{"paper_id":"af511880-b97d-422f-91f4-85fc89375a5d","body_text":"Cocoa ( Theobroma cacao  L.) is a\ntropical crop native to Central and South America, with a global production\nof approximately 5 million tonnes annually. Côte d’Ivoire\nand Ghana are the largest producers, while Brazil ranks sixth worldwide,\nled by the states of Bahia and Pará. \n ,\nCocoa is best known as the primary raw material for chocolate but\nis also processed into products such as cocoa butter, biscuits, cocoa\nhoney, and cocoa powder. Cocoa powder is derived from roasted, pressed,\nand milled nibs, containing on average 20% (w/w) lipids on a dry basis\nand less than 9% moisture. \n ,\nCocoa cultivation\nfaces significant phytosanitary challenges, including\npests such as the red-banded thrips ( Selenothrips rubrocinctus ), brown rot ( Phytophthora  spp.),\nand the fungal pathogen  Moniliophthora perniciosa , responsible for witches’ broom disease, which can reduce\nyields by up to 90% under favorable conditions.  The spread of witches’ broom in the 1980s devastated\ncocoa production in southern Bahia, transforming the region from an\nexporter to an importer and causing profound socioeconomic impacts.\nConsequently, pesticides have become indispensable\nfor pest management\nin cocoa plantations. These agrochemicals include organochlorines,\norganophosphates, carbamates, pyrethroids, and neonicotinoids, among\nothers. \n , \n  However, their widespread use raises concerns\ndue to potential adverse effects on human health and the environment.\nPesticide exposure has been linked to ocular disorders, respiratory\nand endocrine disruptions, increased risks of cancer and neurodegenerative\ndiseases, and reproductive toxicity. \n −\nAccording to the Brazilian Health Regulatory\nAgency (ANVISA), pesticides\nsuch as atrazine, bifenthrin, cyproconazole, metalaxyl, tebuconazole,\ntrifloxystrobin, and permethrin are approved for use in Brazil.  However, among these, only metalaxyl, trifloxystrobin,\nand tebuconazole are authorized by the European Union.  Thus, investigating these contaminants in cocoa\nsamples is of utmost importance, as Brazil is one of the largest cocoa\nexporters, supplying cocoa commodities to South America, the United\nStates, and the European Union.  Furthermore,\nOrganochlorine pesticides, in particular, belong to the class of persistent\norganic pollutants (POPs). Although banned in many countries, they\ncontinue to be used in some developing nations because of their chemical\nstability, resistance to degradation, volatility, and high lipophilicity,\nwhich promote bioaccumulation and neurotoxicity.  Epidemiological evidence also associates their exposure\nwith Parkinson’s disease, certain cancers, diabetes, and endometriosis.\nResidues of pesticides in cocoa have been\nreported in various studies.  Analyzing\npesticide residues in cocoa presents\nanalytical challenges due to the matrix’s complexity, comprising\nhigh lipid content, fatty acids and esters, sugars, polyphenols, and\ncaffeine. These components can interfere with extraction efficiency\nand contaminate analytical instrumentation.  Therefore, efficient sample preparation methods incorporating effective\ncleanup steps are essential for accurate residue determination.\nSeveral techniques have been employed for multiresidue pesticide\nanalysis in cocoa. For instance, Idowu et al.  quantified 14 organochlorines using Soxhlet extraction and silica/Na 2 SO 4  cleanup, with GC ECD detection. Okoffo et al.  determined 13 organophosphates and 9 pyrethroids\nemploying SPE cartridges (Envi-carb/LC-NH 2  and Bond Elute\nC18), acetonitrile extraction, and GC PFPD or GC ECD quantification.\nYusiasih et al.  applied dispersive SPE\nwith PSA, Florisil, and MgSO 4  for pyrethroids, followed\nby GC ECD and GC–MS.\nAlthough these methods are effective,\nthey are often laborious,\ncostly, and generate substantial chemical waste. The QuEChERS (Quick,\nEasy, Cheap, Effective, Rugged, and Safe) method, first introduced\nby Anastassiades et al.,  has become widely\nadopted for multiresidue analysis due to its simplicity and high recovery\nrates. QuEChERS typically involves acetonitrile extraction, followed\nby dispersive SPE cleanup with PSA and MgSO 4 . This approach\nhas been successfully applied to matrices such as tomatoes,  peppers,  rice,  soybeans,  green\nvegetables,  fruits,  eggs,  and cocoa. \n , \n  However, conventional QuEChERS protocols require multiple partitioning\nand cleanup steps, sometimes involving additional reagents.