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Spectrofluorimetric study of the β-cyclodextrin:vitamin K3 complex and determination of vitamin K3
Talanta 53 (2000) 951 – 959 www.elsevier.com/locate/talanta
Spectrofluorimetric study of the b-cyclodextrin:vitamin K3 complex and determination of vitamin K3 J.J. Berzas Nevado, J.A. Murillo Pulgarı´n *, M.A. Go´mez Laguna Department of Analytical Chemistry and Food Technology, Uni6ersity of Castilla-La Mancha, 13071 Ciudad Real, Spain Received 13 March 2000; received in revised form 28 July 2000; accepted 5 September 2000
Abstract Vitamin K3 (menadione) is an oil-soluble vitamin and not naturally fluorescent but yields fluorescence when it is reduced. However, it is possible to yield a fluorescent derivative in the region of 407 nm in aqueous medium when complexed to b-cyclodextrin (CD). A 1:1 stoichiometric ratio and a formation constant of 373 9 34 l mol − 1 were obtained for the binary inclusion complex between menadione and b-CD. The measurements were performed at pH 6.2 adjusted by adding 0.1 mol l − 1 citrate buffer solution and 6.4 × 10 − 3 mol l − 1 of b-CD concentration. The calibration graph was linear over the range 0.1 – 2.0 mg l − 1 with a repeatability of 2.2%; the detection limit was 0.022 mg l − 1 and the limit of quantification limit was 0.073 mg l − 1. The procedure was applied to pharmaceutical formulations. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Vitamin K3; b-cyclodextrin; Fluorescence
1. Introduction Vitamin K3 (2-methyl-1,4-naphthoquinone) is a fat soluble vitamin, which is not naturally fluorescent. Generally vitamin K has an important role as a cofactor in the synthesis of blood clotting and bone mineralization processes. Several methods were described in literature such as colorimetric reaction [1], spectrophotometric [2,3] detection, but the more commonly used ones are the chromatographic techniques auxiliary by UV spectrophotometric detection * Corresponding author. Tel.: +34-926-295300; fax: + 34926-295318. E-mail address: [email protected] (J.A.M. Pulgarı´n).
[4,5] and spectrofluorimetric [6] detection with post-column derivatization based on the reduction of menadione to a fluorescent derivative. Postcolumn reduction with sodium tetrahydroborate (NaBH4) [7], and with columns with powedered zinc [8] have also been reported. Methods such as electrochemical [9,10] and photochemical [11] reduction previous to a fluorimetric detection have also been used. Another way is the polarographic [12] determination of menadione in anhydrous acetonitrile with lithium perchlorate as carrier electrolyte against a calomel reference electrode. British Pharmacopoeia [13] describes a method based on a cerimetric titration of menadione with a previous reduction to the hydroquinone with zinc in hydrochloric acid medium light excluded.
0039-9140/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 0 ) 0 0 5 8 0 - 4
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J.J.B. Ne6ado et al. / Talanta 53 (2001) 951–959
Martı´nez et al. [14] described the determination of a derivative of vitamin K3 (MSB) by using a flow injection assembly provided with a solidphase reactor with immobilized zinc, where MSB was hydrolyzed in an alkaline medium and reduced by zinc in an HCl medium. It is possible for the determination of vitamin K3 based on the reduction with SnCl2 [15] in an ethanolglycerol medium by spectrofluorimetric technique. In all the procedures above mentioned the use of an organic solvent is necessary in order to get the solubilization of the menadione. Cyclodextrines are cycloamyloses, usually constituted by six to eight glucose sub-units, which present an almost conical hydrophobic cavity in which many molecules, organic or inorganic, may fit. The hydrophobic character of the cavity is shown, for example, by the enhancement of fluorescence of certain molecules in the presence of a or b-cyclodextrins (CDs). One of the most important potential applications of CDs is the protection of the guest molecule from the effect of oxidation and photochemical degradation. Saturated and unsaturated fatty acids were first treated in this way [16]. Retinal complex of CD has been described in literature by Lerner et al. [17]. Retinal is insoluble in an aqueous medium and not fluorescent but by a complex of CD it is possible to determine it by a spectrofluorimetric method. The use of CD has been described by Mun˜oz de la Pen˜a et al. [18] in order to get room-temperature phosphorescence from acenaphthene in the presence of 2-bromoethanol. The enhancement of fluorescence of an analyte by micellar solution is well known; that way one proposed to examine the effect on vitamin K3 when a micellar dodecyl sulphate (SDS) solution was used. It was possible to get solubilization of vitamin K3 in an aqueous medium, but no fluorescent signal was obtained. This may be a result of the incorporation of the vitamin into the micellar medium only causing a polarity change and that of CD additionally involving the formation of weak covalent bonds and van der Waals interactions.
The idea of using the formation of inclusion complexes with CDs to increase the solubility in aqueous media is not new and cyclodextrines have also been used to obtain enhancement of fluorescence [19,20] and phosphorescence at room temperature [21,22]. In this work, aimed at improving the luminescence detection of menadione, the effect on vitamin K3 was examined. When b-CD was used it was possible to obtain a fluorescence signal.
