Optimizacija metoda ekstrackcije i određivanja neonikotinoida tečnom hromatografijom u odabranim uzorcima

Abstract

Insekticidi novije generacije, neonikotinoidi, odlikuju se specifičnim načinom delovanja na nervni sistem insekata. Radi dobijanja što brže i kvalitetnije informacije o izloženosti životne sredine ovim insekticidima i količinama njihovih ostataka u hrani potrebno je raspolagati odgovarajućim instrumentalnim metodama za njihovo određivanje. Razvijene su i optimizovane analitičke metode zasnovane na tečnoj hromatografiji za određivanje sedam odabranih neonikotinoida (dinotefurana, nitenpirama, tiametoksama, klotianidina, imidakloprida, acetamiprida i tiakloprida) u medu i likeru od meda. Ispitivana je mogućnost određivanja klotianidina pomoću tečne hromatografije visoke efikasnosti sa detektrorom od niza dioda (HPLC-DAD) primenom kombinacije tečno-tečne i ekstrakcije na čvrstoj fazi iz uzoraka meda. Na osnovu preliminarnih rezultata može se zaključiti da korišćenje faznih-čvrsto kolona u kombinaciji sa tečno-tečnom ekstrakcijom dihlormetanom rezultira prihvatljivim prinosom klotianidina u uzorcima meda pri koncentraciji od oko 0,5 µg g-1 klotianidina. Radi dobijanja većih prinosa odabrana je disperzna tečno-tečna mikroekstrakcija (DLLME) kao tehnika pripreme uzoraka meda. Testirana je upotreba acetonitrila kao disperznog sredstva. Pored hloroforma, korišćen je i dihlormetan kao drugo ekstrakciono sredstvo, kako bi se uporedila efikasnost ekstrakcije. Zabeleženi su prinosi klotianidina od 69,7 i 68,3% u zavisnosti da li je korišćen hloroform, odnosno DHM kao rastvor za ekstrakciju. Može se zaključiti da je prinos ekstrakcije bio povoljniji pri odnosu 0,5 mL ACN i 2,0 mL DHM. Prinosi su se kretali od 68,4% do 92,1%, što je ukazalo da su parametri DLLME ekstrakcije optimalni. Kako bi se detaljnije ispitali ključni parametri DLLME tehnike, korišćena je metodologija površine odziva (RSM), kao i detekcija na osetljivijem kuplovanom masenom detektoru (MS/MS). Optimizovani su HPLC-MS/MS parametri kako bi se obezbedilo zadovoljavajuće hromatografsko razdvajanje i niske granice detekcije (GD, 0,5–1,0 μg kg-1) i određivanja (GO, 1,0–2,5 μg kg-1) ispitivanih neonikotinoida u medu. Upotrebom centralno kompozitnog dizajna konstruisani su kvadratni modeli ispitivanih faktora: zapremine ekstrakcionog (DHM, 1,0–3,0 mL) i disperznog (ACN, 0,0–1,0 mL) sredstva, izračunati statistički parametri i optimizovan proces DLLME upotrebom Derringer-ove funkcije poželjnih odgovora. Upotrebom MMC i SC krivih u opsegu GO–100,0 μg kg-1 ispitan je uticaj matriksa pri čemu zaključeno je da je najveći uticaj matriksa bio na odziv analitičkog signala nitenpirama, dinotefurana i klotianidina. Ispitani su prinosi odabranih neonikotinoida (R, 74,3–113,9%), kao i preciznost metode u uslovima ponovljivosti (RSD, 2,74– 11,8%) i intermedijerne reproduktivnosti (RSD, 6,64–16,2%). Brza (retenciona vremena 1,5–9,9 min) i osetljiva metoda, koja troši malu količinu rastvarača, primenjena je za ispitivanje 15 realnih uzoraka meda različitog cvetnog porekla. Rezultati su pokazali da ispitivani med nije sadržao ostatke ispitivanih neonikotinoida u koncentracijama iznad GD. Dalje istraživanje je bilo usmereno ka