Article pubs.acs.org/JAFC
Analysis of 10 Metabolites of Polymethoxyflavones with High Sensitivity by Electrochemical Detection in High-Performance Liquid Chromatography Jinkai Zheng,†,§,∥ Jinfeng Bi,†,∥ David Johnson,§ Yue Sun,§ Mingyue Song,§ Peiju Qiu,§ Ping Dong,§ Eric Decker,§ and Hang Xiao*,§ †
Institute of Agro-Products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China § Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States ABSTRACT: Polymethoxyflavones (PMFs) have been known as a type of bioactive flavones that possess various beneficial biological functions. Accumulating evidence demonstrated that the metabolites of PMFs, that is, hydroxyl PMFs (OH-PMFs), had more potent beneficial biological effects than their corresponding parent PMFs. To facilitate the further identification and quantification of OH-PMFs in biological samples, the aim of this study was to develop a methodology for the simultaneous determination of 10 OH-PMFs using high-performance liquid chromatography (HPLC) coupled with electrochemistry detection. The HPLC profiles of these 10 OH-PMFs affected by different chromatographic parameters (different organic composition in mobile phases, the concentration of trifluoroacetic acid, and the concentration of ammonium acetate) are fully discussed in this study. The optimal condition was selected for the following validation studies. The linearity of calibration curves, accuracy, and precision (intra- and interday) at three concentration levels (low, middle, and high concentration range) were verified. The regression equations were linear (r > 0.9992) over the range of 0.005−10 μM. The limit of detection for 10 OHPMFs was in the range of 0.8−3.7 ng/mL (S/N = 3, 10 μL injection). The recovery rates ranged from 86.6 to 108.7%. The precisions of intraday and interday analyses were less than 7.37 and 8.63% for relative standard deviation, respectively. This validated method was applied for the analysis of a variety of samples containing OH-PMFs. This paper also gives an example of analyzing the metabolites of nobiletin in mouse urine using the developed method. The transformation from nobiletin to traces of 5-hydroxyl metabolites has been discovered by this effective method, and this is the first paper to report such an association. KEYWORDS: hydroxyl polymethoxyflavones, HPLC, quantification, electrochemical detection, metabolites, nobiletin
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INTRODUCTION
samples generated from animal and human studies that contain multiple OH-PMFs, for example, the metabolites of PMFs. The electrochemical detection (ECD) is a powerful analytical method that can detect electric currents of less than nanoamps generated from oxidative or reductive reactions of test compounds.18−20 OH-PMFs possess at least one phenolic group that is potentially reactive under electrical potential, which makes OH-PMFs good candidates for electrochemical detection. The aim of this study is to develop a fast, simple, and reliable HPLC coupled with ECD method for the assessment of 10 important OH-PMFs (Figure 1), namely, 4′-demethyltangeretin (1), 5-demethyltangeretin (2), 5,4′-didemethyltangeretin (3), 3′-demethylnobiletin (4), 4′-demethylnobiletin (5), 3′,4′-didemethylnobiletin (6), 5-demethylnobiletin (7), 5,3′didemethylnobiletin (8), 5,4′-didemethylnobiletin (9), and 5,3′,4′-tridemethylnobiletin (10). Herein, we report a detailed validation of a HPLC-ECD method for the aforementioned 10 OH-PMFs, and we also successfully utilized this method to analyze urine samples containing multiple OH-PMFs and their metabolites generated from a mouse feeding study.
