Characterization of keto-enol tautomerism of p-hydroxyphenylpyruvic acid using CE with amperometric detection and spectrometric analysis

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1 J. Sep. Sci. 2009, 32, Ying Huang 1,2 Xiaoli Zhang 1 Liangjun Xu 2 Hongqing Chen 2 Guonan Chen 2 1 College of Chemistry and Materials Science, Fujian Normal University, Fuzhou, Fujian, P. R. China 2 Ministry of Education Key Laboratory of Analysis and Detection Technology for Food Safety, and Department of Chemistry, Fuzhou University, Fuzhou, Fujian, P. R. China Received July 20, 2009 Revised September 11, 2009 Accepted September 13, 2009 Research Article Characterization of keto-enol tautomerism of p-hydroxyphenylpyruvic acid using CE with amperometric detection and spectrometric analysis A high-performance CE with amperometric detection (CE-AD) was employed for the kinetic study of keto-enol tautomerism of p-hydroxyphenylpyruvic acid (phpp). Several factors (concentration of b-cyclodextrin (b-cd), concentration and ph of running buffer, separation voltage and injection time) affecting CE-AD were investigated and separation conditions were optimized. The kinetics of phpp was performed in water solution and phosphate solution under different ph and temperature, the homologous ketonization rate constants and half-life were obtained. Also, the activation energy was calculated according to the rate constants under different temperature. The experimental results indicated that b-cd played an important role in the separation, therefore UV spectrometric method was applied for the study of complexation interaction between ketonic phpp and b-cd. The results indicated that the stoichiometric ratio of the phpp b-cd complex was 1:1 and formation constant was determined. The obtained kinetic results are in correspondence with those reported by our group with CE-UV. Keywords: CE with amperometric detection / Keto-enol tautomerism / p-hydroxyphenylpyruvic acid DOI /jssc Introduction p-hydroxyphenylpyruvic acid (phpp) is an important intermediate in the metabolism of tyrosine [1]. The level of phpp in body blood and urine may reflect some diseases such as tyrosinemia [2]. Because of the physiological and clinical significance, chemistry of phpp has been a topic of constant interest. The methods for determination of phpp included flow injection spectrophotometry combined with single chip microcomputer [3], fluorescence quenching [4], GC [5 6], HPLC [7], CE [8 10], and electrochemical technique [1]. The Keto-enol tautomerism of phpp was early investigated by Chi et al., using electrochemical method and UV spectrometry [1]. Recently, Chen and coworkers [8] studied keto-enol tautomerism of phpp by using CE with UV detection. CE with amperometric detection is sensitive, cheap, and selective, which makes it very suitable for analysis of electrochemical active compounds. Although the kinetics of phpp keto-enol tautomerism has been studied by CE-UV, the tautomeric equilibrium is not investigated comprehensively. Moreover, the interaction between phpp and b- cyclodextrin (b-cd) was only qualitatively explained by electrochemical method. Therefore, this article will focus the study on the kinetics of keto-enol tautomerism of phpp by CE-AD and complexation interaction between phpp and b-cd by UV spectrometry. Besides separation conditions optimization, the kinetics in acidic, neutral and basic phosphate solution (ph 5, ph 7, and ph 9, respectively), and under different temperatures (25 401C) was investigated, and the relevant rate constant, half time were obtained. Also, activity energy of the tautomeric process was first calculated according to the rate constants under different temperatures. In addition, stoichiometric ratio of the inclusion complex and formation constant was determined by UV spectrometry. The obtained kinetic results were in correspondence with those of CE-UV reported by our group. Correspondence: Professor Guonan Chen, College of Chemistry and Materials Science, Fujian Normal University, Fuzhou, Fujian, , P. R. China gnchen@fzu.edu.cn Fax: Abbreviations: b-cd, b-cyclodextrin; CE-AD, CE with amperometric detection; phpp, p-hydroxyphenylpyruvic acid 2 Materials and methods 2.1 Apparatus A 730 kv high-voltage DC power supply (Shanghai Institute of Nuclear Research, China) was employed for the separation voltage between the ends of the capillary. Separation capillary was an untreated fused silica capillary

