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Analysis of aesculin and aesculotin in Cortex fraxini by capillary zone electrophoresis
Talanta 52 (2000) 607 – 621 www.elsevier.com/locate/talanta
Analysis of aesculin and aesculotin in Cortex fraxini by capillary zone electrophoresis Hongyi Zhang a,b,c,*, Qianfeng Li a, Zhihong Shi c, Zhide Hu a, Rui Wang b a
Department of Chemistry, Lanzhou Uni6ersity, Lanzhou, Gansu 730000, PR China Department of Biology, Lanzhou Uni6ersity, Lanzhou, Gansu 730000, PR China c Physical and Chemical Center, Henter, Hebei Uni6ersity, Baoding 071002, PR China b
Received 14 October 1999; accepted 27 January 2000
Abstract A simple method for quantitative analysis of aesculin and aesculetin in Cortex fraxini was developed using capillary zone electrophoresis (CZE). Berberin was employed as an internal standard, The running buffer was 6 mM Na2B4O7 and 10 mM NaH2PO4 (pH 6.70). The linear calibration range was 32 – 256 mg ml − 1 (r =0.9996) for aesculin and 23–230 mg ml − 1 (r=0.9993) for aesculetin. The contents of aeculin and aesculetin in C. fraxini were easily determined within 12 min the pKa values of aesculin and asculetin determined by CZE were 6.56 and 5.62, respectively. A simple method for estimation of the temperature inside the capillary during running was also proposed. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Aesculin; Aesculetin; Cortex fraxini; Dissociation constant
1. Introduction Cortex fraxini (Chinese name Qin-pi), a commonly used chinese herbal medicine has been proven to be effective in the treatment of diarrhea and dysentery of intense heat type, especially for dysentery with blood stools, and lung heat syndrome with cough and dyspnea for over 2000 years [1]. Pharmacological action demonstrates that C. fraxini can inhibit the growth of dysentery bacillus and staphylococcus and has the antitus* Corresponding author. Tel.: + 86-931-8911288; fax: +86931-8911100. E-mail address: [email protected] (H. Zhang).
sive and expectorant function. Aesculin, aesculetin fraxetin fraxin and stylosin have been isolated from the plant C. fraxini [2]Fig. 1. Aesulin and aesculetin are the two of the most important bioactive components in C. fraxini. Pharmacological experiment shows that aesculin can prolong the hypnotic effect of hexobarbital in mice and aesculein can exert and antihistaminic effect and relax smooth muscle of guinea pig’s trachea in vitro, and the analgestic effect of aesculetin is stronger than that of aspirin. Aesculin, aesculetin have been considered as indexes for estimation of quality [3]. Consequently the determination of these two components in C. fraxini samples in of interest to many scientists. Several
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H. Zhang et al. / Talanta 52 (2000) 607–621
methods [4], such as paper chromatography, polarography thin layer chromatography and high performance liquid chromatography have been developed for determination of aesculin and aesculentin in plant samples or biological fluids. However, these methods suffer from materials and time consuming since large amounts of organic reagent and many operation steps are often required. The development of high performance capillary electrophoresis (CE) has been reviewed many times [5], and clearly it is continuous to be a very active research area in separation science since this technique often provides higher resolving power, shorter analysis time and lower operational cost than liquid chromatography. Now some research groups are devoting themselves to the study of assay of traditional Chinese medicine by CE [6 – 12]. The aqueous ionization constant (pKa) is a very important physico chemical property in the pharmaceutical industry [13,14] and phytochemistry [15]. Recently, capillary electrophoresis (CE) has been introduced as a method for convenient and precise aqueous pKa determination [16–18]. Compared to the most common methods such as potentiometry and spectroscopy, this method has some outstanding advantages, First, CE requires small amounts of sample at low solute concentration. Second, No precise information about the sample concentration is needed as long as the response of the UV detec-
Fig. 1. Chemical structures of aesculin and aesculetin.
tor offers a good peak. Third the ionization constant for several analytes can be determined simultaneously. As far as we know, however the ionization constants for aesculin and aesculetin are not published and have one been determined using capillary electrophoresis. In this study, based on the systematic determination of the effects of buffer pH and applied voltage on the separation and determination of aesculin and aesuletin the contents of aesculin and aesuletin in C. fraxini and the ionization constants for aesculin and aesculetin were obtained. The temperature inside the capillary during operation was calculated using a simple formula derived by us in this work.
2. Experimental
2.1. Reagents and materials Aesculin, berberin and aesculetin were purchased from National Institute for the Control of Pharmaceutical and Products, Beijing, people’s Republic of China. All other chemicals were of analytical reagent grade. Stock solutions in about 0.8 mg ml − 1 of aesulin and aesculetin were prepared by using 79% methanol (v/v) aqueous solution, Mixed standard solution was prepared by mixing stock solution with destilled water to achieve the desired concentration of standard solution. C. fraxini was purchased from the Chinese herbal store in Baoding, PR China. The buffer used for electrophoresis was 6 mM Na2B4O7 + 10 mM NaH2PO4. 1 MHCl or 1 M NaOH adjusted the value of pH of this buffer to any desired value. C. fraxini ground into a powder was passed through a screen mesh (number 40). About 1.0 g of fine powder was weighed and extracted with 25 ml of 80% methanol for 30 min. Extraction was repeated two times. The mixture of 1.5 ml of berberin with fixed concentration and 6 ml of the combined extracts was diluted with water to 10 ml. This solution was filtered from a 0.45 mm filter and the filtrate was injected directly into CE capillary.
