Dasharath P. Oulkar

Development and interlaboratory validation of a QuEChERS-based liquid chromatography-tandem mass spectrometry method for multiresidue pesticide analysis.

A high-throughput, QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method has been developed and validated for the determination of 191 pesticides in vegetation and fruit samples. Using identical LC analytical column and MS/MS instrumentation and operation parameters, this method was evaluated at the U.S. Food and Drug Administration (FDA), National Research Centre for Grapes (NRCG), India, and Ontario Ministry of the Environment (MOE) laboratories. Method validation results showed that all but 1 of these 191 pesticides can be analyzed by LC-MS/MS with instrument detection limits (IDL) in the parts per trillion (ppt) range. Matrix-dependent IDL studies showed that due to either the low ionization efficiency or matrix effect exerted, 14 of these 191 pesticides could not be analyzed by this method. Method recovery (%R) and method detection limits (MDLs) were determined by the three laboratories using four sample matrices in replicates (N = 4). With >79% of %R data from the fortification studies in the range from 80 to 120%, MDLs were determined in the low parts per billion range with >94% of MDLs in the range from 0.5 to 5 ppb. Applying this method to the analysis of incurred samples showed that two multiple reaction monitoring (MRM) transitions may not be enough to provide 100% true positive identification of target pesticides; however, quantitative results obtained from the three laboratories had an excellent match with only a few discrepancies in the low parts per billion levels. The %R data from the fortification studies were subjected to principal component analysis and showed the majority of %R fell into the cluster of 80% < %R < 120%. Due to the matrix effect exerted by ginseng and peach, outliers were observed at the lowest spiking levels of 10 and 25 ppb. The study also showed that QuEChERS samples should be analyzed as soon as prepared or stored in a freezer to avoid any adverse affect on the analytes evaluated.

Multiresidue analysis of 83 pesticides and 12 dioxin-like polychlorinated biphenyls in wine by gas chromatography-time-of-flight mass spectrometry.

A multiresidue method is described for simultaneous estimation of 83 pesticides and 12 dioxin-like polychlorinated biphenyls (PCBs) in red and white wines. The samples (20mL wine, acidified with 20 mL 1% HCl) were extracted with 10 mL ethyl acetate (+20 g sodium sulphate) and cleaned by dispersive solid-phase extraction (DSPE) with anhydrous calcium chloride and Florisil successively. The final extract (5 mL) was solvent exchanged to 1mL of cyclohexane:ethyl acetate (9:1), further cleaned by DSPE with 25mg primary secondary amine sorbent and analyzed by gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) within 31 min run time. The limits of quantification of most analytes were <or=10-20 microg/L. Acidification of wine prior to extraction prevented hydrolysis of organophosphorous pesticides as well as dicofol, whereas treatment with CaCl(2) minimized the fatty acid co-extractives significantly. Solvent exchange to cyclohexane:ethyl acetate (9:1) further minimized the co-extractives. Recoveries at 5, 10 and 20 ng/mL were >80% for most analytes except cyprodinil, buprofezin and iprodione. The expanded uncertainties at 10 ng/mL were <20% for most analytes. Intra-laboratory precision in terms of Horwitz ratio of all the analytes was below 0.5, suggesting ruggedness of the method. Effectively, the method detection limit for most analytes was as low as up to 1 ng/mL in both red and white wine, except for cyfluthrin and cypermethrin.

Multiresidue Determination and Uncertainty Analysis of 87 Pesticides in Mango by Liquid Chromatography―Tandem Mass Spectrometry

A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method was optimized and validated for the multiresidue analysis of 87 pesticides in mango at the ≤ 10 ng g(-1) level. The method involves extraction of 10 g of homogenized mango samples (+10 mL of water + 1 g of sodium acetate + 10 g of sodium sulfate) with 10 mL of ethyl acetate; cleanup by dispersive solid-phase extraction with a combination of primary secondary amine (PSA, 50 mg), graphitized carbon black (GCB, 25 mg), and anhydrous sodium sulfate (150 mg); and final estimation by LC-MS/MS with multiple reaction monitoring. Direct analysis (no clean up) resulted in significant suppression in ionization of the majority of the test compounds over the electrospray ionization probe. However, clean up with the above combination of PSA + GCB reduced the matrix-induced signal suppressions significantly, and the signals in the cleaned extracts were nearly equivalent to the corresponding solvent standards. Substitution of PSA with florisil also gave equivalent clean up effects. The method was quite rugged as evident from a low Horwitz ratio (mostly <0.5) and low measurement uncertainties at 10 ng g(-1). The limit of quantification was <10 ng g(-1) for all of the pesticides with recoveries within 70-120% for most pesticides even at 2.5 ng g(-1). The method offers a significantly effective, sensitive, cheaper, and safer alternative to the existing methods of multiresidue analysis.

