Plant Protection Products Residues Assessment in the Organic and Conventional Agricultural Production

Abstract

The organic food is progressively enticing purchasers’ attention, as it is recognized to be better than the food produced by the conventional agriculture and more sustainable for the natural environment. Pesticides and their metabolites can enter the human body via food and water. In the food production, over 60 thousand chemical agents are applied, while 90% of the harmful substances are consumed. The organic production is based on the qualitative and healthy food using the natural resources in an ecologically sustainable way. The European Regulations set the maximum pesticide levels (MRLs) in the organic products, which are also regulated by The United States Department of Agriculture in their National program supported by The United States Environmental Protection Agency. It is imperative to bear in mind that in the products from the organic production, the multiple detections cannot be tolerated, i.e., that one product cannot contain more than two detected pesticide residues. In this paper, a multi-residue pesticide method has been developed to determine the pesticides in the agricultural products from the organic and conventional production. In this work, 60 pesticides were analyzed using a simple QuEChERS sample preparation procedure, followed by LC-MS/MS. The tomato, potato, apple, and carrot samples from the organic and conventional products were collected from the market and the pesticide residues assessment comparing the organic to the conventional was done.

1. Introduction

The organic food is increasingly attracting the interest of the consumers, as it is perceived to be healthier than the food produced by the conventional agriculture and more sustainable for the environment [1].

It is well-known that the pesticides and their metabolites can be brought into the human body through food and water. There are many efforts from the EU to achieve sustainable use of these compounds to avoid the increase of pesticide levels in the environment and food [2]. Today, in the food production, over 60 thousand chemical agents are used, whereas 90% of the harmful substances are taken with food. However, the increased use of pesticides is of concern to the agricultural workers and food consumers and threatens the environment [3]. That is why, in the last decades, there have been the growing interest and demand for the organic production [4,5,6,7,8]. The organic production is based on the qualitative and healthy food through the use of natural resources in an ecologically sustainable way [9,10,11]. This way of the agricultural production, different from the conventional, eliminates the application of the pesticides, growth regulators, synthetic minerals, hormones, fertilizers, antibiotics and additives [1,11]. Additionally, the use of genetically modified organisms is forbidden [12,13]. The ban on the synthetic chemical formulations, which are frequently used in the conventional production for the control of weeds, pests and diseases, represents the greatest problem for the organic food producers [14]. The pesticide contamination of organic products can be induced due to the use of water and soil with pesticide residues [15].

The occurrence of pesticide residues in the organic fruit and vegetables is not enough stated in the scientific literature [16,17,18]. Tobin et al. [19] detected one or more pesticide residues in 15 out of 27 tested organic samples, with one pesticide being above the LOQ (imazalil in organic onion, 0.11 mg/kg). In the case of the conventional samples, the pesticide residues were present in 17 out of 27 samples in total, with 12 of them being above the LOQ with the concentrations between 0.01 and 0.154 mg/kg. Out of 136 tested organic samples, the authorized pesticide residues were detected in 4 samples, while the non-authorized pesticides were discovered in 61 samples, which was in accordance with the study from Ireland. Namely, the authors detected the pesticide residues in 15 out of the 27 tested organic samples [2].

The studies conducted in Belgium (1995–2001) determined the presence of the pesticide residues in 12% of organic food samples and 49% of the conventional food samples. The monitoring of the German market from Baden-Wűrttemberg (2002–2009) showed that 88% of the conventional raw materials and 27% of organic product samples contained the pesticide residues. The contamination of the organic crops in some European countries is determined as follows: the Czech Republic 14%, Ireland 11%, Finland 5%, Denmark 3% and New Zealand 22% [20,21]. This statistic is also shown in the last EFSA report [22], where organic food encompassed 6.5% of the total samples [23].

It is important that no specific MRLs are established for the organic food produced in accordance with Regulation (EC) No 2018/848 [24]. The MRLs set in Regulation (EC) No 396/2005 [25] apply to the conventional foodstuff.