H. Zhang et al. / Talanta 52 (2000) 607–621
2.2. Apparatus Electrophoretic experiments were carried out on a Waters Quanta 4000E Capillary Electrophoresis System (Milford, MA, USA). A fused silica capillary with an effective length of 39.7 cm (total length 47.3 cm) and 75 mm I.D. was used for all experiments. Samples were introduced into the capillary by hydrodynamic injection for 10 s. UV detection was employed at a wavelength of 214 nm. Before each analysis the capillary was rinsed under pressure with 0.1 mol 1 − 1 NaOH for 5 min and then equilibrated with the running buffer for 5 min, the electro-osmotic flow (EOF) was measured by using formamide (HCONH2) as a marker. A pHs-3C pH meter (Leici Instrumentation Factory, Shanghai, P.R. China) was used for pH measurements.
8.14 and 8.50. When the pH is lower than 3.09, aesculin, aesculetin and formamide migrate together, since the three analytes are almost complete anionized. When the pH is in the range 3.09–4.47, aesculetin is separated from aesculin and formamide and the latter two are also partly separated at pH 4.47. These results suggest that both aesuletin and aesculin are partly deprotonated when the pH is higher than 3.09. Complete separations of the three analytes are readily achieved in the pH range 5.84–8.50. It is well known that the UV signal of ionizable compound is influenced by the buffer pH since its molar absoprptivity depends on the pH of the medium [20]. As demonstrated in the literature [21], the peak area for species i recorded in the capillary electrophoresis was derived as follows: Ai =
2.3. Calculation The effective mobility, me, was calculated as follows me =
1 1 lV − t te0 V
(1)
where t is the migration time of the analyte (s) te0 is the migration time of an uncharged solute (s) L is the total length of the capillary (cm), l is the length of the capillary between injector and detector and detector (cm) and V is the applied voltage (V).
oi · b · t i Qi × l p · r2
3.1. Effect of pH In capillary zone electrophoresis the, pH of the buffer plays an important role in the separation of ionizable analytes because both the extent of ionization of each individual analyte and the charge of the capillary wall surface are influenced by buffer pH [16,19]. Therefore, manipulation of buffer pH is always a key strategy for optimizing a separation. Fig. 2 shows the electropherograms of aesulin and asculetin using phosphate (20 mmol 1 − 1) as running buffer at pH 4.47, 6.22,
(2)
where oi is the molar absorptivity, ti the migration time for species i, Qi the injection amounts for species i, l the length from the injection end to the detector, b the pathlength of the light. Thus, in order to normalize the bias of peak areas for different solutes, a migration speed correction has to be made by dividing the integrated peak area of solute with its corresponding migration time [22,23]. If the mixture of species i and j are introduced into the capillary the ratio of the corrected area between species i and j, RA, can be expressed as RA=
3. Results and discussion
609
Ai /ti oi · b · Qi /l · p · r 2 oi Qi = = × Aj /tj oj · b · Qj /l · p · r 2 oj Qj
(3)
If the molar absorptivity of species j does not depend on the medium pH in the experimental pH range, Eq. (3) can be simplified into the following form: RA= D · oi
(4)
where D=Qi /oj · Qj is a proportionality constant dependent only on the reference species j and the concentration ratio between species i and j and having nothing to do with the analyte species. Thus the dependence of oi on the buffer pH can be found by observing the changes of RA with duffer pH. Formamide is considered neutral at pHs be-
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Fig. 2. Electropherograms of the mixture of aesculin aesculetin and formamide obtained at various buffers pH values, (A) 4.47; (B) 6.22; (C) 8.14; (D) 8.50. Peaks, 1 = formamide; 2 =aesculin; 3 =aescuetin. Buffer; 6 mM Na2B4O7 +10 mM NaH2PO4; fused silica capillary, 47.3 ×50 mm I.D. (effective length 39.7 cm); applied voltage, 20 kV; injection time 10 s; temperature, 25°C.
H. Zhang et al. / Talanta 52 (2000) 607–621
Fig. 2. (Continued)
611
612
H. Zhang et al. / Talanta 52 (2000) 607–621
Fig. 2. (Continued)
H. Zhang et al. / Talanta 52 (2000) 607–621
Fig. 2. (Continued)
613
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tween 4.47 and 8.50 and its absorportivity keeps constant as the pH changes in the experimental range. The plot of the RA value for aesculin the corrected area ratio between aesculin and for-
mamide against the buffer pH is shown in Fig. 3a. At first, RA is small and keeps nearly constant with increasing the pH. This indicates that the most of aesculin is in the non-ionized molecular
Fig. 3. Plot of the ratio of the corrected peak for aesculin (a) and aesculetin (b) that for formamide (RA) as a function of buffer pH. Operation conditions as in Fig. 2.
H. Zhang et al. / Talanta 52 (2000) 607–621
615
Fig. 4. Plots of the electrophoretic mobilities of aesculin and aesculetin as a function of buffer pH.
state. When pH \6, RA increases rapidly with increasing pH, indicating that both the non ionized molecules and the corresponding ions are present in the species aesculin. However after the ionization perold of aesculin, RA trends to level off showing that aesculin is fully ionized. It can be seen from Fig. 3b that the RA value for aesculetin has the similar result when pH changes. From the above study, it can be found that the relatively high pH has some advantages such as relatively large detection signal and relatively short migration time. A buffer solution of pH 6.70 is chosen in this work because this solution has a relatively large capacity.