2. Experimental
2.1. Reagents Vitamin K3 solutions were prepared by dissolving 50.0 mg of the vitamin K3 in 0.01 M b-CD aqueous solution (purchased from Merck, Darmstadt, Germany) under stirring with a magnetic bar at boiling temperature for 5 min. The use of ultrasounds is inadvisable. A total of 0.01 mol l − 1 of b-CD was prepared by dissolving it in water obtained from a Milli-Q system (Milipore, Milford, MA, USA). Other standard stock solutions were used to prepare working standard solutions by suitable dilutions. Buffer solution of 6.2 pH was prepared by mixing appropriate amounts of citric acid and sodium hydroxide. All experiments were performed with analytical reagent grade chemicals.
2.2. Apparatus Fluorescence measurements were performed on a Perkin-Elmer model LS 50 spectrofluorimeter equipped with a Xenon lamp, connected to an Ataio S 3000 St 386 computer fitted with Perkin-Elmer F.L. Data Manager (FLDM) software and an Epson FX-850 printer. A thermostat Selecta Frigiterm 6000382 system connected to spectroflurimeter, a Crison Model 2001 pH-meter with a glass-saturated calomed combination, an ultrasonic bath and 10.0 mm quartz cells were also used.
J.J.B. Ne6ado et al. / Talanta 53 (2001) 951–959
2.3. Calibration Suitable aliquots containing 0.0025 – 0.050 mg of vitamin K3 were transferred into a series of 25-ml calibrated flasks, 16 ml of b-CD (0.01 mol l − 1) and 5 ml of buffer (0.5 mol l − 1) citrate solution were added to each flask and diluted to volume with water, in order to obtain the calibration graph. Before the measurement was performed it was necessary to wait 30 min so as to obtain a good ratio time-intensity. The measurement of samples with b-CD and buffer solution without vitamin K3 was necessary performed and for every sample it was necessary subtract the blank. For all samples a 313 nm wavelength for the excitation and the measurements were carried out at l = 407 nm for emission.
3. Results and discussion Vitamin K3, which is insoluble in an aqueous medium and does not fluorescence in solution, emits a luminescence in the region 407 nm, even in aqueous medium, when complexed to b-CD. The association constant of b-CD:vitamin K3 was studied. As shown in Fig. 1A, the excitation spectrum for the vitamin K3:b-CD complex exhibits two bands: a sharp one with a maximum at 241 nm and a broad one peaking at 313 nm. As usual, the emission spectrum contains a single band (with maximum fluorescence intensity at 407 nm).
3.1. Factors affecting fluorescence intensity Chemical variables were studied in order to obtain the best measurement conditions and the maximum fluorescence signal. The stability of the inclusion complex between b-CD and vitamin K3 was studied and the dissolution of vitamin K3 in b-CD was investigated to get more reproducible measurements. The vitamin is slowly dissolved in 0.01 M CD solutions, possibly because water molecules within the CD cavity tend to be displaced by the organic molecules. In order to determine the most favourable manner of incorporating vitamin K, the
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influence of stirring, ultrasonication and heating on its dissolution was studied. The efficiency of the inclusion process was determined by measuring the fluorescence intensity; the greater the intensity was, the more efficiently was the inclusion complex assumed to be formed. One experiment involved the application of ultrasounds for 5, 30, 60 and 180 min to four different solutions and measuring their fluorescence intensity over a period of 10 days following preparation. Whichever the ultrasonication time, the fluorescence intensity continued to increase with time; it peaked at day 8 and then levelled off. Accordingly, the use of ultrasounds was discarded. Stirring with a magnetic bar at room temperature for the previous lengths of time also resulted in the fluorescence intensity not peaking immediately after dissolution but 4 days later. The most efficient way of preparing the solutions was to stir them with a magnetic bar while boiling for 5 min. In this way, the fluorescence signal for the solution remained constant for at least 20 h. Consequently, one opted for using this procedure and making fresh solutions on a daily basis. Solutions were thus prepared by dissolving 50.0 mg of the vitamin in 0.01 M CD under stirring with a magnetic bar at boiling temperature for 5 min. The use of ultrasounds is inadvisable. The influence of pH on the fluorescence intensity was studied by adding different amounts of HClO4 or NaOH solutions. Fluorescence intensity of vitamin K3 was constant between pH 4.1 and 7.0 and pH 6.2 was selected as most suitable. pH 6.2 was obtained by adding citrate buffer solutions. The fluorescence intensity of vitamin K3 was not affected by the concentration of the buffer at least during 4 h. A total of 0.1 M concentration of buffer was selected to obtain suitable buffering capacity. The influence of the b-CD concentration on the fluorescence intensity for the complex was examined by changing the concentration from 2.0× 10 − 4 to 7.0 × 10 − 3 M. Higher concentrations of CD could not be tested owing to its poor solubility in water and the need to add a certain volume of buffer prior to making to the mark. As can be
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Fig. 1. (A) Excitation spectrum of complex b-cyclodextrin K3 of 2.0 mg l − 1. (B) Emission spectrum of complex b-CD:K3 of 2.0 mg l − 1.