The maximum residue limits (MRL) in the foodstuff, which represent the maximum residue concentration allowed in the food agricultural commodity, are being controlled by the established legislative framework. Being in accordance with the MRLs is now an obligatory norm for the food security. Depending on the country and the particular commodity, the MRLs can vary, which can be noted in the online databases that contain the summary of their regulatory status in the world [26,27,28].

We are not able to claim that the organic crops do not contain pesticide residues, as well as that they are truly produced according to the good agricultural practice in the organic production, since the products which authenticity of organic origin cannot be confirmed may be found everywhere throughout the market. There is no doubt that the organic products lack the certification, the continuous supply and a proper retail space, while the consumers rightfully expect the certification, quality and product attributes according to their price. Therefore, the aim of this case study was to compare the detected pesticide residues in organic fruit and vegetable samples with those from the conventional production. For this purpose, a monitoring study was conducted based on 92 commercial samples from the conventional (50) and the organic (42) products from 4 different commodity groups (tomato, potato, apple and carrot). The pesticide residues were analyzed using a simple QuEChERS sample preparation procedure, followed by the liquid chromatography coupled with tandem quadrupole mass spectrometry (LC-MS/MS).

2. Materials and Methods

Chemicals and reagents: Acetonitrile and methanol (HPLC grade) were purchased from J.T.Baker (Deventer, Netherlands), acetone was purchased from Merck (Kenilworth, NJ, USA). The QuEChERS extract tubes (Par No. 5982-5650), as well as the dispersive SPE 15 mL kits for fruits and vegetables, EN (Part No. 5982-5056), were purchased from Agilent Technologies (Santa Clara, CA, USA). The water was purified by Mili-Q plus system from Millipore (18.2 MΩ–cm, A10 FOCN53824k, USA). The pesticides (60 active substances) and internal standards (IS, carbofuran-D3 and acetamipride-D3) were obtained from Dr. Ehrenstorfer (Munich, Germany) and Sigma Aldrich (Schnelldorf, Germany) and were prepared in acetone, methanol, or acetonitrile (depending on the solubility of the compound) at the concentration nearest to 1.0 mg/mL. Stock solutions were used to prepare working standard solutions (the mix of 60 pesticide active substances in acetonitrile at 1 and 10 µg/mL) for the calibration. The calibration curves were prepared in the mobile phase as well as matrix-matched calibration (MMC) used in order to minimize the matrix effects because matrix constituents may increase or decrease the analytical signal. MMC was prepared for each matrix separately, namely for tomato, potato, apple and carrot. For obtaining the analytical curves in the solvent and matrix (recovery calibration) the concentration ranged from 0.005 to 0.10 µg/mL.

Sample collection: Tomato, potato, apple and carrot samples from the organic and conventional production for multi-pesticide residues quantification were collected from the Serbian largest cities open markets (Belgrade, Novi Sad, Subotica, Niš, Kragujevac and Čačak) (

Table 1. Number of samples from organic and conventional production.

Commodity Group	Organic Production	Conventional Production
Tomato	10	10
Potato	9	11
Apple	18	21
Carrot	5	8
Total	42	50

) according to SANTE/12682/2019. Randomly sampled units in the amount of 1 kg were rapidly (within one day) transported in the polypropylene bags in the clean containers to the laboratory for the homogenization. In case of each sample the information considering the market location, purchase date and variety has been recorded. Until the moment of the preparation and the analysis, which were carried out within 3 days from the purchase date, the samples were stored at 4 °C.

