http://dx.doi.org/10.5935/0100-4042.20150005 Quim. Nova, Vol. 38, No. 2, 268-273, 2015 MONITORING OF ORGANOPHOSPHOROUS PESTICIDE RESDUES IN SAMPLES OF BANANA, PAPAYA, AND BELL PEPPER Mariana F. Lemos, Mayara F. Lemos, Henrique P. Pacheco and Rodrigo Scherer* Departamento de Farmácia, Universidade Vila Velha, Rua Comissário José Dantas de Melo, 21, 29102-770 Vitória – ES, Brasil Recebido em 11/07/2014; aceito em 16/10/2014; publicado na web em 16/01/2015 Nota Técnica The objective of this study was to monitor 11 organophosphorus pesticides in samples of papaya, bell pepper, and banana, commercialized in the metropolitan area of Vitória (ES, Brazil). The pesticides were determined by an optimized and validated method using high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). All three samples exhibited a matrix effect for most of the pesticides, mainly with signal suppression, and therefore the calibration curves were produced in matrices. Linearity revealed coefficients of determination (r²) greater than 0.9895 for all pesticides and recovery results ranged from between 76% and 118% with standard deviation no greater than 16%. Precision showed relative standard deviation values lower than 19% and HorRat values lower than 0.7, considering all pesticides. Limits of quantification were less than 0.01 mg/kg for all pesticides. Regarding analysis of the samples (50 of each), none of the pesticides exceeded the maximum residue limit determined by Brazilian legislation. Keywords: QuEChERS; Capsicum annuum; Musa spp.; Carica papaya; pesticide analysis. INTRODUCTION Amid the classes of most toxic compounds and with greatest incidence in food in Brazil stand the insecticides, among which the With large, fertile lands and favorable weather for agriculture, organophosphorus compounds are prominent, accounting for more than Brazil is one of the world’s main producers and suppliers of food, 36% of the global market.12 With the rising of the organophosphorus, reaching third place in the global ranking of fruit production (41.5 which are less persistent in the environment, organochlorine pesticides, million tons) in 2010, with significant banana and papaya crops.1 though less hazardous, were substituted for being more resistant in During the same year, the country was one of the largest exporters the nature.13 In particular, organophosphorus are highly neurotoxic, of fruits and vegetables of Latin America, thus contributing to the presenting inhibitory function of cholinesterase, which controls the growth of Brazilian agribusiness, which was corroborated in 2012, nervous system, leading to an elevation in levels of the neurotransmitter when the export revenues raised to 910 million dollars.2 acetylcholine at nerve endings and causing neurobehavioral losses in Among several fruits and vegetables commercialized in Brazil, humans.14,15 Besides, pesticides are connected to other chronic health papaya, banana, and bell pepper achieved great prominence in the issues, such as cancer and adverse reproductive effects, dermatitis, country’s economy. Carica papaya, commonly known as papaya, respiratory problems, and can also be mutagenic and teratogenic.16,17 is produced in large scale in Brazil, which holds the second place In Brazil there are several monitoring programs focusing on in global production, only behind India.3 Moreover, the exports of evaluating food quality and on implementing controlling actions for this fruit accounted for 23 thousand tons, causing it to be the fifth pesticides residues, as an endeavor to minimize the exposure of the most exported product in January/2013.4,5 Similarly, banana (Musa population to these contaminants.18,19 The Program for Analysis of spp.) is one of the most produced and consumed fruits worldwide, Pesticides Residues in Food (PARA), coordinated by the National mainly in Brazil, which has a crop area of 71,253 hectares and ex- Sanitary Surveillance Agency (ANVISA), and the National Program for ports accounting for 82 thousand tons in 2013.4 It is a fruit known Residues and Contaminants Control (PNCRC), ran by the Department for its nutrients that contribute for elevated nutritional and energetic of Agriculture, Livestock, and Supply (MAPA), are responsible for levels.6 Bell pepper (Capsicuum annuum), a vitamin C source and the monitoring of pesticides residues and for instituting maximum rich in minerals, stands among the top ten vegetables consumed in residue limits (MRLs) in food. In recent years, pesticides residues were Brazil and around the world. In 2011 the Brazilian production was found in about 65% of analyzed samples, revealing bell pepper to be approximately 365.7 million tons and its sales accounted for near the vegetable with largest percentage of irregular samples, 90%, and 1.5 billion reais.7 papaya with 20% of unsatisfactory samples.20-22 It is relevant to keep in Fruits and vegetables are crucial for a healthy diet, due to the sight that, in the same year, the most abundant chemical group found in presence of significant amounts of nutrients and minerals. However, crops