2 4156 Y. Huang et al. with 48 cm 25 mm id 370 mm od (Hebei Yongnian Optic Fiber Factory, China). A laboratory-built pre-aligned electrochemical cell, consisting of three electrodes (a 300 mm diameter carbon disc working electrode, a platinum auxiliary electrode and an Ag/AgCl reference electrode), was combined with a BAS LC-4C amperometric detector (Bioanalytical System, West Lafayette, IN, USA). The signal of chromatogram was monitored using a Chromatogram workstation (Model HW-2000, Qianpu Software company, Shanghai, China). The absorption spectra were measured on TU-1900 spectrophotometer (PGeneral, Beijing). A phs- 3C meter (Shanghai Leici Instrument Company, Shanghai, China) was used to measure the ph value of the running buffer. 2.2 Chemicals phpp and b-cd were obtained from Sigma (St. Louis, MO, USA). Other chemicals were of analytical grade. The water was prepared using a Milli-Q Academic/A10 equipment (Millipore, Bedford, MA, USA). Because enolic form of phpp remained very stable in methanol medium, the stock solution of phpp ( mol/l) was prepared by dissolving the required amount of sample in methanol. b-cd was dissolved in water to obtain stock solutions of 10 mmol/l. All of the stock solutions were stored under refrigeration before using, and diluted to the desired concentration with the running buffer. 2.3 Electrophoretic conditions The new capillary was activated with 0.1 mol/l NaOH and water both for 3 h in a washing system. Before each run, the capillary was sequentially rinsed with 0.1 mol/l NaOH and water for 5 min, and running buffer for 10 min in the CE- AD system. The carbon disc electrode was repeatedly polished with emery paper and sonicated in doubly distilled water. Prior to use, all solutions were filtrated using membrane filters (0.22 mm) and degassed by ultrasonic instrument. CE was performed at a separation voltage of 29 kv with a 20 mmol/l Na 2 HPO 4 NaH 2 PO 4 (ph 5 7.0) solution as running buffer. The potential applied to the working electrode was 1.00 V (versus Ag/AgCl). Sample was injected electrokinetically at 29 kv for 10 s. Freshly prepared mol/l phpp water solution was placed in a water bath to maintain a constant temperature. Aliquots (15 ml) were taken at a certain interval time from initiation of the reaction (time 0) to the end of reaction. 2.4 UV spetrometric studies Mixed solutions were prepared with the mole ratio of 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3 between balanced phpp (24 h after prepared) and b-cd. Their absorption (A) at 280 nm was measured using a corresponding b-cd as blank. Synchronously, the absorption (A 0 ) of corresponding phpp solution was measured with 20 mmol/l phosphate buffer (ph 4.00) as blank. We defined DA 1 5 A 0 A. A 100 ml stock solution ( mol/l) of phpp was transferred into eight 10 ml colorimetric tube, and then appropriate amount of 0.01 mol/l b-cd was added to each tube under room temperature. The mixtures were diluted to final volume with 20 mmol/l phosphate buffer (ph 4.00) and shaken thoroughly. The concentration of b-cd in each solution is 0, , , , , , and mol/l, respectively, and their absorptions (DA 2 ) were measured using corresponding phpp solution as blank. 