\nLow-temperature partitioning (LTP) has emerged as an alternative,\nlow-cost cleanup strategy. LTP entails adding a small volume of water\n(commonly ∼2 mL) to the extraction solventtypically\nacetonitrileand subjecting the mixture to freezing. During\nfreezing, matrix interferences are retained in the aqueous phase while\nanalytes remain in the organic phase.  Depending on the matrix and analyte properties, mixtures with ethyl\nacetate or methanol can also be used. \n , \n  The combination\nof QuEChERS with LTP offers the potential to improve selectivity and\nreduce cleanup steps. While LTP has been applied to strawberries,  tomatoes,  lettuce,  butter,  and milk,  to the best of our knowledge, no studies have\nyet evaluated the combination of QuEChERS and LTP for the determination\nof multiclass pesticides in cocoa matrices.\nTherefore, this\nstudy aimed to evaluate the effectiveness of a\nQuEChERS–LTP analytical procedure for the determination of\nmulticlass pesticide residues in cocoa beans. The factors that affect\nthe QuEChERS–LTP procedure efficiency were optimized. After\nvalidation, the procedure was employed to investigate the occurrence\nof 15 multiclass pesticides in cocoa samples.\n\nIn this\nstudy, the factors evaluated were water volume, vortex agitation time,\nand both the type and mass of sorbent ( Table S1 ). According to the Pareto chart of standardized effects ( Figure  \n A), only water volume\nhad a positive and statistically significant effect on the responses.\nIn contrast, extraction time, sorbent type, and sorbent mass were\nnot significant at the 95% confidence level.\n(A,B) Pareto chart of\nstandardized effects obtained from the fractional\nfactorial design of resolution IV (2 4–1 ).\nThe fitted linear model showed no lack of fit at\nthe 5% significance\nlevel, and the residuals were low and randomly distributed ( Figure  \n B). Although sorbent\ntype and mass did not significantly affect the response, extracts\nobtained with PSA appeared clearer ( Figure  \n ). Therefore, 150 mg of PSA and a vortex\nagitation time of 3 min were selected for subsequent experiments.\nExtracts\npurified with PSA and C18 sorbents. Experiments: 1 (C18),\n2 (C18), 4 (C18), 5 (PSA), 6 (PSA), 7 (PSA), 11 (C18/PSA).\nThe miscibility of water in acetonitrile, combined\nwith their distinct\nfreezing points (0 °C and −45 °C, respectively),\nenables the retention of undesirable polar components during freezing,\nthereby minimizing interference of coeluting compounds from cocoa\nmatrix. Based on these considerations, the factor “water volume”\nwas further studied in a univariate way at levels of 2.0 and 3.0 mL.\nThe results exhibited in  Figure S1  show\nthat recovery values for most pesticides remained within the acceptable\nrange when 2 mL of water was added to the samples, whereas 3 mL led\nto overestimated recoveries, frequently exceeding 115%. This effect\ncan be attributed to the strong affinity of water for phenolic compounds\npresent in cocoa  which, at high concentrations,\nmay cause matrix-induced signal enhancement.  Based on these findings, the optimal water volume was set at 2.0\nmL.\nStudies reporting the use of LTP\ngenerally employ a conventional freezer, which typically reaches −18\n± 2 °C as the minimum temperature. In this equipment, effective\nphase separation can require 8 to 24 h. \n − \n \n  In contrast, ultralow\ntemperature freezers (ultrafreezers) can reach −80 °C,\nleading to a substantial reduction in partitioning time. In this study,\ntests performed with the conventional freezer achieved satisfactory\npartitioning after 24 h, whereas in the ultrafreezer, complete partitioning\nwas observed in only 20 min.\nConsidering the analytical response\nin terms of peak area ( Figure  \n ), higher signals were obtained when LTP was performed in\na conventional freezer rather than an ultrafreezer. This behavior\ncan be attributed to the partitioning mechanism.  