Simultaneous determination of pesticide residues and mycotoxins produced by Alternaria spp.

in fresh and processed fruit and vegetables Maria Rosaria Taurino*, Francesco Gallone

Agro.Biolab Laboratory S.p. 240 Km 13,800 - 70018 Rutigliano (Bari) ITALY

INTRODUCTION DISCUSSION RESULTS

Table 1. Alternaria mycotoxins

VISUAL ABSTRACT

METHODS AND MATERIALS CONCLUSIONS

REFERENCES CONTACT

In the framework of this project, we developed a QuEChERS-like analytical method [2] allowing the detection of mycotoxins from Alternaria and, simultaneously, the residues of plant protection products on tomato and tomato-derived matrices (dried tomatoes and tomato-based sauces) and grapes and its processed products (in particular juices). The method consists of a single extraction step followed by the appropriate analysis by liquid chromatography coupled with tandem triple quadrupole mass detectors. The selection of analysed pesticides includes the active ingredients typically found on these matrices, their metabolites and some active ingredients recognized as analytes difficult to determine. The full list of analytes on which we carried the validation procedure for this method is shown in table 2, in which are also reported the transitions monitored by LCMSMS as well as the classes of the different analytes. Noteworthy, both the original active ingredients and their metabolites, (as indicated in the residue definition) were analyzed. In case such as the assessment of spirotetramat or flonicamid on fresh and processed food it is very common to find sensibly higher concentrations of metabolites than of the active principle itself. The instrumental optimisation step was crucial, since analytes like tenuazonic acid, are very difficult to analyse. The optimal chromatographic conditions are obtained using a C18 column for liquid chromatography, with mobile phase A: 1 mM ammonium bicarbonate in water for HPLC/methanol 95: 5 and mobile phase B: Methanol. The extraction step does not envisage purification processes and the performance indicators of the method are optimal, for both toxins (limits of quantification values comprised between 5 and 10 µg/Kg) and for all pesticide residues (LQ comprised between 0,005 and 0,010 mg/kg). The lowest validated spike level meeting the requirements of a recovery within the range 70–120% and a RSDr < 20% was defined as method LOQ (Limit of quantitation) and the calculation was based on the accuracy and precision data, obtained via the recovery study. For calibration curves, in all cases the coefficient of determination (R2) obtained for pesticides and mycotoxins standard solutions prepared in matrix extracts were always above 0,99. Recoveries values for all analyzed mycotoxins and pesticides (calculated by spiking blank matrices of fresh and processed products) were in all cases comprised between 70% and 120%. The accuracy and precision of the method were evaluated through recovery experiments, by spiking with standard solutions a “blank” sample aliquot, at two different concentration levels, as shown in tables 3 and 4, with six replicates at each level. The “blank” samples were also analyzed six times.

The analytical method is fast, simple and rugged, and allows the simultaneous assessment of many classes of analytes, as shown in Table 1. The analytes are residues of pesticides from different chemical classes, their metabolites and mycotoxins. The method applied to real samples led to very interesting informations. In addition to typical pesticide residues found in particular on grapes and its derivatives, on fresh tomato samples, and in particular on transformed products (dried tomatoes and tomato sauces) high concentrations of tenuazonic acid, alternariol and alternariol monomethylether could be detected. Their concentrations, in the case of tenuazonic acid, ranged between 10 and 100 mg/kg, in particular on samples for which a field contamination of the product by Alternaria spp. had been ascertained. As an outlook for the potentialities of the proposed method, we can foresee the inclusion of other important toxins in the list of analytes. The method will be extended to patulin, typically found on fresh and processed food products, such as apples and fruit juices.

Chemicals and reagents Acetonitrile, water, methanol (CHROMASOLV® gradient grade > 99,9%), formic acid (analytical grade), ammonium formiate (analytical grade) anhydrous magnesium sulfate (99,9%), sodium chloride (>99,8%), sodium citrate tribasic dihydrate (>99.0%), and disodium citrate dibasic sesquihydrate (> 99.0%) were purchased from Sigma Aldrich. Buffer salt mixture was prepared weighing 4 g ± 0,2 g of magnesium sulfate anhydrous, 1 g ± 0,05 g of sodium chloride, 1 g ± 0,05 g of trisodium citrate dihydrate and 0,5 g ± 0,03 g of disodium hydrogencitrate sesquihydrate into a PTFE centrifuge tube. HPLC System: SHIMADZU Controller CBM20 A lite Integrated System; Pump: LC20AD; Autosampler model: SIL-20A /HT; Injection Volume: 10 µL; Flux: 0,3 mL/min. Detector: Triple Quadrupole LC-MS/MS Mass Spectrometer API 3200 AB Sciex Instruments. Sample extraction procedure In the optimized extraction procedure, an amount of 10 g of fresh homogenised samples was weighed into a 50 mL PTFE centrifuge tube. For dried sample, an amount of 2 g of weighed and 8,00 g of water was added. A volume of 10 mL acetonitrile and 100 µl of the procedure internal standard solution (about 5 mg/L) were added to the tube, which was shaken with a mechanical shaking machine for 15 minutes. Then, the buffer-salt mixture was added to the tube and an immediate vigorous manual shaking was performed, followed by mechanical shaking for 15 minutes and centrifugation for 5 minutes at > 3000 g. An aliquot of the raw extract was transferred into a vial and employed for LC-MS/MS analysis.

