Letter pubs.acs.org/ac
Quantitative Nonaqueous Capillary Electrophoresis−Mass Spectrometry Method for Determining Active Ingredients in Plant Extracts Jianhui Cheng, Lingyu Wang, Weifen Liu, and David D. Y. Chen* Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, Canada V6T 1Z1 ABSTRACT: Nonaqueous capillary electrophoresis (NACE) is very well suited for online coupling with mass spectrometry due to the relatively high volatility and low surface tension of most organic solvents. Here we present a quantitative NACE-ESI-MS/MS method for separating and determining physcion, chrysophanol, and aloe-emodin in rhubarb. Dantron was used as an internal standard to ensure accuracy and reproducibility in quantitative analyses. Parameters including the pH, background electrolyte (BGE) composition, flow-through microvial chemical modifier solution composition, and modifier solution flow rate were carefully optimized. The developed method was validated by assessing its precision, LODs, and linear range. The contents of physcion, chrysophanol, and aloe-emodin in rhubarb were determined to be 0.22%, 1.0%, and 0.17%, respectively.
albroehl and Jorgenson were the first to demonstrate that capillary electrophoresis (CE) can be performed with nonaqueous solutions by using acetonitrile-based background electrolytes (BGE).1 It has since been shown that nonaqueous CE (NACE) has many advantages over aqueous CE for certain applications. In NACE, an organic solvent or a mixture of organic solvents with dissolved electrolytes are used as the separation buffer. This has several advantages. First, it can easily adjust the selectivity of the separation process because there is a wide variety of available organic solvents with different physicochemical properties.2 Second, many hydrophobic ionic compounds are more soluble in organic solvents than in water. Third, nonaqueous solvents carry weaker currents and experience weaker Joule heating than water during electrophoresis, enabling the use of stronger electric fields, more concentrated buffers and larger capillary internal diameters.3 Fourth, polar interactions are stronger in nonaqueous solutions, which is highly advantageous in chiral separation systems.4 Finally, most organic solvents are more volatile than water and have lower surface tensions, making NACE more suitable than conventional CE for online coupling with mass spectrometry.5 More details in the current state of NACE MS can be found in a recently published review.6 In 2015, Youyou Tu was awarded a Nobel Prize for her pioneering work on traditional Chinese medicine (TCM) herbs, which led to the discovery of the important antimalarial drug artemisinin. Her successes have helped focus worldwide attention on the medicinal potential of extracts from TCM herbs. Rhubarb (Dahuang), the plant used in this study, has a long history in TCM. The pharmaceutically relevant biologically active components in rhubarb are anthraquinones, including chrysophanol, emodin, physcion, aloe-emodin, and rhein.7,8 Previous studies have shown that it is difficult to
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© XXXX American Chemical Society
completely separate physcion, chrysophanol, and aloe-emodin using ordinary capillary zone electrophoresis without using additives, mainly due to their very similar pKa values.9−11 However, those studies were all conducted using aqueous systems, and as noted above, the use of nonaqueous electrophoresis solvents weakens solvophobic interactions and increases the scope for tuning analyte mobility by manipulating electrostatic or donor−acceptor interactions.2 We therefore sought to develop a reliable nonaqueous capillary electrophoresis method for separating aloe-emodin, chrysophanol and physcion in rhubarb extracts. It was demonstrated that the three anthraquinones can be separated using an optimized NACE method and simultaneously quantitated by triple quadrupole mass spectrometry using dantron as an internal standard. To our knowledge, this work is the first to use 100% nonaqueous solvents in both the BGE and the chemical modifier in the flowthrough microvial interface in a NACE-MS system.12,13 The sensitivity of this system is 1−2 orders of magnitude higher than previous NACE-MS studies.
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EXPERIMENTAL SECTION Chemical and Materials. Physcion, chrysophanol, and aloe-emodin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dantron and ammonium acetate were purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile, methanol, ethanol, and triethanolamine (TEA) were purchased from Fisher Scientific (Nepean, ON, Canada). All chemicals were of
Received: December 13, 2016 Accepted: January 6, 2017 Published: January 6, 2017 A
DOI: 10.1021/acs.analchem.6b04944 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
Figure 1. Optimization of the BGE composition. (a) CE-UV results showing the influence of acetonitrile on the migration time (n = 3). Experimental conditions: capillary length, 49 cm; detector length, 39 cm; pHapp, 10.0. (b) CE-ESI-MS results showing that the migration time increases with the concentration of ammonium acetate in the BGE (n = 3). Capillary length, 72 cm; BGE, 20 mM ammonium acetate in 20% acetonitrile; modifier, 1 mM ammonium acetate in ethanol; flow rate, 2.0 μL/min. (c) Absolute areas of MS peaks corresponding to individual compounds from the experiments presented in subfigure in part b).
