Sensitivity improvement of kukoamine determination by complexation with dihydrogen phosphate anions in capillary zone electrophoresis.

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Electrophoresis

20150104

Sensitivity improvement of kukoamine determination by complexation with dihydrogen phosphate anions in capillary zone electrophoresis

Yuan-Yuan Li a, Rui Di a, Wing-Leung Hsu a, Ye-Qing Huang a, Hongyan Sun b,c, Hon-Yeung Cheung a, b *

a

Research Group for Bioactive Products, Department of Biomedical Sciences, City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong SAR, China

b

Key laboratory of Biochip Technology, Shenzhen Biotech and Health Centre, City University of Hong Kong, Shenzhen, 518057, China

c

Department of Biology and Chemistry, City University of Hong Kong

Received: 23-Jan-2015; Revised: 23-Mar-2015; Accepted: 10-Apr-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/elps.201500030. This article is protected by copyright. All rights reserved.


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Abstract

A novel complexation between kukoamines and dihydrogen phosphate ions (DPI) during capillary zone electrophoresis (CZE) was discovered to improve the UV signal of kukoamine by around 30-fold. This complexation formed by electric current was attributed to the hydrogen bonding of hydroxyl and amino (or amide) groups between the analyte and electrolyte anions. The established CZE method is low-cost, easy to operate, and eco-friendly, and it was shown to be superior to HPLC in terms of separation capability, efficiency, specificity, and sensitivity. We believe that our CZE method can be applied as an alternative to HPLC for kukoamine assay. The approach described here can be also extended for analyzing other compounds with similar functional groups.

Keywords: Capillary Zone Electrophoresis (CZE); Kukoamines; Dihydrogen phosphate ion (DPI); Complexation; Sensitivity

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1 Introduction Kukoamines, mainly kukoamines A and B, are spermine alkaloids specifically distributed in the Solanaceae family, such as Lycii Cortex (LyC), potato tubers, tomatoes, and tobacco [1-4]. Owing to prominent bioactivities like anti-hypertension [2], anti-lipid peroxidation

and

lipoxygenase

[5],

and

anti-sepsis

[6-7],

kukoamines

and

kukoamine-containing natural extracts have attracted great interest for use in drugs, functional foods, and nutritional supplements [4, 8]. It is therefore highly desirable to develop simple and efficient methods to determine the compounds for quality inspection and pharmacokinetics research. Since kukoamines contain multiple amino groups which can cause peak tailing and poor resolution on C18 column, the isomers of kukoamines are not easily separated thoroughly using the conventional HPLC method [4, 9]. In the last thirty years, the capillary electrophoretic (CE) technique has been established as a popular analytical method and applied in many fields [10-12], owing to its distinguished separation performance, high efficiency, extremely low sample- and solvent-consumption, and eco-friendliness. As the amino groups in kukoamines are easily ionized under acidic conditions, these compounds are particularly suitable to analysis by capillary zone electrophoresis (CZE). Nevertheless, as a micro-analytical method, the short light-path-length in detector has an intrinsic shortcoming in terms of sensitivity, which severely hinders its application [13]. Substantial efforts have been made to improve the sensitivity of CE mainly including 1) using ultra-sensitive detectors (i.e., MS, LIF detector) [14, 15]; 2) pre-concentration of the analytes by off-line procedures (like solid phase extraction (SPE) and liquid-liquid extraction (LLE)) [16, 17] or on-line “stacking” procedures (i.e., field-amplified sample stacking (FASS), field-amplified sample injection (FASI), and large-volume sample stacking (LVSS)) [18]; and 3) improving the signal response of analytes through derivatization or complexation (off-line or online) [19, 20]. The first approach relies This article is protected by copyright. All rights reserved.