Degradation kinetics and safety evaluation of tetraconazole and difenoconazole residues in grape

BACKGROUND Tetraconazole and difenoconazole are triazole fungicides with proven bioefficacy against grapevine powdery mildew disease. In the present work, the authors explored the residue dynamics of these two compounds in grapes and determined their preharvest intervals (PHIs) corresponding to multiple field applications at recommended and double rates considering the most critical use pattern in Indian viticulture. A confirmatory residue analysis method was validated for trace-level determination of both the compounds. RESULTS Dissipation of both the fungicides followed non-linear two-compartment first + first-order rate kinetics. Tetraconazole and difenoconazole dissipated with PHIs of 12.5 and 25.5 days at recommended rates and of 28.5 and 38.5 days at double application rates respectively. On all the sampling days, the residues were below the maximum permissible intake, indicating consumer safety. The residues in the grape samples drawn from the farms where these two fungicides were applied, maintaining the above PHIs, were below their respective MRLs. CONCLUSION The rate of degradation of tetraconazole was faster than that of difenoconazole. Thus, the growers will have the choice of using these new chemicals for the management of powdery mildews in succession, difenoconazole at early growth stages, followed by tetraconazole during the last month before harvest. The recommendations of PHIs proved to be effective in minimizing residues in farm grape samples. Thus, this work is of high significance to the grape industry of India, and will support the registration of these new fungicides for effective management of powdery mildews with minimum residue problems.

Protocol for an electrospray ionization tandem mass spectral product ion library: development and application for identification of 240 pesticides in foods.

Modern determination techniques for pesticides must yield identification quickly with high confidence for timely enforcement of tolerances. A protocol for the collection of liquid chromatography (LC) electrospray ionization (ESI)-quadruple linear ion trap (Q-LIT) mass spectrometry (MS) library spectra was developed. Following the protocol, an enhanced product ion (EPI) library of 240 pesticides was developed by use of spectra collected from two laboratories. A LC-Q-LIT-MS workflow using scheduled multiple reaction monitoring (sMRM) survey scan, information-dependent acquisition (IDA) triggered collection of EPI spectra, and library search was developed and tested to identify the 240 target pesticides in one single LC-Q-LIT MS analysis. By use of LC retention time, one sMRM survey scan transition, and a library search, 75-87% of the 240 pesticides were identified in a single LC/MS analysis at fortified concentrations of 10 ng/g in 18 different foods. A conventional approach with LC-MS/MS using two MRM transitions produced the same identifications and comparable quantitative results with the same incurred foods as the LC-Q-LIT using EPI library search, finding 1.2-49 ng/g of either carbaryl, carbendazim, fenbuconazole, propiconazole, or pyridaben in peaches; carbendazim, imazalil, terbutryn, and thiabendazole in oranges; terbutryn in salmon; and azoxystrobin in ginseng. Incurred broccoli, cabbage, and kale were screened with the same EPI library using three LC-Q-LIT and a LC-quadruple time-of-flight (Q-TOF) instruments. The library search identified azoxystrobin, cyprodinil, fludioxinil, imidacloprid, metalaxyl, spinosyn A, D, and J, amd spirotetramat with each instrument. The approach has a broad application in LC-MS/MS type targeted screening in food analysis.

Quantitative Screening of Agrochemical Residues in Fruits and Vegetables by Buffered Ethyl Acetate Extraction and LC-MS/MS Analysis

A buffered ethyl acetate extraction method is proposed for the simultaneous analysis of 296 agrochemicals in a wide range of fruit and vegetable matrices by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimized quantity of acetate buffer (1% acetic acid + 0.5 g of sodium acetate per 10 g of sample) adjusted the pH of each test matrix to 5-6, which in turn significantly improved recoveries of acidic and basic compounds. The role of diethylene glycol (used in the evaporation step) on signal suppression of certain compounds was evaluated, and its quantity was optimized to minimize such an effect. The method was validated in grape, mango, drumstick, bitter gourd, capsicum, curry leaf, and okra as per the DG-SANCO/12571/2013 guidelines. Recoveries in the fortification range of 1-40 μg/kg were within 70-120% with associated relative standard deviations below 20% for most of the compounds. The method has potential for regulatory and commercial applications with a generic approach.

