Elsevier

Journal of Chromatography A

Volume 1217, Issue 5, 29 January 2010, Pages 705-714
Journal of Chromatography A

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

https://doi.org/10.1016/j.chroma.2009.12.017Get rights and content

Abstract

A microwave-assisted extraction (MAE) and ultra high performance liquid chromatography coupled with diode array detection and time-of-flight mass spectrometry (UHPLC-DAD-TOF-MS) method was developed for simultaneous determination of 14 phenolic compounds in the root of Pueraria lobata (Wild.) Ohwi and Pueraria thomsonii Benth. Operational conditions of MAE were optimized by central composite design (CCD). The optimized result was 65% ethanol as extraction solvent, 17 mL of extraction volume, 100 °C of extraction temperature and 2 min of hold time. A Zorbax SB C18 (50 mm × 4.6 mm I.D., 1.8 μm) and gradient elution were used during the analysis. The chromatographic peaks of 14 investigated compounds in samples were successfully identified by comparing their retention time, UV spectra and TOF mass data with the reference substances. All calibration curves showed good linearity (r > 0.9997) within the test ranges. The intra-day and inter-day variations were less than 1.77% and 2.88%, respectively. The developed method was successfully applied to determine the investigated compounds in 10 samples of Radix Puerariae Lobatae and Radix Puerariae Thomsonii, respectively. The result indicated that MAE and UHPLC-DAD-TOF-MS system might provide a rapid method for the quality control of Radix Puerariae.

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).

References (44)

  • Y.J. Lin et al.

    Biochem. Biophys. Res. Commun.

    (2009)
  • A. Ryokkynen et al.

    Anim. Reprod. Sci.

    (2006)
  • Y. Ishimi et al.

    Bone

    (2002)
  • Z. Yu et al.

    Cancer Lett.

    (2006)
  • F.H. Lo et al.

    Biomed. Pharmacother.

    (2007)
  • H.Y. Zhang et al.

    Cell Biol. Int.

    (2008)
  • J. Bo et al.

    J. Neurosci. Res.

    (2005)
  • S. Malaivijitnond et al.

    J. Ethnopharmacol.

    (2006)
  • M.G. Wade et al.

    Food Chem. Toxicol.

    (2003)
  • W. Cherdshewasart et al.

    Phytomedicine

    (2008)
  • R.W. Jiang et al.

    J. Ethnopharmacol.

    (2005)
  • M.H. Lee et al.

    J. Food Chem.

    (2007)
  • W. Cherdshewasart et al.

    J. Pharm. Biomed. Anal.

    (2007)
  • S.B. Chen et al.

    J. Chromatogr. A

    (2006)
  • G. Chen et al.

    J. Chromatogr. A

    (2001)
  • M. Careri et al.

    J. Chromatogr. A

    (2007)
  • Y. Hu et al.

    Chem. Eng. Process.

    (2008)
  • Z.K. Guo et al.

    Anal. Chim. Acta

    (2001)
  • H.Q. Huang et al.

    J. Pharm. Biomed. Anal.

    (2009)
  • M.T. Ren et al.

    J. Pharm. Biomed. Anal.

    (2008)
  • G.H. Li et al.

    Asian Chem. Lett.

    (2009)
  • S.F. Osman et al.

    Phytochemistry

    (1983)
  • Cited by (103)

    • Global identification and quantitative analysis of representative components of Xin-Nao-Kang Capsule, a traditional Chinese medicinal formula, by UHPLC-Q-TOF-MS and UHPLC-TQ-MS

      2021, Journal of Pharmaceutical and Biomedical Analysis
      Citation Excerpt :

      Consequently, F40 was identified as quercetin by comparing retention time, MS and MS/MS spectra data with authentic standard. By comparing with the reference standards and available literature [3,6,10,11,19,21,23–26], flavone aglycones were easy to eliminate the small neutral fragments such as H2O (18.0106 Da), CH3 (15.0234 Da), CO (27.9948 Da), CO2 (43.9898 Da), and/or the combination of these mentioned above, and usually obeyed the Retro Diels-Alde (RDA) fragmentation mechanism to produce the RDA fragment ion at m/z 153 Da in the positive mode or m/z 151 Da in negative mode. Finally, another 13 flavone aglycones (F17, F27, F28, F30, F38, F39, and F42-F48) were characterized in XNKC (Table S1).

    View all citing articles on Scopus
    View full text