 0–8% ACN as a mobile phase [20–22]. This fact significantly limited applicability of the method for routine analysis. water miscible solvents and aqueous solution of inorganic components as buffering agents [20–22. Several wavelengths in UV spectral range (190–400 nm) were tested for the detection using a mixture of water-soluble vitamins B (thiamine. were compared on the basis of retention factor of very neighbouring vitamin peaks (thiamine and nicotinic acid.9 (see Fig. 3A). nicotinic acid. 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. pantothenic acid.2. 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.010% trifluoroacetic acid/methanol mobile phase dramatically influenced chromatographic separation of water-soluble vitamins (l-ascorbic acid.9 of the TFA aqueous solution.29]. The retention factor was calculated as k = (tR − tM )/tM .2) at pH 3. pyridoxine.27].0 ± 0. 0. the symmetry factor and the peak area) were evaluated in our experiments. The baseline separation of the water-soluble vitamins was also achieved using mobile phases consisting of relatively low concentrations of organic.1. Separation optimisation and chromatographic column selection Primarily it was necessary to select the most suitable wavelength for the simultaneous detection of water. Majority of studied compounds pertained to interim of good symmetric chromatographic peak (symmetry factor. It is well known that trifluoroacetic acid (TFA) is a very strong acid and the very suitable buffering agent.25. 3. . The best chromatographic separation of the water-soluble vitamins was obtained at the highest content of inorganic part (99. Three basic chromatographic parameters (the retention factor. 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. 1. 2A. The evaluation was based on the peak area of the water-soluble vitamins measured in the pH-range from 3.010% trifluoroacetic acid with methanol at different ratios as another type of the mobile phase. A type of chosen chromatographic column has a significant influence on separation of vitamins. Beyond good chromatographic separation. retinol acetate and trans-retinol.28.9 of aqueous phase (Fig. In our work we attempted to simultaneously analyse water.60 B.24. 2B). of the water-soluble vitamins are well detectable in Fig. Klejdus et al.2). We defined the symmetry of peaks by a symmetry factor equal to one for absolutely symmetrical chromatographic peak.2 were considered as highly symmetric (graph is shown in Fig. 266 and 280 nm for the following experiments [7. 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.2 to 5.

column temperature 20 ◦ C.9.010% was the most suitable for the baseline separation. detection wavelength of DAD is 280 nm. Klejdus et al. 90 and 95% (Fig. Ansys Technologies. then decreased to 2:98 during the next 10 min and finally linearly increased up to 92:8 from 32 to 35 min. 0. respectively.6. A less efficient and significantly slower separation (about 15% longer than at 20 ◦ C) was observed at 15 ◦ C. 2.020 and 0. The influence of different concentration of trifluoroacetic acid (0. pKa1 ≈ 0) and markedly improved efficiency of chromatographic separation of the vitamins. the results were irreproducible: the peaks were broad and poorly resolved. / Analytica Chimica Acta 520 (2004) 57–67 61 Fig.015.5. 3 ␮m particle size. We found the highest frequency of MPf exceeding the critical values at pH 3. mobile phase consisted of 0. the symmetry factor and the peak area as the basic criteria.and fat-soluble vitamins (100 ␮g ml−1 of each) on pH values. 0.005.7 ml min−1 .9) value was selected as the most advisable for separation of water-soluble vitamins.max × 100 (%).3 and 5. 3. MetaChem Polaris C18-A column.010. 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. HPLC–UV parameters: MetaChem Polaris C18-A (150 mm × 4. PA. Symbols of individual vitamins in figures are given in Table 1. TFA probably influenced acid-base equilibrium of studied vitamins due to its acid-base properties (very strong acid. We tested the impact of four different temperatures (15.1 we calculated relative sensitivities (as a maximal peak factor—MPf ) according to the formula: MPf = PA /PA. Autosampler injection was 3 ␮l. Primarily. Vitamin concentrations were 100 ␮g ml−1 . 3. 25 and 30 ◦ C) using the retention factor. CA.B. USA. for comparison of peak areas of individual vitamins at pH = 3. 20.9 (Fig.7. isocratic flow rate of 0. 0. (v/v)) and was constant in the first 2 min. 4. Effect of temperature on vitamin separation The column temperature affects efficiency of chromatographic separation too. for complete set of fatand water-soluble vitamins we compared the total number of MPf values exceeding value of MPf = 80. 3C). 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 .6 mm.max is the highest peak area of an individual vitamin in the whole range of pH values.4.030%) on separation of water-soluble vitamins was studied. Torrance. Influence of trifluoroacetic acid on vitamin separation We assumed that the content of TFA in the mobile phase seriously influenced chromatographic separation of individual compounds. PA is the peak area of individual vitamin at the given pH value. 3. gradient profile started at 92:8 (TFA:methanol. Dependences of retention (A and C) and symmetry (B and D) factors of water.010% TFA and methanol. 3. Furthermore. 3C) for all vitamins thus the pH (3.

