3326 Yuanqi Lu1,2,3 Hong Bai2 Chunyan Kong1 Hao Zhong2∗ Michael C. Breadmore4 1 Analysis
and Testing Centre, Dezhou University, Dezhou, P. R. China 2 Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan, P. R. China 3 Key Laboratory of Coordination Chemistry and Functional Materials in Universities of Shandong, Dezhou University, Dezhou, P. R. China 4 Austrialian Centre for Research on Separation Science, School of Chemistry, University of Tasmania, Hobart, Tasmania, Australia
Received August 19, 2013 Revised September 16, 2013 Accepted September 23, 2013
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Research Article
Analysis of brazilin and protosappanin B in sappan lignum by capillary zone electrophoresis with acid barrage stacking A method was developed to determine brazilin and protosappanin B in natural products by CE after acid barrage stacking. The optimum conditions were as follows: a BGE of 20 mM sodium tetraborate (pH 9.2) containing 6% v/v of methanol, hydrodynamic injection (0.5 psi, 65 s) followed by hydrodynamic injection of 150 mM citric acid (pH 2.3; 0.5 psi, 22 s), and separated with +25 kV. Under these conditions, brazilin and protosappanin B were separated with a sample-to-sample time less than 13 min and detection limits of 0.28 g/mL and 0.15 g/mL, respectively. The applicability of the developed method was demonstrated by the detection of brazilin and protosappanin in methanol extract of sappan lignum. Keywords: Acid barrage stacking (ABS) / Brazilin / CE / Protosappanin B DOI 10.1002/elps.201300402
1 Introduction Sappan lignum, also called “sumu” in Chinese, is the dried heartwood of Caesalpinia sappan L. It is known to promote blood circulation and remove blood stasis, reducing swelling and pain, antibiosis, and diminish inflammation, antitumor, cholagogue; and has been used as an emmenagogue, hemostatic, and anti-inflammatory agent for the treatment of contusion and thrombosis in traditional Chinese medicine since ancient times [1]. The chemical constituents of sappan lignum have been studied by some research groups [2–4] using an array of chromatographic and spectroscopy methods, and these studies determined that the active constituents are homoisoflavonoids, chalcones, brazilins, and protosappanins [5, 6]. Currently, the routine method for the constituent analysis of sappan lignum is HPLC [5, 7–11]. Brazilin and protosappanin B (structures shown in Fig. 1) are two active pharmaceutical components of sappan lignum, which have anti-inflammation and antioxidation properties making their correct determination important. Chen et al. have determined these in the water extracts of sappan lignum by HPLC [11], while Zhao et al. used an MeOH extract [12]. Although the constituents in the water decoction (water
Correspondence: Dr. Michael C. Breadmore Austrialian Centre for Research on Separation Science, School of Chemistry, University of Tasmania, Hobart, Tasmania, 7001, Australia E-mail: [email protected]
extracts) of traditional Chinese medicine are in closer agreement with those used in medicinal practice, the solubility of brazilin and protosappanin B in water is much lower than in methanol, which means much more extractant is needed and a concentration step is required before analysis. The Chinese Pharmacopoeia also lists an HPLC method for determination of brazilin and protosappanin B in the MeOH extract and advises that the contents of the two constituents should not be less than 0.5% [10]. CE, with the advantages of low consumption of samples and chemicals, short analysis times, and high resolution, has received considerable interests in traditional Chinese medicine analysis [13, 14]. But due to its short optical length, CE has the inherent problem of low sensitivity that limits its applicability. To enhance the sensitivity, many online concentration methods have been proposed. These online concentration methods can be classified into the following three groups: those based on changes in migration due to a conductivity difference [15–18]; a difference in pH between the sample zone and the buffer zone (sometimes the concentration zone) [19–24], and those based on the association between the analytes and a moving electrolyte component [25, 26]. These can also be combined in a number of ways to produce additional more complex and more powerful enrichment approaches [27–30]. Acid barrage stacking (ABS) exploits changes in ionization with pH to concentrate weak acids [21, 23]. This is achieved by injecting a high concentration of low pH
∗
Abbreviations: ABS, acid barrage stacking; MAE, microwaveassisted extraction C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Additional corresponding author: Dr. Hao Zhong, E-mail: [email protected]
Colour Online: See the article online to view Figs. 2–5 in colour. www.electrophoresis-journal.com
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Figure 1. Structures of the analytes.
solution after a large volume of sample has been injected. When the separation voltage is applied, the anionic components in the sample migrate to the inlet and when they enter the acidic region, they stop and stack on the interface between the sample and acid zones. This method is similar to dynamic pH junction, but is more flexible because it allows the acidic solution for concentration to be quite different to that of the separation electrolyte. This approach also has the advantage of being tolerant of high salt in the sample and has been utilized to analyze amino acids in rat brain microdialysate, rat serum, and human saliva [23]. Feng et al. used CE with ABS to determine genistein in the traditional Chinese medicine Frucuts sophorae [21]. Here a CE method with ABS is developed and validated for the determination of brazilin and protosappanin B in sappan lignum after microwave-assisted extraction (MAE). The sensitivity was enhanced by 62-fold and 111-fold for brazilin and protosappanin B, respectively.