3.2. Determination of pKa 6alues Fig. 4 shows a plot of the electrophoretic mobility versus pH for aesculin and aesculetin along with the superimopsed curve fit to the following equation [16]: me =
Ka ×m− A [H+]+ Ka
(5)
where me is the electrophoretic mobility of aesculin or aesculetin at a given pH m− A is the mobility of the anionic form of aesculin and aesculetin
and Ka is the ionization constant. The pKa values of aesculin and aesculetin listed in Table 1 have not been compared with any literature values, since they do not exist in some common handbooks. However the fact that the more stable the conjugate anion of an acid, the stronger its acidity is, supports the rationality of the values determined in this work. The conjugate anions formed from aesculin and aesculetin at relatively high pH are stabled compared with the anion of phenol, since the latter has a relatively small conjugation system, The pKa value for phenol is about 10. Therefore, is can be concluded that the values of pKa for aesculin and aesculetin must be less than 10. From the molecular structures of aesculin and aesculetin, is easier to deprotonate than aesculin because it has two OH groups in ortho position that can form a hydrogen bond. Table 1 a pKa and m− A for aesculin and aesculetin Analytes
pKa
m− A
Aesculin Aesculetin
6.56 5.62
−2.27 −3.22
a
Mobility in 10−4 cm2 v−1 s−1.
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616
Table 2 The results of linear regression for effect of voltage Analyte
Aesulin Aesculetin Formamide
Intercept
1.515 3.402 1.087
Slope ( f ) Experimental
Ideal
128.390 285.882 91.746
86.310 120.815 268.870
Correlation
e:f
0.9996 0.9991 0.9997
0.0118 0.0119 0.0119
Fig. 5. Plots of migration time as a function of reciprocal applied voltage for formamide under ideal (a) and real (b) conditions.
3.3. Effect of applied 6oltage The influence of applied voltage is studied. As derived by Jorgenson [24], when net mobility me + me0 is independent on the applied voltage, the migration time of a solute should be inversely and linearly proportional to the applied voltage t=
l ·L 1 × me +me0 V
(6)
Consequently, under this ideal condition, the linear plot of migration time as the function of the reciprocal applied voltage should approximately pass through the point (0, 0). However, our experimental results show that there is a non-zero
intercept for the linear plot (see Table 2). As explained in our previous week [25], the non-zero intercept in the linear plot of the migration time versus the reciprocal applied voltage under nonideal condition is attributed to the thermal effect that tends to shorten migration time. It is assumed that the Joule heating under the influence of 5 kV the applied voltage is small enough to be negligible. Therefore, the value me + me0 in ideal condition can be calculated according to the migration time in 5 kV obtained from the linear regression equation. The linear dependence of migration time for formamide on the reciprocal of the applied voltage under ideal and real conditions is shown in Fig. 5. When the applied voltage is V, the migration time in ideal condition, t1(6 )
H. Zhang et al. / Talanta 52 (2000) 607–621
and the experimental migration time, t2(6 ) can be calculated from the ideal linear equation and the linear regression equation, respectively. The analyte migration time can be expressed as [26] t=
h ·l 2 · za ×f(k · a) o · E · zc + 3
n
3.4. Application
(8)
where A and B are constants characterizing the properties of the running buffer. Therefore, log t= log
A ·l B + (o · E · [zc +(2 · za/3) · f(k · a)]) T (9)
If one assumes that the variation of o, za, zc and f(ka) are negligible compared with the variations of h with temperature, Eq. (9) implies that the logarithm of migration time is linearly related to the reciprocal of the temperature inner the capillary at a constant voltage. Therefore the temperature inside the capillary at any applied voltage V can be expressed as follows T2(V ) =
B log((t2(6 ))/(t1(6 ))) + (B/T1(6 ))
respectively. The average temperature is 35.6°C. As the electric current almost keeps constant at various pH conditions, the acid dissociation constant listed in Table 1 is obtained at about 35.6°C.
(7)
where h is the viscosity coefficient; o the dielectric constant of the medium; za,zc the zeta potential of the analyte and the inner wall of the capillary, respectively, k the reciprocal of the analyte double layer thickness, a ‘radium’ of the analyte. The viscosity of the buffer has the following simplified relationship with temperature [26]: h=A · 10B/T
617
(10)
Theoretically, Eq. (8) shows that log h should have a linear relationship with the absolute temperature (T). The slope of the straight line should be equal to the constant B. From the viscosities of water at various temperature [27], it is found that log h has a linear relationship with the absolute temperature in the range 275 – 373 K. The correlation coefficient is 0.990 and the slope B, is 791.0 When T1 is regarded to be equal to the thermostated temperature, 25°C. The T2 values based on the migration behavior of aesculin aesculetin and formamide are 308.63, 308.56 and 308.62,
As demonstrated previously by some authors [28,29], the approach for obtaining improved precision in capillary electrophoresis is added various markets to samples as aids for monitoring shifts in the electroosmotic flow or electroosmotic flow or electrophoretic mobilities of solute, During the course of the ten injections the average time is 4.94 min for aesculin and 11.16 min for aesculetin the relative standard deviation (R.S.D.%) for migration time range is 9 1.41% for aesculin and 9 2.35% for aesculetin. In the case, migration time is not a reliable indicator for the identification of a particular compound. To help correct for the variability in the migration times, a migration time ratio is calculated for aesculin and aesculetin during each run. To finish this task transforming migration time into relative migration time, we have to select a compound as the internal standard. From our experiment, we find that no peak appeared before formamide, which is regarded as neutral compound in the electropherogram of the mixture of formamide and the extract of a C. fraxini sample. This result indicates that on positively charged compound is found under the optimum conditions. Thus, we select berberin as the internal standard. Because berberin is a positively charged compound the addition of berberin to both standard mixture and the extract of a C. fraxini sample does not cause any interference to original separation. Our experimental results show that the average relative migration is 2.23 for aesculin and 5.00 for aesculetin and the relative precision of the migration time ratio is 90.54% for aesculin and 9 0.83% for aesculetin. These R.S.D. values are much smaller than those for the migration times. For quantification of aesculin and aesculetin, berberin is also chosen as an internal standard. The peak area ratios of aesculin and aesculetin to berberin are calculated. The calculated curve for
618
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Fig. 6. Capillary electripherogram of a standard mixture. Operation conditions as in Fig. 2, except that pH is at 6.70. Peaks, 1 = berberin; 2 =aesculin; 3 =aesculetin.