Polymethoxyflavones (PMFs) are a unique class of bioactive flavonoids that are mainly found in citrus fruits such as sweet orange and tangerine, especially in their peels.1 Increasing numbers of studies have demonstrated various beneficial biological functions of PMFs, for example, anti-inflammatory, anticarcinogenic, antiatherogenic, antiviral, and antioxidative effects.2 We and others have identified several metabolites of PMFs after their oral administration in mice.3−5 It was found that these metabolites are mainly hydroxyl PMFs (OH-PMFs). Interestingly, accumulating evidence has shown that OH-PMFs have potent biological activities,6−13 and some of them were much stronger that those produced by their corresponding parent compounds.3−5 OH-PMFs can be obtained by isolation from natural sources, such as citrus peels, and by chemical synthesis.14 Several methods have been developed for the quantification of PMFs, including gas chromatography (GC),15 supercritical liquid chromatography (SFC),16 and highperformance liquid chromatography (HPLC) with UV detection.17 However, there is no effective analytical method available for the simultaneous quantification of multiple OHPMFs with quite similar chemical structures. With increasing interests in the beneficial bioactivities of PMFs, it is important to develop highly sensitive methods to analyze biological © 2015 American Chemical Society
Received: Revised: Accepted: Published: 509
November 17, 2014 December 30, 2014 January 1, 2015 January 1, 2015 DOI: 10.1021/jf505545x J. Agric. Food Chem. 2015, 63, 509−516
Article
Journal of Agricultural and Food Chemistry
Figure 1. Chemical structures of 10 OH-PMF analogues.
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min (100% B), 30 min (100% B), 30.1 min (10% B), and 35 min (10% B). pH Values of Mobile Phases. The pH values of mobile phases A and B were adjusted to different values in the range of 2.0−7.0 by adding different volumes of TFA. The effects of pH on the sensitivity, retention time, and peak resolution of 10 OH-PMFs were determined and compared. Concentrations of Ammonium Acetate. In the mobile phases, ammonium acetate was used as a buffer system that can affect the sensitivity of the ECD. Five different concentrations of ammonium acetate (0, 25, 50, 75, and 100 mM) were used to determine their effects on the ECD of 10 OH-PMFs. Validation of HPLC-ECD Method. The stock solutions (10 mM) of each standard compound were prepared in DMSO and stored at 4 °C, and the final test solutions with a series of concentrations were prepared by diluting the stock solution with 50% methanol just before every experiment. Linearity, limit of quantification (LOQ), and limit of detection (LOD), and precision of the current method were determined. To assess the linearity of the method, calibration curves were constructed with eight concentrations of each OH-PMF (0.005, 0.01, 0.05, 0.5, 5, 10, 20, and 50 μM) in the test solutions. Calibration curves were obtained by plotting the responses (peak areas) of 10 OHPMF standards against their respective concentrations using a linear least-squares regression. The slope, intercept, and correlation coefficient (r2) were calculated for each OH-PMFs using a regression equation. The sensitivity of the method was determined by the LOD and LOQ. LOD and LOQ were the lowest measured concentrations with signal-to-noise (S/N) ratios of of 3 and 10, respectively. A known amount of standard OH-PMFs (at low, medium, and high concentrations of 0.05, 0.5, and 5 μM, respectively) was added to mouse urine, and overall recoveries were determined by the standard addition method. Intraday and interday precisions were determined for both retention time and peak area at low (0.05 μM), medium (0.5 μM), and high (5 μM) concentrations of OH-PMFs. Quantification of OH-PMFs in Biological Samples. The optimized HPLC-ECD method was utilized to quantify OH-PMFs in the urine of mice after they were fed nobiletin, a polymethoxyflavone found in citrus fruits. Fifteen CF-1 male mice (7 weeks of age) were fed nobiletin at 500 ppm in standard AIN-93G powder diet for 1 week. The urine was collected using Tecniplast metabolic cages (5 mice per cage) during the last 12 h of feeding. Two hundred microliters of collected urine sample was extracted with 500 μL of ethyl acetate three times. The combined ethyl acetate extracts were