Partitioning depends on cooling and the subsequent freezing\nof the aqueous phase. In the conventional freezer, the slower freezing\nrate provides sufficient time for the system to approach thermodynamic\nequilibrium, favoring analyte diffusion between phases. Under these\nconditions, analyte molecules have more time to migrate and distribute\noptimally before complete solidification. In contrast, the rapid freezing\nin the ultrafreezer may trap analytes in a nonideal distribution,\nreducing the efficiency of the partitioning process  ( Figure  \n ). According to these observations, the conventional freezer was\nselected for subsequent experiments.\nComparison of chromatographic peak areas\nof pesticides after partitioning\nin the conventional freezer versus the ultrafreezer.\nThe performance\nparameters achieved during validation of the analytical methodology\nare presented in  Table  \n .\nSurrogate standard.\nThe determination coefficients ( R \n 2 )\nobtained for all matrix-matched calibration curves were satisfactory,\nranging from 0.9985 (metalaxyl) to 0.9995 (trifloxystrobin). The calibration\ncurves for diazinon, metalaxyl, cyproconazole I, and permethrin exhibited  R \n 2  values <0.9990 and were therefore evaluated\nby ANOVA ( p  < 0.05). The results indicated no\nevidence of lack of fit for these curves.\nThe obtained LOD and\nLOQ values ranged from 4.16 to 6.95 μg\nkg –1  and from 13.9 to 23.1 μg kg –1 , respectively. Recent studies have reported concentrations of some\ntarget pesticides in cocoa samples within the range of 10.0–200\nμg kg –1 . \n , \n  This comparison\nindicates that the LOQ values achieved with the proposed method are\nadequate for the quantification of pesticide residues in cocoa products.\nMoreover, with the exception of α-HCH, the LOQs fall within\nthe maximum residue limits (MRLs) established by European Union regulations.\nWith respect to precision, the RSD values\nfor repeatability and\nintermediate precision ranged from 5.7% to 15.7% and from 13.1% to\n20.8%, respectively. Accordingly, all pesticides investigated in this\nstudy exhibited RSD values below 21% for both repeatability and intermediate\nprecision, which is considered acceptable at the evaluated concentration\nlevels.\nRegarding ME, it was observed\nthat all studied pesticides exhibited\nME > 1.10, which indicates a signal enhancement induced by the\ncocoa\nmatrix. The values for ME ranged from 1.61 (atrazine) to 20.5 (cyproconazole)\n( Table  \n ). The ME arises\nprimarily from competition between analytes and matrix constituents\nfor active sites within the chromatographic system, particularly the\nsilanol groups present in the injector.  A commonly employed strategy to mitigate ME involves constructing\ncalibration curves in the presence of analyte protectants. \n − \n \n  These protectants, compounds that simulate matrix components, interact\nwith active sites in the chromatographic system, such as silanol groups,\nthrough hydrogen bonding. \n , \n  Consequently, the signal\nof the analyte in the presence of analyte protectants is significantly\nincreased. However, in this study, calibration curves prepared in\nan analyte-free cocoa matrix solution provided a better analytical\nresponse than those prepared in an analyte protectant solution in\nacetonitrile ( Figure  \n ). Therefore, matrix-matched calibration curves were selected to\ncorrect for the matrix effect instead of analyte protectants.\nInfluence of\nmatrix components on the analytical response of tebuconazole\ncalibration curves prepared in solvent, matrix extract, and with analyte\nprotectants.\nConcerning trueness, recoveries at the lowest concentration\nlevel\n(25 μg kg –1 ) ranged from 62.4% (dimetachlor)\nto 117% (metalaxyl), whereas at the highest concentration level (50\nμg kg –1 ), they ranged from 81.8% (cyproconazole)\nto 120% (metalaxyl) ( Table  \n ). Depending on matrix complexity and analyte concentration\nin the spiked sample, recoveries between 60% and 120% are generally\nconsidered acceptable. Therefore, for cocoa beans, this analytical\nprocedure was considered accurate.\nTable S4  presents a comparison\nbetween\nthe analytical performance of the proposed QuEChERS–LPT procedure\nand conventional methods for pesticide extraction from cocoa beans.\nThe proposed procedure provided LOQ and recovery values comparable\nto those reported for established extraction techniques, while enabling\nthe simultaneous determination of a wide range of chemical classes,\nincluding chloroacetamides, strobilurins, phenylamides, organochlorines,\norganophosphates, pyrethroids, thiocarbamates, triazines, and triazoles.