H. Zhang et al. / Talanta 52 (2000) 607–621
619
Fig. 7. The typical capillary electropherogram of the extract of a C. fraxini sample. Operation conditions as in Fig. 2 except that pH is at 6.70. Peaks, 1 = berberin; 2 = aesculin; 3 = aesculetin.
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aesculin is constructed in the range of 32 –256 mg ml − 1 with the linear regression equation and correlation coefficient being as follows: Y= 0.0678X +0.2520
(r =0.9996)
where Y is the peak area ratio of aesculin to berberin and X is the concentration (mg ml − 1). Similarly, a linear relationship between the concentration of aesculetin and its corresponding peak area ratio of aesculetin to berberin is found in the range of 23 – 230 mg ml − 1. The calibration curve is as follows: Y=0.2477X + 0.3938
infer that these substances, either have COOH groups or have OH groups, and they are anions in this condition.
(r = 0.9993)
where Y is the peak area ratio of aesculetin to berberin and X is the concentration (mg ml − 1). The limits of detection (LODs) calculated as the analyte concentration that produces a peak three times higher than the baseline noise are 20 mg ml − 1 for aesculin and 19 mg ml − 1 for aesculetin. For the sample treated with the method described in the Section 2, the peak area of the selected compound to berberin was 8.40 for aesculin and 28.56 for aesculetin. From these ratio the contents of aesculin and aesculetin is 1.00% (m m − 1) and 0.95% (m m − 1). Different amounts of aesculin and aesculetin standards are weighed and added to a C. fraxini sample known aesculin and aesculetin contents and the mixture are extracted and analyzed with the method described above. The recovery is 98.3% for aesculin and 98.0% for aesculetin The R.S.D. of recoveries for aesculin and aesculetin are 3.31 and 3.84%, respectively. Fig. 6 presents and electropherogram of a standard mixture of aesculin aesculetin and berberin. Fig. 7 gives the electropherogram of extract of crude C. fraxini. Although the electropherogram of the real sample is quite simple, this does not exclude the possibility for the existence of other compounds with weak absobance at the set detection wavelength, 214 nm. The method of capillary zone electrophoresis can separate the mixture into different zones in electropherogram according to their charge characters. Under the conditions described in this work the elution order should be as follows; ion, neutral and anion. Thus although the peaks (4, 5 and 6) can not be identified the information from our experiment can help us
4. Conclusion The use of berberin as the internal standard for CZE to quantify the amounts of aesculin and aesculetin has been developed. Because the internal standard was mixed with samples in fixed ratio the peak ratios are always constant regardless of the variation of the injected amounts. In addition the pKas for aesculin and aesculetin are easily obtained on the basis of the changes of their electrophoretic mobilities, with pH. This proposed method is simple, economic and rapid and can provide more information compared with the method recommended by pharmacopoeia of P.R. China
Acknowledgements This work is financially supported by Post-doctoral Foundation of People’s Republic of China.
References [1] J.S. Li, Study on the Identification of Chinese Traditional Medicine, Shanghai Science and Technology Press, 1995, p. 316. [2] X.S. Guo, Y.Z. Zhang, Acta Pharmac. Sinica 18 (1983) 434. [3] Pharmacopoeia Committee of People’s Republic of China, Pharmacopoeia of People’s Republic of China (vo1. 1), Chemistry Industry Publisher, 1995, p. 241. [4] X.S. Guo, Y.X. Zhang, Acta Pharmac. Sinica 18 (1983) 446. [5] L.S.C. Robert III, Anal. Chem. 68 (1996) 569R. [6] H. Zhang, Z. Hu, G. Yang, et al., Chromatographia 49 (1999) 219. [7] H. Zhang, Z. Hu, G. Yang, et al., Anal. Lett. 30 (1997) 2327. [8] Z. Zhang, Z. Hu, G. Yang, Chromatographia 44 (1997) 162. [9] C.M. Liu, Y.M. Tzeng, J. Chromatogr. A 809 (1998) 258. [10] S.J. Sheu, Huaxue 54 (1996) 55. [11] Y.R. Ku, Y.T. Lin, J.H. Lin, et al., J. Chromatogr. A 805 (1998) 301.