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seen from Fig. 2, the fluorescence intensity increased with increasing CD concentration up to about 0.006 M, above which it remained constant at its peak value. A concentration of 6.4 × 10 − 3 M was adopted as the most suitable for analytical purpose. The data collected in this experiment were subsequently used to determine the formation constant of the inclusion complex. Another factor that affects fluorescence intensity is temperature. Thus, fluorescence intensity shows a decrease as temperature increases from 3 to 70°C. Temperature coefficient was of the order of 1.49% °C − 1. This can be ascribed to an increase in temperature increasing the kinetic energy of the molecules and hence the probability of their colliding; as a result, radiation less deactivation through internal conversion prevailed and the fluorescence quantum efficiency decreased [23].
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Therefore, the use of a thermostat is recommended, choosing a measurement temperature of 20°C, as an average room temperature. In these conditions, the influence of vitamin concentration on fluorescence intensity was studied. A linear relation between fluorescence intensity and vitamin K3 concentration in the range from 0.1 to 2.0 mg l − 1 can be observed. Higher concentrations exhibit a non-linear influence with a decreasing slope up to 0.01 M that remains constant up to 0.1 M. Beyond this vitamin concentration, the fluorescence decreases through inversion.
3.2. Association constant of b-CD–K3 complex It was not possible to measure the fluorescence intensity in aqueous solutions because the vitamin
Fig. 2. Influence of b- cyclodextrin concentration on the emission fluorescence of the binary complex b-CD – K3.
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Fig. 3. Double reciprocal plot for the b-cyclodextrin – K3 complex. The nonlinear fit of the data assuming the formation of a 1:1 b-CD – K3 complex and curved line assuming the formation 1:2 b-CD – K3 complex.
is water-insoluble; as noted earlier, however, the fluorescence signal decreased with decreasing CD concentration. This, together with the fact that the vitamin exhibits no native fluorescence in other solvents, led one to assign the vitamin a zero fluorescence in water on the basis of the Benesi–Hildebrand [24] procedure for determining the complex stoichiometry and formation constant. Two possible stoichiometries were considered for the inclusion complex, namely: 1:1 and 2:1. Assuming that b-CD forms a 1:1 inclusion complex with K3, the following expression can be written: K3 + b-CD X b-CD – K3
(1)
The formation constant of the complex (K) is given by: K=[CD – K3]/[CD][K3]
(2)
where [CD], [K3] and [CD–K3] are equilibrium concentrations. The direct relation between the observed fluorescence intensity enhancement (F− F0) and the using expression to that b-CD concentration is given by: F− F0 = (F − F0)K[CD]0/(1+ K[CD]0)
(3)
where [CD]0 denotes the initial CD concentration. F0 denotes the fluorescence intensity of vitamin K3 in the absence of b-CD. F denotes the fluorescence intensity when all of the vitamin K3 molecules are essentially complexed with b-CD. F is the observed fluorescence at each CD concentration tested. By typical double-reciprocal plots (method of Benesi–Hilderbrand) 1/F − F0 = 1/(F − F0)K[CD]0 + 1/(F − F0)
(4)
J.J.B. Ne6ado et al. / Talanta 53 (2001) 951–959
Evidence for the existence of 1:1 complex was obtained by fitting a double-reciprocal plot (1/F − F0 vs. 1/[CD]0) as can be seen in Fig. 3. In order to effectively check the determination the stoichiometric ratio, assuming 1:2 inclusion complex with b-CD, has been studied. The following expression can be written: K3 + 2b-CD X (b-CD)2 – K3
(5)
The formation constant of the complex (K%) is given by: K%= [(b-CD)2 – K3]/([CD]0)2[K3]
(6)
If [CD]0 \ \ \(b-CD – K3)2 \ \ \b-CD – K3, then the following expression is obtained: 1/F − F0 =1/(F −F0)K%([CD]0)2 +1/(F − F0) (7) when 1/F −F0 versus ([CD]0)2 is represented (Fig. 3) the non linearity can be observed. As a conclusion the stoichiometry of the complex is 1:1. The initial parameter estimates needed for the non-linear regression method were obtained from linear plots (Fig. 3).The calculated association constant is K =373 9 34 l mol − 1. This suggests the formation of a complex between b-CD and K3 in solution just as it was assumed. A possible inclusion of vitamin K3 into b-CD must be an axial inclusion because b-CD has a inside cavity of 8 A, , and naphthalene, which is the aromatic structure of vitamin K3 (2-methyl-1,4naphthoquinone), and can not be included into b-CD by equatorial way with a measure of 8.4 A, of length and 6.8 A, of height and can not include into b-CD by equatorial way (8.4 A, ) while an axial way is possible. The inclusion of b-CD – K3 complex will be partial, because the unsubstituted benzene ring will be inside the b-CD cavity while the substitute benzene ring by cetones groups (1,4) and methyl group (2) will be outside the b-CD. The proposed hypothetical model is shown in Fig. 4.