2 Materials and methods 2.1 Instrumentation CE separations were carried out in a P/ACE MDQ CE system with a photodiode array detector for absorbance measurements at 254 nm (Beckman Coulter, Fullerton, CA, USA). Uncoated fused-silica capillaries were purchased from Polymicro Technologies (Phoenix, AZ, USA). The capillary was 60.2 cm × 50 m id with an effective length of 50.0 cm. The temperature of the capillary was kept at 25⬚C. The CE system was interfaced with a computer and controlled using the Beckman 32 karat software (version 7.0). New capillaries were flushed with 1 M NaOH for 20 min, 18.2 m⍀·cm water for 20 min, and BGE for 20 min. Each day, capillaries were flushed with 0.1 M NaOH for 10 min, 18.2 m⍀·cm water for 10 min, and BGE for 10 min. To maintain good repeatability, the capillary was flushed between each separation with water and the BGE for 2 and 4 min, respectively. The BGE comprised 20 mM of sodium tetraborate (pH 9.2) containing 6% v/v MeOH. The BGE was prepared freshly each day, sonicated for 5 min, and filtered through a 0.45 m membrane filter before use. 2.2 Chemicals Brazilin and protosappanin B were prepared in Institute of Materia Medica, Shandong Academy of Medical Sciences, C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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and their purity was not less than 99% [31]. Other chemicals, unless otherwise stated, were all of analytical grade. Water of 18.2 m⍀·cm was from a CascadaTM Lab Water System (Pall Life Science, China). A standard solution of 1000 g/mL of each analyte was prepared in MeOH. A mixed standard solution of the two analytes was prepared at a concentration of 500 g/mL in MeOH. The working standards were prepared daily by diluting the mixed standard solution with 20 mM borax-NaH2 PO4 buffer (pH 8.0) containing 6% v/v MeOH. All solutions were stored in dark containers at 4⬚C.
2.3 ABS and enhancement factor calculation After filling the capillary with BGE (20 psi, 4 min), the sample was injected hydrodynamically with a positive pressure (0.5 psi, 65 s; 6.6% of the length to the window), followed by a hydrodynamic injection of 150 mM citric acid (0.5 psi, 22 s; 2.2% of the length to the window). A voltage of +25 kV was applied to stack and separate the ions. The enhancement factor was calculated by dividing the detection limits obtained with ABS with the detection limits obtained from normal hydrodynamic sample injection (0.5 psi, 5 s, 0.4% of capillary volume).
2.4 Preparation of sappan lignum sample Sappan lignum sample was purchased from Auguo Chinese medicine market (Anguo, Hebei). It was dried at 60⬚C for 6 h and was then pulverized using mill. A total of 0.5 g of the powder was dispersed in 25.00 mL of MeOH and exposed to the 450 W microwave irradiation for 4 min. After cooling, the extract was filtered with a medium-speed filter paper (⌽ = 9 cm, Hangzhou Fuyang Special Paper, China). The MeOH extracts were made to 25.00 mL with MeOH. Before analysis, 100 L of this extract was diluted with 20 mM BoraxNaH2 PO4 (pH 8.0) buffer to 5 mL after 200 L MeOH was added.
3 Results and discussion 3.1 Separation optimization Borate can form negatively charged boronate esters with compounds that have two adjacent hydroxyl groups [32]. This can be used to enhance solubility and also as was used in this work, to provide some enhanced charge to facilitate separation by electrophoresis. The first step was therefore to optimize the pH, BGE concentration, and the concentration of MeOH. Keeping the BGE concentration at 20 mM, the effect of pH on the separation was investigated over the range of 7.0– 9.5 adding either NaH2 PO4 (to ensure adequate buffering) or NaOH to the 20 mM sodium tetraborate. The results show www.electrophoresis-journal.com
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tion as the buffer concentration increased. At the same time, the capillary current increased and there was a noticeable increase in detection limit when the BGE concentration was higher than 20 mM, presumably due to more pronounced joule heating, which made the baseline noise higher (from 0.020 to 0.026 mAU). As a compromise between resolution, analysis time, and sensitivity, 20 mM was selected as the optimum borax concentration. To further improve the separation, the addition of MeOH to the BGE was investigated in the 0–8% v/v range. As MeOH was increased the resolution improved, however, the mobility of the analytes decreased prolonging the analysis time. The addition of 6% v/v MeOH was found to improve the resolution without compromising analysis time and was used for all further experiments. The optimum BGE was therefore 20 mM sodium tetraborate, pH 9.20 with 6% v/v MeOH.