Samples extraction and clean-up procedures: The agricultural samples were extracted by the QuEChERS method described by Anastassiades et al. [15] and Bursić et al. [29]. For the extraction, the homogenized samples (10.0 g) were weighed into a polypropylene centrifuge tube (50 mL) and spiked with 100 µL of ISs. Next, 10 mL of acetonitrile were added, and the mixture was shaken vigorously for 1 min using a vortex mixer. A liquid-liquid partitioning step was performed by adding the QuEChERS extraction kit to the tube and the solution was stirred again for 1 min. After that the mixture was centrifuged for 5 min (at 4000 rpm–1900 g). After the centrifugation, the clean-up step was done based on which an aliquot of 6 mL was transferred to a 15 mL polypropylene centrifuge tube containing dispersive SPE kits for fruits and vegetables. The extract was vigorously shaken for 1 min and centrifuged for 5 min at 4000 rpm (1900 g). Finally, an aliquot of supernatant was filtrated through a PTFE 0.45 µm filter and transferred to a vial followed by injecting into the LC-MS/MS.

LC-MS/MS analysis: The detection and quantification were performed by the liquid chromatography tandem mass spectrometry equipped with the electrospray ionization (LC(ESI)-MS/MS), 6410B Agilent Technologies. In terms of chromatographic conditions, a Zorbax Eclipse XDBC18 column (50 mm × 4.6 mm id 1.8 µm) was used and kept at 25 °C. The mobile phase consisted of the gradient using methanol with 0.1% formic acid (solvent A) and 0.1% formic acid in water (solvent B), with the following gradient: 0 min−90% B; 2 min−90% B; 15 min 20% B; 20 min−15% B; 25 min−5% B and then returning to the initial conditions in 5 min. The total run time was 30 min. The flow rate of the mobile phase was 0.4 mL/min and the volume of 5 µL of sample extract was injected into the column. In terms of mass spectrometry, the MS source temperature was set at 350 °C, nitrogen gas flow 10 L/min and nebulizer pressure 40 psi. The data acquisition in the multiple reaction monitoring mode (MRM) was optimized after direct infusion of each pesticide. The instrument uses MassHunter software (vB.06.00, Agilent Technologies, Santa Clara, CA, USA) for the acquisition and quantification [30].

Method validation: All the validation parameters were evaluated following the Document N° SANTE/12682/2019 [31]. The analytical curves linearity was evaluated by injecting the analytical solutions prepared in the solvent and the matrix (tomato, potato, apple and carrot–matrix match calibration-MMC) at 0.005, 0.01, 0.05 and 0.1 µg/mL. The recovery was obtained by spiking the samples with a known amount of the mixture solution in the concentration range at 0.005 and 0.1 mg/kg. For each concentration five replicates were performed. The limit of detection (LOD) was approximated in the MRM mode analysis as the lowest concentration level that yielded a signal-to-noise ratio S/N ratio greater than 5. The limit of quantification (LOQ) of the method was set on 0.005 mg/kg as the most common default LOQ value for pesticide residues, i.e., which is below the MRLs for most pesticides in food [32].

3. Results and Discussion

The fragmentation of the protonated molecular ion obtained by LC-MS/MS in the positive electrospray ionization (ESI+) of the examined pesticides is given in

Table 2. MRM transitions, fragmentation, and collision energies.