3.3. Determination of 6itamin K3 The excitation spectra were performed at 313 nm wavelength excitation. The scan speed and slit
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Fig. 4. Inclusion complex.
values were optimized at 240 nm min − 1 and 10.0 nm, respectively. The measurements were performed at 313 nm for excitation and 407 nm for emission. Under these conditions the calibration graph was constructed from the emission spectra for standards containing between 0.1 and 2.0 mg l − 1 of vitamin K3 which has been dissolving in b-CD. Linear regression equation (y= a+mx) for vitamin K3 was obtained. The slope, intercept, determination coefficient, S.D., detection limit and quantification limit obtained for the determination of vitamin K3 by conventional fluorescence are summarized in Table 1. The significance of the intercept on the y axis of the obtained regression line was evaluated by applying the Student t-test at 95% confidence level. In the calibration graphs the intercept is negligible, since the experimental tvalue is smaller than the critical t-value, as can be seen in Table 1. In order to study the repeatability of the method, a series of ten standard samples containing 0.30 mg l − 1 of vitamin K3 were prepared, with the results of 0.279 mg l − 1 and 3.51% for the S.D. and the relative error respectively, when the conventional fluorescence was used.
Table 1 Statistical parameters of calibration graph Slope Intercept Determination coefficient (r 2) Slope standard deviation Intercept standard deviation Experimental ‘t’ value Theoretical ‘t’ value New slope Detection limit Quantification limit
208.5 −1.63 0.999 2.8 3.2 0.505 2.365 207.3 0.022 0.073
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Table 2 Results obtained by the application of the proposed method to pharmaceutical preparations Commercial preparation
Theoretical value (mg l−1)
Experimental value (mg l−1)
Recovery (%)
Vitaencil C.K.P.
0.624 0.689 0.745 0.676 0.743 0.851
0.635 0.639 0.749 0.695 0.783 0.869
101.76 100.54 100.67 102.81 105.38 102.09
BIO-HUBBER
3.4. Applications In order to study the validity of the method, it was applied to the determination of vitamin K3 in commercial preparations: Vitaendil C.K.P. from the ‘Wassermann’ Laboratory (whose composition per tablet is: ascorbic acid 75.0 mg; menadione 5.0 mg; vitamin P 25.0 mg; starch wheat 44.85 mg and lactose); BIO-HUBBER from ‘ICN Ibe´rica, S.A.’ Laboratory (whose composition per tablet is: neomicine (sulphate) 25.0 mg; bacitracine (zinc) 1.000 U.I.; streptomicine 50.0 mg; sulfadiazine 30.0 mg; pectine 50.0 mg; menadione 20.0 mg; sodium saccharine 10.0 mg; starch wheat 50.0 mg and lactose. Before the proposed method was applied to real samples, the effect of all the compounds which accompanying menadione in pharmaceuticals preparations, on the determination of the vitamin was studied. Three determinations were carried out for each of these pharmaceuticals preparations at three different concentrations. The pharmaceutical preparations were dissolved such as it is described in Section 2. The recovery percentages are summarized in Table 2. Recoveries achieved by means of the method proposed are in accordance with the real content of vitamin K3 in commercial preparations.
4. Conclusions The idea of using the formation of inclusion complexes with b-CD to increase the solubility in aqueous media and to obtain luminescence has been developed satisfactorily.
As a conclusion b-CD is a good host to promote the fluorescence of vitamin K3 in an aqueous solution and it is possible to use a molecular fluorescence technique for the determination of vitamin K3. The method is simple, sensitive and selective. It is possible to carry out the determination without any previous separations or tedious extraction and without other auxiliary techniques. The proposed method is more sensitive and selective than direct spectrophotometric methods and HPLC methods with UV detection [2–5]; it also surpasses some spectrofluorimetric methods for the same purpose [7,8,11,14]. Unlike reported fluorimetric methods, it requires no derivatization — usually reduction — reaction prior to the determination proper [7,8,11,14], which results in greater simplicity and precision for the method.
References [1] H. Kofler, Helv. Chim. Acta 28 (1945) 702. [2] Y. Ruan, F. Duan, Y. Ning, X. Fan, Fenxi Huaxue 16 (1988). [3] M.L. Iskander, H.A.A. Mediem, L.H. Khulil, Anal. Lett. 28 (1995) 1513. [4] K. Kaufft, H.Z. Dmckemanm, Lebensm. Unters. Farsch. 22 (1984) 89. [5] U. Manz, R. Mauer, Int. J. Vitam. Nutr. Res. 52 (1982) 248. [6] K. Abe, O. Hiroshima, K. Ishibashi, M. Ohmae, K. Kawabe, G. Katsui, Yakugakizasshi 99 (1979) 1992. [7] A.J. Speek, J. Schrijver, W.H.P. Schreurs, J. Chromatogr. 301 (1984) 441. [8] S.M. Billedeau, J. Chromatogr. 472 (1989) 371. [9] Y. Haroon, D.S. Bacon, A.J. Sadowski, Biomed. Chromatogr. 2 (1987) 4. [10] L.Z. Wang, C.S. Ma, X.L. Zhang, L. Xu, Microchem. J. 50 (1994) 101.