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0 acetic acid
tartaric acid
citric acid
phosphoric acid
Figure 2. Comparison of different acids as acid barrage on preconcentration effects. Conditions: 60 cm × 50 m (50 cm to detector), +25 kV; 254 nm; 20 mM sodium tetraborate, pH 9.20 with 6% v/v MeOH. Hydrodynamic injection of sample 65 s at 0.5 psi, followed by hydrodynamic injection of barrage acid (pH 2.3) for 22 s at 0.5 psi.
3.2 ABS optimization ABS exploits the change in ionization of the analytes due to the pH difference between the sample and the acid barrage zone to lower the mobility of the analytes and to narrow the sample band. So the sample matrix and the composition of the acid barrage zone, namely the type, concentration, and pH as well as the injection times for both sample and barrage acid, were optimized.
that the electrophoretic mobility of the two analytes increases as the pH of the buffer is increased due to the increased ionization of the analytes, and that there was a change in migration order at a pH of 8.0. In addition, the EOF remained relatively constant due to changes in ionic strength countering the increase in pH. When the pH was equal to or higher than 9.0, the two analytes were well separated. As sodium tetraborate buffers well at a pH of 9.20, which is close to the pH of 20 mM sodium tetraborate at room temperature, and there was adequate resolution between the two compounds at this pH, this was used for all further experiments. The effect of BGE concentration was investigated next over the range of 10–30 mM. The results show that there was a slight reduction in the electrophoretic mobility of brazilin and protosappanin B, with a slight improvement in resolu-
3.2.1 Effects of sample matrix Borate buffers are frequently used in preparing CE samples [23]. In this work, 20 mM sodium tetraborate (the same concentration as the BGE) was applied as the sample matrix, and the effect of pH was investigated in the pH range of
B
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1000 200 20
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Figure 3. Effects of injection time on (A) peak height and (B) peak area of brazilin and protosappanin B. Conditions as for Fig. 2, except the injection time that are shown in the figure. The barrage zone time was adjusted with each sample injection time to maintain a ratio of 1:3.
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7.0–9.5. The results showed that the peak heights of the analytes increased with the increase of the pH, but after the pH was equal to or higher than 8.5, the peak height decreased quickly, believed to be due to the degradation of the analytes at high pH. When prepared at pH 8.0, the RSD (%) of peak height for ten consecutive injections (one injection per hour) were 3.59 and 3.25% for brazilin and protosappanin B, respectively. The good repeatability signifies that the two analytes are stable in the sample matrix for 10 h.
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Any type of acid that is able to maintain a low pH can in theory be used as barrage acid. Keeping the barrage zone pH at 2.3, different acids including 150 mM of citric acid (pKa = 3.13, 4.76, 6.40), phosphoric acid (pKa = 2.12, 7.2, 12.36), acetic acid (pKa = 4.74), and tartaric acid (pKa = 3.04) were examined. The results are shown in Fig. 2 that clearly shows that citric acid produced the highest peak heights for brazilin and protosappanin B. The concentration of citric acid was then investigated from 50 to 200 mM. The peak heights first increased with the increase of the citric acid concentration until 150 mM after which they decreased. At the same time, the migration time of the analytes decreased with the increase of citric acid concentration. This can be explained by the fact that more barrage acid reacts with the analyte anions and thus increase the stacking efficiency as barrage acid concentration increased. On the other hand, the analytes will migrate fast with the increase of barrage acid concentration that will reduce the separation time. One hundred and fifty micromolar citric acid was chosen as a compromise.
3.2.3 Effects of the ratio for acid injection to sample injection and injection time The effects of the acid injection to sample injection ratio was investigated from 1:1 (same length) to 1:4 (sample four times longer than the barrage zone). As the ratio was increased, the stacking efficiency increased; however above 1:3, the resolution for the two analytes deteriorated significantly indicating insufficient capillary length to stack and separate the analytes.
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Figure 4. Electropherograms of (A) normal injection and (B) ABS: Conditions (A): 100 g/mL standards dissolved in methanol, 0.5 psi for 5 s injection. (B) 4.0 g/mL standards solved in 20 mm Borax with 6% v/v MeOH (pH 8.0) injection, 0.5 psi for 65 s followed by 22 s of 150 mM citric acid (pH 2.3). For both separations, the BGE was 20 mm sodium tetraborate with 6% v/v methanol (pH 9.20), 60 cm × 50 m id capillary (50 cm to detector), +25 kV. Peak 1 is Brazilin and peak 2 is protosappanin B.
Keeping the injection ratio at 1:3, the sample and barrage injection times were varied, and the sample time was varied from 25 to 75 s. The results are shown in Fig. 3 with the peak area increasing linearly but the peak height leveling off after 65 s. From 65 to 75 s, the peak areas still increase, while the peak heights did not change considerably. Furthermore, the peaks start to broaden at 75 s.