Table 1. Structures, Mass Spectrometric Parameters, and pKa Values of Rhubarb Anthraquinoid
cmpd
R1
R2
[M − H]−
fragments
MS/MS ions
collision energy
pKa
Physcion Chrysophanol Aloe-emodin Dantron
OCH3 CH3 CH2OH H
CH3 H H H
283 253 269 239
R1 + R2 R1 + CO R1 CO
240 225 240 211
−30 −40 −38 −45
7.89, 8.56 7.94, 8.59 7.80, 8.46 9.09
solvent was removed under reduced pressure. The crude solid extract was then redissolved in 10 mL of methanol.14 Capillary Electrophoresis. CE analyses of the working standards and real samples were performed with a PA800+ system (Beckman Coulter Inc., Fullerton, CA) using an uncoated fused capillary (72 cm total length × 50 μm i.d. and 360 μm o.d., Polymicro Technologies, Phoenix, AZ). Before each experiment, the capillary was flushed with 0.1 M NaOH for 5 min, 0.1 M HCl for 5 min, deionized water for 5 min, and the BGE solution for 5 min. To maintain reproducibility, the capillary was also flushed with BGE solution for 1 min before each run. Sample solutions were injected over 3 s at a pressure of 0.3 psi. The high voltage power supply was set at 20 kV. Time programs were controlled using the 32 Karat software package to ensure reproducible injections and infusions of the solutions. Mass Spectrometry. CE−MS analyses were performed using an API 4000 triple quadrupole mass spectrometer (AB SCIEX, Concord, ON, CA) in MRM mode. A flow-through microvial interface developed in our laboratory was used in all experiments. The flow rate of the modifier solution was set at 2.0 μL/min using a syringe pump (Harvard Apparatus, MA). The ESI voltage was set to −4.0 kV, and the mass spectrometry parameters were controlled and optimized using the Analyst software package. MS/MS mode was initially used to optimize the collision energies. The optimized collision energies and the key MS properties of the studied compounds are shown in Table 1.
analytical grade or better and were used without further purification. Standard Solution Preparation. The background electrolyte (BGE) consisted of 80% methanol and 20% acetonitrile with 20 mM ammonium acetate (NH4AC) (see Figure 1). The system’s pH was adjusted by adding TEA (VBGE/VTEA = 25:1). The chemical modifier solution consisted of 0.2 mM NH4AC in ethanol and was used for detection in negative ESI mode. Stock solutions were prepared by dissolving appropriate amounts of each compound in methanol to obtain final concentrations of 0.38 mg/mL for aloe-emodin, 0.17 mg/mL for chrysophanol, 0.10 mg/mL for physcion, and 0.12 mg/mL for the internal standard, dantron. Mixed standards were prepared by mixing aloe-emodin, chrysophanol and physcion (V1/V2/V3 = 1:1:1), and working solutions were prepared by diluting the working standard to the desired concentrations. The internal standard was then added to the prepared working standards (VIS/Vsample = 1:4). Real Sample Preparation. Rhubarb was ground into a powder, and 0.1505 g of the powder was dissolved in 25 mL of methanol in a round bottomed flask. The flask’s weight was then recorded, and the solution was refluxed for 1 h. After it was cooled to room temperature, extra methanol was added to compensate for any lost mass. A 10 mL aliquot of the resulting solution was combined with 10 mL of 8% aqueous HCl and sonicated for 2 min, after 10 mL of CHCl3 was added and the resulting mixture was refluxed again for 1 h. The CHCl3 layer was then separated, and another 10 mL of CHCl3 was added, refluxed, and separated. After a third cycle of this extraction process, the three CHCl3 fractions were combined and the B
DOI: 10.1021/acs.analchem.6b04944 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Figure 2. Optimization of chemical modifier. (a) Concentration of ammonium acetate in the modifier influences the migration time (n = 3). (b) Concentration of ammonium acetate in modifier changes the peak height of MS signals (n = 3). (c) The migration time increases as the flow rate of modifier rises (n = 3). (d) The absolute areas of MS signals decrease when flow rate increases (n = 3). All experiments were conducted under the optimum conditions.