1. EFSA Journal 2011; 9 (10): 2407

2. M. Anastassiades, S. J. Lehotay, D. Stajnbaher and F. J. Schenck (2003), Journal of AOAC International, vol. 86, n° 2, pp. 412-431

3. Lilia Lohrey, Stefanie Marschik, Benedikt Cramer and Hans-Ulrich Humpf, J. Agric. Food Chem. 2013, 61, 114−120

Maria Rosaria Taurino Agro.Biolab Laboratory Email: qualita@agrobiolabitalia.it Phone: +39 080 477.07.62 Website: www.agrobiolabitalia.it

The need to obtain reliable, reproducible and complete analytical results in an always more rapid timeframe is becoming a “must” for laboratories providing food control services. In this respect, in addition to the detection of residues of plant protection treatments (normed by the 396/2005 Regulation) as well as to the mycotoxins (normed by the 1881/2006 Regulation), it has become mandatory, for foodstuff circulation within international markets, to assess the presence of other compounds, not yet regulated. This analysis can provide an index of quality and of proper application of good agricultural practices in foodstuff production. The Alternaria is a very common genus of microfungi which contaminates a large number of field crops causing major problems during the postharvest stage. The contaminated foods are cereals, several kinds of fruit (grapes, blueberries, peaches, cherries, citrus fruits such as lemons and oranges, apples) and vegetables (cucurbits, peas, onions, cabbages), causing: leaf spotting; fruit, bulb and blossom rotting; insurgence of blight. The Alternaria genus produces 71 known mycotoxins and phytotoxins. Important Alternaria toxins which contaminate food are: alternariol, alternariol monomethylether, tentoxin and tenuazonic acid. The aforementioned toxins are subject of high attention from the European food safety authorities [1], also for their characteristics in terms of toxicity. Alternariol (AOH) is cytotoxic, fetotoxic and teratogenic; alternariol monomethylether (AME) has necrotic, fetotoxic, teratogenic and mutagenic effects. Tenuazonic acid (TeA) possesses the highest toxicity among all Alternaria mycotoxins.

ANALITE Q1 Q3 DP EP CE CXP MODALITY CLASS OF CONTAMINANT 2,4-D 1 219 160,9 -19 -10 -14 -10 ESI NEGATIVE Auxins 2,4-D 2 219 124,9 -19 -10 -34 -26

B N O A 1 201 143 -19 -10 -14 -10 ESI NEGATIVE Auxins B N O A 2 201 115 -19 -10 -34 -26

BENTAZONE 1 239,1 132 -56 -10 -32 -10 ESI NEGATIVE Unclassified herbicides BENTAZONE 2 239,1 197 -56 -10 -24 -12

BIXAFEN 1 412 280 -85 -11 -30 -8 ESI NEGATIVE Anilide fungicides BIXAFEN 2 412 131 -85 -11 -35 -8

CHLORANTRANILIPROLE 1 484 203,9 -10 -5 -15 -10 ESI NEGATIVE Pyrazole insecticides CHLORANTRANILIPROLE 2 484 147,1 -10 -5 -40 -7

DICHLORPROP 1 233 161 -19 -10 -14 -10 ESI NEGATIVE Phenoxypropionic herbicides DICHLORPROP 2 233 125 -19 -10 -34 -10 DICLOSULAM 1 404 181 -16 -10 -22 -10 ESI NEGATIVE Sulfonanilide herbicides DICLOSULAM 2 404 153 -16 -10 -22 -10

DIFLUBENZURON 1 309 155,9 -26 -10 -12 -10 ESI NEGATIVE Benzoylphenylurea chitin synthesis inhibitors DIFLUBENZURON 2 309 288,9 -26 -10 -8 -16

FLUAZINAM 1 462,9 415,8 -9 -10 -24 -26 ESI NEGATIVE Pyridine fungicides FLUAZINAM 2 462,9 397,9 -9 -10 -20 -24

FLUDIOXONIL 1 247 125,9 -56 -10 -42 -14 ESI NEGATIVE Pyrrole fungicides FLUDIOXONIL 2 247 169 -56 -10 -42 -24

FORCHLORFENURON 1 246 91 -19 -10 -40 -3 ESI NEGATIVE Growth stimulators FORCHLORFENURON 2 246 127 -19 -10 -20 -2