MATERIALS AND METHODS
Reagents and Chemicals. Organic solvents, including methanol (MeOH), acetonitrile (ACN), tetrahydrofuran (THF), and trifluoroacetic acid (TFA), were of HPLC grade and obtained from Fisher Scientific. Ammonium acetate was a product of EMD Chemicals Inc. (Gibbstwon, NJ, USA). 4′-Demethyltangeretin (1), 5-demethyltangeretin (2), 5,4′-didemethyltangeretin (3), 3′-Demethylnobiletin (4), 4′demethylnobiletin (5), 3′,4′-demethylnobiletin (6), 5-demethylnobiletin (7), 5,3′-didemethylnobiletin (8), 5,4′-didemethylnobiletin (9), and 5,3′,4′-tridemethylnobiletin (10) were synthesized following previously published methods.3,4 The purity of these 10 OH-PMFs was >98%, and their chemical structures have been confirmed by MS and NMR.3,4 Instrumentation and Chromatographic Conditions. The CoulArray HPLC system (Chelmsford, MA, USA) consisted of a binary solvent delivery system (model 584), an autosampler (model 542), and a CoulArray Multi-Channel EC detector (model 6210) (Waters, Milford, MA, USA).21 Instrument control and data processing were performed with CoulArray 3.06 software. An Ascentis RP-Amide reversed-phase HPLC column (15 cm × 4.6 mm id, 3 μm) (Sigma-Aldrich, St. Louis, MO, USA) was used. Flow rate was 1.0 mL/ min. The temperature of the autosampler was set to 4 °C. The injection volumes were between 10 and 50 μL. The optimal elution conditions were as follows: mobile phase A, 75% water, 20% acetonitrile, 5% THF, and 50 mM ammonium acetate; mobile phase B, 50% water, 40% acetonitrile, 10% THF, and 50 mM ammonium acetate (pH values of both mobile phases were adjusted to 3.0). The linear solvent gradient consisted of 10% B at the beginning, 50% B at 5.0 min, 70% B at 15.0 min, 90% B at 25.0 min, 100% B at 25.1 min, and 100% B at 30.0 min. Re-equilibration duration was 5 min between individual runs. Two EC detection cells (each contains four channels) were used, and the detecting potentials were set at 100, 200, 300, 400, 500, 600, and 700 mV. Optimization of Chromatographic Conditions. Elution Conditions. To obtain optimal resolution of 10 OH-PMFs simultaneously, two complex mobile phases A and B were used: A, 75% water, 20% acetonitrile, 5% THF, and 50 mM ammonium acetate; B, 50% water, 40% acetonitrile, 10% THF, and 50 mM ammonium acetate. Both mobile phases were adjusted to pH 3.0. Four different elution conditions were compared, that is, (i) isocratic elution with 40% A and 60% B; (ii) isocratic elution with 50% A and 50% B; (iii) isocratic elution with 60% A and 40% B; and (iv) gradient elution, 0 min (10% B), 5 min (50% B), 15 min (70% B), 25 min (90% B), 25.1 510
DOI: 10.1021/jf505545x J. Agric. Food Chem. 2015, 63, 509−516
Article
Journal of Agricultural and Food Chemistry
Figure 2. HPLC profiles of 10 OH-PMFs analyzed using four different elution conditions, that is, three isocratic elution conditions (i.e., 60, 50, and 40% mobile phase B) and one gradient elution condition (0 min, 10% B; 5 min, 50% B; 15 min, 70% B; 25 min, 90% B; 25.1 min, 100% B; 30 min, 100% B). The composition of mobile phase A was 75% water, 20% acetonitrile, 5% THF, and 50 mM ammonium acetate (pH 3.0); the composition of mobile phase B was 50% water, 40% acetonitrile, 10% THF, and 50 mM ammonium acetate (pH 3.0). Different EC potentials were used for detection (100 mV, in red; 300 mV, in green; 400 mV, in black; 600 mV, in blue). Each peak was labeled with corresponding OH-PMF 1−10 (injection volume = 10 μL; sample concentration = 0.5 μM).
Table 1. Retention Time (Minutes) of OH-PMFs 1−10 Eluted by Different Elution Conditions elution condition 60% B 50% B 40% B gradient elution a
1 3.15 4.51 7.88 12.95
2 a
6.58 12.07 26.91 27.65
3 5.25 8.91 18.52 22.90
4 a
5 b
2.80 3.74a 6.16 10.70
6 b
2.80 3.93a 6.63 11.35
2.49 3.30 5.28 9.46
7 a
4.84 8.10 16.42 21.20
8 a
4.22 6.78 13.01 18.50
9 a
4.51 7.37 14.56 19.80
10 a
3.87a 6.01 11.29 16.90
The peak cannot be separated with others from baseline. bThe peak completely overlapped with another one.
