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  • Ultraviolet UV spectroscopy is commonly used for screening p


    Ultraviolet (UV) spectroscopy is commonly used for screening potential ADA inhibitors [15]. In the UV method, the ADA reaction is calculated by measuring the decrease in absorbance of the substrate at 265 nm or increase in absorbance of the product at 248 nm. However, AD and inosine share a very close maximum UNC669 wavelength. Thus, exact evaluation of the product formation at the usually selected wavelength is difficult. In addition, the reaction is uncertain and is frequently interfered by the background color of compounds or plant extracts. The use of the UV method to screen potential ADA inhibitors usually yields false positive or negatives results. To overcome these drawbacks, many methods, such as radiative method [16], Berthelot method [17], capillary electrophoresis (CE) [18], high-performance liquid chromatography (HPLC) [19], and aptasensor method [20], have been developed for screening potential ADA inhibitors. However, these methods still exhibit some shortcomings. The radioactive method is effective but is inconvenient to use and is environmentally toxic [16]. The Berthelot method measures the amount of liberated NH3 but suffers from lack of accuracy due to the interference of exogenous NH3 [17]. Given its high efficiency separations and short analysis times, CE has been widely used to evaluate ADA activity. However, the measuring sensitivity of the method is very low [18]. HPLC is also applied in ADA analysis to separate the substrate and the product. Despite its superior repeatability, it requires intensive sample pretreatment and long running time [19]. The aptasensor approach allows for exclusive analysis of the substrate AD and indirect detection of ADA activity. Despite its low detection limit, this method requires many labeled molecules and relevant testing instruments, which result in high costs and limitation for the general laboratory without these conditions [20]. Therefore, convenient and sensitive methods for the assay of ADA activity should be developed. UPLC–MS with high sensitivity and selectivity is especially applicable for detecting enzyme activities [21]. Orbitrap is a new developed mass analyzer in high-resolution mass spectrometry (HRMS). It promises high mass resolution and mass accuracy, wide dynamic range, and good duty cycle and sensitivity [22]. Therefore, this work developed a selective, convenient, quick, and sensitive ultra-high-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometric (UHPLC-Q-Orbitrap HRMS) method for detecting and screening ADA inhibitors. The direct product (inosine) from the selective substrate (AD) catalyzed by ADA in the incubation was quantitated. The method not only involves the classic principle of ADA activity assay in vitro but also performs rapid and sensitive screening of inhibitors and avoids the interferences of the matrix effects as much as possible.
    Results and discussion
    Conflict of interest
    Acknowledgments This work was supported by the Key projects of National Natural Science Foundation of China (Grant No. 81530101), the National Nature Science Foundation of China (Grant No. 81703681), the Shanghai Sailing Program (Grant No. 17YF1419800), and the Budget Project of Shanghai University of Traditional Chinese Medicine (Grant No. 2016YSN12) awarded to Professors Chang-hong Wang and Ping Liu for financial support of this study.
    Introduction Mycotoxins are considered toxic secondary metabolites naturally produced by several filamentous fungi [1]. Fungi are commonly found in grains used to feed several species of animals, however the production of mycotoxins occurs when there is a stressful environment to these agents, such as alterations on temperature or humidity [2]. Among the different types of mycotoxins, the aflatoxin B1 (AFB1) is the most pathogenic, and it is produced mainly by species of Aspergillus flavus and Aspergillus parasiticus[3]. Recently, a study demonstrated that mycotoxins in chickens negatively affect growth rate, feed conversion, and reproductive efficiency of these animals, causing damage in different tissues, such as liver, spleen, heart, and bursa of Fabricius [4], and thus, increase the cost of production for poultry industries. These alterations in tissue weight and size contribute to economic losses in the poultry industry [5], [6], [7].