关键词: 麦角生物碱毒素, 牧草, 固相支撑液液萃取, 超高效液相色谱-串联质谱

Abstract:

Toxic alkaloids， especially ergot alkaloid toxins， present in forage grass and pose serious health hazards to humans and livestock. Hence， a method for simultaneously detecting these dangerous plant toxins is needed. This study established a rapid method for detecting ten ergot alkaloid toxins using solid-phase supported liquid-liquid extraction technology combined with ultra performance liquid chromatography-tandem mass spectrometry. Forage samples were extracted with 8.0 mL of acetonitrile containing 1% formic acid. The extraction mixture was vortexed and then centrifuged at 8 000 r/min for 5 min， after which 1.0 mL of the supernatant was mixed with 3.0 mL of water and vortexed for 0.5 min. The entire mixture was transferred to a solid-phase supported liquid-liquid extraction column and allowed to stand for 10 min. The column was eluted with 6.0 mL of ethyl acetate and the collected eluate was evaporated under nitrogen at temperatures below 40 ℃. The residue was dissolved in 1 mL of acetonitrile-water （1∶3， volume ratio， containing 0.5% formic acid）， and separated using an Acquity UPLC BEH C₁₈ column （100 mm×2.1 mm， 1.7 μm）. A solution of 0.5% formic acid in water containing 0.25 mmol/L ammonium acetate was used as mobile phase A， while 0.5% formic acid in acetonitrile was used as mobile phase B. The following gradient elution program was used： 0-1.0 min， 90%B； 1.0-5.0 min， 90%B-75%B； 5.0-10 min， 75%B-50%B； 10-12 min， 50%B-10%B； 12-14 min， 10%B； 14-16 min， 10%B-90%B. Ten ergot alkaloid toxins （including three pairs of isomers： ergocornine， ergocristine， ergocristinine， ergocryptine， and ergocryptinine） were effectively separated following gradient elution. Positive electrospray-ionization mode and multiple reaction monitoring （MRM） scanning was used for detection， with an external standard curve used for quantification purposes. The ten ergot alkaloid toxins exhibited good linear relationships within their respective linear ranges （r²>0.995）. Alfalfa， Leymus chinensis， oats， and silage corn exhibited LODs of 0.1–2.3 μg/kg for the ten ergot alkaloid toxins， with LOQs of 0.4–7.3 μg/kg. The developed method exhibited overall recovery rates of between 66.3% and 116.7%， with relative standard deviations of less than 9.9%. The matrix effect mainly manifested itself in the form of matrix suppression， with silage corn exhibiting a significantly stronger matrix effect than the other three types of forage. Silage corn exhibited a strong matrix effect in more than 50% of samples， while alfalfa， oats， and Leymus chinensis showed relatively low matrix effects， with more than 63% being weak. The developed method is simple to operate， provides accurate results， and exhibits minimal interference； hence， it is suitable for simultaneously screening and confirming ergot alkaloid toxins in forage， thereby providing technical support for monitoring forage quality， while also expanding the applicability of solid-phase supported liquid-liquid extraction technology in the toxin-detection field.

Key words: ergot alkaloid toxins, forage grass, solid-phase supported liquid-liquid extraction, ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）