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, 11 (9), e0163797
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Simultaneous Quantification of Antioxidant Compounds in Phellinus Igniarius Using Ultra Performance Liquid Chromatography-Photodiode Array Detection-Electrospray Ionization Tandem Mass Spectrometry

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Simultaneous Quantification of Antioxidant Compounds in Phellinus Igniarius Using Ultra Performance Liquid Chromatography-Photodiode Array Detection-Electrospray Ionization Tandem Mass Spectrometry

Dan Shou et al. PLoS One.

Abstract

Natural antioxidants are widely used in the life sciences. Phellinus igniarius is a historically used natural antioxidant containing a variety of active compounds. Phenols, particularly Inoscavin A and Hypholomine B, are found in the high concentrations. Better quantitative methods are needed to perform quality control in order to support further research of this mushroom. An ultra-performance liquid chromatography method coupled to photodiode-array detection and an electrospray ionization tandem mass spectrometry method (UPLC-PAD-MS) was developed to simultaneously quantify Inoscavin A and Hypholomine B levels in the medicinal fungus Phellinus igniarius. The two compounds were quantified using UPLC-PAD and UPLC-MS. The methods were accurate (mean accuracy for spiked matrix ranged from 101.5% to 105.8%), sensitive (limit of detection ranged from 0.28 to 1.14 mg L-1) and precise (the relative standard deviations ranged from 0.13 to 2.8%). Inoscavin A and Hypholomine B were purified using high-speed counter-current chromatography (HSCCC), structural evaluated to meet the request of standard substances. UPLC separation was performed on a reversed-phase C18 column using gradient elution with acetonitrile and 0.1% formic acid over 10 min. The developed method was successfully applied to determine Inoscavin A and Hypholomine B in twelve Phellinus igniarius samples of different origins and the results showed that it was suitable for the analysis of these active components in Phellinus igniarius samples.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The structure of Inoscavin A and Hypholomine B.
Fig 2
Fig 2. The effects of column temperature.
The separation conditions were the mobile phase consisted of (A) 0.1% formic acid in water and (B) ACN containing 0.1% formic acid, linear gradient from 20% to 30% B (0–8 min), and isocratic at 30% B (8–10 min), at a flow rate of 0.3 mL min-1.
Fig 3
Fig 3. UPLC-PAD chromatograms of (A) Standard; (B) blank sample; (C) sample spiked with Inoscavin A (20 mg L-1); (D) sample spiked with Hypholomine B (90 mg L-1).
Hypholomine B (1), Inoscavin A (2). The separation conditions were the mobile phase consisted of (A) 0.1% formic acid in water and (B) ACN containing 0.1% formic acid, linear gradient from 20% to 30% B (0–8 min), and isocratic at 30% B (8–10 min), at a flow rate of 0.3 mL min-1.
Fig 4
Fig 4. MRM chromatograms of (A) blank sample and sample spiked with Inoscavin A (20 mg L-1); (B) blank sample and sample spiked with Hypholomine B (90 mg L-1).
Hypholomine B (1), Inoscavin A (2).
Fig 5
Fig 5. HPLC-UV chromatogram. of (A) blank sample; (B) sample spiked with Inoscavin A (20 mg L-1); (C) sample spiked with Hypholomine B (90 mg L-1).
Hypholomine B (1), Inoscavin A (2).

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Grant support

This study was financially supported by National Natural Science Foundation of China (Grant No. 81603349), Zhejiang Provincial Natural Science Foundation (Grant No. LQ15H280008), Science and Technology Plan of Zhejiang Province (No. 2016ZB008, 2016C33097). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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