The apparent pH (pHapp), the concentration of the electrolyte, and the content of acetonitrile BGE solution were optimized with respect to migration time, peak shape, resolution (especially the resolution of aloe-emodin and chrysophanol), and sensitivity. The accuracy of pH measurements with pH electrodes may be lower for nonaqueous systems than for water-based systems. We therefore use pHapp to denote pH values obtained with the pH meter. The influence of pHapp on the separation was assessed by using BGE solutions spanning the pHapp range 9.0− 11.0. Aloe-emodin and chrysophanol were poorly resolved at pHapp values below 10.0. Raising the pHapp above 10.0 improved the resolution but increased the compounds’ migration times. We therefore chose pHapp = 10.0 (VBGE/ VTEA = 25:1) as our working pHapp because it provided acceptable resolution and reasonably short migration times. To reduce the analysis time, experiments were performed with varying proportions of acetonitrile (0%, 20%, 40%) in the BGE solution. Adding acetonitrile dramatically reduced the analytes’ migration times without sacrificing resolution. However, the acetonitrile also dissolved the capillary’s polyimide coating. This was not a serious problem when using UV detection because the only consequence was a reduction in the capillary’s lifetime. However, in the CE-MS experiments, the dissolved polymer had a tendency to block the flow through the microvials and to reduce the current. An acetonitrile content of 20% was ultimately chosen to achieve a good balance between analysis duration and experimental stability.
Figure 3. Calibration curves for the three anthraquinones. The relative concentration of each compound (see Table 2) is plotted against its relative peak area. Each data point represents the average of five tests.
Table 2. Concentrations of Working Solutions (μg/mL) real concn (μg/mL) no.
physcion
chrysophanol
aloeemodin
relative concn
dantron (μg/mL)
1 2 3 4 5
27 20 14 6.8 3.4
88 66 44 22 11
51 38 26 13 6.4
8 6 4 2 1
24 24 24 24 24
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RESULTS AND DISCUSSION Optimization of NACE Conditions. NACE proved to be a good method for separating anthraquinones after optimization. C
DOI: 10.1021/acs.analchem.6b04944 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry Table 3. Precision of the NACE-ESI-MS/MS Method intraday (RPA, n = 5) no. Pa Ca Aa Pa Ca Aa Pa Ca Aa
5
3
1 a
interday (RPA, n = 9)
intraday (MT, n = 5)
interday (MT, n = 9)
mean
RSD %
mean
RSD %
mean
RSD %
0.219 ± 0.008 0.38 ± 0.02 0.081 ± 0.004 0.87 ± 0.02 1.53 ± 0.07 0.33 ± 0.04 1.86 ± 0.06 3.36 ± 0.09 0.72 ± 0.04
4 6 5 2 4 10 6 6 5
0.21 ± 0.02 0.38 ± 0.02 0.082 ± 0.006 0.81 ± 0.06 1.52 ± 0.08 0.34 ± 0.03 1.8 ± 0.2 3.3 ± 0.3 0.71 ± 0.07
9 6 7 8 5 8 11 11 10
15.64 ± 0.03 16.53 ± 0.04 17.05 ± 0.03 15.7 ± 0.1 16.6 ± 0.1 17.1 ± 0.1 15.68 ± 0.03 16.58 ± 0.03 17.11 ± 0.02
0.2 0.2 0.2 0.9 0.8 0.8 0.2 0.2 0.1
mean
RSD %
± ± ± ± ± ± ± ± ±
1 1 2 1 1 1 1 1 1
15.9 16.8 17.3 15.9 16.8 17.3 15.8 16.8 17.3
0.2 0.2 0.3 0.2 0.2 0.2 0.2 0.2 0.2
P, physcion; C, chrysophanol; A, aloe-emodin.