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on significant investment in equipment, which significantly increases the cost. The second method improves the sensitivity by increasing the actual amount of analytes injected, which may sacrifice the separation performance. In contrast, the third approach is considered to be more direct and efficient, through amplifying the signal of analyte by installation of chromophore or other detectable groups. [20] Nevertheless, in recent years, much attention was paid to the first two approaches, particularly the pre-concentration methods like “stacking” and SPE [18], while the third approach based on on-line derivatization was seldom discussed, especially in the field of food and drug analysis.[11, 12] In this study, we present an on-line complexation between kukoamine and dihydrogen phosphate ion (DPI) during electrophoresis. This complex is shown to be effective in improving the UV signal for kukoamines. On the basis of this complex, a sensitive CZE method is established and validated to determine kukoamines. This method is comparable with the conventional HPLC method in terms of separation performance, sensitivity, precision, reproducibility, and working efficiency, and was suggested as an alternative to HPLC in kukoamine assay. Our work could be used as a reference for other methods.


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2 Methods 2.1 Materials and Reagents Methanol (HPLC grade) was purchased from Labscan Asia (Bangkok, Thailand). Trifluoroacetic acid (HPLC grade) was purchased from Fluka (Buchs, Switzerland). Acetic acid (HPLC grade) and phosphoric acid (85 %, HPLC grade) was purchased from International Laboratory (San Bruno, USA). Disodium hydrogen phosphate and sodium acetate in ultra pure grade were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultra high pure (UHP) water was prepared by a Millipore Milli Q-Plus system (Millipore, Bedford, MA, USA). All other chemicals were of analytical grade. The standard chemical kukoamines A and B (>98% purity) were purified by our group. Five batches of Lycii Cortex were collected from different cultivation places in China (i.e., Jiangxi, Ningxia, Shanxi, Neimeng and Gansu). The collected herbs from Ningxia were identified as the dried root bark from Lycium barbarum and the others were from L. chinense. 2.2 Apparatus Capillary electrophoresis was carried out on a P/ACE MDQ electrophoresis system with photodiode array detection (DAD) (Beckman Instruments, Fullerton, CA, USA). The fused-silica capillary tube with size 50 μm (id) × 60.2 cm (50 cm active length) was also purchased from Beckman. HPLC analyses were performed on an Agilent 1260 system (Agilent, USA) equipped with an on-line degasser, a binary bump, and a diode array detector (DAD). An Agilent Zorbax C18 SB-AQ column (250 mm × 4.6 mm id, 5μm) was used for separation. The UV spectra of analytes were scanned by a UV-260 Ultraviolet Spectrometer (Shimadzu, Japan). The pH was measured with a HI 8424 Microcomputer pH meter (Hanna Instruments, Porto, Portugal). Plant samples were sonicated in a Transsonic TS 540 Tank


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(Lab-Line Instruments, Troisdorf, Germany). 2.3 Sample preparation Five hundred mg of accurately weighed herbal powder was immersed in 10.0 mL extracting solvent (50% methanol aqueous solution containing 0.5% acetic acid) for 1 h, and then sonicated in an ultrasonic bath (350W) for 30 min. After a centrifugation at 5000 rpm for 10 min, the supernatant was transferred to a 25 mL volumetric flask and the precipitate was re-extracted with 10 mL the solvent again. After centrifugation, the supernatant was combined with the first extractive solution. Then, another 5 mL extracting solvent was adopted to wash the containers and the washing solution was transferred into the same volumetric flask and filled to the mark. The prepared solution was subjected to pass 0.22 μm pore size filter before HPLC analysis. The solution was 1:1 (v/v) diluted with the running buffer (as described for the electrophoresis method) and then filtered for CE analysis. 2.4 Method for CE The capillary tube was rinsed in the following order: 0.1 M NaOH (30 psi, 5 min), UHP water (30 psi, 5 min), and running buffer (30 psi, 5 min) before each run. The running buffer (100 mL) was made by 72.5 mL A (1.66% phosphoric acid aqueous solution, v/v) and 27.5 mL B (500 mM disodium hydrogen phosphate solution), which is according to the standard procedure for Phosphate Buffered Saline (PBS) at pH 2.0 in the compendium [21]. The separation voltage was 25 kV, and the capillary temperature was 25°C. The sample solution was loaded by pressure injection for 10 s at 0.5 psi. The detection wavelength was set at 205 nm. The prepared buffer was stored in a sealed container, and the running buffer was replaced every ten runs.


