Study On The Simultaneous Determination Of 19 Mycotoxins In Feed And Feedstuffs By High Performance Liquid Chromatography Tandem Mass Spectrometry

Mycotoxins are toxic secondary metabolities prouduced by molds in suitable growing condition,which are serious threat to human health and life safety due to their genetic toxicity,carcinogenicity,teratogenicity on humans and animals,and they also will cause kidney poisoning,abnormal reproductive system and suppress the immune response.Currently,the contamination of mycotoxin widely existed in feed and feedstuffs have become a global problem while just small kinds of mycotoxins was involved in our country?s national standard testing method and the detection efficiency was low.Therefore,it is very important to develop a sensitive,rapid,accurate,reliable,and in line with international standards method to determine mycotoxins in feed and feedstuffs simultaneously.In this paper,the sample pretreatment method and detection method were researched with the detection object of 19 mycotoxins in feed and feedstuffs.The main conclusions were shown as below.Firstly,the conditions of mass spectrometry and chromatographic in the process of simultaneous determination of 19 mycotoxins in feed and feedstuffs were optimized.In this method,electrospray ionization source?ESI?and multiple reaction monitoring mode?MRM?were chosen.In order to obtain optimized conditions for each mycotoxin,it was decided to use the ESI⁺and ESI^-mode in two seprate chromatographic analytes due to polarity switching during the chromatographic run was not possible with large number of mycotoxins.Mass spectra parameters of precursor ions,product ions and collision energy were optimized and the effect of chromatographic conditions such as the type of chromatographic column,mobile phase and injection volume were researched meanwhile.Chromatographic column was Waters C₁₈?5?m,150 mm?4.6 mm i.d?.Mobile phase gradient elution was used in both ESI⁺and ESI^-mode.The mobile phases of ESI⁺mode were A-methanol and B-10 mmol/L ammonium formate containing 0.1%formic acid aqueous solution,and the mobile phases of ESI^-mode were A-acetonitrile and B-water.The 19 mycotoxins can be completely separated under optimized conditions.Secondly,the QuEChERS technology was used in pretreatment for feed and feedstuffs,and the influence factors such as extraction agent,extraction time,salting-out agent and adsorbent were optimized.It was confirmed that the extractant was 10 m L acetonitrile and the extraction time was 30 min.0.5 g Anhydrous magnesium sulfate and 1.5 g sodium chloride were chosen as salting-out agent,and150 mg C₁₈,20 mg PSA and 150 mg.anhydrous magnesium sulfate were chosen as adsorbent.In order to increase the extraction efficiency of acetonitrile,the 0.3%formic acid was added in water.The solution was centrifuged in high speed before detection rather than filtered because of the membrane adsorption.There was matrix induced signal suppression in both feed and feedstuffs.In order to reduce the matrix effect,the blank matrix matching curve of the compound feed,concentrate feed and premix feed were adopted as make working curve,and the blank matrix matching quantitative calculation of wheat was chosen as working curve for all feedstuffs.After obtained the optimized conditions of chromatography,mass spectrometry and pretreatment conditions,the high performance liquid chromatography tandem mass spectrometry?HPLC-MS/MS?method to determine 19 mycotoxins in feed and feedstuffs simultaneously was developed with good linear,low detection limit,high accuracy and precision.For all mycotoxin,the linear correlation coefficients?r?were from 0.9921 to 0.9996 and the linear range was from 0.05?g/L to 200?g/L.The limit of detection?LOD?and the limit of quantification?LOQ?for feed were in the range of0.12³.85?g/kg,and 0.40¹0.8?g/kg respectively,the average recovery ranged from 80.6%to 94.0%,the relative standard deviations?RSD?were 7.1%¹3.7%.The LOD and LOQ for feedstuffs were in the range of 0.10³.4?g/kg and 0.25¹0.0?g/kg respectively,the average recovery ranged from 83.6%to 95.4%,the RSD were 6.2%¹2.6%.In order to examine the applicability of the methods,the quality control samples and actual samples were determined,and the method was compared with enzyme-linked immunoassay.It was proved that the HPLC-MS/MS method was suitable for the simultaneous determination of mycotoxins in feed and feedstuffs.