J.J.B. Ne6ado et al. / Talanta 53 (2001) 951–959 [11] M.F. Lefevere, R.W. Frei, H.M.T. Scholten, U.A. Brinkman, Chromatographia 15 (1982) 459. [12] Pharmaceutical Chemistry. In: H.J. Roth (Ed.), Drug Analysis, vol. 2, Ellis Horwood, Clichester, 1988, p. 111 [13] British Pharmacopeia 1999, H.M. Stationary Office, London, 1993, p. 410. [14] J. Martı´nez Calatayud, I. Gil Torro, J.V. Garcia Mateo, Analyst 122 (1997) 139. [15] J.J. Berzas Nevado, J.A. Murillo Pulgarı´n, M.A. Gome´z Laguna, Analyst 123 (1998) 287. [16] W. Schlenk, D.M. Sand, J.A. Tillotson, J. Am. Chem. Soc. 77 (1955) 3587. [17] D.A. Lerner, B. Castillo del, S. Mun˜oz-Botella, Anal. Chim. Acta 227 (1989) 297.
.
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[18] A. Mun˜oz de la Pen˜a, I. Dura´n-Mera´s, F. Salinas, I.M. Warner, T.T. Ndou, Anal. Chim. Acta 255 (1991) 351. [19] J.A. Arancibia, G.M. Escandar, Analyst 124 (1999) 1833. [20] I. Velaz, M. Sa´nchez, A. Zornoza, N. Goyenechea, Biomed. Chromatogr. 13 (1999) 155. [21] A. Mun˜oz de la Pen˜a, M.C. Mahedero, A. EspinosaMansilla, A. Bautista-Sanchez, M. Reta, Talanta 48 (1999) 15. [22] M. Algarra Gonza´lez, M. Herna´ndez Lo´pez, Analyst 123 (1998) 2217. [23] G.G. Guilbault, Practical Fluorescence, Marcel Dekker, New York, 1990. [24] K.A. Connors, Binding Constants. The Measurements of Molecular Complex Stability, Wiley, New York, 1987.
Spectrofluorimetric study of the b-cyclodextrin:vitamin K3 complex and determination of vitamin K3 J.J. Berzas Nevado, J.A. Murillo Pulgarı´n *, M.A. Go´mez Laguna Department of Analytical Chemistry and Food Technology, Uni6ersity of Castilla-La Mancha, 13071 Ciudad Real, Spain Received 13 March 2000; received in revised form 28 July 2000; accepted 5 September 2000
Abstract Vitamin K3 (menadione) is an oil-soluble vitamin and not naturally fluorescent but yields fluorescence when it is reduced. However, it is possible to yield a fluorescent derivative in the region of 407 nm in aqueous medium when complexed to b-cyclodextrin (CD). A 1:1 stoichiometric ratio and a formation constant of 373 9 34 l mol − 1 were obtained for the binary inclusion complex between menadione and b-CD. The measurements were performed at pH 6.2 adjusted by adding 0.1 mol l − 1 citrate buffer solution and 6.4 × 10 − 3 mol l − 1 of b-CD concentration. The calibration graph was linear over the range 0.1 – 2.0 mg l − 1 with a repeatability of 2.2%; the detection limit was 0.022 mg l − 1 and the limit of quantification limit was 0.073 mg l − 1. The procedure was applied to pharmaceutical formulations. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Vitamin K3; b-cyclodextrin; Fluorescence
1. Introduction Vitamin K3 (2-methyl-1,4-naphthoquinone) is a fat soluble vitamin, which is not naturally fluorescent. Generally vitamin K has an important role as a cofactor in the synthesis of blood clotting and bone mineralization processes. Several methods were described in literature such as colorimetric reaction [1], spectrophotometric [2,3] detection, but the more commonly used ones are the chromatographic techniques auxiliary by UV spectrophotometric detection * Corresponding author. Tel.: +34-926-295300; fax: + 34926-295318. E-mail address: [email protected] (J.A.M. Pulgarı´n).
[4,5] and spectrofluorimetric [6] detection with post-column derivatization based on the reduction of menadione to a fluorescent derivative. Postcolumn reduction with sodium tetrahydroborate (NaBH4) [7], and with columns with powedered zinc [8] have also been reported. Methods such as electrochemical [9,10] and photochemical [11] reduction previous to a fluorimetric detection have also been used. Another way is the polarographic [12] determination of menadione in anhydrous acetonitrile with lithium perchlorate as carrier electrolyte against a calomel reference electrode. British Pharmacopoeia [13] describes a method based on a cerimetric titration of menadione with a previous reduction to the hydroquinone with zinc in hydrochloric acid medium light excluded.