Residue analysis of fipronil and difenoconazole in okra by liquid chromatography tandem mass spectrometry and their food safety evaluation.

A liquid chromatography tandem mass spectrometry (LC-MS/MS) based method is reported for simultaneous analysis of fipronil (plus its metabolites) and difenoconazole residues in okra. The sample preparation method involving extraction with ethyl acetate provided 80-107% recoveries for both the pesticides with precision RSD within 4-17% estimated at the limits of quantification (LOQ, fipronil=1ngg(-1), difenoconazole=5ngg(-1)) and higher fortification levels. In field, both the pesticides dissipated with half-life of 2.5days. The estimated pre-harvest intervals (PHI) for fipronil and difenoconazole were 15 and 19.5days, and 4 and 6.5days at single and double dose of field applications, respectively. Decontamination of incurred residues by washing and different cooking treatments was quite efficient in minimizing the residue load of both the chemicals. Okra samples harvested after the estimated PHIs were found safe for human consumption.

Rate of degradation of λ-cyhalothrin and methomyl in grapes (Vitis vinifera L.)

Rates of degradation of λ-cyhalothrin and methomyl residues in grape are reported. The dissipation behavior of both insecticides followed first-order rate kinetics with similar patterns at standard and double-dose applications. Residues of λ-cyhalothrin were lost with pre-harvest intervals (PHI) of 12.0–12.5 and 15.0–15.5 days, corresponding to the applications at 25 and 50 g a.i. ha−1, respectively. In the case of methomyl, residues were lost with PHI of 55.0 and 61.0 days, following applications at 1 and 2 kg a.i. ha−1, respectively. The PHI, recommended on the basis of the experimental results, was shown to be effective in minimizing residue load of these insecticides below their maximum residue limits (MRLs) in vineyard samples.

Residue dissipation and processing factor for dimethomorph, famoxadone and cymoxanil during raisin preparation.

A method was validated for the simultaneous analysis of the residues of dimethomorph, famoxadone and cymoxanil in grape and raisin matrix by ethyl acetate based extraction and liquid chromatography tandem mass spectrometric analysis. Field studies were conducted to evaluate the dissipation rate kinetics and processing factor (PF) for these pesticides during raisin making. Residue data during the drying process were best fitted to 1st+1st order rate kinetics with half-life ranging between 8-9 days for dimethomorph, 12-13 days for famoxadone and 9-10 days for cymoxanil at single dose (SD) and double dose (DD), respectively. PF values calculated were 1.03 and 1.14 for dimethomorph, 1.95 and 2.09 for famoxadone, and 1.99 and 1.35 for cymoxanil at SD and DD, respectively. PF value >1 indicates concentration of the residues during raisin making. The residues of detected pesticides in market samples of raisins were devoid of any risk of acute toxicity related to dietary exposure.

Biodegradation of Profenofos by Bacillus subtilis Isolated from Grapevines (Vitis vinifera)

The biodegradation of profenofos, an organophosphorus insecticide, by four Bacillus subtilis strains, namely, DR-39, CS-126, TL-171, and TS-204, isolated from grapevines or grape rhizosphere was studied in liquid culture, on grape berries, and in vineyard soil. Each of the four B. subtilis strains enhanced the degradation of profenofos in all three matrices. Degradation rate constants were best obtained by first + first-order kinetics module. In nutrient broth spiked with 5 μg/mL profenofos, inoculation with B. subtilis strains DR-39, CS-126, TL-171, and TS-204 reduced the half-life (DT50) of profenofos to 4.03, 3.57, 2.87, and 2.53 days, respectively, from the DT50 = 12.90 days observed in the uninoculated control. In Thompson Seedless grapes sprayed with profenofos at a field dose of 1250 mL ai/ha, the DT50 values were 1.07, 1.00, 2.13, and 2.20 days in grapes inoculated with B. subtilis strains DR-39, CS-126, TL-171, and TS-204, respectively, as compared to 2.20 days in uninoculated grapes. These four B. subtilis strains also enhanced the degradation of profenofos in autoclaved soil (DT50 = 5.93, 7.47, 6.00, and 4.37 days) and in nonautoclaved soil (DT50 = 0.87, 2.00, 2.07, and 2.43 days) amended with 5 μg/g profenofos from the half-lives of 17.37 and 14.37 days in respective uninoculated soils. Growth dynamic studies indicated that all four B. subtilis strains were able to establish and proliferate on berries and soil equally well in the presence or absence of profenofos. Degradation product 4-bromo-2-chlorophenol was identified by GC-MS. Strain DR-39 was most effective in the natural environments of grape and soil.