LUFENURON 1 509 326 -35 -10 -20 -10 ESI NEGATIVE Benzoylphenylurea chitin synthesis inhibitors LUFENURON 2 509 339 -35 -10 -20 -10

MeptylDinocap 1 295,1 163 -25 -10 -51 -5 ESI NEGATIVE Dinitrophenol fungicides MeptylDinocap 2 295,1 134 -25 -10 -51 -5

METAFLUMIZONE 1 505,2 302,2 -20 -11 -20 -17 ESI NEGATIVE Unclassified insecticides METAFLUMIZONE 2 505,2 328 -20 -11 -20 -17

PROTHIOCONAZOLE 1 342 100 -15 -10 -15 -11 ESI NEGATIVE Conazole fungicides PROTHIOCONAZOLE 2 342 125 -15 -10 -20 -11

DINOSEB 1 239,1 134 -44 -10 -52 -10 ESI NEGATIVE INTERNAL STANDARD (Dinitrophenol herbicides) DINOSEB 2 239,1 193 -44 -10 -30 -12

SULFENTRAZONE 1 385 307 -69 -10 -30 -8 ESI NEGATIVE Anilide herbicides; triazolone herbicides SULFENTRAZONE 2 385 199 -69 -10 -45 -10

TFNA 1 190 69 -41 -8 -47 -11 ESI NEGATIVE Flonicamid metabolite TFNA 2 190 126 -41 -8 -32 -7 TFNG 1 247 183 -50 -10 -16 -11 ESI NEGATIVE Flonicamid metabolite TFNG 2 247 163 -50 -10 -21 -9

TRICLOPYR 1 253,9 195,8 -15 -10 -15 -11 ESI NEGATIVE Pyridine herbicides TRICLOPYR 2 253,9 217,8 -15 -10 -9 -11

TRIFLUMURON 1 357 154 -16 -10 -14 -10 ESI NEGATIVE Benzoylphenylurea chitin synthesis inhibitors TRIFLUMURON 2 357 175,9 -16 -10 -22 -10 Tenuazonic acid 1 196 138,9 -70 -10 -28 -7 ESI NEGATIVE Alternaria toxin Tenuazonic acid 2 196 112,1 -70 -10 -32 -5

Alternariol 1 257,1 215 -45 -10 -20 -3 ESI NEGATIVE Alternaria toxin Alternariol 2 257,1 146,7 -45 -10 -20 -3

Alternariol MME 1 271,1 256,2 -45 -10 -20 -3 ESI NEGATIVE Alternaria toxin Alternariol MME 2 271,1 228,2 -45 -10 -20 -3

Tentoxin 1 413,5 271,2 -45 -11 -20 -3 ESI NEGATIVE Alternaria toxin Tentoxin 2 413,5 214,8 -45 -11 -20 -3

ACETAMIPRID 1 223 126 34 12 27 6 ESI POSITIVE Pyridylmethylamine neonicotinoid insecticides ACETAMIPRID 2 223 90,1 34 12 45 4

AZOXISTROBIN 1 404,4 372,5 25 10 21 4 ESI POSITIVE Methoxyacrylate strobilurin fungicides AZOXYSTROBIN 2 404,4 344,7 25 10 37 4

BOSCALID 1 343,1 307,1 55 5 29 4 ESI POSITIVE Anilide fungicides BOSCALID 2 343,1 140,1 55 5 29 3

CARBENDAZIM 1 192,1 160 27 11 25 3 ESI POSITIVE Benzimidazole fungicides CARBENDAZIM 2 192,1 132 27 12 43 3

CHLOTHIANIDIN 1 250,1 169,1 22 8 20 3 ESI POSITIVE Neonicotinoid insecticides; thiametoxam metabolite CHLOTHIANIDIN 2 250,1 131,9 22 8 20 3 CYFLUFENAMID 1 413 295 20 8 21 14 ESI POSITIVE Amide fungicides CYFLUFENAMID 2 413 359 20 8 18 17

CYMOXANIL 1 199,1 111,1 15 8 26 6 ESI POSITIVE Aliphatic nitrogen fungicides CYMOXANIL 2 199,1 128,2 15 8 13 6 CYPRODINIL 1 226,1 93,3 62 7 55 4 ESI POSITIVE Anilinopyrimidine fungicides CYPRODINIL 2 226,1 77,3 59 7 67 5

DELTAMETRINA 1 523,1 280,6 25 5 25 4 ESI POSITIVE Pyrethroid ester insecticides DELTAMETRINA 2 523,1 181,3 25 5 63 3

DIFENOCONAZOLE 1 406,1 251,1 30 12 38 5 ESI POSITIVE Conazole fungicides DIFENOCONAZOLE 2 406,1 337,1 20 9 25 5 DIMETHOMORPH 1 388,1 301,1 41 10 27 3 ESI POSITIVE Morpholine fungicides DIMETHOMORPH 2 388,1 165 41 10 43 3