Primarily we selected profile that started at 92:8 and was constant in the first 2 min.62 B. Different ratios of 0. riboflavin and folic acid) and thioctic acid and p-aminobenzoic acid. The symmetry factors. cyanocobalamin. The other water-soluble vitamins and p-aminobenzoic acid and thioctic acid were added to group of vitamins B in the next experiments.9 and methanol (88:12. The time of the initial interval of isocratic flow varied from 2 to 8 min. 92:8. (C) Other conditions are the same as in Fig. Gradient elution Gradient and isocratic elution profiles were used for the water-soluble vitamins separation [20–22. 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. Klejdus et al. respectively. we tested the applicability of combined isocratic and linear gradient profile of the mobile phase on the separation of water-soluble vitamins (B group). then decreased to 2:98 during the next 6 min. 95:5 and 97:3.21]. Next we tested different time intervals in which the composition of mobile phase was kept constant (isocratic elution). Thus different linear gradient profiles were tested. was kept constant for the next 20 min and finally linearly increased up to 92:8 in the last 5 min.010% aqueous TFA at pH 3. Increasing dispersion of the chromatographic peaks was observed at the higher ratios of water phase (95:5). Dependence of peak areas of water. The baseline separation of B vitamins was achieved but the conditions were not very suitable for separation of other water-soluble vitamins (pantothenic acid.6. 3.010% aqueous TFA at pH 3. 2. The mobile phase consisted of 0. isocratic elution at 2:98 in the next 20 min and final linear increase up to .9 and methanol (in ratio 92:8). 90 and 95%. The retention factors and the symmetry factors decreased with the decreasing TFA ratio. Mixtures of inorganic salts with organic solvents (acetonitrile) were most frequently applied as the mobile phase [20. In our experiments. for the very effective and fast separation of water-soluble vitamins and used in all further experiments. After that period the optimised linear gradient profile consisting of decrease from 95:5 TFA:methanol in the first 6 min up to 2:98. 3.(A) and fat-soluble (B) vitamins on pH and the total number of MPf values exceeding MPf = 80.31]. / Analytica Chimica Acta 520 (2004) 57–67 Fig. respectively) were tested for the initial isocratic elution.

5A). thioctic acid and water-soluble vitamins were used as a criterion of separation efficiency. riboflavin. thiamine. nicotinamide. cyanocobalamin. ␣-tocopherol. Klejdus et al. D3. The chromatographic behaviour of both sub-groups differed seriously. pyridoxine. 4A.7. thioctic acid and folic acid) and polar ones (l-ascorbic acid. The optimised conditions of chromatographic separation of water-soluble substances allowed the very efficient and highly sensitive separation of l-ascorbic acid. / Analytica Chimica Acta 520 (2004) 57–67 63 92:8 ratio in 5 min was applied in each run. . nicotinic acid. thioctic acid and p-aminobenzoic acid (100 ␮g ml−1 ) in the first 15 min. Chromatographic behaviour of fat-soluble vitamins (trans-retinol.32]. p-aminobenzoic acid. When the initial interval of isocratic elution was excessively prolonged. Resulted chromatographic gradient profile started at 95:5 (0. thiamine. 3.9): methanol was studied in the next part of our experiment. cyanocobalamin. tocopherol-acetate. Optimised linear gradient profile 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. K1. pyridoxine and nicotinamide). cyanocobalamin.3tR per min) with increasing time of isocratic flow of mobile phase. The symmetry factor was nearly one when period of isocratic elution lasted 4 min (not shown). retinol-acetate. 2. Separation of fat-soluble vitamins Separation of some fat-soluble vitamins was described in literature [24. K2-isomer. then decreased to 2:98 during the next 6 min. riboflavin and folic acid) and thioctic acid and p-aminobenzoic acid increased. too. folic acid. Vitamin concentrations were 100 ␮g ml−1 . nicotinic acid.010% TFA (pH 3. The symmetry factors and the retention time differences between the last peak of B-group vitamins and the peaks of p-aminobenzoic acid.B. 4. The well-separated chromatographic signals at 280 nm are shown in Fig.010% TFA:methanol) and was constant in the first 4 min. pantothenic acid. was constant for next 20 min and finally linearly increased up to 98:2 in the last 5 min (Fig.28. pyridoxal. Based on the chromatographic behaviour the group of water-soluble substances could be divided into two distinct sub-groups—low-polar compounds (pantothenic acid. The symmetry factors were changing. K1-isomer and K2) using 0. riboflavin. Other conditions are the same as in Fig.(A) and fat-soluble (B) vitamins at 280 nm. D2. pyridoxal. the retention times of some vitamins (pantothenic acid. Both groups of vitamins were linearly drifting (2. HPLC–UV chromatograms of water.