3 Results and discussion J. Sep. Sci. 2009, 32, Kinetic study of keto-enol tautomerism of phpp with CE-AD Optimization of separation condition Several factors, such as the concentration of b-cd, concentration and ph of running buffer, separation voltage and injection time were investigated in order to optimize the electrophoresis condition. Our previous report [8] declared that good separation and shortened time was obtained when using b-cd as additive. Therefore, the CE behavior of tautomers in the existence of b-cd was examined in our study. It can be seen from Fig. 1 that the addition of b-cd would shorten the migration time of the tautomers. Thus the effects of the concentration of b-cd in the range of 08 mmol/l was examined. It can be known that with the increasing of the concentration of b-cd, the migration time is decreased, and when the concentration is higher than 6 mmol/l, the peak shape of enolic phpp is unsymmetrical. Considering the migration time and peak shape, 6 mmol/l was chosen as the optimum concentration of b-cd. Based on the fact that b-cd played an important role in separation, inclusion complex between phpp and b-cd was deduced, and their interaction has been explained by using electrochemical method [8]. In order to further confirm this interaction, UV spectrometry was applied to study the complex interaction between phpp and b-cd in this article, and this will be discussed later in Section 3.4. In addition, the effects of concentration and ph of the phosphate buffer solution on separation were investigated, and the results showed that the migration time of analyte increased with the concentration of the running buffer in the range of mmol/l. The peak shape of enolic phpp was broadening with the increase of buffer concentration. Therefore, 20 mmol/l was selected as the optimal concentration of running buffer. The experimental results also indicated that the migration time was decreased with

3 J. Sep. Sci. 2009, 32, Electrodriven Separations 4157 enolic part and ketonic part remained unchanged during the tautomerization. So the kinetics of enolic phpp was investigated according to the change of second peak (enolic form). Our previous report [8] indicated that the tautomerism of phpp is a first-order reaction and the kinetic equation is as follows: ln C enol ¼ kt ln C 0 ð1þ where C 0 is the initial concentration of phpp and C enol is the concentration of enolic phpp at a tautomerism time t. The rate constant k can be obtained by the slope of the straight line plotted with ln C enol against t Study on the kinetics of keto-enol tautomerism of phpp Figure 1. Effects of the concentration of b-cd. Peak 1 refers to oxidation of phenol OH group on working electrode. Peak 2 refers to oxidation of C==C in side chain of the enolic phpp. increasing of ph in the range of Considering the peak shape and analytic time, ph 7.0 was chosen as the optimum ph when the kinetics was studied in aqueous solution. Furthermore, ph 5.0, 7.0 and 9.0 were utilized to measure the rate constants while the kinetics was investigated in phosphate buffer. Moreover, the effects of separation voltage (20 29 kv) and injection time (5 15 s) were also examined. It was found that the higher voltage was benefited for shorter migration time, and did not cause the increase of the baseline noise and the broadening of the peak shape, so 29 kv was chosen as the separation voltage. The effects of injection time were studied in the range of 5 15 s at 29 kv, and the results showed that the peak current would be increased with increasing of injection time. However, when injection time was longer than 10 s, the peak current was not increased much and the peak exhibited a significant broadening. So 10 s was selected as the injection time subsequently Keto-enol tautomerism of phpp As described in reference [1] and [8], there are two tautomers of phpp, i.e. enolic and ketonic form in the aqueous solution, and the enolic form would convert spontaneously to the ketonic form in aqueous solution (Fig. 2). Figure 3 shows the conversion process of the two tautomers in different reaction time. Peak 2 refers to oxidation of C==C in side chain of the enolic phpp on working electrode, thus its height decreases gradually with the ketonization time increases (i.e. the decreasing amount of enolic phpp). Peak 1 refers to oxidation of phenol OH group. Since enolic and ketonic form both contain phenol OH group, the total amount of OH group is the same during the conversion. Thus the height of peak 1 which is contributed both by In order to determine the linearity between the enolic phpp concentration and its peak area, a series of concentrations of phpp were tested under the optimized condition. The