dried under vacuum. Dried samples were dissolved in 1000 μL of 50% methanol for HPL-ECD analysis using the optimized method. The identities and concentrations of OH-PMFs (as metabolites of nobiletin) in mouse urine were determined by comparing the HPLC profiles of urine samples with those of OH-PMF standards and their corresponding standard curves. Statistical Analysis. All analyses were performed in triplicate, and the results are presented as means ± standard deviation of three independent experiments.
conditions are listed in Table 1. The isocratic elution with 60 or 50% of mobile phase B did not produce satisfactory separation of the 10 OH-PMFs, which was evidenced by overlapping peaks of different OH-PMFs, especially compounds 4 and 5. In contrast, isocratic elution with 40% mobile phase B resulted in good separation of all 10 OH-PMFs. Nevertheless, the retention times of the 10 OH-PMFs were not evenly distributed, and compounds 1 and 4−6 were eluted within only 2.6 min when eluted with 40% mobile phase B. A potential drawback of the quick elution of compounds 1 and 4−6 is that any other compounds (e.g., contaminants or impurities) with similar retention times may have a good chance to interfere with one or more of them. The gradient elution (0 min, 10% B; 5 min, 50% B; 15 min, 70% B; 25 min, 90% B; 25.1 min, 100% B; 30 min, 100% B) produced the best separation of all 10 OHPMFs within 30 min. Moreover, much better sensitivities can be obtained under gradient elution condition. For example, the sensitivity of compound 1 under gradient elution increased 8fold in comparison with those under isocratic elution with 40% mobile phase B. Therefore, the gradient elution was selected as the optimal elution conditions. Optimization of the pH of Mobile Phases. The pH of the mobile phase often significantly affects the nature of chromatograms, especially the sensitivity of ECD. In this section, we determine the effects of mobile phase pH on the retention time, sensitivity, and peak resolution of all OH-PMF. The initial pH values of mobile phases A and B were 7.15 and 7.34, respectively. Their pH values were adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 by adding different volumes of TFA. The gradient elution condition was used in this section. As shown in Figure 3, varying pH values of the mobile phase had only
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RESULTS AND DISCUSSION Optimization of HPLC-ECD Method. Different OHPMFs may have different electrochemical reactivities and retention times under different chromatographical conditions. Therefore, it is necessary to determine the impact of multiple influencing factors (pH value, salt concentration, and polarity of eluents) to obtain optimal separation and sensitivity for all 10 OH-PMFs. The column was equilibrated for at least 2 h with new mobile phases when different mobile phases were used. Optimization of Elution Condition. On the basis of our previous experience in analyzing PMFs by HPLC,4,21 we developed two complex mobile phases A and B to optimize the elution conditions. Mobile phase A consisted of 75% water, 20% acetonitrile, 5% THF, and 50 mM ammonium acetate, and mobile phase B consisted of 50% water, 40% acetonitrile, 10% THF, and 50 mM ammonium acetate. In this section, the pH of mobile phases A and B was adjusted to 3.0. The effects of pH on the HPLC-ECD results are investigated in the following section. Four elution conditions were used to analyze a mixture of 10 OH-PMFs at 1 μM each. The HPLC profiles of 10 OHPMFs under different elution conditions are shown in Figure 2, and the retentions times of OH-PMFs under different elution 511
DOI: 10.1021/jf505545x J. Agric. Food Chem. 2015, 63, 509−516
Article
Journal of Agricultural and Food Chemistry
Figure 3. Effects of pH of mobile phases on HPLC profiles of 10 OH-PMFs. The pH values of both mobile phases A and B were adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, or 7.0. Different EC potentials were used for detection (100 mV, in red; 300 mV, in green; 400 mV, in black; 600 mV, in blue). Each peak was labeled with corresponding OH-PMFs 1−10 (injection volume = 10 μL; sample concentration = 0.5 μM).