\nMoreover, the incorporation of LPT as a cleanup step represents a\nclear advantage, as it simplifies multiple operations and reduces\nreagent consumption. Nonetheless, the long partitioning time (24 h)\nrequired in a conventional freezer, along with the relatively low\nrecoveries for polar pesticides such as dimetachlor and diazinon,\nmust be considered limitations of this procedure.\nFifteen samples were analyzed\nin triplicate, comprising two from the State of Pará and 12\nfrom the State of Bahia, located in the North and Northeast regions\nof Brazil, respectively. Among the 15 pesticides investigated, only\npermethrin isomers were quantified in four samples ( Table  \n ). The herbicide molinate was\nalso detected in three samples; however, its concentration was below\nLOQ. Permethrin isomers concentrations ranged from 17.1 ± 0.3\nto 49.6 ± 1.8 μg kg –1 . These values are\nbelow the MRL established by European Union regulation, which is 100\nμg kg –1 .  Although\npermethrin is not permitted in the European Union,  it remains authorized for use as insecticide in Brazil,\nparticularly in crops such as cotton, rice, coffee, citrus, cabbage,\nbeans, tobacco, maize, soybean, tomato, wheat, and grape.  However, no MRL values have been established\nfor cocoa beans under Brazilian legislation. \n Table  \n  shows the\nconcentration of detected compounds in the cocoa samples.\nOnly compounds detected in at least\none sample are reported.\nConcentrations < LOQ.\nTo assess method performance for pesticides not detected\nin the\ncocoa samples, one of the analyzed samples was fortified with a mixed\nstandard solution at concentrations of 50, 250, and 500 μg kg –1 . The fortified analytes were subsequently detected\nin the chromatograms presented in  Figures S2–S4 , indicating good response of the proposed method.\n\nThe QuEChERS–LTP procedure was successfully\ndeveloped for\nthe determination of 16 multiclass pesticide residues in cocoa beans.\nFor the first time, ultralow temperature (−80 °C) was\napplied to accelerate the low-temperature partitioning process during\npesticide extraction from cocoa beans. Nevertheless, conventional\nfreezing at −12 °C proved to be more effective for the\nextraction of the target analytes.\nEvaluation of the matrix\neffect demonstrated that matrix-matched\nanalytical curves, constructed in an analyte-free cocoa bean extract,\nwere more effective in compensating for matrix interferences than\nthe use of analyte protectants.\nThe pyrethroid permethrin was\nquantified in four cocoa samples.\nHowever, the concentrations did not exceed the MRL established by\nEuropean Union legislation.\nOverall, the QuEChERS–LTP\nprocedure proved to be a promising\napproach for pesticide residue determination, as it requires smaller\nvolumes of solvents and reagents, involves fewer cleanup and extraction\nsteps, and shows strong potential for application to other complex\nmatrices.\n\nIndividual stock solutions of\nanalytical standards were prepared in methanol: atrazine- d \n 5  (850 mg L –1 ), bifenthrin (1870 mg\nL –1 ), cyproconazole (1020 mg L –1 ), diazinon (1880 mg L –1 ), dimethachlor (1477 mg\nL –1 ), disulfoton (2450 mg L –1 ),\nethion (2204 mg L –1 ), molinate (2010 mg L –1 ), permethrin (1824 mg L –1 ), and tebuconazole (1070\nmg L –1 ). A mixed standard solution (Mix A) at 10\nmg L –1  was prepared by appropriate dilutions. Another\nmixed solution (Mix B) containing atrazine, metalaxyl, and trifloxystrobin\nwas also prepared in methanol (10 mg L –1 ). All standards\nwere purchased from AccuStandard (New Haven, USA) with ≥97%\npurity.\nAn additional reference mixture containing 16 organochlorine\npesticides (EPA 46960-U, Sigma-Aldrich, USA) was dissolved in hexane/toluene\n(2000 mg L –1 ) and diluted to 100 mg L –1  in hexane.\nAcetonitrile (99.8%), methanol (99.9%), and  n -hexane\n(98.5%) were obtained from Merck (Darmstadt, Germany). Sodium chloride\n(99.8%) was purchased from Sigma-Aldrich, and anhydrous sodium sulfate\n(99%) from Merck. Ultrapure water was produced using an ultrapure\nwater system (18.2 MΩ cm, <3 ppb TOC; Merck Millipore, Germany).\nPSA sorbent (primary-secondary amine, 70 Å) was obtained from\nSigma-Aldrich, and C 18  sorbent (55–105 μm)\nfrom Waters (USA).