7A and B. Identification and determination of vitamins in real samples Furthermore we tested the elaborated analytical chromatographic method for the determination of water. The mixture was used for construction of calibration curves (peak areas versus compound concentration) by stepwise dilution with ACS water. gradient profile) were observed for fat-soluble vitamins in contrast to the water-soluble substances. Vitamin concentrations were 100 ␮g ml−1 . 2. Nestlé BEBA and Fortuna silueta) were separated and determined by the same procedure. 7C. Other conditions are the same as in Fig. 266 nm (C) and 280 nm (D).64 B. Gradient profile (A. / Analytica Chimica Acta 520 (2004) 57–67 vitamins in methanol [13]. Declared and found concentrations are compared in Table 2. quantification limits (for 3.9. 3B). Equations of bisectors.8. Klejdus et al. Simultaneous HPLC–UV chromatograms of water. Typical HPLC–UV chromatogram of the mixture of water. Less pronounced changes in the chromatographic parameters (the retention factor. Chromatogram of vitamin separation and determination in Fortuna milk is shown in Fig. TFA concentration. Peak areas increased at pH 3. The well-separated chromatographic peaks of fat-soluble vitamins at 280 nm are shown in Fig. 3. 3. respectively). Simultaneous determination of water-soluble and fat-soluble vitamins A standard mixture of the vitamins and other compounds (100 ␮g ml−1 of each) was prepared before analyses. 5. Declared and found concentrations are compared in Table 3. The symmetry factor was approximating the criterion at pH value 3.9 and then they were constant or they slightly increased (Fig. The identity (retention time) of all individual detectable vitamins (water-soluble and fat-soluble) was confirmed by standard addition and HPLC–MS detection methods [33].and fat-soluble vitamins and other substances (100 ␮g ml−1 ) using the combined isocratic and linear gradient profile is presented in Fig.9 and the negative values of the symmetry factors at other pH values confirmed peak tailing Fig. R2 . pharmaceutical preparation and fortified drinks. respectively. column temperature and TFA concentration influenced the retention factor less then 30% (not shown). pH value was 3.9. mobile phase methanol) for simultaneous vitamin separation that started at 95:5 (0. The wavelength at 280 nm was selected as the most suitable for detection and quantification of vitamins in the further experiments. The baselines of the full scan separations follow the gradient profile (Fig. 101 and 95% were determined. Variations in pH. 4B. respectively) and concentration range (10–1000 ␮g ml−1 ) are given in Table 1.and fat-soluble vitamins at three detection wavelengths: 230 nm (B). An average recovery of 100 and 99% were determined. then decreased to 2:98 during the next 10 min and finally linearly increased up to 95:5 from 32 to 35 min is shown on (A). 2C and D). There are a lot of additives of different origin and characteristics (see lists above) in such samples that make vitamin determination more difficult.9. Vitamins in homogenised samples (Soja milk. food supplements. The adverse effects of gradient elution profile disappear (see Fig.010% TFA:methanol) and was constant in the first 4 min. 5D) at longer wavelengths (≥280 nm). Average recoveries for individual food samples (Soja milk.and fat-soluble vitamins and thioctic and p-aminobenzoic acids in a more complicated matrix represented by foods. Fig. Nestlé BEBA and Fortuna silueta) of 103. respectively. column temperature.S/N and 10. the symmetry factor and the peak area) with changes in separation conditions (pH. . 6. Vitamins in homogenised sample of pharmaceutical preparation (B-Komplex) and food supplement (multivitamin drink) were separated and determined by HPLC–UV (Fig.S/N criterion. 5) of the simultaneous separations of waterand fat-soluble vitamins at shorter wavelengths (230 and 266 nm) and the optimised chromatographic conditions. The maximum of the peak area was obtained again at pH value about 3. detection limits.