0039-9140/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 0 ) 0 0 5 8 0 - 4
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J.J.B. Ne6ado et al. / Talanta 53 (2001) 951–959
Martı´nez et al. [14] described the determination of a derivative of vitamin K3 (MSB) by using a flow injection assembly provided with a solidphase reactor with immobilized zinc, where MSB was hydrolyzed in an alkaline medium and reduced by zinc in an HCl medium. It is possible for the determination of vitamin K3 based on the reduction with SnCl2 [15] in an ethanolglycerol medium by spectrofluorimetric technique. In all the procedures above mentioned the use of an organic solvent is necessary in order to get the solubilization of the menadione. Cyclodextrines are cycloamyloses, usually constituted by six to eight glucose sub-units, which present an almost conical hydrophobic cavity in which many molecules, organic or inorganic, may fit. The hydrophobic character of the cavity is shown, for example, by the enhancement of fluorescence of certain molecules in the presence of a or b-cyclodextrins (CDs). One of the most important potential applications of CDs is the protection of the guest molecule from the effect of oxidation and photochemical degradation. Saturated and unsaturated fatty acids were first treated in this way [16]. Retinal complex of CD has been described in literature by Lerner et al. [17]. Retinal is insoluble in an aqueous medium and not fluorescent but by a complex of CD it is possible to determine it by a spectrofluorimetric method. The use of CD has been described by Mun˜oz de la Pen˜a et al. [18] in order to get room-temperature phosphorescence from acenaphthene in the presence of 2-bromoethanol. The enhancement of fluorescence of an analyte by micellar solution is well known; that way one proposed to examine the effect on vitamin K3 when a micellar dodecyl sulphate (SDS) solution was used. It was possible to get solubilization of vitamin K3 in an aqueous medium, but no fluorescent signal was obtained. This may be a result of the incorporation of the vitamin into the micellar medium only causing a polarity change and that of CD additionally involving the formation of weak covalent bonds and van der Waals interactions.
The idea of using the formation of inclusion complexes with CDs to increase the solubility in aqueous media is not new and cyclodextrines have also been used to obtain enhancement of fluorescence [19,20] and phosphorescence at room temperature [21,22]. In this work, aimed at improving the luminescence detection of menadione, the effect on vitamin K3 was examined. When b-CD was used it was possible to obtain a fluorescence signal.
2. Experimental
2.1. Reagents Vitamin K3 solutions were prepared by dissolving 50.0 mg of the vitamin K3 in 0.01 M b-CD aqueous solution (purchased from Merck, Darmstadt, Germany) under stirring with a magnetic bar at boiling temperature for 5 min. The use of ultrasounds is inadvisable. A total of 0.01 mol l − 1 of b-CD was prepared by dissolving it in water obtained from a Milli-Q system (Milipore, Milford, MA, USA). Other standard stock solutions were used to prepare working standard solutions by suitable dilutions. Buffer solution of 6.2 pH was prepared by mixing appropriate amounts of citric acid and sodium hydroxide. All experiments were performed with analytical reagent grade chemicals.
2.2. Apparatus Fluorescence measurements were performed on a Perkin-Elmer model LS 50 spectrofluorimeter equipped with a Xenon lamp, connected to an Ataio S 3000 St 386 computer fitted with Perkin-Elmer F.L. Data Manager (FLDM) software and an Epson FX-850 printer. A thermostat Selecta Frigiterm 6000382 system connected to spectroflurimeter, a Crison Model 2001 pH-meter with a glass-saturated calomed combination, an ultrasonic bath and 10.0 mm quartz cells were also used.
J.J.B. Ne6ado et al. / Talanta 53 (2001) 951–959
2.3. Calibration Suitable aliquots containing 0.0025 – 0.050 mg of vitamin K3 were transferred into a series of 25-ml calibrated flasks, 16 ml of b-CD (0.01 mol l − 1) and 5 ml of buffer (0.5 mol l − 1) citrate solution were added to each flask and diluted to volume with water, in order to obtain the calibration graph. Before the measurement was performed it was necessary to wait 30 min so as to obtain a good ratio time-intensity. The measurement of samples with b-CD and buffer solution without vitamin K3 was necessary performed and for every sample it was necessary subtract the blank. For all samples a 313 nm wavelength for the excitation and the measurements were carried out at l = 407 nm for emission.
3. Results and discussion Vitamin K3, which is insoluble in an aqueous medium and does not fluorescence in solution, emits a luminescence in the region 407 nm, even in aqueous medium, when complexed to b-CD. The association constant of b-CD:vitamin K3 was studied. As shown in Fig. 1A, the excitation spectrum for the vitamin K3:b-CD complex exhibits two bands: a sharp one with a maximum at 241 nm and a broad one peaking at 313 nm. As usual, the emission spectrum contains a single band (with maximum fluorescence intensity at 407 nm).
