Study On The Determination Methods Of Alternaria Toxins In Citrus And Fruit Juices

Mycotoxins are secondary metabolites of filamentous fungi that naturally occur in fruits, vegetables and cereals, which affect their quality and economic value. The presence of these compounds is of high concern for human health due to their toxicological profiles, such as teratogenesis, nephrotoxicity and immunotoxicity. Citrus is one of the most important fruit in the world and occupies an important position in fruit industry. China is one of major original centers of citrus, and its planting area and yield are the highest in the world. Citrus fruits like citrus reticulata, oranges, pomelos and fruit juices, such as orange juice, apple juice, grape juice play an important part of residents’daily diet. As a result, it’s meaningful to detect mycotoxins in citrus and fruit juices.In this paper, the types and harm of Alternaria toxins to fruits and vegetables, the techniques of sample preparation, and the detection methods of Alternaria toxins were summarized. Importantly, the application of high performance liquid chromatography (HPLC), high performance liquid chromatography-tandem spectrum (LC-MS/MS), QuEChERS, and DLLME in the detection of Alternaria toxins were introduced. On the basis of current trends of QuEChERS and dispersive liquid-liquid microextraction (DLLME), three new detection methods were developed and applied to real samples, and the results were satisfactory. The details and results are as follows:A method was developed for the simultaneous determination of four mycotoxins (aletrnariol monomethyl ether, tentoxin, alternariol and citrinin) in citrus fruits using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with optimized QuEChERS. The sample was extracted with acetonitrile, salted out with anhydrous MgSO4and NaCl, cleaned up with C18and then analyzed using UPLC-MS/MS in multiple reaction monitoring (MRM) mode with electrospray ionization. The separation of the target mycotoxins was performed on an ACQUITY UPLC BEH C18liquid chromatography column using a mobile phase of acetonitrile and0.1%formic acid aqueous solution in gradient elution and the matrix-matched external standard calibration was used for quantitation. This method made a good linarity in the5.0-200μg/L with correlation coefficients more than0.9927(R2), and limits of detection (LODs) were in the range of0.2-0.7μg/kg. The average rescoveries of tentoxin, aletrnariol monomethyl ether, alternariol and aitrinin in different matrix (citrus pulp, peel and whole fruits) at three spiked concentration levels were ranged from78.0%to103.3%, with relative standard deviations (RSDs) of2.6%-10.6%. This method is simple, quick, accurate and sensitive and suitable for the quick confirmation and quantitative determination of trace4mycotoxins in citrus fruits.A novel method was developed for the simultaneous determination of3alternaria toxins (alternariol, alternariol methyl ether and tentoxin) in fruit juices (orange, apple, pear, grape and peach juices) using ultra high performance liquid chromatography-photo diode array (UPLC-PDA) combined with DLLME. The factors including types and volumes of extractive solvents, extraction time and salinity which affect the extractive efficiency of DLLME were optimized. Under the optimized conditions, enrichment factors in theory were50. This method made a good linarity in the5.0-200μg/L with R2>0.9943, and LODs were in the range of1.0-3.0μ.g/kg. The average rescoveries of alternariol, alternariol methyl ether and tentoxin at two spiked concentration levels were ranged from69.8%-110.5%, with RSDs of2.7%-9.4%. This method is simple, quick and cheap and suitable for the quick confirmation and quantitative determination of trace3alternaria toxins in a large number of fruit juice samples.A novel method was developed for the simultaneous determination of3alternaria toxins in fruit juices using UPLC-PDA combined with a technique composed of quick, easy, cheap, rugged and safe (QuEChERS) and DLLME sample preparation. The sample was extracted with acetonitrile, salted out with anhydrous MgSO4and NaCl, cleaned up with C18. The extract from QuEChERS and chloroform were used as dispersive solvent and extractive solvent in DLLME respectively. Under the optimized conditions, the enrichment factors were10in theory. This method made a good linarity in the5.0-200μg/L with R2>0.9969, and LODs were in the range of3.0-10.0μ.g/kg. The average resvoveries of alternariol, alternariol methyl ether and tentoxin at two spiked concentration levels were ranged from78.4%-106.0%, with RSDs of1.9%-9.2%. The result indicated that this method was suitable for confirmation and quantitative determination of trace3alternaria toxins in fruit juices in13min with good purifying effects.