Pesticide	Molecular Formula	M g/Mol	Precursor ion m/z	Product ion m/z	Frag (V)	CE (V)	Rt (min)
Acetamiprid	C10H11ClN4	223	223.0 223.0	125.8 55.7	120 120	10 10	11.45
Azoxystrobin	C22H17N3O5	403	404.1 404.1	372.0 344.1	100 100	9 25	13.17
Aldicarb	C7H14N2O2S	190	213 213	116 89	120 120	10 15	13.90
Azinphos-ethyl	C12H16N3O3PS2	346	346 346	132 77.1	120 120	16 16	6.20
Bitertanol	C20H23O2N3	337.4	338 338	145 117	120 120	20 30	7.68
Dimethomorph	C21H22ClNO4	388.1	388.1 388.1	301.1 165	120 120	30 20	17.30
Epoxiconazole	C17H13ClFN3O	329.7	330.1 330.1	121 101	130 130	21 50	18.13
Ethiofencarb	C11H15NO2S	225.3	226.1 226.1	164.1 107	80 80	5 5	14.95
Fenarimol	C17H12Cl2N2O	331.2	331 331	268 81	80 80	10 25	18.40
Fenoxycarb	C17H19NO4	301.4	302.1 302.1	116.1 88	100 100	5 20	18.30
Fenpropathrin	C22H23NO3	349. 4	350.1 350.1	125 97	135 135	24 34	7.51
Fenpropimorph	C20H33NO	303.5	304.2 304.2	147.1 57.2	120 100	30 28	5.53
Fluroxypyr-meptyl	C15H21Cl2FN2O3	367.2	367 367	254.9 181	80 80	11 32	7.44
Flusilazole	C16H15F2N3Si	315.4	316.1 316.1	247.1 165	110 110	12 20	18.21
Flutriafol	C16H13F2N3O	301.2	302.1 302.1	70.2 123.1	100 100	18 29	5.24
Phoxim	C12H15N2O3PS	298.3	299 299	129 77	80 80	10 20	17.52
Hexaconazole	C14H17Cl2N3O	314.2	314.1 314.1	159 70.1	100 130	20 17	18.80
Imazalil	C14H14Cl2N2O	297.1	297.1 297.1	255 159	100 100	15 23	14.80
Imidacloprid	C9H10ClN5O2	255.7	256 256	208.7 174.6	100 100	15 20	11.60
Indoxacarb	C22H17ClF3N3O7	527.8	528.1 528.1	203 150	120 120	36 16	18.80
Isoproturon	C12H18N2O	206.3	207 207	78 123	135 135	17 17	12.40
Carbaryl	C12H11NO2	201.2	202.1 202.1	145 127	100 100	10 35	15.50
Carbendazim	C9H9N3O2	191.1	192.1 192.1	160.1 132	104 104	18 34	9.35
Carbofuran	C12H15NO3	221.2	222.1 222.1	165.1 123	90 90	20 15	15
Carboxin	C12H13NO2S	235.3	236 236	87 143	120 120	20 20	6.19
Carbosulfan	C20H32N2O3S	380.5	381.2 381.2	118.1 160.1	31 31	33 22	5.52
Clothianidin	C6N5H8SO2Cl	249.6	250 250	169.1 132.1	90 90	10 15	11.80
Kresoxim-methyl	C18H19NO4	313.3	336.2 336.2	246.2 229.2	120 120	15 15	18.40
Quintozene	C6Cl5NO2	295.3	237 237	143 119	30 30	10 10	13.61
Myclobutanil	C15H17ClN4	288.7	289.2 289.2	125.1 70.2	150 150	20 15	17.78
Linuron	C9H10Cl2N2O2	249.0	249 249	182 160	70 70	18 18	9.72
Malathion	C10H19O6PS2	330.3	331.1 331.1	127 99	90 90	5 21	17.6
Metalaxyl	C15H21NO4	279.3	280.2 280.2	220.1 192.1	120 120	10 15	16.3
Metamitron	C10H10N4O	202.2	203.1 203.1	175 104	115 115	14 22	12.78
Methidathion	C6H11N2O4PS3	302.3	303 303	165 127	120 120	10 20	12.76
Methiocarb	C11H15NO2S	225.3	226.1 226.1	169 121	62 62	6 18	17.36
Metconazole	C17H22ClN3O	319.8	320 320	125 70	100 100	20 20	18.88
Methoxyfenozide	C22H28N2O3	368.5	369.2 369.2	149.1 133	100 90	20 25	17.2
Methomyl	C5H10N2O2S	162.2	163.1 163.1	106 88	80 80	5 5	9.8
Nicosulfuron	C15H18N6O6S	410.4	411 411	182 106	100 100	32 32	4.57
Oxadixyl	C14H18N2O4	278.3	279.1 279.1	219.1 133.3	80 80	10 15	14.35
Oxamyl	C7H13N3O3S	219.2	237.1 237.1	90 72	60 60	5 10	9
Pencycuron	C19H21ClN2O	328.8	329.1 329.1	125.1 99.1	120 130	38 35	17.62
Pymetrozine	C10H11N5O	217.2	218 218	105 78	120 100	30 20	3.61
Pyraclostrobin	C19H18ClN3O4	387.8	388.1 388.1	194 163	100 100	10 10	18.6
Pyrimethanil	C12H13N3	199.2	200.1 200.1	107.1 82.1	136 136	26 30	16
Pirimiphos-methyl	C11H20N3O3PS	305.3	306 306	164 108	20 20	20 39	7.49
Pirimicarb	C11H18N4O2	238.2	239.2 239.2	182.1 72	120 120	15 20	12
Pyriproxyfen	C20H19NO3	321.3	322.1 322.1	227.1 185.1	120 120	10 10	20
Prochloraz	C15H16Cl3N3O2	376.6	376 376	308 266	80 80	10 10	18.39
Propamocarb	C9H20N2O2	188.2	189.1 189.1	102 144	120 100	20 20	1.82
Propiconazole	C15H17Cl2N3O2	342.2	342.1 342.1	159 69	120 120	20 20	18.60
Propyzamide	C12H11Cl2NO	236.3	256.1 256.1	190 173	120 120	23 31	5.98
Propoxur	C11H15NO3	209.2	210.1 210.1	168.1 111	60 60	5 10	15.10
Spiroxamine	C18H35NO2	297.4	298 298	144 100	120 100	32 20	5.44
Tebufenpyrad	C18H24ClN3O	333.8	334.2 334.2	145.1 117	175 175	24 32	19.70
Tebuconazole	C16H22ClN3O	307.8	308.1 308.1	125 70	100 100	25 25	18.58
Tefluthrin	C17H14ClF7O2	418.7	177 177	137 127	10 10	15 15	14.99
Thiodicarb	C10H18N4O4S3	354.4	355.1 355.1	108 88	80 80	10 15	15.50
Thiacloprid	C10H9ClN4S	252.7	253 253	186 126	110 110	10 20	13.40
Trifloxystrobin	C20H19F3N2O4	408.3	409.1 409.1	206.1 186.1	120 120	10 15	18.95