2 2.24 30.2 2.8x − 67 y = 2.59 8.1 277.3 12. Inset shows the gradient profile (A.6x + 3. b 0.9887 0.68x + 15 y = 2.8 2.49 188.6 40.08 21.0097x + 0.4x − 26 y = 7.2 572.93 4.9988 0.1 y = 0.9 2.B. Simultaneous HPLC–UV determination of water.03 3.69 22.6 3.3 y = 10x − 188 y = 0.9983 0.9939 0.05 2884 76.48 157.0 2.9880 0.7x − 58 R2 . Fig.1 3.S.9 424.9974 0.88x − 1.2 y = 7.30 LOQ (ng ml−1 )d 85.9980 0.9977 0.16 324. .1 2.9990 0.9991 0.5x − 11 y = 2.56 21.3 2.98 28.67 3.7 2. / Analytica Chimica Acta 520 (2004) 57–67 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 Riboflavin Thiamine Thioctic acidf ␣-Tocopherol Tocopherol acetate Trans-retinol a b c d e f 65 tR (min)a 2.8x − 21 y = 9.7 y = 1.2x + 291 y = 2.9979 0.0 12.57 25.7 34.6 2.99 R.5 3.00 25.37 9.9 171.9958 0.77 30.47 129.0 1.8 2.18 394.43 22.8 y = 7.1 Symbols in figures · · · ×· · · · · · –· · · · · · ᮀ· · · —᭛— · · · ᭜· · · —ᮀ— · · · +· · · — — ··· ··· —–— · · · ᭡· · · — · · · ᭹· · · —|— ··· —᭺— —᭜— —᭿— —᭡— — ×— · · · ᭺· · · · · · ᭛· · · — ··· Retention times in min.31 15.55 41.55 97.9995 0.S/N).52 865.6 83.0x − 8.61 1316 115.1 526.19 13.3 2.83 3.83 8.5 40.5 1. Non-vitaminous substances.9952 0.71 55.6x − 16 y = 3.07 14.13 y = 4.4x − 4.9975 0.4 41.9986 0.9941 LOD (ng ml−1 )c 25.0x − 30 y = 0. Regression coefficients.37 7.17 12.99 Regression equation y = 2.9 431.9992 0.46x − 1.40 16.0 657. Limits of quantitation (10.78 127. mobile phase methanol). Limits of detection (3.9 118.1 139.64 626.9 2.95 70.4x − 57 y = 1.9998 0.6x − 80 y = 4.3x − 40 y = 13x − 28 y = 0. Other conditions are the same as in Fig.and fat-soluble vitamins at 280 nm.9988 0.D.9990 0.98 5.1 3. 6. Klejdus et al.1 54.S/N).47 23. Relative standard deviations.2 2.41 16.6 8.9936 0. 5. (%)e 2.59 17.2 2.4 35.10 197.4 23.7x − 30 y = 10x − 68 y = 2.4 101.

9 ± 0.3 ND ND ND ND Vitamin drink (mg per 100 g powder) Declared 7. B-Komplex (A).7 9.29 ± 0.0 36.87 ± 0. 5.0 60.7 ± 0.0 25.07 ± 0.00 18 nd 1 × 10−3 60 nd 0.0 Found 6. / Analytica Chimica Acta 520 (2004) 57–67 Fig.59 ± 0.0 ± 0. Klejdus et al.0 90.01 0. 7.3 ± 0.3 4±1 12.1 ± 0.57 21 Found 0.2 ± 0.0 1.002 (5. 15 15 10 25 50 nd nd nd nd Found 14.8 8±1 9.66 B.025 575.8 ± 0.0 8.95 3.2 (2. food supplement.0 × 10−3 9.40 2.68 ± 0.2 ND 2.4 ± 0.40 nd 2.3 ± 0.3 13 4.2) × 10−2 1.4 Table 3 Declared and found concentrations of vitamins in food samples (n = 5) Vitamins Fortuna silueta (mg per 100 ml) Declared Thiamine Riboflavin Pyridoxine Pantothenic acid Nicotinamide ␣–Tocopherol Cyanocobalamin l-Ascorbic acid Folic acid Trans-retinol Vitamin K1 0.7) × 10−4 46 ± 3 0.4 3.5 10.4 1. 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 Riboflavin Pyridoxine Pantothenic acid Nicotinamide ␣-Tocopherol Cyanocobalamin l-Ascorbic acid Folic acid nd: not declared.5 × 10−4 48 0.5) × 10−3 (9.28 0.03 0. Vitamin drink (B) and food sample.9 ± 0.5 7. .0 0.1 ND (8 ± 2) × 10−4 58 ± 3 ND 0.0 ± 0.05 0.08 1.and fat-soluble vitamins at 280 nm in pharmaceutical preparation.32 0.1 ± 0.1 0. ND: not found.72 1.7 15.03 ND ND Nestl´ e BEBA (mg per 100 g powder) Declared 1.8 ± 0.04 nd nd Found 0.09 ND ND 0.03 22 ± 1 nd: not declared. Fortuna silueta (C).60 2.8 ± 2.2 ± 0.39 ± 0. Simultaneous HPLC–UV determination of water.02 1.14 0.9 53.00 nd nd 0.8 nd Found 1.7 36 ± 2 89 ± 2 59 ± 1 0.26 ± 0.030 ± 0.1 ± 1. ND: not found.0 3. Other conditions are the same as in Fig.9 ± 0.2 16.3 ± 1.2 ± 0.2 1.2 ND Soja milk (mg per 100 g powder) Declared 0. 8 ± 0.16 ± 0.5 ± 0.00 6.

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