Optimization of Ammonium Acetate Concentration. For the mobile phase for use with EC detection, a buffered system that can provide ionic strength for good electron transfer is required in mobile phase.22−24 In our study, ammonium acetate was used as a buffer system for EC detection.25 The gradient elution condition was used in this section. Five different concentrations of ammonium acetate (0, 25, 50, 75, and 100 mM) were prepared in both mobile phases A and B with pH 3.0. As shown in Figure 5, the concentration of ammonium acetate did not have a significant effect on the retention times of OH-PMFs, but the addition of ammonium acetate greatly improved the symmetry and sharpness of peaks of OH-PMFs. Most interestingly, when the concentration of ammonium acetate increased from 0 to 25 mM and then to 50 mM, the sensitivity of EC detection was significantly enhanced. However, further increase of ammonium acetate concentration to 75 and 100 mM caused no further enhancement of sensitivity, and it even decreased sensitivity for some OHPMFs. On the basis of these results, the optimal concentration of ammonium acetate was determined to be 50 mM, and this condition was used for the rest of the study described below. Method Validation. Using the optimized chromatography conditions, we constructed the calibration curves for all 10 OHPMFs by plotting the peak areas against a series of concentrations of prepared standards (0.005, 0.01, 0.05, 0.5, 5, 10, 20, and 50 μM, respectively; Figure 6). On the basis of the EC potential where the maximum sensitivities of different OH-PMFs were observed, we selected the potentials of 600, 300, 300, 400, 400, 100, 300, 300, 300, and 100 mV for analyzing OH-PMFs 1−10, respectively. Satisfactory linearity was observed when the concentrations of OH-PMFs were within the range of 0.005−10 μM (Figure 6). The results on linear regression of standard curves of all 10 OH-PMFs (slope, intercept, correlation coefficient, and linear range) are reported in Table 2. The linearities of the standard curves for all 10 OHPMFs were all >0.9992, suggesting good linearity within a relatively wide concentration range (0.005−10 μM, 2000-fold) for all 10 OH-PMFs. The limits of detection (LOD) and the limits of quantification (LOQ) were determined at signal-to-
marginal effects on the retention times. To a certain extent, the peak symmetry and sharpness were improved when the pH decreased. Most importantly, the sensitivity of ECD was increased when the pH values of mobile phases were decreased. For example, as shown in Figure 4, at a potential of 400 mV,
Figure 4. Effects of pH value on peak areas (nC) of compound 5 under different EC potentials (mV). The pH values of both mobile phases A and B were adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, or 7.0. EC potentials were set at 100, 200, 300, 400, 500, 600, and 700 mV (injection volume = 10 μL; sample concentration = 0.5 μM; n = 3).
the mobile phase with pH 2.0 produced the highest sensitivity, followed by pH 3.0 and 4.0. A similar trend can be observed for other OH-PMFs. It is worth mentioning that at high pH, that is, pH 7.0, the sensitivity of ECD of compounds 6 and 10 became so low that they cannot be even detected at 1 μM concentration. Although pH 2.0 produced the highest overall sensitivity, we selected pH 3.0 as the optimal pH value for the mobile phases because high acidity of the mobile phase may cause damage to the stationary phase and therefore shorten the working life of the HPLC columns. 512
DOI: 10.1021/jf505545x J. Agric. Food Chem. 2015, 63, 509−516
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Journal of Agricultural and Food Chemistry
Figure 5. Effects of the concentrations of ammonium acetate (0, 25, 50, 75, and 100 mM) in the mobile phases on the HPLC profiles of 10 OHPMFs. Different EC potentials were used for detection (100 mV, in red; 300 mV, in green; 400 mV, in black; 600 mV, in blue). Each peak was labeled with corresponding OH-PMFs 1−10 (injection volume = 10 μL; sample concentration = 0.5 μM).
Figure 6. Calibration curves of OH-PMFs 1−10 (gradient elution; injection volume = 10 μL).
noise ratios (S/N) of 3 and 10, respectively. The results showed that the LODs of the 10 PMFs ranged from 8 to 37 pg per injection (10 μL) at a signal-to-noise ratio of 3:1, and the LODs ranged from 25 to 119 ng at a signal-to-noise ratio of 10:1. Recovery rates of the extraction and detection method were obtained by analyzing the mouse urine samples that had been spiked with known amounts of OH-PMFs. As shown in Table 2, the recovery rates of different OH-PMFs ranged from 86.6 to 108.7% with the RSD (%, relative standard deviations) of
