UPLC-MS/MS Detection of 10 Bufotoxins in Biological Tissues
The detection method for simultaneously analyzing 10 bufotoxins(arenobufagin,gammabufotalin,resibufagin,desacetylcinobufagin,resibufogenin,cinobufotalin,bufotalin,cinobufagine,bufalin,cinobufaginol)in biological tissues samples was established using ultra-high performance liquid chromatography-quadrupole/electrostatic field orbital trap high resolution mass spectrometry(UPLC-Q/Exactive MS)with liver as the sample.0.8 mL of acetonitrile was added in 0.2 g of liver to precipitate proteins,and then was purified with a hybrid solid phase column.ACQUITY UPLC HSS T3 column(2.1 mm×100 mm×1.8 µm)was selected.The mobile phase A consisted of 0.1%formic acid in water and B consisted of acetonitrile with a gradient elution program for separation.The mass spectrum was analyzed by electrospray ion source with positive ion mode.The detection limit of 10 bufotoxins in liver could be less than 10 ng/mL,and the lowest could reach 0.1 ng/mL.These 10 bufotoxins showed good linearity within the range of 20 ng/mL to 400 ng/mL(R2＞0.996).The extraction recoveries of 10 bufotoxins from liver ranged from 62.9%to 93.2%,and the matrix effect ranged from 90.9%to 109.0%.The intraday precision was within 9.2%,and the interday precision was within 14.6%.The stabilities of 10 bufotoxins in the matrix were good under repeated freezing condition,thawing condition and room temperature condition,and after extraction processing,with the RSD of concentration all within 15%.After gastric administration,the distribution order of content in male rats was as follows:liver>lung>heart>kidney>genital organ>muscles;in female rats,the distribution order of content was as follows:lung>genital organ>liver>heart>kidney>muscles.This study established a detection method for bufotoxins in biological tissues and revealed the distribution pattern of bufotoxins in animal bodies tentatively,providing a reference for the examination of bufotoxins and the selection of related specimens.
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