H. Zhang et al. / Talanta 52 (2000) 607–621 [12] H.K. Wu, W.C. Chuang, S.J. Sheu, J. Chromatogr. A 803 (1998) 179. [13] A. Afdeef, J.E.A. Comer, S.T. Thomson, Anal. Chem. 65 (1993) 42. [14] L.Z. Benet, J.E. Goyan, J. Pharm. 56 (1967) 665. [15] B.T. Grayson, D.A. Kleier, Pestic. Sci. 30 (1990) 67. [16] H. Zhang, Z. Hu, G. Yang, et al., J. Liq. Chromatogr. Rel. Technol. 21 (1998) 2465. [17] C.E. Lin, C.C. Hsueh, W.C. Lin, et al., J. Chromatogr. A 746 (1996) 295. [18] C.E. Lin, C.C. Chen, J. Chromatogr. A 731 (1996) 299. [19] H. Zhang, T. Ding, Z. Hu, Fenxi Huaxue 25 (1997) 318. [20] D.K. An, Pharmaceticl Analysis, Jinan Press, Jinan, 1990, p. 63.
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[21] J.N. Vander Moolen, H.F.M. Boelens, H. Poppe, et al., J. Chromatogr. A 744 (1996) 103. [22] X. Huang, M.J. Gordon, R.N. Zare, Anal. Chem. 60 (1988) 375. [23] K.D. Altria, Chromatographia 35 (1993) 117. [24] J.W. Jorgenson, Trends Anal. Chem. 9 (1984) 51. [25] H. Zhang, L. Jia, Z. Hu, Anal. Chim. Acta 342 (1997) 59. [26] N. Chen, L. Wang, Y.K. Zhang, J. Liq. Chromatogr. 16 (1993) 3069. [27] J.A. Dean, Lange’s Handbook of chemistry, 13th edn, Mc Graw Hill Book Company, 1985, pp. 10 – 97. [28] F.A. Gomez, L.Z. Avila, Y.H. Chu, et al., Anal. Chem. 66 (1994) 1785. [29] J. Yang, S. Bose, D.S. Hage, J. Chromatogr. A 735 (1996) 209.
Analysis of aesculin and aesculotin in Cortex fraxini by capillary zone electrophoresis Hongyi Zhang a,b,c,*, Qianfeng Li a, Zhihong Shi c, Zhide Hu a, Rui Wang b a
Department of Chemistry, Lanzhou Uni6ersity, Lanzhou, Gansu 730000, PR China Department of Biology, Lanzhou Uni6ersity, Lanzhou, Gansu 730000, PR China c Physical and Chemical Center, Henter, Hebei Uni6ersity, Baoding 071002, PR China b
Received 14 October 1999; accepted 27 January 2000
Abstract A simple method for quantitative analysis of aesculin and aesculetin in Cortex fraxini was developed using capillary zone electrophoresis (CZE). Berberin was employed as an internal standard, The running buffer was 6 mM Na2B4O7 and 10 mM NaH2PO4 (pH 6.70). The linear calibration range was 32 – 256 mg ml − 1 (r =0.9996) for aesculin and 23–230 mg ml − 1 (r=0.9993) for aesculetin. The contents of aeculin and aesculetin in C. fraxini were easily determined within 12 min the pKa values of aesculin and asculetin determined by CZE were 6.56 and 5.62, respectively. A simple method for estimation of the temperature inside the capillary during running was also proposed. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Aesculin; Aesculetin; Cortex fraxini; Dissociation constant
1. Introduction Cortex fraxini (Chinese name Qin-pi), a commonly used chinese herbal medicine has been proven to be effective in the treatment of diarrhea and dysentery of intense heat type, especially for dysentery with blood stools, and lung heat syndrome with cough and dyspnea for over 2000 years [1]. Pharmacological action demonstrates that C. fraxini can inhibit the growth of dysentery bacillus and staphylococcus and has the antitus* Corresponding author. Tel.: + 86-931-8911288; fax: +86931-8911100. E-mail address: [email protected] (H. Zhang).
sive and expectorant function. Aesculin, aesculetin fraxetin fraxin and stylosin have been isolated from the plant C. fraxini [2]Fig. 1. Aesulin and aesculetin are the two of the most important bioactive components in C. fraxini. Pharmacological experiment shows that aesculin can prolong the hypnotic effect of hexobarbital in mice and aesculein can exert and antihistaminic effect and relax smooth muscle of guinea pig’s trachea in vitro, and the analgestic effect of aesculetin is stronger than that of aspirin. Aesculin, aesculetin have been considered as indexes for estimation of quality [3]. Consequently the determination of these two components in C. fraxini samples in of interest to many scientists. Several
0039-9140/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 0 ) 0 0 3 4 3 - X
608
H. Zhang et al. / Talanta 52 (2000) 607–621
methods [4], such as paper chromatography, polarography thin layer chromatography and high performance liquid chromatography have been developed for determination of aesculin and aesculentin in plant samples or biological fluids. However, these methods suffer from materials and time consuming since large amounts of organic reagent and many operation steps are often required. The development of high performance capillary electrophoresis (CE) has been reviewed many times [5], and clearly it is continuous to be a very active research area in separation science since this technique often provides higher resolving power, shorter analysis time and lower operational cost than liquid chromatography. Now some research groups are devoting themselves to the study of assay of traditional Chinese medicine by CE [6 – 12]. The aqueous ionization constant (pKa) is a very important physico chemical property in the pharmaceutical industry [13,14] and phytochemistry [15]. Recently, capillary electrophoresis (CE) has been introduced as a method for convenient and precise aqueous pKa determination [16–18]. Compared to the most common methods such as potentiometry and spectroscopy, this method has some outstanding advantages, First, CE requires small amounts of sample at low solute concentration. Second, No precise information about the sample concentration is needed as long as the response of the UV detec-
Fig. 1. Chemical structures of aesculin and aesculetin.
tor offers a good peak. Third the ionization constant for several analytes can be determined simultaneously. As far as we know, however the ionization constants for aesculin and aesculetin are not published and have one been determined using capillary electrophoresis. In this study, based on the systematic determination of the effects of buffer pH and applied voltage on the separation and determination of aesculin and aesuletin the contents of aesculin and aesuletin in C. fraxini and the ionization constants for aesculin and aesculetin were obtained. The temperature inside the capillary during operation was calculated using a simple formula derived by us in this work.
