Simultaneous determination of ochratoxin a and pesticide residues in grapes and wine by LC-MS/MS

Abstract

A fast, selective and sensitive reversed-phased liquid chromatography with tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantification of 41 pesticide residues and ochratoxin A (ОТА) in grapes and wine. For increasing sensitivity of mass spectrometer multiple reaction monitoring (MRM) was transferred into time segmented method, dynamic multiple reaction monitoring (dMRM). Grape and wine extraction and purification for both, pesticide residues and OTA, were performed with a modified Quick Easy Cheap Effective Rugged and Safe (QuEChERS) method. For OTA LC-MS/MS analysis acetonitrile extract was directly injected into system, and for pesticide analysis extracts were purified with dispersive solid phase extraction for pigmented samples (black grapes and red wine) and for fruits and vegetables (white grapes and wine). MS/MS conditions were optimized in order to increase selectivity, selecting the corresponding product ions (precursor-to-fragment m/z 404 → 239 and m/z 404 → m/z 221) for quantification and identification. For pesticide residues, MS/MS aquisition parameters, SRM transitions, fragment and colision energies, from database were used. For method validation, the performance of the method was assessed and compared with the European Commission (EC) Regulations 401/2006 for OTA and with the SANTE/11813/2017 document for pesticide residues, by studying the linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effect, accuracy and precision. Linearity for quantification pesticide residues in grapes and wine was performed in mobile phase and in sample matrixes (0.005 – 0.500 mg/kg) and by standard addition (0.010 – 0.200 mg/kg). Correlation coefficients for most pesticides, in all four matrixes, were over 0.99. Linearity for OTA quantification in grapes was performed in acetonitrile and in the sample matrixes (0.001 – 0.050 μg/ml) and in acetonitrile and in wine matrixes (0.0005 – 0.010 μg/ml).

Razvijena je brza, selektivna i osetljiva tečno-hromatografska metoda sa masenom spektrometrijom (LC-MS/MS) za istovremeno kvantitativno određivanje ostataka 41 aktivne supstance pesticida i ohratoksina A (OTA) u grožđu i vinu. Zbog povećanja osetljivosti masenog spektrometra za ispitivane analite postavljena multirezidualna metoda (MRM) je prebačena u vremenski segmentisanu metodu, tzv. metodu u dinamičkom modu (dMRM). Ekstrakcija i prečišćavanje analita iz uzoraka grožđa i vina su rađene modifikovanom Quick Easy Cheap Effective Rugged and Safe (QuEChERS) metodom. Za analizu OTA acetonitrilni ekstrakt je direktno analiziran LC-MS/MS tehnikom, dok je prečišćavanje ekstrakta za analizu ostataka rađeno pomoću kolona za disperziju na čvrstoj fazi za pigmentisane uzorke (crno grožđe i crveno vino) i za voće i povrće (belo grožđe i belo vino). Za detekciju OTA uslovi masenog spektrometra su optimizovani u cilju povećanja selektivnosti izborom jona sa najosetljivijim odgovorom (m/z 404  239 i m/z 404  221) za kvantifikaciju i identifikaciju. Za detekciju aktivnih supstanci pesticida akvizicioni parametri masenog spektrometra, SRM prelazi, fragmentacione energije i kolizione energije su preuzete iz baze podataka. U cilju razvoja metode ispitani su parametri linearnost odgovora detektora, granica detekcije (LOD), granica merenja (LOQ), matriks efekat, tačnost, preciznost i pouzdanost, a dobijene vrednosti su upoređene sa kriterijumima Uredbe 401/2006 za OTA i SANTE/11813/2017 dokumenta za aktivne supstance pesticida. Linearnost metode za ostatake u grožđu i vinu je ispitivana u mobilnoj fazi i matriksima uzoraka (0,005 do 0,500 mg/kg) i metodom standardnog dodatka (0,010 - 0,200 mg/kg)...