3.2.4 Analytical performance of ABS Under the optimum conditions, an ABS-CZE separation of the two analytes is shown in Fig. 4B and compared to a normal injection as shown in Fig. 4A. The sample-to-sample time was less than 13 min. Intraday variations (n = 5) in migration time were between 0.63 and 0.86% RSD, and for peak area were between 3.16 and 3.47% and those for interday repeatability (n = 5) were 1.49–1.63% and 3.82–4.78%. The detection limits and calibration parameters are summarized in Table 1. The sensitivity enhancement factors were 62 and 111 for brazilin and protosappanin B, respectively, with LODs of 0.28 and
Table 1. The regression equations in ABS
Compounds
Regression equationa)
Correlation coefficient
Linear range (g/mL)
Detection limitsb) (g/mL)
LOQc) (mg/g)
EFd)
Brazilin Protosappanin B
y = 531.42x − 19.76 y = 1490.58x + 659.88
0.9983 0.9978
1.0∼20.0 0.6∼10.0
0.28 0.15
2.3 1.3
62 111
a) In the regression equation, the x value is the concentration of analytes (g/mL), the y value is the peak area. b) Detection limits were based on three times noises. c) Limits of quantitation were based on ten times noises. d) Enhancement factor, which was calculated by dividing LOD with hydrodynamic sample injection (0.5 psi, 5 s) by those when ABS was performed.
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0.15 g/mL, based on three times noise. The calibration was linear over the range of 1.0–20 g/mL and 0.6–10 g/mL for brazilin and protosappanin B, respectively.
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MAE has been proven to be a powerful sample extraction technique due to its ability to reduce the volume of extraction solvents and extraction time, improve the reproducibility and recovery of analytes, and increase sample throughput [33]. According to the literature, MeOH was used as the extraction solvent [12]. The effects of extraction temperature, microwave power, and microwave time and extractant volume were investigated.
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The effects of extraction temperature were investigated in 30–70⬚C range. The results showed that peak area of the two analytes in the sample increased when the temperature became higher. But after 60⬚C, this increase became trivial, so 60⬚C was chosen as the extraction temperature.
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3.3.3 Effects of extractant volume With 0.5 g sample, the effect of extraction solvent volume was investigated from 5 to 30 mL range. As shown in Fig. 5C, the extraction efficiency of the analytes increased up to 25 mL after which it remained constant and this volume was used for all further extractions. 3.4 Real sample analysis The recovery of the method was determined with the addition of the standards (comparable to 5.0 mg/g in the solid sample) in the real sample solution, with result of 89.9 and 101.4% for brazilin and protosappanin B, respectively (Table 2). This is adequate for their analysis demonstrating the potential applicability of this method to analyze brazilin and protosappanin B in the traditional Chinese medicine. The developed C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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The effects of microwave power and microwave time on the extraction efficiency were investigated in the 150–550 W and 2–6 min range. The results are shown in Fig. 5A and B. The extraction efficiencies of the two analytes generally increases with the microwave power and irradiation time from 150 to 450 W and 2 to 4 min. When the microwave power is greater than 450 W or the time greater than 4 min, the extraction efficiency decreased slowly, which may be due to degradation of the analytes; 450 W for 4 min was selected as the optimum.
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Figure 5. Effects of (A) microwave time, (B) microwave power, and (C) methanol volume on the extraction efficiency (sappan lignum from Guangxi). Conditions are the same as Fig. 4B Table 2. Results of recovery (n = 5)
Compounds
Original (mg/g)
Added (mg/g)
Found (mg/g)
RSD (%)
Recovery (%)
Brazilin Protosappanin B
6.53 5.04
5.00 5.00
11.03 10.11
3.88 3.51
89.92 ± 8.50 101.40 ± 7.01
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0.0008
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Figure 6. Electropherograms of the extracts of sappan lignum from (A) Guangxi and (B) Yunnan. Condition are the same as Fig. 4B. peak identity the same as Fig. 4. Table 3. Results of sample analysis (n = 5)
5 References
Compounds
Brazilin (mg/g)
RSD (%)
Protosappanin B (mg/g)
RSD (%)
Guangxi Yunan
6.53 10.15
4.69 4.83
5.04 5.10
3.95 4.12
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method was applied to analyze brazilin and protosappanin B in sappan lignum. Figure 6 shows the electropherogram from the extract of sappan lignum from Guangxi(a) and Yunnan(b). It can be clearly seen that the brazilin and protosappanin B are present at concentrations well above the LOQ in two samples. The results, shown in Table 3, establish that the brazilin and protosappanin B content was above 0.5%, indicating the authenticity of these sappan lignum samples.
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4 Concluding remarks A method for the determination of brazilin and protosappanin B by CE with online concentration with ABS was developed. The sensitivity was improved by 62- and 111-fold giving detection limits between 0.28 and 0.15 g/mL for brazilin and protosappanin B, respectively. The method was shown to be suitable for the detection of brazilin and protosappanin B in sappan lignum. This work was supported by funding from the Key Technologies R&D Programme of Shandong Provine (2010GSF10615) and the Natural Science Foundation of China (81072544). MCB would like to acknowledge the Australian Research Council (DP0984745) for funding and a QEII fellowship. The authors have declared no conflict of interest.