\nAn analyte-free matrix solution was obtained\nby successive extraction\nof 2 g of dried cocoa beans with acetonitrile, followed by cleanup\nusing the proposed analytical procedure. The resulting extract was\ninjected into the GC–MS system to verify the presence of target\npesticides, and no coeluting compounds were detected. This analyte-free\nsolution was subsequently employed to prepare matrix-matched calibration\ncurves with eight concentration levels (1.00–100 μg L –1 ).\nPesticide separation and identification\nwere performed on a Shimadzu GC-MS-QP2010SE system (Kyoto, Japan)\nequipped with an AOC-20i autosampler. Separation was achieved using\nan Agilent DB-5MS capillary column (30 m × 0.25 mm × 0.25\nμm) with a stationary phase of 5% phenyl/95% dimethylpolysiloxane.\nThe instrumental conditions were based on Nascimento et al.  and they are summarized in  Table  \n .\nFor quantification, the base peak was used, while\ntwo additional\nions were monitored for confirmation ( Table S2 ) in accordance with SANTE guidelines.  A GC–MS/SIM chromatogram illustrating the separation of all\ntarget pesticides is presented in  Figure  \n .\nExtracted ion chromatogram (EIC) in SIM mode\nof the target pesticides\nobtained from the injection of a mixed standard solution at 100 μg\nL –1 . Analytes are listed in order of elution: [1]\nMolinate (6.18 min); [2] α-HCH (8.15 min); [3] atrazine- d \n 5  (8.69 min); [4] atrazine (8.75 min); [5] diazinon\n(9.31 min); [6] disulfoton (9.67 min); [7] dimethachlor (10.60 min);\n[8] metalaxyl (11.25 min); [9]  p , p ′-DDE (15.82 min); [10] cyproconazole I (16.50 min); [11]\ncyproconazole II (16.56 min); [12]  p , p ′-DDD (17.29 min); [13] ethion (17.34 min); [14] trifloxystrobin\n(18.43 min); [15] tebuconazole (19.13 min); [16] bifenthrin (20.47\nmin); [17] permethrin I (23.92 min); [18] permethrin II (24.18 min).\nFor compounds exhibiting stereoisomerism, such\nas cyproconazole\nand permethrin, multiple peaks corresponding to individual isomers\nwere detected, each of which was integrated separately during data\nprocessing.  The concentration reported\nin the samples represents the sum of all isomer contributions. The\nchemical structures of all pesticides analyzed in this study are presented\nin  Table S3 .\nFifteen cocoa samples\nwere collected between 2023 and 2024 in Brazil, specifically from\nthe municipalities of Ilhéus (14°47′20″\nS, 39°02′58″ W) and Ituberá (13°43′58″\nS, 39°08′15″ W) in Bahia, and from the state of\nPará (1°27′0″ S, 48°30′0″\nW). The cocoa beans were stored under refrigeration (−18 °C)\nuntil milling in an A11 benchtop grinder (IKA, São Paulo, Brazil).\nThe ground material was then packaged in polypropylene bags and maintained\nunder the same refrigeration conditions until analysis.\nThe\nmain factors influencing the extraction efficiency of pesticide residues\nin complex matrices such as cocoa include the volume and type of extraction\nsolvent, ionic strength, sorbent mass and water volume, and extraction\ntime. \n , ,\nFollowing\npreliminary tests to assess the chemical system under study, a screening\nof variables was necessary to identify those with the significant\neffect on analytical response. Initially, a fractional factorial design\nof resolution IV (2 4–1 ) was employed to evaluate\nthese factors.\nThe factors investigated\nto optimize the QuEChERS–LTP methodology\ncombined with low-temperature partitioning for cocoa powder samples\nwere water volume (0.5–2.0 mL), vortex extraction time (1.0–5.0\nmin), and sorbent mass (50.0–150 mg). To evaluate the effect\nof sorbent type on the cleanup efficiency of cocoa extracts, PSA and\nC18 were included in the experimental design matrix as categorical\n(qualitative) factors ( Table S3 ). This\napproach has been adopted in recent studies. \n − \n \n  The total solvent\nvolume was fixed at 10.0 mL, and the sample mass at 2.0 g. All experiments\nwere performed in randomized order, including triplicates at the central\npoint.\nThe peak area of each pesticide was used as experimental\nresponse.\nDue to the large number of analytes, a multiresponse approach was\napplied,  as expressed in  eq  \n \n \n 1 \n R M = R ( X 1 ) L R ( X 1 ) + R ( X 2 ) L R ( X 2 ) + . . . + R ( X n ) / L R ( X n ) \n where  R ( Xn ) represents the peak area in a specific experiment and LR­( Xn ) corresponds to the largest peak area observed among\nall experiments for that pesticide.