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To investigate and use the different constituents in Bofonis Corium distinguishing from Bufonis Venenum based on ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive Orbitrap MS) coupled with chemometrics.
UPLC analysis was performed on a Waters Atlantis T3 (150 mm×2.1 mm, 3 μm) column with gradient elution of acetonitrile and 0.1% formic acid at a flow rate of 0.3 mL·min-1. The column temperature was set at 30 ℃. UPLC-Q-Exactive Orbitrap MS was chosen for data collection in both positive and negative ion modes. The potential differential ions were obtained by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The Xcalibur 3.0 data processing system was utilized to extract the secondary fragmentation details of the differential ions. Subsequently, the specificity of differential ions was confirmed through the multiple reaction monitoring (MRM) mode of AB SCIEX 6500+ triple quadrupole mass spectrometry. Identification of adulterated Bofonis Corium in Bufonis Venenum was performed by applying the ion pairs with good specificity.
After the research and verification, a total of five distinction pairs (m/z: 377.5→243.2, 172.1；330.4→170.9, 127.0；313.4→201.2, 171.0；452.4→280.2, 298.2 and 614.7→332.4, 281.3) were discovered, and a method for the examination of Bofonis Corium in Bufonis Venenum was established and methodologically investigated by applying the information of the different ions. The constructed method had reasonable specificity and durability. Each ion pair showed an excellent linear relationship within a specific range, with correlation coefficients above 0.996 7. The RSDs of the precision test were in the range of 1.3%-4.1%. The RSDs of the repeatability test were in the range of 1.3%-3.2%. Five batches of market samples of Bufonis Venenum were determined according to the above process, and two batches of samples showed the presence of Bofonis Corium components but did not exceed the proposed 5% adulteration limit.
In this study, a rapid method for detecting discrepancy information based on UPLC-Q-Exactive Orbitrap MS combined with chemometrics is presented, and the discrepant information is applied to establish an identification method for adulterated Bofonis Corium in Bufonis Venenum.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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