2. Experimental
2.1. Reagents and materials Aesculin, berberin and aesculetin were purchased from National Institute for the Control of Pharmaceutical and Products, Beijing, people’s Republic of China. All other chemicals were of analytical reagent grade. Stock solutions in about 0.8 mg ml − 1 of aesulin and aesculetin were prepared by using 79% methanol (v/v) aqueous solution, Mixed standard solution was prepared by mixing stock solution with destilled water to achieve the desired concentration of standard solution. C. fraxini was purchased from the Chinese herbal store in Baoding, PR China. The buffer used for electrophoresis was 6 mM Na2B4O7 + 10 mM NaH2PO4. 1 MHCl or 1 M NaOH adjusted the value of pH of this buffer to any desired value. C. fraxini ground into a powder was passed through a screen mesh (number 40). About 1.0 g of fine powder was weighed and extracted with 25 ml of 80% methanol for 30 min. Extraction was repeated two times. The mixture of 1.5 ml of berberin with fixed concentration and 6 ml of the combined extracts was diluted with water to 10 ml. This solution was filtered from a 0.45 mm filter and the filtrate was injected directly into CE capillary.
H. Zhang et al. / Talanta 52 (2000) 607–621
2.2. Apparatus Electrophoretic experiments were carried out on a Waters Quanta 4000E Capillary Electrophoresis System (Milford, MA, USA). A fused silica capillary with an effective length of 39.7 cm (total length 47.3 cm) and 75 mm I.D. was used for all experiments. Samples were introduced into the capillary by hydrodynamic injection for 10 s. UV detection was employed at a wavelength of 214 nm. Before each analysis the capillary was rinsed under pressure with 0.1 mol 1 − 1 NaOH for 5 min and then equilibrated with the running buffer for 5 min, the electro-osmotic flow (EOF) was measured by using formamide (HCONH2) as a marker. A pHs-3C pH meter (Leici Instrumentation Factory, Shanghai, P.R. China) was used for pH measurements.
8.14 and 8.50. When the pH is lower than 3.09, aesculin, aesculetin and formamide migrate together, since the three analytes are almost complete anionized. When the pH is in the range 3.09–4.47, aesculetin is separated from aesculin and formamide and the latter two are also partly separated at pH 4.47. These results suggest that both aesuletin and aesculin are partly deprotonated when the pH is higher than 3.09. Complete separations of the three analytes are readily achieved in the pH range 5.84–8.50. It is well known that the UV signal of ionizable compound is influenced by the buffer pH since its molar absoprptivity depends on the pH of the medium [20]. As demonstrated in the literature [21], the peak area for species i recorded in the capillary electrophoresis was derived as follows: Ai =
2.3. Calculation The effective mobility, me, was calculated as follows me =
1 1 lV − t te0 V
(1)
where t is the migration time of the analyte (s) te0 is the migration time of an uncharged solute (s) L is the total length of the capillary (cm), l is the length of the capillary between injector and detector and detector (cm) and V is the applied voltage (V).
oi · b · t i Qi × l p · r2
3.1. Effect of pH In capillary zone electrophoresis the, pH of the buffer plays an important role in the separation of ionizable analytes because both the extent of ionization of each individual analyte and the charge of the capillary wall surface are influenced by buffer pH [16,19]. Therefore, manipulation of buffer pH is always a key strategy for optimizing a separation. Fig. 2 shows the electropherograms of aesulin and asculetin using phosphate (20 mmol 1 − 1) as running buffer at pH 4.47, 6.22,
(2)
where oi is the molar absorptivity, ti the migration time for species i, Qi the injection amounts for species i, l the length from the injection end to the detector, b the pathlength of the light. Thus, in order to normalize the bias of peak areas for different solutes, a migration speed correction has to be made by dividing the integrated peak area of solute with its corresponding migration time [22,23]. If the mixture of species i and j are introduced into the capillary the ratio of the corrected area between species i and j, RA, can be expressed as RA=
3. Results and discussion
609
Ai /ti oi · b · Qi /l · p · r 2 oi Qi = = × Aj /tj oj · b · Qj /l · p · r 2 oj Qj
(3)
If the molar absorptivity of species j does not depend on the medium pH in the experimental pH range, Eq. (3) can be simplified into the following form: RA= D · oi
(4)
where D=Qi /oj · Qj is a proportionality constant dependent only on the reference species j and the concentration ratio between species i and j and having nothing to do with the analyte species. Thus the dependence of oi on the buffer pH can be found by observing the changes of RA with duffer pH. Formamide is considered neutral at pHs be-
610
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Fig. 2. Electropherograms of the mixture of aesculin aesculetin and formamide obtained at various buffers pH values, (A) 4.47; (B) 6.22; (C) 8.14; (D) 8.50. Peaks, 1 = formamide; 2 =aesculin; 3 =aescuetin. Buffer; 6 mM Na2B4O7 +10 mM NaH2PO4; fused silica capillary, 47.3 ×50 mm I.D. (effective length 39.7 cm); applied voltage, 20 kV; injection time 10 s; temperature, 25°C.