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2.5 Influence of buffer system on electrophoretic behavior and signal response of kukoamines The electrophoretic behavior of kukoamines and their UV signal in CZE were compared under two buffer systems with different concentrations of each. The investigated concentrations of acetate buffer solution (ABS) were from 150 to 498 mM (pH 3.6), and the concentrations for PBS were from 120 mM to 564 mM (pH 2.0). The concentrations were calculated with the [CH3COO]- and [H2PO4]- in the buffer system, respectively (As shown in Supporting Information section 1.2). The electrophoretic behaviors were related to the electrophoretic mobility (EM) with equation 1 [22]: μep = μ =

Ld L t 1 V tm

(1)

where μep is the EM of analyte, tm is the migration time measured directly from the chromatogram, Lt is the total length of capillary, Ld is the length of capillary between injection and detection, and V is the applied voltage. The UV signal was reflected by peak areas, which were calculated automatically by 32 Karat™ Version 8.0 (Beckman Instruments, Fullerton, CA, USA). A thermodynamic double-reciprocal equation [23, 24] (equation 2) was used to calculate the formation constant (K) between the host and guest molecules (ions).

1 1 1 = +  ( A c  A f) K[H 2 PO 4 ] Ac  A f A ep  A f

(2)

where Aep is peak area of the analyte tested; Af is peak area of the analyte in free state where complex was not formed (calculated as the average peak area of analyte tested in acetic buffer system); Ac is the peak area of analyte in a theoretical equilibrium of complexation; and [H2PO4]- is the concentration of dihydrogen phosphate ion (DPI). A straight line was


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derived by plotting 1 / (Aep–Af) versus 1/ [H2PO4]-. The values of K and Ac were calculated from the intercept and the slope of the straight line. All the experiments were carried out in triplicates. The influence of pH ranging from 1.5 to 7.0 on the UV signal response was investigated. The concentration of DPI in the PBS buffers was kept constant at 287 mM and the acidity was adjusted by phosphoric acid. 2.6 Method for HPLC The mobile phase consisted of a 0.1% TFA aqueous solution (A) and methanol (B). With a flow rate of 1.00 mL min-1, a linear change from 12% B to 22% B within 35 min was set in the program for gradient elution. The column temperature was 40°C, the injection volume was 10 μL and the detection wavelength was 280 nm. 2.7. Method validation The calibration curve was derived by plotting the peak area of individual analyte versus each concentration. The limit of detection (LOD) and quantification (LOQ) for each analyte were determined at about 3 and 10 times the signal-to-noise ratio (S/N), respectively. The precision test was performed by analyzing a standard solution containing kukoamines A and B with six replicates in the same day (intra-day precision) and different days (inter-day precision). The precision, including the peak area precision and migration (retention) time precision, was calculated with relative standard deviation (RSD, %) of the tests. A reproducibility test was performed by determination of five replicated samples with the proposed methods; the reproducibility was calculated with the RSD of contents obtained from measurements. The sample recovery was performed by adding known amounts of standards into an accurately weighed herbal sample (the weighted amount was half of that in the proposed sample). The mixed sample was extracted and analyzed using the method mentioned above. For each concentration, three replicate experiments with the whole analysis process were performed. Recovery was calculated with the following equation: Recovery (%) = 100 × (amount found-original amount) /amount spiked.