razvijanju i optimizaciji HPLC-DAD analitičke metode upotrebom DLLME i QuEChERS tehnika za pripremu uzoraka za određivanje 7 neonikotinoida u uzorcima meda. U ovom delu istraživanja optimizovani su i hromatografski parametri, upotrebom RSM sa Box-Behnken-ovim dizajnom i Derringer-ovom funkcijom poželjnih odgovora. Od ispitivanih neonikotinoida dinotefuran i imidakloprid su bili u najvećoj meri izloženi uticaju matriksa, bez obzira na proceduru pripreme uzoraka. Može se istaći da je uticaj matriksa na analitički signal dinotefurana bio izraženiji u slučaju MS/MS, apostrofirajući manju robusnost ove metode određivanja. Prinosi neonikotinoida su bili (R, 73,1–118,3%), preciznost u uslovima ponovljivosti (RSD, 3,28–10,40%) i intermedijerne reproduktivnosti (RSD, 6,45–17,70%), a granice detekcije (GD, 1,5–2,5 µg kg-1) i određivanja (GO, 5,0–10,0 µg kg-1). Metoda je primenjena za ispitivanje 7 neonikotinoida u 104 uzorkameda različitog cvetnog porekla sa teritorije Autonomne Pokrajine Vojvodine. Detektovano je prisustvo tiakloprida, imidakloprida i tiametoksama u količinama koje su bile ispod MDK RS i EU. Analizirani su uzorci likera od meda - medice. Upoređivane su dve tehnike pripreme uzoraka, DLLME i QuEChERS i primenjeni optimizovani hromatografski uslovi i MS/MS parametri. U slučaju nitenpirama, dinotefurana i tiametoksama uticaj matriksa bio je najizraženiji. Metoda je validovana određivanjem prinosa neonikotinoida (R, 69,2–113,4%), preciznosti u uslovima ponovljivosti (RSD, 3,21–12,81%) i intermedijerne reproduktivnosti (RSD, 9,11–16,63%), kao i granice detekcije (GD, 0,5–2,5 µg kg-1) i određivanja (GO, 1,0–10,0 µg kg-1). Analizom 10 komercijalno dostupnih likera od meda otkriveno je prisustvo klotianidina i tiakloprida, evčzokinotš z na neophodnost daljeg kontrolisanja ovog proizvoda na prisustvo neonikotinoida. Ispitana je mogućnost uklanjanja odabranih neonikotinoida (dinotefurana, klotianidina i tiakloprida) iz vodene sredine (reke Dunav). Ispitivanje efikasnosti 6 različitih vrsta uklanjanja odabranih neonikotinoida (u prisustvu prirodne insolacije u laboratorijskim uslovima, sa dodatkom H2O2, sa dodatkom MWCNT, sa dodatkom MWCN+H 2O2, sa dodatkom Fe-MWCNT, sa dodatkom Fe-MWCNT+H2O2) vršeno je upotrebom prethodno razvijene HPLC-MS/MS metode. Krive uklanjanja odabranih neonikotinoida, pokazale su da tokom 60 minuta pri prirodnoj insolaciji u laboratorijskim uslovima koncentracija smanjenje oko 25%. Analitički signal dinotefurana dobijen u prisustvu H2O2 pod istim uslovima ukazuje na uklanjanje ciljnog analita od oko 40%, tiakloprida od oko 70%, a klotianidina u potpunosti. Testirana je adsorpcija ciljnog analita na višezidnim ugljeničnim nanocevima (MWCNT). Ovim postupkom može da se ukloni oko 30% dinotefurana, oko 50% klotianidina i 60% tiakloprida. U kombinaciji sa H2O2 , MWCNT pokazuju bolju sposobnost uklanjanja za 15–50% u zavisnosti od ispitivanog neonikotinoida. Upotreba Fe-MWCNT i njihova kombinacija sa H2O2 otvorila je mogućnost za dalja ispitivanja mehanizma uklanjanja. Ustanovljeno je nastajanje intermedijera kojima odgovaraju m/z od 117,5 i 140,6 u slučaju razgradnje dinotefurana u sistemima sa H2O2, MWCNT+H2O2, Fe-MWCNT+H2O2 i klotianidina u sistemu Fe-MWCNT+H2O2.