was the organophosphorus one, with 38% nonconformities.20-22 at the same time they may contain hazardous substances, such as In spite of the existence of the above-mentioned programs, they pesticides.8 Agricultural procedures with them still the most common only monitor small quantities of samples at each region, and that is way in order to achieve food production in adequate quantities, as why they do not present the thorough reality of the incidence of pes- they are an efficient tool against pests that can jeopardize production ticides in food. Therefore, the objective of this paper is to validate a and lead to food shortage.9 Between 2007 and 2012, the amount of method using sample preparation by QuEChERS method23 and high pesticides used in crops was 346.6 thousand tons, making Brazil the performance liquid chromatography with tandem mass spectrometry world’s leader in pesticide commerce in 2010 and the second largest (HPLC-MS/MS) to monitor organophosphorus residues in samples market for pesticides in 2012, the first being the United States.10,11 of banana, papaya, and bell pepper from ten different market from at Vitória and Vila Velha cities (Espírito Santo, Brazil), during a *e-mail: [email protected] three-month period. Vol. 38, No. 2 Monitoring of organophosphorous pesticide resdues in samples of banana, papaya, and bell pepper 269 EXPERIMENTAL rate was constant at 0.8 mL min-1. The chromatography was coupled to a mass spectrometer with triple quadrupole (API3200, Applied Chemicals and reagents Biosystems), operating in positive (+5500 V) ionization mode. Ion source temperature was kept at 600 ºC and nitrogen was used as HPLC grade methanol and acetonitrile were purchased from collision gas. Data were collected by multiple reaction monitoring Tedia (Rio de Janeiro, Brazil) and J.T. Baker (Phillipsburg, USA), (MRM) and processed by the Analyst™ 5.0 software. respectively. Ultrapure water was obtained from a Direct-Q 3 UV system (Millipore, France). The analyzed organophosphorus stan- HPLC-MS/MS parameters optimization dards were bought from Absolute Standards in ampoules with 200 Due to its sensitivity and selectivity for trace analysis in com- µg mL-1 of each pesticide (azinphos-methyl, mevinphos, sulprofos, plex matrixes, liquid chromatography is a widespread technique to demeton-S-methyl sulfone, diazinon, etoprophos, fensulfothion, determine pesticides in large scale using MRM. As a way to obtain fenthion, coumaphos, dichlorvos, and tetrachlorvinphos). Formic maximum sensitivity to identify and quantify the target compounds, acid was purchased from Synth (Brazil), whereas acetic acid was the optimization of all mass spectrometer parameters was performed obtained from Cromoline (Brazil). For the QuEChERS method, for each analyte in a 0.8 µg mL-1 acetonitrile solution with 0.1% anhydrous magnesium sulfate, primary secondary amine (PSA), (v/v) acetic acid. Optimization of the parameters is displayed and sodium acetate (NaOAc), bought from VETEC (Brazil), Sigma in Table 1. Aldrich (USA), and Panreac (EU), respectively. Method validation Samples Method performance was evaluated according to the reference From September to November, 2013, 50 samples of Cavendish document DOQ-CGCRE-008, by the National Institute of Metrology, bananas, 50 of papaya, and 50 of green bell pepper were purchased Normalization, and Industrial Quality.25 Evaluated analytical param- from markets and open markets at the metropolitan area of Vitória (ES, eters were: selectivity (matrix effect), linearity, precision (repeat- Brazil). Samples were collected every fifteen days in polyethylene ability and intermediate precision), accuracy (recovery), and limits bags to store 1.0 kg of each sample (randomly chosen) and conducted of detection (LOD) and quantification (LOQ). The validation process immediately to the laboratory for the analyses. was developed using spiked samples and pesticide-free samples as blank (with previously confirmed absence of pesticides in samples Extraction of banana, papaya, and bell pepper). Samples of banana, papaya, and bell pepper were extracted Selectivity and matrix effect according to the QuEChERS method described by AOAC Official Selectivity assesses the studied substance in presence of other Method 2007.01.24 Each unit of the studied food, from its respective interfering compounds in the sample. Matrix effect is observed by sample, was cut in four pieces. Two were discarded and the remain- the increase or decrease of the detector response of an analyte in the ing ones were homogenized in a food processor. Part of the ground matrix extract when compared to the same substance analyzed in an sample was stored under refrigeration for retest. A 15 g portion was organic solvent. This evaluation was done by comparing detector inserted into a 50 mL Falcon tube and then 15 mL of acetonitrile responses (peak areas), analyzing a standard solution of pesticides with 1% acetic acid, 6 g of anhydrous MgSO4, and 1.5 g of NaOAc in solvent (acetonitrile 0.1% (v/v) acetic acid) and in extracts of each were added to the system.