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3 Results and discussion 3.1. Complexation between DPI and kukoamines in CZE 3.1.1 DPI-kukoamine complex facilitates UV signal enhancement Kukoamines A and B are spermine alkaloids with pKa at 11.23 and 11.12, which were calculated according to the method in literature [25]. To assess the influence of background electrolytes (BGE) on the signal of kukoamines in CZE-UV, two conventional acidic-buffer systems, acetate buffer solution (ABS) and phosphate buffer solution (PBS), were used and compared. As shown in Fig 1A, when the PBS was adopted, the peak areas of both KA and KB were enhanced with the increment of DPI concentrations. This phenomenon, however, was not found in the ABS system. Furthermore, the double reciprocal regression of the DPI concentrations against the peak areas indicated a linear relation (R>0.998), an excellent fit with the pseudo-second-order kinetic model (Fig 1B). This suggested that there was complexation between kukoamines and DPI and this interaction amplified the UV absorbance. According to the regression, the formation constants (K, L mol-1) for KA and KB were calculated as 2.50 and 7.78, respectively (Table 1). The calculated parameter Ac which represents signal intensity of analytes in complexation equilibrium was 2.063 × 105 for KA and 1.502 × 105 for KB, respectively (Table 1). This value is about 20- to 30-fold more abundant than without the complexation. To shed more light on the complexation in CZE, the influence of pH on the signal intensity of kukoamines was also investigated with regard to the same DPI concentration in the BGE. As shown in Fig. 1C, the peak areas of KA and KB stay constant regardless of pH. This demonstrated that ion concentration rather than pH is the key factor influencing the signal amplification.


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3.1.2 Hydrogen-bond interaction contributes to complexation in CE To investigate the molecular interaction in electrophoresis, the UV spectra of analytes were recorded by a diode-array detector (DAD) with different DPI concentrations. As shown in Fig. 2A, the signal intensities at 190-210 nm were enhanced with the DPI concentration. This wavelength range corresponds to the characteristic band for DPI in the UV spectrum, which indicated the complex formation between DPI moiety and kukoamines during the electrophoresis. In contrast, the absorption at 280 nm was unaffected, which indicated that the complexation did not occur on the site of phenolic groups. Therefore we reckon that the complexation may be formed on the site of amides or amines in kukoamine (Fig 3). Similarly, the UV spectra of kukoamines were also recorded by an ultraviolet spectrometer without electric current. Unlike the phenomenon observed in electrophoresis, the UV signal at 190-210 nm did not increase with the DPI concentration. This indicated that the complex was not formed by simply mixing kukoamine with PBS. Therefore, the electric current is an indispensable factor for the complexation. On the basis of the above results, we hypothesized that the hydrogen bond [26] was the major force contributing to the complexation. In this study, the DPI, which contain active protons and electronegative oxygen atoms, played the roles of proton donator and receptor simultaneously to interact with the amide and amine groups in kukoamines (Fig. 3). Literatures have shown that a similar hydrogen bond effect can occur in anion receptors containing bis-thioureas unit for recognition of DPI [27-29]. It was also pointed out that stability of the forming complex can be influenced by many factors, e.g., the substitutions connected with the thioureas unit, as well as the molecular geometry [28, 29]. Therefore, we hypothesize that the attraction between DPI and kukoamine molecules in aqueous solution is too weak to form a stable complex. Thus the UV signal cannot be observed by simply mixing. However, under strong voltage (25 kV) during electrophoresis, the hydrogen bond interaction This article is protected by copyright. All rights reserved.

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was strengthened and the complex was stable, resulting in strong UV signal. We reckon that there are two possible reasons for the formation of the stable DPI and kukoamine complex: 1) polarization of host-guest molecules in electric field enhanced the electrostatic force; 2) geometry changes of kukoamines in electric field facilitated a better molecular fit. To the best of our knowledge, this work is the first to utilize complexation between analyte and electrolyte anion via hydrogen-bond to enhance

sensitivity in CZE.