H. Zhang et al. / Talanta 52 (2000) 607–621
Fig. 2. (Continued)
611
612
H. Zhang et al. / Talanta 52 (2000) 607–621
Fig. 2. (Continued)
H. Zhang et al. / Talanta 52 (2000) 607–621
Fig. 2. (Continued)
613
614
H. Zhang et al. / Talanta 52 (2000) 607–621
tween 4.47 and 8.50 and its absorportivity keeps constant as the pH changes in the experimental range. The plot of the RA value for aesculin the corrected area ratio between aesculin and for-
mamide against the buffer pH is shown in Fig. 3a. At first, RA is small and keeps nearly constant with increasing the pH. This indicates that the most of aesculin is in the non-ionized molecular
Fig. 3. Plot of the ratio of the corrected peak for aesculin (a) and aesculetin (b) that for formamide (RA) as a function of buffer pH. Operation conditions as in Fig. 2.
H. Zhang et al. / Talanta 52 (2000) 607–621
615
Fig. 4. Plots of the electrophoretic mobilities of aesculin and aesculetin as a function of buffer pH.
state. When pH \6, RA increases rapidly with increasing pH, indicating that both the non ionized molecules and the corresponding ions are present in the species aesculin. However after the ionization perold of aesculin, RA trends to level off showing that aesculin is fully ionized. It can be seen from Fig. 3b that the RA value for aesculetin has the similar result when pH changes. From the above study, it can be found that the relatively high pH has some advantages such as relatively large detection signal and relatively short migration time. A buffer solution of pH 6.70 is chosen in this work because this solution has a relatively large capacity.
3.2. Determination of pKa 6alues Fig. 4 shows a plot of the electrophoretic mobility versus pH for aesculin and aesculetin along with the superimopsed curve fit to the following equation [16]: me =
Ka ×m− A [H+]+ Ka
(5)
where me is the electrophoretic mobility of aesculin or aesculetin at a given pH m− A is the mobility of the anionic form of aesculin and aesculetin
and Ka is the ionization constant. The pKa values of aesculin and aesculetin listed in Table 1 have not been compared with any literature values, since they do not exist in some common handbooks. However the fact that the more stable the conjugate anion of an acid, the stronger its acidity is, supports the rationality of the values determined in this work. The conjugate anions formed from aesculin and aesculetin at relatively high pH are stabled compared with the anion of phenol, since the latter has a relatively small conjugation system, The pKa value for phenol is about 10. Therefore, is can be concluded that the values of pKa for aesculin and aesculetin must be less than 10. From the molecular structures of aesculin and aesculetin, is easier to deprotonate than aesculin because it has two OH groups in ortho position that can form a hydrogen bond. Table 1 a pKa and m− A for aesculin and aesculetin Analytes
pKa
m− A
Aesculin Aesculetin
6.56 5.62
−2.27 −3.22
a
Mobility in 10−4 cm2 v−1 s−1.
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616
Table 2 The results of linear regression for effect of voltage Analyte
Aesulin Aesculetin Formamide
Intercept
1.515 3.402 1.087
Slope ( f ) Experimental
Ideal
128.390 285.882 91.746
86.310 120.815 268.870
Correlation
e:f
0.9996 0.9991 0.9997
0.0118 0.0119 0.0119
Fig. 5. Plots of migration time as a function of reciprocal applied voltage for formamide under ideal (a) and real (b) conditions.
3.3. Effect of applied 6oltage The influence of applied voltage is studied. As derived by Jorgenson [24], when net mobility me + me0 is independent on the applied voltage, the migration time of a solute should be inversely and linearly proportional to the applied voltage t=
l ·L 1 × me +me0 V
(6)
Consequently, under this ideal condition, the linear plot of migration time as the function of the reciprocal applied voltage should approximately pass through the point (0, 0). However, our experimental results show that there is a non-zero
intercept for the linear plot (see Table 2). As explained in our previous week [25], the non-zero intercept in the linear plot of the migration time versus the reciprocal applied voltage under nonideal condition is attributed to the thermal effect that tends to shorten migration time. It is assumed that the Joule heating under the influence of 5 kV the applied voltage is small enough to be negligible. Therefore, the value me + me0 in ideal condition can be calculated according to the migration time in 5 kV obtained from the linear regression equation. The linear dependence of migration time for formamide on the reciprocal of the applied voltage under ideal and real conditions is shown in Fig. 5. When the applied voltage is V, the migration time in ideal condition, t1(6 )
H. Zhang et al. / Talanta 52 (2000) 607–621
and the experimental migration time, t2(6 ) can be calculated from the ideal linear equation and the linear regression equation, respectively. The analyte migration time can be expressed as [26] t=
h ·l 2 · za ×f(k · a) o · E · zc + 3
n
3.4. Application
(8)
where A and B are constants characterizing the properties of the running buffer. Therefore, log t= log
A ·l B + (o · E · [zc +(2 · za/3) · f(k · a)]) T (9)
If one assumes that the variation of o, za, zc and f(ka) are negligible compared with the variations of h with temperature, Eq. (9) implies that the logarithm of migration time is linearly related to the reciprocal of the temperature inner the capillary at a constant voltage. Therefore the temperature inside the capillary at any applied voltage V can be expressed as follows T2(V ) =
B log((t2(6 ))/(t1(6 ))) + (B/T1(6 ))
respectively. The average temperature is 35.6°C. As the electric current almost keeps constant at various pH conditions, the acid dissociation constant listed in Table 1 is obtained at about 35.6°C.
