[UPLC-Q-TOF-MS/MS qualitative analysis and HPLC determination of triterpenoids in Ganoderma lucidum spores]

Ganoderma spores are the dry and mature spores of G. lucidum or G. sinense of Polyporaceae, with strong immunity-improving, anti-tumor, and anti-oxidant activities. Triterpenoids are one of the main active components in Ganoderma spores, while the method for determining the content of triterpenoids remains to be developed. In this paper, UPLC-Q-TOF-MS/MS was employed to qualitatively analyze the triterpenoids in G. lucidum spores, and an HPLC method was established for simultaneous determination of five triterpenoids in the spores. The comparison on the retention time, ultraviolet spectrum, and fragmentation with the reference substance in the negative ion mode revealed nine triterpenoids in G. lucidum spores. These nine triterpenoids were ganoderic acid I, ganoderenic acid C, ganoderic acid C2, ganoderic acid C6, ganoderic acid G, ganoderenic acid B, ganoderic acid B, ganoderic acid A, and ganoderic acid H. Three triterpenoids from the spores were inferred by comparison with the available articles, including lucidenic acid E,(Z)-6-\[(3S,5R,7S,10S,12S,13R,14R,17R)-12-acetoxy-3,7-dihydroxy-4,4,10,13,14-pentamethyl-11,15-dioxo-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenteno \[α\] phenanthrene-17-yl\]-2-methyl-4-oxoheptyl-5-enoic acid, and ganoderin A. With Ultimate HPLC XS-C_(18) column(4.6 mm×250 mm, 5 μm) as the stationary phase and acetonitrile-0.072% phosphoric acid solution as the mobile phase, gradient elution was carried out at a flow rate of 1.2 mL·min~(-1), detection wavelength of 257 nm, and column temperature of 40 ℃. These conditions enabled the simultaneously quantitative analysis of the five triterpenoids, and the results of methodological investigation showed that the method met the requirements of content determination. Ganoderenic acid C, ganoderic acid C2, ganoderic acid C6, ganoderenic acid B, and ganoderic acid H showed a good linear relationship(R~2>0.999) within the concentration ranges of 0.93-16.08, 3.44-23.88, 4.28-59.40, 1.40-16.16, and 3.53-61.20 μg·mL~(-1), respectively, with the average recovery rate of 79.53%-93.60% and RSD of 4.7%-7.6%. The content of the five triterpenoids in 15 batches of raw spores and 4 batches of sporoderm-broken spores from different habitats was within the ranges of 0.44-16.25 μg·g~(-1) and 0.52-3.57 μg·g~(-1), respectively, which showed a large difference. The method established in this paper was simple and reliable and could be used for the accurate determination of triterpenoids in G. lucidum spores, which provides a methodological basis for the quality evaluation of G. lucidum spores.