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and Testing Centre, Dezhou University, Dezhou, P. R. China 2 Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan, P. R. China 3 Key Laboratory of Coordination Chemistry and Functional Materials in Universities of Shandong, Dezhou University, Dezhou, P. R. China 4 Austrialian Centre for Research on Separation Science, School of Chemistry, University of Tasmania, Hobart, Tasmania, Australia
Received August 19, 2013 Revised September 16, 2013 Accepted September 23, 2013
Electrophoresis 2013, 34, 3326–3332
Research Article
Analysis of brazilin and protosappanin B in sappan lignum by capillary zone electrophoresis with acid barrage stacking A method was developed to determine brazilin and protosappanin B in natural products by CE after acid barrage stacking. The optimum conditions were as follows: a BGE of 20 mM sodium tetraborate (pH 9.2) containing 6% v/v of methanol, hydrodynamic injection (0.5 psi, 65 s) followed by hydrodynamic injection of 150 mM citric acid (pH 2.3; 0.5 psi, 22 s), and separated with +25 kV. Under these conditions, brazilin and protosappanin B were separated with a sample-to-sample time less than 13 min and detection limits of 0.28 g/mL and 0.15 g/mL, respectively. The applicability of the developed method was demonstrated by the detection of brazilin and protosappanin in methanol extract of sappan lignum. Keywords: Acid barrage stacking (ABS) / Brazilin / CE / Protosappanin B DOI 10.1002/elps.201300402
1 Introduction Sappan lignum, also called “sumu” in Chinese, is the dried heartwood of Caesalpinia sappan L. It is known to promote blood circulation and remove blood stasis, reducing swelling and pain, antibiosis, and diminish inflammation, antitumor, cholagogue; and has been used as an emmenagogue, hemostatic, and anti-inflammatory agent for the treatment of contusion and thrombosis in traditional Chinese medicine since ancient times [1]. The chemical constituents of sappan lignum have been studied by some research groups [2–4] using an array of chromatographic and spectroscopy methods, and these studies determined that the active constituents are homoisoflavonoids, chalcones, brazilins, and protosappanins [5, 6]. Currently, the routine method for the constituent analysis of sappan lignum is HPLC [5, 7–11]. Brazilin and protosappanin B (structures shown in Fig. 1) are two active pharmaceutical components of sappan lignum, which have anti-inflammation and antioxidation properties making their correct determination important. Chen et al. have determined these in the water extracts of sappan lignum by HPLC [11], while Zhao et al. used an MeOH extract [12]. Although the constituents in the water decoction (water
Correspondence: Dr. Michael C. Breadmore Austrialian Centre for Research on Separation Science, School of Chemistry, University of Tasmania, Hobart, Tasmania, 7001, Australia E-mail: [email protected]
extracts) of traditional Chinese medicine are in closer agreement with those used in medicinal practice, the solubility of brazilin and protosappanin B in water is much lower than in methanol, which means much more extractant is needed and a concentration step is required before analysis. The Chinese Pharmacopoeia also lists an HPLC method for determination of brazilin and protosappanin B in the MeOH extract and advises that the contents of the two constituents should not be less than 0.5% [10]. CE, with the advantages of low consumption of samples and chemicals, short analysis times, and high resolution, has received considerable interests in traditional Chinese medicine analysis [13, 14]. But due to its short optical length, CE has the inherent problem of low sensitivity that limits its applicability. To enhance the sensitivity, many online concentration methods have been proposed. These online concentration methods can be classified into the following three groups: those based on changes in migration due to a conductivity difference [15–18]; a difference in pH between the sample zone and the buffer zone (sometimes the concentration zone) [19–24], and those based on the association between the analytes and a moving electrolyte component [25, 26]. These can also be combined in a number of ways to produce additional more complex and more powerful enrichment approaches [27–30]. Acid barrage stacking (ABS) exploits changes in ionization with pH to concentrate weak acids [21, 23]. This is achieved by injecting a high concentration of low pH
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Abbreviations: ABS, acid barrage stacking; MAE, microwaveassisted extraction C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Additional corresponding author: Dr. Hao Zhong, E-mail: [email protected]
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Figure 1. Structures of the analytes.
solution after a large volume of sample has been injected. When the separation voltage is applied, the anionic components in the sample migrate to the inlet and when they enter the acidic region, they stop and stack on the interface between the sample and acid zones. This method is similar to dynamic pH junction, but is more flexible because it allows the acidic solution for concentration to be quite different to that of the separation electrolyte. This approach also has the advantage of being tolerant of high salt in the sample and has been utilized to analyze amino acids in rat brain microdialysate, rat serum, and human saliva [23]. Feng et al. used CE with ABS to determine genistein in the traditional Chinese medicine Frucuts sophorae [21]. Here a CE method with ABS is developed and validated for the determination of brazilin and protosappanin B in sappan lignum after microwave-assisted extraction (MAE). The sensitivity was enhanced by 62-fold and 111-fold for brazilin and protosappanin B, respectively.