3.1. Factors affecting fluorescence intensity Chemical variables were studied in order to obtain the best measurement conditions and the maximum fluorescence signal. The stability of the inclusion complex between b-CD and vitamin K3 was studied and the dissolution of vitamin K3 in b-CD was investigated to get more reproducible measurements. The vitamin is slowly dissolved in 0.01 M CD solutions, possibly because water molecules within the CD cavity tend to be displaced by the organic molecules. In order to determine the most favourable manner of incorporating vitamin K, the
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influence of stirring, ultrasonication and heating on its dissolution was studied. The efficiency of the inclusion process was determined by measuring the fluorescence intensity; the greater the intensity was, the more efficiently was the inclusion complex assumed to be formed. One experiment involved the application of ultrasounds for 5, 30, 60 and 180 min to four different solutions and measuring their fluorescence intensity over a period of 10 days following preparation. Whichever the ultrasonication time, the fluorescence intensity continued to increase with time; it peaked at day 8 and then levelled off. Accordingly, the use of ultrasounds was discarded. Stirring with a magnetic bar at room temperature for the previous lengths of time also resulted in the fluorescence intensity not peaking immediately after dissolution but 4 days later. The most efficient way of preparing the solutions was to stir them with a magnetic bar while boiling for 5 min. In this way, the fluorescence signal for the solution remained constant for at least 20 h. Consequently, one opted for using this procedure and making fresh solutions on a daily basis. Solutions were thus prepared by dissolving 50.0 mg of the vitamin in 0.01 M CD under stirring with a magnetic bar at boiling temperature for 5 min. The use of ultrasounds is inadvisable. The influence of pH on the fluorescence intensity was studied by adding different amounts of HClO4 or NaOH solutions. Fluorescence intensity of vitamin K3 was constant between pH 4.1 and 7.0 and pH 6.2 was selected as most suitable. pH 6.2 was obtained by adding citrate buffer solutions. The fluorescence intensity of vitamin K3 was not affected by the concentration of the buffer at least during 4 h. A total of 0.1 M concentration of buffer was selected to obtain suitable buffering capacity. The influence of the b-CD concentration on the fluorescence intensity for the complex was examined by changing the concentration from 2.0× 10 − 4 to 7.0 × 10 − 3 M. Higher concentrations of CD could not be tested owing to its poor solubility in water and the need to add a certain volume of buffer prior to making to the mark. As can be
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Fig. 1. (A) Excitation spectrum of complex b-cyclodextrin K3 of 2.0 mg l − 1. (B) Emission spectrum of complex b-CD:K3 of 2.0 mg l − 1.
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seen from Fig. 2, the fluorescence intensity increased with increasing CD concentration up to about 0.006 M, above which it remained constant at its peak value. A concentration of 6.4 × 10 − 3 M was adopted as the most suitable for analytical purpose. The data collected in this experiment were subsequently used to determine the formation constant of the inclusion complex. Another factor that affects fluorescence intensity is temperature. Thus, fluorescence intensity shows a decrease as temperature increases from 3 to 70°C. Temperature coefficient was of the order of 1.49% °C − 1. This can be ascribed to an increase in temperature increasing the kinetic energy of the molecules and hence the probability of their colliding; as a result, radiation less deactivation through internal conversion prevailed and the fluorescence quantum efficiency decreased [23].
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Therefore, the use of a thermostat is recommended, choosing a measurement temperature of 20°C, as an average room temperature. In these conditions, the influence of vitamin concentration on fluorescence intensity was studied. A linear relation between fluorescence intensity and vitamin K3 concentration in the range from 0.1 to 2.0 mg l − 1 can be observed. Higher concentrations exhibit a non-linear influence with a decreasing slope up to 0.01 M that remains constant up to 0.1 M. Beyond this vitamin concentration, the fluorescence decreases through inversion.
3.2. Association constant of b-CD–K3 complex It was not possible to measure the fluorescence intensity in aqueous solutions because the vitamin
Fig. 2. Influence of b- cyclodextrin concentration on the emission fluorescence of the binary complex b-CD – K3.
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Fig. 3. Double reciprocal plot for the b-cyclodextrin – K3 complex. The nonlinear fit of the data assuming the formation of a 1:1 b-CD – K3 complex and curved line assuming the formation 1:2 b-CD – K3 complex.
is water-insoluble; as noted earlier, however, the fluorescence signal decreased with decreasing CD concentration. This, together with the fact that the vitamin exhibits no native fluorescence in other solvents, led one to assign the vitamin a zero fluorescence in water on the basis of the Benesi–Hildebrand [24] procedure for determining the complex stoichiometry and formation constant. Two possible stoichiometries were considered for the inclusion complex, namely: 1:1 and 2:1. Assuming that b-CD forms a 1:1 inclusion complex with K3, the following expression can be written: K3 + b-CD X b-CD – K3
(1)
The formation constant of the complex (K) is given by: K=[CD – K3]/[CD][K3]
(2)
where [CD], [K3] and [CD–K3] are equilibrium concentrations. The direct relation between the observed fluorescence intensity enhancement (F− F0) and the using expression to that b-CD concentration is given by: F− F0 = (F − F0)K[CD]0/(1+ K[CD]0)
(3)
where [CD]0 denotes the initial CD concentration. F0 denotes the fluorescence intensity of vitamin K3 in the absence of b-CD. F denotes the fluorescence intensity when all of the vitamin K3 molecules are essentially complexed with b-CD. F is the observed fluorescence at each CD concentration tested. By typical double-reciprocal plots (method of Benesi–Hilderbrand) 1/F − F0 = 1/(F − F0)K[CD]0 + 1/(F − F0)
(4)
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Evidence for the existence of 1:1 complex was obtained by fitting a double-reciprocal plot (1/F − F0 vs. 1/[CD]0) as can be seen in Fig. 3. In order to effectively check the determination the stoichiometric ratio, assuming 1:2 inclusion complex with b-CD, has been studied. The following expression can be written: K3 + 2b-CD X (b-CD)2 – K3
(5)
The formation constant of the complex (K%) is given by: K%= [(b-CD)2 – K3]/([CD]0)2[K3]
(6)
If [CD]0 \ \ \(b-CD – K3)2 \ \ \b-CD – K3, then the following expression is obtained: 1/F − F0 =1/(F −F0)K%([CD]0)2 +1/(F − F0) (7) when 1/F −F0 versus ([CD]0)2 is represented (Fig. 3) the non linearity can be observed. As a conclusion the stoichiometry of the complex is 1:1. The initial parameter estimates needed for the non-linear regression method were obtained from linear plots (Fig. 3).The calculated association constant is K =373 9 34 l mol − 1. This suggests the formation of a complex between b-CD and K3 in solution just as it was assumed. A possible inclusion of vitamin K3 into b-CD must be an axial inclusion because b-CD has a inside cavity of 8 A, , and naphthalene, which is the aromatic structure of vitamin K3 (2-methyl-1,4naphthoquinone), and can not be included into b-CD by equatorial way with a measure of 8.4 A, of length and 6.8 A, of height and can not include into b-CD by equatorial way (8.4 A, ) while an axial way is possible. The inclusion of b-CD – K3 complex will be partial, because the unsubstituted benzene ring will be inside the b-CD cavity while the substitute benzene ring by cetones groups (1,4) and methyl group (2) will be outside the b-CD. The proposed hypothetical model is shown in Fig. 4.