DIMETOATE 1 230 125 14 10 29 3 ESI POSITIVE Organothiophosphate insecticides and nematicides DIMETOATE 2 230 198,8 14 10 13 3 FLONICAMID 1 230 203 70 10 26 9 ESI POSITIVE Unclassified insecticides FLONICAMID 2 230 148 70 10 37 6

LINURON 1 249,1 159,9 30 10 26 7 ESI POSITIVE Phenylurea herbicides LINURON 2 249,1 181,9 30 10 26 7

METALAXYL 1 280,1 220 49 12 19 12 ESI POSITIVE Anilide fungicides METALAXYL 2 280,1 159,9 49 11,5 31 8

PROPAMOCARB 1 189,2 102 16 10 23 4 ESI POSITIVE Carbamate fungicides PROPAMOCARB 2 189,2 144 16 10 17 6

PROPARGITE 1 368,1 175,1 10 9 21 3 ESI POSITIVE Sulfite ester acaricides PROPARGITE 2 368,1 231,2 10 9 17 3

PYRACLOSTROBIN 1 388,1 194 9 7 19 10 ESI POSITIVE Carbanilate fungicides PYRACLOSTROBIN 2 388,1 163 9 6,5 29 6

SPINOSIN A1 732,5 142,1 49 11 37 3 ESI POSITIVE Spinosyn insecticides SPINOSIN A2 732,5 98,3 20 12 96 3 SPINOSIN D1 746,5 142,2 61 11,5 39 3 ESI POSITIVE Spinosyn insecticides SPINOSIN D2 746,5 98,1 61 10,5 79 3

SPIROTETRAMAT 1 374,3 216,2 40 10 45 14 ESI POSITIVE Tetramic acid insecticides SPIROTETRAMAT 2 374,3 302,3 40 10 24 14

SPIROTETRAMAT ENOL 1 302 216 79 9 40 3 ESI POSITIVE Spirotetramate metabolite SPIROTETRAMAT ENOL 2 302 270 79 9 36 5 SPIROTETRAMAT ENOL

GLUCOSIDE 1 464 302 40 10 30 3 ESI POSITIVE Spirotetramate metabolite

SPIROTETRAMAT ENOL GLUCOSIDE 2 464 270 40 10 50 3

SPIROTETRAMAT KETHOYDROXY 1 318 300 58 10 24 5 ESI POSITIVE Spirotetramate metabolite

SPIROTETRAMAT KETHOYDROXY 2 318 268 58 10 32 4

SPIROTETRAMAT MONOHYDROXY 1 304 254 68 10 30 4 ESI POSITIVE Spirotetramate metabolite

SPIROTETRAMAT MONOHYDROXY 2 304 211 68 10 30 3

TEBUTAM 1 234,2 91 21 10,5 29 6 ESI POSITIVE INTERNAL STANDARD (amide herbicides) TEBUTAM 2 234,2 192 21 10,5 19 10

THIAMETHOXAM 1 292 211 20 10 15 3 ESI POSITIVE Nitroguanidine neonicotinoid insecticides THIAMETHOXAM 2 292 181 54 8,5 31 3

Toxin Name Formula CAS

Alternariol 3,7,9-Trihydroxy-1-methyl-6H-dibenzo[b,d]pyran-6-one

!

! !!

!

641-38-3

Alternariol monomethyl

ether

3,7-Dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one

!

!

!!

!

23452-05-3

Tentoxin Cyclo[N-methyl-L-alanyl-L-leucyl-

(αZ)-α,β-didehydro-N-methylphenylalanylglycyl]

!

! !!

!

28540-82-1

Tenuazonic acid

(5S)-3-Acetyl-1,5-dihydro-4-hydroxy-5-[(1S)-1-methylpropyl]-

2Hpyrrol-2-one

!

! !!

!

610-88-8

!

Chromatograms of tenuazonic acid showing monitored fragments ions (fresh tomato)

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Chromatograms of Alternariol monomethyl ether showing monitored fragments ions (fresh tomato)

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Chromatograms of Alternariol showing monitored fragments ions (fresh tomato)

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Chromatograms of Tentoxin showing monitored fragments ions (fresh tomato)

Alternaria alternata spores

Fresh and processed food products typically contaminated by Alternaria toxins and pesticide residues

Tomato plant affected by Alternaria

Chromatograms of DINOSEB (dinitrophenol herbicide, used as internal standard) showing monitored fragments ions (fresh tomato)

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Table 2. Analites, analytical parameters and class of contaminant

Tables 3 and 4. Analytical performances obtained using the described method on four different matrices: tablegrapes, fruit

juice, tomato and dried tomatoes