. The selected reaction monitoring mode (SRM) was carried out to obtain the maximum sensitivity for each pesticide detection, while the confirmation of pesticides, two SRM transitions and a correct ratio between the optimized SRM transitions abundance were used taking into account the matching of the Rt (pesticide retention time).

The obtained results indicate a good response linearity in the range of 0.005 to 0.1 µg/mL for all the investigated analytes. Therefore, the method is selective, showing good linearity, expressed by the values of determination coefficient (r²)>0.99 for all 60 pesticides. The matrix effect (ME) was estimated on matrix and solvent calibration graph slopes and it indicated that tomato, potato, apple and carrot matrix have a strong influence on 60 pesticides. The ME was compensated with MMC.

The LOQ as the lowest concentration that will be detected and quantified by an outstanding analytical method with sufficient precision and accuracy was established on 0.005 mg/kg for every pesticide and was confirmed experimentally. The LODs were calculated by MassHunter software and all the values were in the range of 0.001 to 0.003 mg/kg.

The recovery studies were appraised at two levels, spiking blank tomato, apple, carrot and potato samples at 0.01 and 0.1 mg/kg in five replicates (Figure 1). The 53 out of 60 analyzed pesticides showed the recovery ranging from 67.4 to 118.5%. The obtained results are in accordance with those published by Mao et al. [18], whose values for recovery varied from 61.6 to 119.4%. The repeatability, expressed as a relative standard deviation (%RSD), was between 1.87 and 14.73%. Broadly, the accuracy and precision results were tolerable to all investigated pesticides, according to the Document N° SANTE/12682/2019 [31].