3.3. Determination of 6itamin K3 The excitation spectra were performed at 313 nm wavelength excitation. The scan speed and slit
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Fig. 4. Inclusion complex.
values were optimized at 240 nm min − 1 and 10.0 nm, respectively. The measurements were performed at 313 nm for excitation and 407 nm for emission. Under these conditions the calibration graph was constructed from the emission spectra for standards containing between 0.1 and 2.0 mg l − 1 of vitamin K3 which has been dissolving in b-CD. Linear regression equation (y= a+mx) for vitamin K3 was obtained. The slope, intercept, determination coefficient, S.D., detection limit and quantification limit obtained for the determination of vitamin K3 by conventional fluorescence are summarized in Table 1. The significance of the intercept on the y axis of the obtained regression line was evaluated by applying the Student t-test at 95% confidence level. In the calibration graphs the intercept is negligible, since the experimental tvalue is smaller than the critical t-value, as can be seen in Table 1. In order to study the repeatability of the method, a series of ten standard samples containing 0.30 mg l − 1 of vitamin K3 were prepared, with the results of 0.279 mg l − 1 and 3.51% for the S.D. and the relative error respectively, when the conventional fluorescence was used.
Table 1 Statistical parameters of calibration graph Slope Intercept Determination coefficient (r 2) Slope standard deviation Intercept standard deviation Experimental ‘t’ value Theoretical ‘t’ value New slope Detection limit Quantification limit
208.5 −1.63 0.999 2.8 3.2 0.505 2.365 207.3 0.022 0.073
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Table 2 Results obtained by the application of the proposed method to pharmaceutical preparations Commercial preparation
Theoretical value (mg l−1)
Experimental value (mg l−1)
Recovery (%)
Vitaencil C.K.P.
0.624 0.689 0.745 0.676 0.743 0.851
0.635 0.639 0.749 0.695 0.783 0.869
101.76 100.54 100.67 102.81 105.38 102.09
BIO-HUBBER
3.4. Applications In order to study the validity of the method, it was applied to the determination of vitamin K3 in commercial preparations: Vitaendil C.K.P. from the ‘Wassermann’ Laboratory (whose composition per tablet is: ascorbic acid 75.0 mg; menadione 5.0 mg; vitamin P 25.0 mg; starch wheat 44.85 mg and lactose); BIO-HUBBER from ‘ICN Ibe´rica, S.A.’ Laboratory (whose composition per tablet is: neomicine (sulphate) 25.0 mg; bacitracine (zinc) 1.000 U.I.; streptomicine 50.0 mg; sulfadiazine 30.0 mg; pectine 50.0 mg; menadione 20.0 mg; sodium saccharine 10.0 mg; starch wheat 50.0 mg and lactose. Before the proposed method was applied to real samples, the effect of all the compounds which accompanying menadione in pharmaceuticals preparations, on the determination of the vitamin was studied. Three determinations were carried out for each of these pharmaceuticals preparations at three different concentrations. The pharmaceutical preparations were dissolved such as it is described in Section 2. The recovery percentages are summarized in Table 2. Recoveries achieved by means of the method proposed are in accordance with the real content of vitamin K3 in commercial preparations.
4. Conclusions The idea of using the formation of inclusion complexes with b-CD to increase the solubility in aqueous media and to obtain luminescence has been developed satisfactorily.
As a conclusion b-CD is a good host to promote the fluorescence of vitamin K3 in an aqueous solution and it is possible to use a molecular fluorescence technique for the determination of vitamin K3. The method is simple, sensitive and selective. It is possible to carry out the determination without any previous separations or tedious extraction and without other auxiliary techniques. The proposed method is more sensitive and selective than direct spectrophotometric methods and HPLC methods with UV detection [2–5]; it also surpasses some spectrofluorimetric methods for the same purpose [7,8,11,14]. Unlike reported fluorimetric methods, it requires no derivatization — usually reduction — reaction prior to the determination proper [7,8,11,14], which results in greater simplicity and precision for the method.
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