3.2 Influence of electrolytes on electrophoretic behaviors The migration behaviors of kukoamines in two different BGE were investigated. As shown in Fig. 1D, kukoamines presented almost the same migration behavior in both ABS and PBS. The decrease in electrophoretic mobility with the increment of electrolyte can be explained as the comprehensive effect of the increment of viscosity and conductivity in BGE. From another aspect, the extension of migration time was beneficial for the resolution improvement of the neighboring peaks (Fig. S1). Although ABS and PBS presented almost the same influence on separation, PBS was adopted as the running buffer since it can also improve the UV response for the analytes. 3.3 Analytical performance of the CZE and HPLC Parallel experiments were performed to assess the performance of the CZE and HPLC methods for kukoamine assay. As shown in Fig. 4 and Table 2, CZE outperformed HPLC and displayed better peak resolution, symmetry, and theoretical plates, which suggested the superior separation capability of CZE. Moreover, the CZE method is around 10-fold more sensitive than HPLC in LOD and LOQ. This result demonstrated that the complexation between DPI and kukoamine has significantly improved the sensitivity of CZE. The remarkably lower consumption of sample and solvent (Table 2) also demonstrated that CZE is more economical and eco-friendly than HPLC. Method validation tests suggested that CE is comparable with HPLC in precision (Table S1, S2), reproducibility, and recovery (Table This article is protected by copyright. All rights reserved.

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S3), which indicates that it can fully satisfy the requirements of food and drug analysis. 3.4. Determination of kukoamines in LyC raw material and extracts The established CZE and HPLC methods were applied to analyze kukoamines in LyC and the extracts. Fig. 4A and 4B showed the representative chromatograms and electrophoretograms for kukoamine standards and real samples, respectively. It was found that CZE displayed much better selectivity than HPLC for kukoamines in the real sample, as only the cation showed a migration in the electrophoresis and the influence from sample matrix was effectively removed. The comparison of determination results between HPLC and CZE (Table S4) indicated that CZE was capable of giving as consistent results as HPLC. Thus, it can be used as an alternative method for kukoamine assay in food and drug analysis.

4 Conclusions remarks A novel complexation between kukoamine and DPI during electrophoresis was discovered to be effective in amplifying the UV signal for kukoamines. On the basis of electric current, the complexation was ascribed to the hydrogen bonding of hydroxyl and amino (or amide) groups between analyte and DPI. Due to the complexation, the detection limit of CZE was enhanced to 10-fold, which is much more sensitive than the current HPLC method. Because of the other advantages of CZE in separation capability, efficiency, specificity, and eco-friendness, it could serve as an alternative to HPLC for kukoamine assay. Our approach could also be utilized for analyzing other analytes with similar functional groups. The authors are thankful for funding support from the Department of Health, Hong Kong Government SAR, China, to a project (CityU No. 9210029) on the Hong Kong Chinese Materia Medica Standards (HKCMMS). We would like to give thanks to Dr. David C. K. Chiu, Dr. Hongli Wen, and Dr. Haixing Wang for their suggestions on manuscript revision, and Mr. William T. Mahan for proofreading.


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A.S., Gaspar, A., Dawod, M., Quirino, J.P., Electrophoresis 2013, 34, 29-54. [19] Hai, X., Nauwelaers, T., Busson, R., Adams, E., Hoogmartens, J., Schepdael, A. Van, Electrophoresis 2010, 31, 3352-3361. [20] Oliver, J.D., Gaborieau, M., Hilder, E.F., Castignolles, P., J. Chromatogr. A 2013, 1291, 179-186. [21] The State Pharmacopoeia Commission of RP China. Pharmacopoeia of the People’s Republic of China, 9rd ed; Chemical Industry Press: Beijing, 2010. [22] Lin, C.E., Lin, S.L., Liao, S.W., Liu, Y.C., J. Chromatogr. A 2004, 1032, 227-235. [23] Wren, S.A.C., Rowe, R.C., J. Chromatogr. A 1993, 635, 113-118. [24] Li, Y.Y., Zhang, Q. F., Sun, H., Cheung, N., Cheung, H.Y., Talanta 2013, 105, 393-402. [25] Wang, D., Yang, G., Song, X., Electrophoresis 2001, 22, 464-469. [26] Arunan, E., Desiraju, G.R., Klein, R.A, Sadlej, J., Scheiner, S., Alkorta, I., Clary, D.C., Crabtree, R.H., Dannenberg, J.J., Hobza, P., Kjaergaard, H.G., Legon, A.C., Mennucci, B., Nesbitt, D.J., Pure Appl. Chem. 2011, 83, 1619–1636. [27] Pandian, T.S., Cho, S.J., Kang, J., J. Org. Chem. 2013, 78, 12121-12127. [28] Nishizawa, S., Bühlmann, P., Iwao, M., Umezawa, Y., Tetrahedron Lett. 1995, 36, 6483-6486. [29] Bühlmann, P., Nishizawa, S., Xiao, K.P., Umezawa, Y., Tetrahedron 1997, 53, 1647-1654.