2 Materials and methods 2.1 Instrumentation CE separations were carried out in a P/ACE MDQ CE system with a photodiode array detector for absorbance measurements at 254 nm (Beckman Coulter, Fullerton, CA, USA). Uncoated fused-silica capillaries were purchased from Polymicro Technologies (Phoenix, AZ, USA). The capillary was 60.2 cm × 50 m id with an effective length of 50.0 cm. The temperature of the capillary was kept at 25⬚C. The CE system was interfaced with a computer and controlled using the Beckman 32 karat software (version 7.0). New capillaries were flushed with 1 M NaOH for 20 min, 18.2 m⍀·cm water for 20 min, and BGE for 20 min. Each day, capillaries were flushed with 0.1 M NaOH for 10 min, 18.2 m⍀·cm water for 10 min, and BGE for 10 min. To maintain good repeatability, the capillary was flushed between each separation with water and the BGE for 2 and 4 min, respectively. The BGE comprised 20 mM of sodium tetraborate (pH 9.2) containing 6% v/v MeOH. The BGE was prepared freshly each day, sonicated for 5 min, and filtered through a 0.45 m membrane filter before use. 2.2 Chemicals Brazilin and protosappanin B were prepared in Institute of Materia Medica, Shandong Academy of Medical Sciences, C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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and their purity was not less than 99% [31]. Other chemicals, unless otherwise stated, were all of analytical grade. Water of 18.2 m⍀·cm was from a CascadaTM Lab Water System (Pall Life Science, China). A standard solution of 1000 g/mL of each analyte was prepared in MeOH. A mixed standard solution of the two analytes was prepared at a concentration of 500 g/mL in MeOH. The working standards were prepared daily by diluting the mixed standard solution with 20 mM borax-NaH2 PO4 buffer (pH 8.0) containing 6% v/v MeOH. All solutions were stored in dark containers at 4⬚C.
2.3 ABS and enhancement factor calculation After filling the capillary with BGE (20 psi, 4 min), the sample was injected hydrodynamically with a positive pressure (0.5 psi, 65 s; 6.6% of the length to the window), followed by a hydrodynamic injection of 150 mM citric acid (0.5 psi, 22 s; 2.2% of the length to the window). A voltage of +25 kV was applied to stack and separate the ions. The enhancement factor was calculated by dividing the detection limits obtained with ABS with the detection limits obtained from normal hydrodynamic sample injection (0.5 psi, 5 s, 0.4% of capillary volume).
2.4 Preparation of sappan lignum sample Sappan lignum sample was purchased from Auguo Chinese medicine market (Anguo, Hebei). It was dried at 60⬚C for 6 h and was then pulverized using mill. A total of 0.5 g of the powder was dispersed in 25.00 mL of MeOH and exposed to the 450 W microwave irradiation for 4 min. After cooling, the extract was filtered with a medium-speed filter paper (⌽ = 9 cm, Hangzhou Fuyang Special Paper, China). The MeOH extracts were made to 25.00 mL with MeOH. Before analysis, 100 L of this extract was diluted with 20 mM BoraxNaH2 PO4 (pH 8.0) buffer to 5 mL after 200 L MeOH was added.
3 Results and discussion 3.1 Separation optimization Borate can form negatively charged boronate esters with compounds that have two adjacent hydroxyl groups [32]. This can be used to enhance solubility and also as was used in this work, to provide some enhanced charge to facilitate separation by electrophoresis. The first step was therefore to optimize the pH, BGE concentration, and the concentration of MeOH. Keeping the BGE concentration at 20 mM, the effect of pH on the separation was investigated over the range of 7.0– 9.5 adding either NaH2 PO4 (to ensure adequate buffering) or NaOH to the 20 mM sodium tetraborate. The results show www.electrophoresis-journal.com
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tion as the buffer concentration increased. At the same time, the capillary current increased and there was a noticeable increase in detection limit when the BGE concentration was higher than 20 mM, presumably due to more pronounced joule heating, which made the baseline noise higher (from 0.020 to 0.026 mAU). As a compromise between resolution, analysis time, and sensitivity, 20 mM was selected as the optimum borax concentration. To further improve the separation, the addition of MeOH to the BGE was investigated in the 0–8% v/v range. As MeOH was increased the resolution improved, however, the mobility of the analytes decreased prolonging the analysis time. The addition of 6% v/v MeOH was found to improve the resolution without compromising analysis time and was used for all further experiments. The optimum BGE was therefore 20 mM sodium tetraborate, pH 9.20 with 6% v/v MeOH.
