A rapid method for simultaneous determination of 14 phenolic compounds in Radix Puerariae using microwave-assisted extraction and ultra high performance liquid chromatography coupled with diode array detection and time-of-flight mass spectrometry
Introduction
Radix Puerariae (RP) has been widely used as herbal medicine and dietary supplement in eastern Asia [1], [2]. It includes dried Radix Puerariae Lobatae (RPL) and Radix Puerariae Thomsonii (RPT) [3]. The phenolic compounds in RP have been demonstrated to have multiple pharmacological activities, such as effect on reproductive organ development [4], prevention of bone loss [5], anti-cancer action [6], [7], neuroprotective effect [8], [9], estrogenic activity [10], [11] and anti-oxidative activity [12], [13]. Therefore, it is necessary to develop a method for the rapid identification and quantification of these phenolic compounds. Up to now, a number of extraction methods, including low temperature soaking [14], [15], ultrasonic extraction (UE) [13], [16], [17], [18], reflux extraction (RE) [3], [19], [20] and pressurized liquid extraction (PLE) [16], [21] have been developed for extraction of phenolic compounds from RP. But these methods usually need long extraction time and large amount of solvent consumption. Meanwhile, high performance thin-layer chromatography (HPTLC) [18], high performance capillary electrophoresis (HPCE) [19], [20] and high performance liquid chromatography (HPLC) [1], [21], [22], [23], [24] were used to analyze phenolic compounds in RP. However, these technologies suffered from long analysis time [1], [18], [21], [22], [23], [24], low resolution [18], low sensitivity [18] and/or few analytes [1], [19], [21], [22].
As a fast and effective extraction method, microwave-assisted extraction (MAE) was first reported by Ganzler et al. [25]. And then it was widely used in sample preparation like extracting isoflavones from soybean [26] and drying isoflavones extract from RPL [27]. Modern physical chemistry studies indicated that the large dielectric constant solvent, such as water and ethanol, absorbs microwave energy and produces intense molecular vibration, which leads to simultaneous heating up of whole solvent and samples [28]. Thus, MAE using water and ethanol as a mixture solvent could obtain high extraction efficiency. Comparing with other techniques such as PLE, RE and UE, MAE reduces extraction time, solvent consumption and increases extraction efficiency. According to the previous report, MAE was applied to the extraction of RP [29]. However, some chemical properties of puerarin, such as solubility, were different from isoflavones aglycones, puerarin was chosen as the only evaluating indicator to optimize the extraction condition, which could not provide the comprehensive optimum extraction condition for isoflavones glycosides and aglycones in RP.
The analyses of Chinese medicines (CMs) generally cost long time due to the complicated matrix. Fortunately, ultra high performance liquid chromatography (UHPLC) has been proved to be a rapid chromatographic analytic tool, which performed multi-component analysis with satisfactory separation, good resolution and sensitivity [30]. Nowadays, mass spectrometry (MS) has been widely used for identification of chemical components in CMs. Especially, time-of-flight mass spectrometry (TOF-MS) has various advantages including high resolution, accurate mass measurement and high sensitivity [31]. Thus, UHPLC coupled with TOF-MS may provide a rapid qualitative and quantitative analysis method for CMs.
In this paper, it was the first time to report a MAE coupled with UHPLC-DAD-TOF-MS system for rapid determination of the major components in Radix Puerariae. Furthermore, the contents of 14 phenolic compounds, namely puerarin-4′-O-glucoside, puerarin-3′-methyoxy-4′-O-glucoside, daidzein-4′,7-O-glucoside, puerarin, mirificin, daidzin, 6″-O-xylosylpuerarin, 3′-methoxypuerarin, genistin, sophoraside A, ononin, daidzein, genistein and formononetin, in RPL and RPT were also compared.
Section snippets
Chemicals, reagents and materials
Methanol and formic acid (HPLC grade) for UHPLC analysis were purchased from Merck (Darmstadt, Germany). Absolute ethanol (AR grade) used for extraction purpose was obtained from Riedel-de Haën (Seeize, Germany). Deionized water was purified by a Millipore Milli-Q purification system (Millipore, Bedford, MA, USA).
Puerarin, daidzin, daidzein and genistein were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The 10 phenolic compounds,
Optimization of MAE
Optimizing MAE conditions should consider the interaction of different extraction factors and the linear relationship between response and variables. In order to reveal the complicated interaction and relationship, a statistical analysis method, central composite design was selected to optimize MAE parameters. The overall desirability (OD) [37], the geometric mean of the contents of 14 target compounds were used as marker to evaluate the extraction efficiency.
Before CCD optimizing MAE
Conclusions
A rapid qualitative and quantitative method for simultaneous determination of 14 major constituents in two Radix Puerariae species by UHPLC-DAD–TOF-MS was developed. In this study, MAE showed outstanding extraction efficiency compared with other conventional extraction methods. Meanwhile, six unknown chromatographic peaks were tentatively identified by accurate mass measurement of TOF-MS. The results indicated that MAE combined with UHPLC analysis was an excellent method for the quality
Acknowledgements
We are grateful to Mr. Z.M. Qian from our institute for his expert technical assistance. The research was supported by grants from Macao Science and Technology Development Fund (049/2005/A-R1).
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