results of the regression equations and correlation coefficients are summarized in Table 1. The kinetics of keto-enol tautomerism of phpp was studied in different media with initial concentration of phpp mol/l. The correlation coefficients and rate constants are calculated and presented in Table 2. It is obvious that with the increasing of ph value, the rate constant is increased, and the ketonization rate constant in phosphate buffer is larger than that in aqueous solution. The results are accorded with those reported by our group with CE-UV [8] (Table 2). This is probably because the activation energy for intermediate formation under basic conditions is less, and thus the reaction proceeds at a higher rate. The kinetics of keto-enol tautomerism would be affected by temperature. Hence, in this article, the kinetics of ketoenol tautomerism of phpp was studied under 25, 30, 35 and 401C, respectively. The phpp was dissolved in 20 mmol/l phosphate buffer (ph 7.0), and the initial concentration of phpp utilized in this study was mol/l. The linearity between the ketonic phpp concentration and its peak area was presented in Table 1 (condition 2). A CH C COOH CH 2 C COOH OH OH B OH Figure 2. Keto-enol tautomerization of phpp. (A) Enolic form; (B) ketonic form. O

4 4158 Y. Huang et al. The rate equations and constants under four different temperatures are given in Table 3. The result showed that tautomerization process under high temperature is faster J. Sep. Sci. 2009, 32, than that under low temperature. Rate increases with temperature may be due to increased collisions between molecules. According to Arrhenius equation: ln k ¼ ln A E a ð2þ RT The correlation equation between ln k and T was obtained and, activation energy (E a ) was calculated. The result is as follows: Ln k ¼ 8644:3=T þ 25:386 E a KJ/mol. 3.2 Study on the complexation of phpp with b-cd Stoichiometry of phpp b-cd complex Figure 3. Electropherogram of enol-keto tautomerization of phpp in aqueous solution. Reaction time for (a), (b), (c), (d) is 5, 115, 200 and 244 min, respectively. Peak 1 and Peak 2 are the same as in Fig. 1. Conditions: 20 mmol/l phosphate buffer (ph ) including different concentrations of b-cd; separation voltage: 29 kv; injection time: 10 s; [phpp]: mol/l. Figure 4 shows that the absorption peak of ketonic phpp is at 280 nm, while b-cd has no absorption. The maximum absorption is decreased in the presence of b-cd. In order to assess the stoichiometry of the complex, equimolar series method [11] was applied. The mole ratio contraposing maximal DA 1 (A 0 A) was the stoichiometric ratio of phpp b-cd complex. Figure 5 shows the continuous variation plot of phpp and b-cd. It can be observed that the maximum absorbance is reached at mole fraction of 0.5, which indicates a 1:1 stoichiometry for the phpp b-cd complex. Table 1. Regression equations and correlation coefficients of phpp under different media a) Conditions Regression equation Correlation coefficient Linear range (mol/l) 1 Y X Y X Y X Y X a) Conditions: (i) Running buffer was 20 mmol/l phosphate buffer (ph 5.0) and phpp was dissolved in this buffer; (ii) running buffer was 20 mmol/l phosphate buffer (ph 7.0) and phpp was dissolved in this buffer; (iii) running buffer was 20 mmol/l phosphate buffer (ph 9.0) and phpp was dissolved in this buffer; (iv) running buffer was 20 mmol/l phosphate (ph 7.0) and phpp was dissolved in water. Table 2. Rate equations and constants of phpp in different media a) Conditions Regression equation Correlation coefficient Ketonization constant b) (per min) Half-life (min) 1 ln C t ln C t (0.0276) 27.8 (25) 3 ln C t (0.0596) 14.1 (12) 4 ln C t (0.061) (114) a) Conditions: (i) Running buffer was 20 mmol/l phosphate buffer (ph 5.0) and phpp was dissolved in this buffer; (ii) running buffer was 20 mmol/l phosphate buffer (ph 7.0) and phpp was dissolved in this buffer; (iii) running buffer was 20 mmol/l phosphate buffer (ph 9.0) and phpp was dissolved in this buffer; (iv) running buffer was 20 mmol/l phosphate (ph 7.0) and phpp was dissolved in water. b) The values in parentheses are derived from [8].