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Figure 2. Comparison of different acids as acid barrage on preconcentration effects. Conditions: 60 cm × 50 m (50 cm to detector), +25 kV; 254 nm; 20 mM sodium tetraborate, pH 9.20 with 6% v/v MeOH. Hydrodynamic injection of sample 65 s at 0.5 psi, followed by hydrodynamic injection of barrage acid (pH 2.3) for 22 s at 0.5 psi.
3.2 ABS optimization ABS exploits the change in ionization of the analytes due to the pH difference between the sample and the acid barrage zone to lower the mobility of the analytes and to narrow the sample band. So the sample matrix and the composition of the acid barrage zone, namely the type, concentration, and pH as well as the injection times for both sample and barrage acid, were optimized.
that the electrophoretic mobility of the two analytes increases as the pH of the buffer is increased due to the increased ionization of the analytes, and that there was a change in migration order at a pH of 8.0. In addition, the EOF remained relatively constant due to changes in ionic strength countering the increase in pH. When the pH was equal to or higher than 9.0, the two analytes were well separated. As sodium tetraborate buffers well at a pH of 9.20, which is close to the pH of 20 mM sodium tetraborate at room temperature, and there was adequate resolution between the two compounds at this pH, this was used for all further experiments. The effect of BGE concentration was investigated next over the range of 10–30 mM. The results show that there was a slight reduction in the electrophoretic mobility of brazilin and protosappanin B, with a slight improvement in resolu-
3.2.1 Effects of sample matrix Borate buffers are frequently used in preparing CE samples [23]. In this work, 20 mM sodium tetraborate (the same concentration as the BGE) was applied as the sample matrix, and the effect of pH was investigated in the pH range of
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Figure 3. Effects of injection time on (A) peak height and (B) peak area of brazilin and protosappanin B. Conditions as for Fig. 2, except the injection time that are shown in the figure. The barrage zone time was adjusted with each sample injection time to maintain a ratio of 1:3.
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7.0–9.5. The results showed that the peak heights of the analytes increased with the increase of the pH, but after the pH was equal to or higher than 8.5, the peak height decreased quickly, believed to be due to the degradation of the analytes at high pH. When prepared at pH 8.0, the RSD (%) of peak height for ten consecutive injections (one injection per hour) were 3.59 and 3.25% for brazilin and protosappanin B, respectively. The good repeatability signifies that the two analytes are stable in the sample matrix for 10 h.
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Any type of acid that is able to maintain a low pH can in theory be used as barrage acid. Keeping the barrage zone pH at 2.3, different acids including 150 mM of citric acid (pKa = 3.13, 4.76, 6.40), phosphoric acid (pKa = 2.12, 7.2, 12.36), acetic acid (pKa = 4.74), and tartaric acid (pKa = 3.04) were examined. The results are shown in Fig. 2 that clearly shows that citric acid produced the highest peak heights for brazilin and protosappanin B. The concentration of citric acid was then investigated from 50 to 200 mM. The peak heights first increased with the increase of the citric acid concentration until 150 mM after which they decreased. At the same time, the migration time of the analytes decreased with the increase of citric acid concentration. This can be explained by the fact that more barrage acid reacts with the analyte anions and thus increase the stacking efficiency as barrage acid concentration increased. On the other hand, the analytes will migrate fast with the increase of barrage acid concentration that will reduce the separation time. One hundred and fifty micromolar citric acid was chosen as a compromise.
3.2.3 Effects of the ratio for acid injection to sample injection and injection time The effects of the acid injection to sample injection ratio was investigated from 1:1 (same length) to 1:4 (sample four times longer than the barrage zone). As the ratio was increased, the stacking efficiency increased; however above 1:3, the resolution for the two analytes deteriorated significantly indicating insufficient capillary length to stack and separate the analytes.
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Figure 4. Electropherograms of (A) normal injection and (B) ABS: Conditions (A): 100 g/mL standards dissolved in methanol, 0.5 psi for 5 s injection. (B) 4.0 g/mL standards solved in 20 mm Borax with 6% v/v MeOH (pH 8.0) injection, 0.5 psi for 65 s followed by 22 s of 150 mM citric acid (pH 2.3). For both separations, the BGE was 20 mm sodium tetraborate with 6% v/v methanol (pH 9.20), 60 cm × 50 m id capillary (50 cm to detector), +25 kV. Peak 1 is Brazilin and peak 2 is protosappanin B.
Keeping the injection ratio at 1:3, the sample and barrage injection times were varied, and the sample time was varied from 25 to 75 s. The results are shown in Fig. 3 with the peak area increasing linearly but the peak height leveling off after 65 s. From 65 to 75 s, the peak areas still increase, while the peak heights did not change considerably. Furthermore, the peaks start to broaden at 75 s.