According to the validation parameters, LC-MS/MS is a suitable technique for the qualitative and quantitative analysis of 60 pesticide residues in selected matrices-samples. TIC and MRM chromatograms of the pesticides determined in the apple samples from the organic production are given in Figure 1 and Figure 2.

The results presented in Figure 3 and Figure 4 show the pesticide residues in the investigated samples from the organic (Figure 3) and conventional production (Figure 4) with no detections (meaning<LOD), the samples with the determinations below LOQ, the determinations compliant with the MRLs and the determinations exceeding the MRLs.

The EU-harmonized MRLs are set for more than 500 pesticides covering 370 food products/food groups. A default MRL of 0.01 mg/kg is applicable for pesticides not explicitly mentioned in the MRL legislation. The Regulation (EC) No 396/2005 [25] imposes on the Member States the obligation to carry out the controls to ensure that the food placed on the market is compliant with the legal limits. For the organic food items produced following Regulation (EC) No 834/2007 [33] no specific MRLs are established. However, in the Regulation (EC) No 396/2005 [25] in Article 18 it is stated that 0.01 mg/kg is the MRL value for those products for which no specific MRL is set out in Annexes II or III, or for the active substances not listed in Annex IV. The value of 0.01 mg/kg is the usually accepted MRL for organic products. According to Regulation (EC) No 834/2007 [33], the plant protection products should only be used if they are compatible with the objectives and principles of the organic production following the provisions laid down in Article 16(3)(c). Regulation (EC) No 889/2008 [20] lays down the detailed rules for the implementation of Council Regulation (EC) No 834/2007 [33] on organic production and labelling of the organic products. It defines the restricted list of plant protection products that may be used in the organic farming. Most of these substances are exempted from the setting of legal limits under Regulation (EC) No 396/2005 [25], as these substances are listed in Annex IV of the MRL regulation. The EOCC (European Organic Certifiers Council) is an organization of the organic certifiers in Europe. The EOCC has formed a “task force residues”, which developed the “EOCC pesticide residues guideline”, and presented it to the public in 2012. This guideline also follows the BNN (Bundesverband Naturkost Naturwaren) concept of the orientation value of 0.010 mg/kg, but the value is called ’action level’. This guideline emphasizes the procedural aspects in which certifiers should handle pesticide residues. Together with this guideline, the ’EOCC task force residues’ has also published a discussion paper in which the possibilities of applying a maximum pesticide level for the organic products are discussed [34]. This maximum level is called ’critical level’. The task force proposed that the critical level might be set at the value of 10% of the MRL, but does not insist on this particular value. It is extremely important to bear in mind the fact that in the products from the organic production the multiple detections cannot be tolerated, i.e., that one product cannot contain more than two detected pesticide residues concerning the BNN.

The most detected pesticides from the conventional production were fluopyram, difenoconazole, metalaxyl, pyrimethanil, azoxystrobin, boscalid, cyprodinil, pyraclostrobin and delthametrine. The concentrations were in the range from 0.003 to 0.154 mg/kg. In the samples from the organic production the most frequently detected were fluopyram, difenoconazole, azoxystrobin, boscalid and cyprodinil.

According to Montiel-León et al. [35] the pesticides of great concern these days imply carbamates, neonicotinoids, organophosphates and triazines.

The similar results to those obtained in our case study were published by Mao et al. [18], where the conventional vegetable samples contained multiple pesticide residues compared with those in the organic vegetable samples and most of these residues were detected at higher levels in the conventional than in the organically produced vegetables.

According to Mansour et al. [36], the organic potato tubers sampled from the market have had higher pesticide residue levels than those collected from a specific organic farm. Therefore, along with our results, these findings may give an indication that the data obtained from a single supervised farm may not reflect the market quality where the products from the different agricultural producers could be found. Although the pesticide residues uptake from soils depended on plant variety, the preparation of the products for sale on the market could have a significant influence. For example, Zohair et al. [37] emphasized that washing and peeling carrots or potatoes removed 52–100% of the contaminant residues, which also varied with the crop type and the contaminant amount and properties.