5 J. Sep. Sci. 2009, 32, Electrodriven Separations 4159 Table 3. Rate equations and constants of phpp under different temperatures a) Temperature (1C) Regression equation Correlation coefficient Ketonization constant (per min) Half-life (min) 25 ln C t ln C t ln C t ln C t a) Conditions: Running buffer was 20 mmol/l phosphate buffer (ph 5.0) and phpp was dissolved in this buffer. Absorbance a b 0.05 c Wavelength/nm Figure 4. UV absorption spectra of phpp and b-cd. Conditions: (a) [phpp]: mol/l; (b) mol/l [phpp] mol/l [b-cd]; c. [b-cd]: mol/l Formation constant K s of the complexation of phpp with b-cd Δ A [phpp] / ([phpp] + [β - CD]) Figure 5. Continuouse variation plot of phpp and b-cd complexes. PBS: 20 mmol/l, ph 5 4. Absorbance Absorbance Wavelength/nm Wavelength/nm Figure 6. UV absorption spectra of phpp and b-cd complexes. Conditions: 20 mmol/l phosphate buffer (ph4.0); [phpp]: mol/l; [b-cd] 1-8: 0, , , , ,3 10 3,4 10 3, mol/l. The formation constant (K s ) of the complexation of phpp and b-cd can be calculated from the absorption data by modified Benesi Hildebrand equation [12, 13]: 1 1 ¼ DA 2 K s De½GŠ 0 ½HŠ þ 1 ð3þ De½GŠ 0 where [G] 0 was the initial concentration of phpp and [H] was the concentration of b-cd. K s was formation constant for phpp b-cd complexes. DA 2 was the absorbance change in the absorption spectra of the phpp by the addition of b- CD. De was the difference in the molar absorptivity between free and complexed phpp. The straight lines obtained by plotting (1/DA 2 ) versus (1/[H]) were utilized to evaluate K s values from the intercept and the slope. Figure 6 shows UV absorption spectra of phpp in the absence and presence of b-cd. It was noted that the absorption decreased with the increasing of b-cd concentration. Similar results have been gained with complexes of ibuprofen with b-cd [14]. Figure 7 shows the plot of 1/DA 2 versus 1/[b-CD]. It exhibits good linearity, which implies that the complexes have a stoichiometry of 1:1. The stability constant K s obtained from the intercept/slope ratio of Fig. 7 was calculated to be L/mol

6 4160 Y. Huang et al. 1/ΔA /C(β-CD) Figure 7. Double reciprocal plot for phpp in the presence of b-cd. 4 Concluding remarks In this study, we developed a CE-AD method for the kinetic study of keto-enol tautomerism of phpp. The influence of media (acid, neutral and basic), temperature, ph on the keto-enol tautomerism was investigated. The ketonization rate constant under different aqueous solution and temperature and activation energy was calculated. The result indicated that tautomerization proceeds faster in basic solution and in phosphate buffer solution, those results are in accordance with our previous work [8]. Higher temperature also speeds up the keto-enol tautomerization. In addition, UV spectrometric method was applied to study complexation interaction between ketonic phpp and b-cd. The complex showed a 1:1 stoichiometry, with a formation constant of L/mol. This project was supported by the National Nature Sciences Foundation of China ( , ), and the Program of Science Foundation of Science and Technology Department of Fujian Province, P. R. China (2005K013). The authors have declared no conflict of interest. 5 References J. Sep. Sci. 2009, 32, [1] Chi, Y., Duan, J. P., Qi, X. Z., Chen, G. N., Bioelectrochem 2003, 60, [2] Zhang, C. Y., Chinese Medical Encyclopedia, Biochemistry, Shanghai Science and Technology Press, Shanghai 1987, [3] Wang, J., Xue, J. G., Fu, M. G., J. Nanchang University 2004, 28, [4] Wu, F., Fu, M. G., Wei, X. S., Spectroscopy Spectral Anal. 2001, 21, [5] Deutsch, J. C., J. Chromatogr. B 1997, 690, 1 6. [6] Niwa, T. J., Chromatography 1986, 379, [7] Cherlet, M., De Backer, P., Croubels, S., J. Chromatogr. A 2006, 1133, [8] Huang, L., Huang, Y., Chi, Y. W., Lin, J. M., Yu, L. S., Xu, L. J., Chen, G. N., J. Chromatogr. A 2007, 1175, [9] Huang, Y., Jiang, X., Wang, W., Duan, J., Chen, G., Talanta 2006, 70, [10] Chen, G., Chi, Y., Wu, X., Duan, J., Li, N., Anal. Chem. 2003, 75, [11] Job, P., Ann. Chim. 1928, 9, 113. [12] Jurgen, K., Charles, A. H., Chem. Eng. Sci. 2001, 56, [13] Pickering, P. J., Chaudhuri, J. B., Chem. Eng. Sci. 1997, 52, [14] Chow, D. D., Karara, A. H., Int. J. Pharm. 1986, 28,