\nIn\nthe conventional freezing step, a Brastemp freezer (São Paulo,\nBrazil) operating at −12 ± 2 °C was used, and optimization\nwas performed by varying the freezing time to 6, 12, and 24 h. For\nultralow temperature partitioning, a laboratory ultrafreezer (Thermotemp\nUltra Refrigeration, São Paulo, Brazil) set at −80 ±\n2 °C was employed, with optimization conducted by testing freezing\ntimes of 10, 20, 60, and 120 min.\nIn a 50 mL Falcon tube, 2.0 g of sample was weighed using an analytical\nbalance (AUX320, Shimadzu, Kyoto, Japan). A total of 2.0 g of salt\n(NaCl and Na 2 SO 4  in a 1:1 w/w ratio) was added,\nfollowed by an aliquot of the atrazine- d \n 5  solution (surrogate standard) to yield a final concentration of\n250 μg kg –1 . A mixture of acetonitrile (8.0\nmL) and ultrapure water (2.0 mL) was then added. The system was vortexed\n(K40-10208, Kasvi, São Paulo, Brazil) for 3.0 min at 2500 rpm\nand centrifuged (MPW-351R, MPW Med. Instruments, Warsaw, Poland) for\n10.0 min at 15 °C and 10,000 rpm. After phase separation, approximately\n6.0 mL of the supernatant was transferred to a Falcon tube containing\n150.0 mg of PSA sorbent. The mixture was vortexed again for 3.0 min\nat 2500 rpm and centrifuged for an additional 10.0 min under the same\nconditions. The tubes were then placed in the freezer for 24 h. Once\nthe aqueous phase had frozen, the organic extract containing the analytes\nwas collected, filtered through a Cytiva Whatman MiniUniprep G2 vial\nwith a 0.20 μm membrane filter (Marlborough, Massachusetts,\nUSA), and injected into the GC–MS system ( Figure  \n ).\nSchematic representation\nof the extraction procedure using the\nQuEChERS–LTP procedure for pesticide extraction in cocoa powder\nsamples.\nThe analytical\nmethod was validated following IUPAC guidelines for “In-house”\nvalidation.  The following performance\nparameters were evaluated: selectivity, linear range, linearity, limit\nof detection (LOD), limit of quantification (LOQ), matrix effect (ME),\nprecision and trueness.\nMatrix-matched calibration curves were\nprepared in an analyte-free cocoa matrix solution at eight concentration\nlevels, ranging from 1.00 to 100 μg L –1 , with\ntriplicate measurements at each level. To assess the matrix effect\n(ME), two additional calibration curves were constructed over the\nsame concentration range: one in acetonitrile and another in a solution\nof analyte protectants ( l -glucono-δ-lactone and sorbitol),\n10 mg L –1  in methanol/ethyl acetate (1:1, v v –1 ).\nLinearity was\nevaluated according to significance of  R \n 2 , and the lack-of-fit test assessed by ANOVA ( p  <\n0.05). The ANOVA was applied for curves with  R \n 2  ≤ 0.9990.\nThe limits\nof detection and quantification were calculated based\non the calibration data, with LOD estimated as LOD = 3 × (SB/ a ) and LOQ as LOQ = 10 × (SB/ a ), where\nSB represents the standard deviation of the intercept and “ a ” is the slope of the calibration curve.\nThe matrix effect (ME), reflecting the\ninfluence of coextracted\ncomponents on the analytical signal, was evaluated by comparing the\nslopes of calibration curves prepared in solvent and in blank matrix\nextract. The ME was calculated as ME = slope matrix /slope solvent . An ME < 0.9 indicates signal suppression, while\nan ME > 1.1 indicates signal enhancement.\nPrecision was expressed as repeatability and intermediate\nprecision.\nRepeatability was assessed as the relative standard deviation (RSD)\nfrom ten consecutive injections of a single sample fortified with\nall analytes at 250 μg kg –1  ( n  = 10). Intermediate precision was determined by calculating the\nRSD of ten injections performed over three consecutive days ( n  = 30).\nOwing to absence of certified reference materials\nfor pesticides\nin cocoa beans, the trueness of the analytical procedure was assessed\nby spiking/recovery assays. For this purpose, 2 g of dried and milled\ncocoa samples were fortified at two concentration levels (25 and 50\nμg kg –1 ) and processed according to the proposed\nmethod.\nExperimental data were processed using\nStatistica 7.0 (Statsoft, USA) and OriginPro 2024 (OriginLab, USA).\nThe obtained mathematical models for experimental design and linearity\nof the analytical curves were evaluated by analysis of variance (ANOVA,  p  < 0.05) at a significance level of 5% to assess both\nmodel significance and lack of fit.","source_license":"CC-BY-4.0","license_restricted":false}