3.2.4 Analytical performance of ABS Under the optimum conditions, an ABS-CZE separation of the two analytes is shown in Fig. 4B and compared to a normal injection as shown in Fig. 4A. The sample-to-sample time was less than 13 min. Intraday variations (n = 5) in migration time were between 0.63 and 0.86% RSD, and for peak area were between 3.16 and 3.47% and those for interday repeatability (n = 5) were 1.49–1.63% and 3.82–4.78%. The detection limits and calibration parameters are summarized in Table 1. The sensitivity enhancement factors were 62 and 111 for brazilin and protosappanin B, respectively, with LODs of 0.28 and
Table 1. The regression equations in ABS
Compounds
Regression equationa)
Correlation coefficient
Linear range (g/mL)
Detection limitsb) (g/mL)
LOQc) (mg/g)
EFd)
Brazilin Protosappanin B
y = 531.42x − 19.76 y = 1490.58x + 659.88
0.9983 0.9978
1.0∼20.0 0.6∼10.0
0.28 0.15
2.3 1.3
62 111
a) In the regression equation, the x value is the concentration of analytes (g/mL), the y value is the peak area. b) Detection limits were based on three times noises. c) Limits of quantitation were based on ten times noises. d) Enhancement factor, which was calculated by dividing LOD with hydrodynamic sample injection (0.5 psi, 5 s) by those when ABS was performed.
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0.15 g/mL, based on three times noise. The calibration was linear over the range of 1.0–20 g/mL and 0.6–10 g/mL for brazilin and protosappanin B, respectively.
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MAE has been proven to be a powerful sample extraction technique due to its ability to reduce the volume of extraction solvents and extraction time, improve the reproducibility and recovery of analytes, and increase sample throughput [33]. According to the literature, MeOH was used as the extraction solvent [12]. The effects of extraction temperature, microwave power, and microwave time and extractant volume were investigated.
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The effects of extraction temperature were investigated in 30–70⬚C range. The results showed that peak area of the two analytes in the sample increased when the temperature became higher. But after 60⬚C, this increase became trivial, so 60⬚C was chosen as the extraction temperature.
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3.3.3 Effects of extractant volume With 0.5 g sample, the effect of extraction solvent volume was investigated from 5 to 30 mL range. As shown in Fig. 5C, the extraction efficiency of the analytes increased up to 25 mL after which it remained constant and this volume was used for all further extractions. 3.4 Real sample analysis The recovery of the method was determined with the addition of the standards (comparable to 5.0 mg/g in the solid sample) in the real sample solution, with result of 89.9 and 101.4% for brazilin and protosappanin B, respectively (Table 2). This is adequate for their analysis demonstrating the potential applicability of this method to analyze brazilin and protosappanin B in the traditional Chinese medicine. The developed C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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The effects of microwave power and microwave time on the extraction efficiency were investigated in the 150–550 W and 2–6 min range. The results are shown in Fig. 5A and B. The extraction efficiencies of the two analytes generally increases with the microwave power and irradiation time from 150 to 450 W and 2 to 4 min. When the microwave power is greater than 450 W or the time greater than 4 min, the extraction efficiency decreased slowly, which may be due to degradation of the analytes; 450 W for 4 min was selected as the optimum.
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Figure 5. Effects of (A) microwave time, (B) microwave power, and (C) methanol volume on the extraction efficiency (sappan lignum from Guangxi). Conditions are the same as Fig. 4B Table 2. Results of recovery (n = 5)
Compounds
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Added (mg/g)
Found (mg/g)
RSD (%)
Recovery (%)
Brazilin Protosappanin B
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11.03 10.11
3.88 3.51
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Figure 6. Electropherograms of the extracts of sappan lignum from (A) Guangxi and (B) Yunnan. Condition are the same as Fig. 4B. peak identity the same as Fig. 4. Table 3. Results of sample analysis (n = 5)
5 References
Compounds
Brazilin (mg/g)
RSD (%)
Protosappanin B (mg/g)
RSD (%)
Guangxi Yunan
6.53 10.15
4.69 4.83
5.04 5.10
3.95 4.12
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method was applied to analyze brazilin and protosappanin B in sappan lignum. Figure 6 shows the electropherogram from the extract of sappan lignum from Guangxi(a) and Yunnan(b). It can be clearly seen that the brazilin and protosappanin B are present at concentrations well above the LOQ in two samples. The results, shown in Table 3, establish that the brazilin and protosappanin B content was above 0.5%, indicating the authenticity of these sappan lignum samples.
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4 Concluding remarks A method for the determination of brazilin and protosappanin B by CE with online concentration with ABS was developed. The sensitivity was improved by 62- and 111-fold giving detection limits between 0.28 and 0.15 g/mL for brazilin and protosappanin B, respectively. The method was shown to be suitable for the detection of brazilin and protosappanin B in sappan lignum. This work was supported by funding from the Key Technologies R&D Programme of Shandong Provine (2010GSF10615) and the Natural Science Foundation of China (81072544). MCB would like to acknowledge the Australian Research Council (DP0984745) for funding and a QEII fellowship. The authors have declared no conflict of interest.
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