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Figure 1. Influence of background electrolytes on signal response and electrophoretic behaviors of kukoamines. (A) Influence of concentration of acetate ion (solid symbols) and dihydrogen phosphate ions (DPI) (hollow symbols) on peak areas of kukoamines A (circle) and B (triangle); (B) Double-reciprocal plot of peak areas against concentration of DPI for KA (circle) and KB (triangle); (C) Effects of pH of phosphate buffer on peak areas of kukoamines A (circle) and B (triangle); (D) Influence of concentration of acetate (solid symbols) and phosphate (hollow symbols) ion on electrophoretic mobility of KA (circle) and KB (triangle).


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Figure 2. Change of UV spectra of kukoamine regarding to PBS concentrations with (A) and without (B) electric current. Colors represent different PBS concentrations: green, 200 mM; blue, 100 mM; red, 50 mM; black, 25 mM. The dotted line box indicates the characteristic wavelength range responsible for DPI.


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Figure 3. The proposed complexation between kukoamines and DPI during electrophoresis. 1), complex with kukoamine B; 2), complex with kukoamine A.


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Figure 4. Representative chromatograms (or electropherograms) of standard solution (Std) and Lycii cortex samples (LyC) obtained by HPLC (A) and CZE (B). Conditions: A) Separation of HPLC was carried out on an Agilent Zorbax C18 SB-AQ column (250 mm × 4.6 mm id, 5μm) with TFA aqueous solution (0.1%, v/v) and methanol as mobile phase; B) Separation of CE was carried on a fused-silica capillary tube with size 50 μm (id) × 60.2 cm (50 cm active length) and run by a phosphate buffered saline at pH 2.0. Details of other conditions were described in Methods.


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Table 1. Formation constants (K) of analytes and dihydrogen phosphate ions and the peak areas in free and complex states (n = 3) Analytes

Regressiona)

K (L mol-1)c) 2.500 ± 0.000

Af d)

Ac e)

Y=2 × 10-6 X +5 × 10-6

R2 (n = 7)b) 0.997

KA

6278 ± 139

206 278 ± 323

KB

Y=9 × 10-7 X + 7 × 10-6

0.991

7.778 ± 0.001

7316 ± 149

150 173 ± 767

a) The linear regressions were obtained through plotting the double-reciprocal of peak areas against the concentrations of DPI. b) R2, correlation coefficient (n=7) calculated on the basis of the regression (the plot was shown in Fig 1B). c) K, formation constant calculated from Eq.(2). d) Af, the peak areas obtained without complexation formed. Af here equal to the peak areas of analytes obtained with acetate buffer as running electrolyte. e) Ac, the peak area of analytes in the complex state when the ideal equilibrium is achieved.



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Table 2 Performance of HPLC and CE in determination of kukoamines Parameters a,b)

Resolution Tailing factor b) Theoretical Plates b) Migration/retention time (min) c) Precision (RSD, %)

CE KA 4.99 1.04 112 305 12.60

1.08 137 675 11.38

HPLC KA 3.65 1.08 29 369 26.47

KB

KB 1.24 27 308 24.27

retention time

0.57 d), 0.57 e)

0.53 d), 0.53 e)

1.29 d), 1.81 e)

0.36 d), 1.43 e)

peak area

3.09 d), 5.22 e)

3.14 d), 5.43 e)

1.55 d), 1.95 e)

0.60 d), 0.78 e)

0.996 3.52

0.998 4.58

0.998 2.46

0.999 0.99

98.97~113.39 0.117 0.583

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