Neonicotinoid insecticides, as one of the fastest growing new generation of insecticides, have contributed to a significant reduction of toxicity for the environment; therefore, monitoring and determination of trace levels of the neonicotinoids in honey are necessary and demands highly efficient, selective and sensitive analytical techniques. The objective of the present work was to develop a rapid, sensitive, optimized and accurate analytical method based on liquid chromatography for determining seven neonicotinoid insecticides, dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid, acetamiprid and thiacloprid in honey and honey liqueur samples. The possibility for determination of clothianidin in honey samples was investigated by HPLC with a diode array detector (HPLC-DAD). Based on preliminary results, it can be concluded that the use of a solid-phase column in combination with a liquid-liquid extraction with dichloromethane results in an acceptable recovery of clothianidin in the samples with a clothianidin concentration of about 0.5 µg g-1. After obtaining low recovery of clothianidin, dispersed liquid-liquid microextraction (DLLME) was selected as a technique for the preparation of honey samples.. The adequacy of acetonitrile as a dispersing agent was investigated. Besides the chloroform, a dichloromethane was used as a second extracting agent , in order to compare the relative efficiency of the extraction solvents. It can be concluded that the extraction recovery (68.4–92.1%) was more favorable with the use of 0.5 mL ACN and 2.0 mL DHM. Furthermore, LC-MS/MS parameters were optimized to unequivocally provide good chromatographic separation, low detection (LOD, 0.5–1.0 μg L−1) and quantification (LOQ, 1.0–2.5 μg L−1) limits for acetamiprid, clothianidin, thiamethoxam, imidacloprid, dinotefuran, thiacloprid and nitenpyram in honey samples. Using different types (chloroform, dichloromethane) and volumes of extraction (1.0–3.0 mL) and dispersive (acetonitrile; 0.0–1.0 mL) solvent and by mathematical modeling it was possible to establish the optimal sample preparation procedure. Matrix-matched calibration and blank honey sample spiked in the concentration range of LOQ–100.0 μg kg−1 were used to compensate the matrix effect and to fulfill the requirements of SANCO/12495/2011 for the accuracy (R 74.3–113.9%) and precision (expressed in terms of repeatability (RSD 2.74–11.8%) and within-laboratory reproducibility (RSDs 6.64–16.2%)) of the proposed method. The rapid (retention times 1.5–9.9 min), sensitive and low solvent consumption procedure described in this work provides reliable, simultaneous, and quantitative method applicable for the routine laboratory analysis of seven neonicotinoid residues in 15 real honey samples. Neonicotinoid residues were not detected in any of the investigated samples. The objective of next study was to develop and optimize HPLC-DAD analytical method with dispersive liquid-liquid microextraction (DLLME) and QuEChERS sample preparation procedures for the simultaneously analysis of seven neonicotinoids in honey samples. The liquid chromatographic conditions were optimized by response surface methodology with Box-Behnken design and the global Derringer´s desirability. The optimized method was validated to fulfill the requirements of SANCO/12495/2011 standard for both sample pretreatment procedures providing results for accuracy (R, 73.1–118.3%), repeatability (RSD, 3.28–10.40%) and within-laboratory reproducibility (RSD, 6.45–17.70%), limits of detection (LOD, 1.5–2.5 gµ kg-1) and quantification (LOQ, 5.0–10.0 µg kg-1). For the first time, more than 100 honey samples collected from all 7 counties of Autonomous Province of Vojvodina were analyzed. The presence of thiacloprid, imidacloprid and thiametoxam was discovered in a small number of samples. The objective of next study was to develop an optimized LC-MS/MS analytical method with DLLME and QuEChERS procedures for analysis of 7 neonicotinoids in honey liqueur. The method was validated to fulfill the requirements of SANCO/12495/2011 for both sample pretreatment procedures providing results for accuracy (R, 69.2–113.4% for DLLME; 71.8–94.9% for QuEChERS), precision (RSD expressed in terms of repeatability (3.21–10.20% for DLLME; 4.19–12.81% for QuEChERS) and within-laboratory reproducibility (9.11–16.63% for DLLME; 11.32–16.40% for QuEChERS)), limits of detection (LOD, 0.5–1.5 gµ L-1 for DLLME; 1.0–2.5 gµ L-1 for QuEChERS) and quantification (LOQ, 1.0–5.0 gµ L-1 for DLLME; 2.5–10.0 µg L-1 for QuEChERS). Analysis of real honey liqueur samples obtained from local markets showed the presence of clothianidin or thiacloprid in four of the analyzed samples, therefore implicating the necessity of ongoing control of this type of traditional product. Removal of selected neonicitinoid insecticides - dinotefuran, clothianidin and thiacloprid using MWCNT and H2O2 from Danube water matrix was investigated. Efficiency of different systems for neonicotinoids removal (under natural insolation in laboratory, with H2O2, with MWCNT, with MWCNT+ H2O2, with Fe-MWCNT, with Fe-MWCNT+H2O2) was evaluated with developed LC-MS/MS method. Analysis of degradation rates revealed loss of 25% of the initial neonicotinoid concentration under natural insolation in the laboratory conditions during 60 min. Addition of chemical agent H2O2 promoted loss of 40% of the initial dinotefuran, 70% of thiacloprid concentration and total removal of clothianidin under same conditions. With the addition of MWCNT concentration of dinotefuran, clothianidin and thiacloprid decayed for 30, 50 and 60%, respectively. Iron modification of MWCNT in combination with H2O2 increased the removal rate of selected neonicotinoid for 15–50%. Presence of intermediates was discovered in systems of dinotefuran with H2O2, MWCNT+H2O2, e-MWCNT+H2O2 and of clothianidin in systems with Fe-MWCNT+H2O2 with m/z of 117,5 and 140,6.