Considering the fact that our samples were taken simultaneously during a week in April, the interesting fact that should not be neglected is the seasonal dynamic of pesticide residue levels. According to Mansour et al. [36], the highest pesticide residue peaks in the conventional potato production were noticeably raised in August, December, February and April, and for the organic potatoes in September. The total pesticide contamination level showed different arrangements: winter > summer > fall > spring in the conventional and fall > summer > winter > spring in the organic potato production.

The tomato, carrot and potato samples are considered to be the organic products based on the pesticide residues. However, the analyzed organic apple sample contained six pesticide residues, with the pyrimethanil and pyraclostrobin residues above the MRLs (for the conventional production) of 0.05 and 0.02 mg/kg, respectively. This sample cannot implement the state established in SANTE/11945/20, as well as IFOAM [38], which allows the pesticide residue detection concerning the measurement of the uncertainty of 50% because we have detections of six pesticide residues.

The apple samples from the conventional production contained four pesticide residues, with azoxystrobin concentration over MRL of 0.01 mg/kg [32]. The conventional tomato and potato did not contain pesticide residues, all detections were under the LOQs. The carrot sample contained fluopyram and difenoconazole with residues being below the MRLs.

Montiel-León et al. [39] conducted the research on 37 samples of apples and determined that 57% of the tested samples contained at least one of the studied pesticides. The most common detected pesticide was acetamiprid, with the detection frequency being 41% and the maximum concentration of 24 μg/kg in the case of the Cortland apple, which was sampled from the conventional production. They also detected carbendazim (detection frequency of 19%), carbaryl (3%) and simazine (5%), as well as some other neonicotinoids: clothianidin (detection frequency of 3%), imidacloprid (16%) and thiacloprid (5%). Their research also comprised the analysis of the tomato samples, the results of which showed that 17% of the tested samples contained at least one of the studied pesticides, all of which were classified as neonicotinoids. The acetamiprid was detected in one sample (detection frequency of 3%) at 16 μg/kg, dinotefuran was found in two samples (concentrations of 13 and 20 μg/kg), while the imidacloprid was registered in 10% of the tested tomato samples (concentrations of 7.6, 10 and 11 μg/kg).

The analysis of the pesticide residues in food is subject to constant modification owing to matrix complexity, low concentrations of the compounds of interest and the increasing number of pesticides approved for use [40]. Namely, LC coupled with a QQQ tandem mass spectrometer, working in the multiple reaction monitoring (MRM) mode is the most frequently applied platform used in the analysis of pesticide residues in food. The most important advantages of validated LC-MS/MS in this study include high sensitivity and selectivity, short duration of analysis, which enables the separation and determination of a considerable number of compounds (60 pesticides with internal standard) during a single analytical run. The obtained results indicate good response linearity in the range of 0.005 to 0.1 µg/mL for all 60 pesticides (r²)>0.99. The MMC reduces the matrix effect on the quantification results, especially taking into account that the amount of pesticide residues is in/on the trace levels. Very low LOQ set on 0.005 mg/kg for every pesticide, with the LODs values in the range of 0.001 to 0.003 mg/kg, potentiate the quantification of pesticide residues in the organic food below the 0.01 mg/kg. Additionally, the recovery studies on two spiking levels (0.01 and 0.1 mg/kg) indicate that 88.3% of the investigated pesticides have the recovery in the interval from 67.4 to 118.5%, with the RSD between 1.87 and 14.73%.

The obtained results of the present study provide an indication regarding the pesticide residues in the organic apples. However, they cannot be responsible for the de-characterization of apples as an organically-produced commodity. The amount of the analyzed samples is not high; still, the results of our results accentuate the need for the constant monitoring of the products from the organic, as well as from the conventional production.