(7)
where h is the viscosity coefficient; o the dielectric constant of the medium; za,zc the zeta potential of the analyte and the inner wall of the capillary, respectively, k the reciprocal of the analyte double layer thickness, a ‘radium’ of the analyte. The viscosity of the buffer has the following simplified relationship with temperature [26]: h=A · 10B/T
617
(10)
Theoretically, Eq. (8) shows that log h should have a linear relationship with the absolute temperature (T). The slope of the straight line should be equal to the constant B. From the viscosities of water at various temperature [27], it is found that log h has a linear relationship with the absolute temperature in the range 275 – 373 K. The correlation coefficient is 0.990 and the slope B, is 791.0 When T1 is regarded to be equal to the thermostated temperature, 25°C. The T2 values based on the migration behavior of aesculin aesculetin and formamide are 308.63, 308.56 and 308.62,
As demonstrated previously by some authors [28,29], the approach for obtaining improved precision in capillary electrophoresis is added various markets to samples as aids for monitoring shifts in the electroosmotic flow or electroosmotic flow or electrophoretic mobilities of solute, During the course of the ten injections the average time is 4.94 min for aesculin and 11.16 min for aesculetin the relative standard deviation (R.S.D.%) for migration time range is 9 1.41% for aesculin and 9 2.35% for aesculetin. In the case, migration time is not a reliable indicator for the identification of a particular compound. To help correct for the variability in the migration times, a migration time ratio is calculated for aesculin and aesculetin during each run. To finish this task transforming migration time into relative migration time, we have to select a compound as the internal standard. From our experiment, we find that no peak appeared before formamide, which is regarded as neutral compound in the electropherogram of the mixture of formamide and the extract of a C. fraxini sample. This result indicates that on positively charged compound is found under the optimum conditions. Thus, we select berberin as the internal standard. Because berberin is a positively charged compound the addition of berberin to both standard mixture and the extract of a C. fraxini sample does not cause any interference to original separation. Our experimental results show that the average relative migration is 2.23 for aesculin and 5.00 for aesculetin and the relative precision of the migration time ratio is 90.54% for aesculin and 9 0.83% for aesculetin. These R.S.D. values are much smaller than those for the migration times. For quantification of aesculin and aesculetin, berberin is also chosen as an internal standard. The peak area ratios of aesculin and aesculetin to berberin are calculated. The calculated curve for
618
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Fig. 6. Capillary electripherogram of a standard mixture. Operation conditions as in Fig. 2, except that pH is at 6.70. Peaks, 1 = berberin; 2 =aesculin; 3 =aesculetin.
H. Zhang et al. / Talanta 52 (2000) 607–621
619
Fig. 7. The typical capillary electropherogram of the extract of a C. fraxini sample. Operation conditions as in Fig. 2 except that pH is at 6.70. Peaks, 1 = berberin; 2 = aesculin; 3 = aesculetin.
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aesculin is constructed in the range of 32 –256 mg ml − 1 with the linear regression equation and correlation coefficient being as follows: Y= 0.0678X +0.2520
(r =0.9996)
where Y is the peak area ratio of aesculin to berberin and X is the concentration (mg ml − 1). Similarly, a linear relationship between the concentration of aesculetin and its corresponding peak area ratio of aesculetin to berberin is found in the range of 23 – 230 mg ml − 1. The calibration curve is as follows: Y=0.2477X + 0.3938
infer that these substances, either have COOH groups or have OH groups, and they are anions in this condition.
(r = 0.9993)
where Y is the peak area ratio of aesculetin to berberin and X is the concentration (mg ml − 1). The limits of detection (LODs) calculated as the analyte concentration that produces a peak three times higher than the baseline noise are 20 mg ml − 1 for aesculin and 19 mg ml − 1 for aesculetin. For the sample treated with the method described in the Section 2, the peak area of the selected compound to berberin was 8.40 for aesculin and 28.56 for aesculetin. From these ratio the contents of aesculin and aesculetin is 1.00% (m m − 1) and 0.95% (m m − 1). Different amounts of aesculin and aesculetin standards are weighed and added to a C. fraxini sample known aesculin and aesculetin contents and the mixture are extracted and analyzed with the method described above. The recovery is 98.3% for aesculin and 98.0% for aesculetin The R.S.D. of recoveries for aesculin and aesculetin are 3.31 and 3.84%, respectively. Fig. 6 presents and electropherogram of a standard mixture of aesculin aesculetin and berberin. Fig. 7 gives the electropherogram of extract of crude C. fraxini. Although the electropherogram of the real sample is quite simple, this does not exclude the possibility for the existence of other compounds with weak absobance at the set detection wavelength, 214 nm. The method of capillary zone electrophoresis can separate the mixture into different zones in electropherogram according to their charge characters. Under the conditions described in this work the elution order should be as follows; ion, neutral and anion. Thus although the peaks (4, 5 and 6) can not be identified the information from our experiment can help us
4. Conclusion The use of berberin as the internal standard for CZE to quantify the amounts of aesculin and aesculetin has been developed. Because the internal standard was mixed with samples in fixed ratio the peak ratios are always constant regardless of the variation of the injected amounts. In addition the pKas for aesculin and aesculetin are easily obtained on the basis of the changes of their electrophoretic mobilities, with pH. This proposed method is simple, economic and rapid and can provide more information compared with the method recommended by pharmacopoeia of P.R. China
Acknowledgements This work is financially supported by Post-doctoral Foundation of People’s Republic of China.
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