Table 4. LODs, Linear Ranges, and Contents of Each Anthraquinoid in Rhubarb As Determined by NACE-ESI-MS/MS P C A
regression equation
adjusted R2
LOD (ppb)
linear range (μg/mL)
RPAreal
contents in rhubarb
y = 0.232x − 0.0307 y = 0.413x − 0.0764 y = 0.0904x − 0.0157
0.9978 0.9988 0.9973
84 180 210
2.7−54 2.9−176 5.1−102
0.7145 1.767 0.1041
0.22% 1.0% 0.17%
establish a stable spray, we used the flow-through microvial interface developed by our group to deliver a chemical modifier solution. The modifier helps to limit postcolumn band broadening and improve the analytes’ ionization efficiency. Mixtures of methanol, acetonitrile, ethanol, and isopropanol were tested as modifiers. A mixture of ethanol and ammonium acetate (0.2 mM) was ultimately selected because it gave strong signals and was environmentally friendly. Usefully, the ammonium acetate in the modifier also reduced the analytes’ migration times: ammonium acetate concentrations of 0.1−5 mM were tested, and the lower concentrations were found to yield shorter migration times (Figure 2). The optimum modifier flow rate would minimize the dilution of the analytes and support stable electrospray formation. Flow rates between 1.0 and 4.0 μL/min were tested. Although 1.0 μL/min provided the best signal intensities, the current was less stable than that achieved at higher flow rates and peak shapes were not as good. Higher flow rates gave more stable currents and better peak shapes, but the signal intensity decreased as the flow rate increased. We therefore selected a rate of 2.0 μL/min for subsequent experiments. Method Validation. The method’s analytical parameters including its precision, limits of detection (LODs), and the linearity of its calibration curve were evaluated for the purposes of validation. Working solutions of five concentrations were analyzed 5 times each to create a calibration curve. Each working solution contained 24 μg/mL dantron as an internal standard, and the target analytes were quantitated by their average relative peak areas (RPA) to the internal standard. Dantron was chosen as the internal standard because of its similar structure and properties to the tested anthraquinones, and because later experiments indicated that there was no detectable dantron in the rhubarb samples (Figure 4). Calibration curves for each target analyte were obtained by plotting their RPA values against their concentrations (Figure 3). The corresponding linear regressions, adjusted R-squared values, LODs, and linear ranges are listed in Table 4. The method’s precision was evaluated by computing the intra- and interday variation in the high, intermediate, and low concentrations of the analytes. The relative standard deviation
Figure 4. Electropherograms of a real rhubarb extract with and without internal standard.
The effect of varying the ammonium acetate concentration in the BGE solution was studied by testing concentrations between 10 and 20 mM. Concentrations below 15 mM provided faster analyses but poorer signal intensity. Higher concentrations increased the current and the time required to complete an analysis but provided better signal intensities and better resolutions. Raising the ammonium concentration above 15 mM caused only minor signal enhancement. However, because this did not greatly increase the migration time, we ultimately decided to use an ammonium concentration of 20 mM. Optimization of the Modifier Solution. A stable electrospray is required for mass spectrometric analysis. To D
DOI: 10.1021/acs.analchem.6b04944 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry for the migration time (MT) and RPA were below 2% and 2− 11%, respectively (Table 3). The optimized method was used to analyze the levels of physcion, chrysophanol, and aloe-emodin in rhubarb. A real sample was tested three times, and the average contents of the three target analytes in this sample are listed in Table 4. Figure 4 presents typical electropherograms of the real sample with and without the internal standard, clearly showing that there is no dantron in the sample with no added internal standard.
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CONCLUSIONS Three rhubarb anthraquinones have been successfully separated by CZE using dantron as an internal standard. The developed method is practically straightforward and requires no additive, sensitive (having low LODs), fast (analyses can be completed within 20 min), and precise (based on its inter- and intraday RSD values). The successful quantitation of closely related rhubarb anthraquinones clearly demonstrates the power of NACE-MS and its ability to separate compounds that would be challenging to differentiate by other means.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
David D. Y. Chen: 0000-0002-3669-6041 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by grants from SCIEX Separations (Brea, CA) and the Natural Sciences and Engineering Research Council of Canada. J.C. and L.W. acknowledge Mitacs Accelerate PhD Fellowships sponsored by PromoChrom Inc. and Lipont Pharmaceuticals Inc., respectively.
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REFERENCES
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