Validation and application of a method for determination of multi-class pesticides in muscle chicken breast fillets using QuEChERS extraction and GC/MS

Cereals are the main component of animal feed for chickens. Among its cereal or cereal-derived components are oil products, grain mill products, corn, soybeans, starch products and dried plant products. Nowadays, growing cereals requires the use of a wide range of insecticides, fungicides and herbicides. The use of pesticides to prevent crop disease has increased significantly in recent years (21). Although the use of pesticides brings many benefits to agriculture, their residues can appear in poultry tissue and poultry products and some can affect human and animal health (15). Short-term exposure to pesticides does not have a significant harmful effect, while long-term exposure to certain pesticides can cause malignant diseases, cardiovascular diseases and damage to vital organs (5). Certain types of pesticide and their formulations may be used in different stages of cereal crop cultivation and during storage and can thus contaminate underground and surface water (6). Consequently, pesticide residues can be transferred to all compartments of the environment and thus reach poultry meat (12). Contamination of poultry may result both from direct exposure to pesticides in feed and water and from indirect exposure through environmental contamination. Chickens are also exposed to pesticides by inhaling them through contaminated air. Recently, much attention has been given to the importance of monitoring meat for the presence of pesticides. While many pesticide residues cannot be fully metabolised in livestock and poultry, or are excreted in their original form in feces, certain residues remain and can be detected in tissue and fat. The occurrence of pesticides in the fat tissues of goats, sheep and cattle has been observed, where deltamethrin in sheep samples and hexachlorobenzene in cattle samples were present above the limit of quantification (11). In recent years, there has been a large number of documented detection of residues in chicken meat samples (20, 23, 29).

Poultry meat is the main protein source and the poultry industry is expanding in Serbia every year. Because of possible contamination, which is a general global concern around meat and animal products for both human and animal health, residue surveillance is necessary. Chicken is a good and cheap source of proteins, vitamins and minerals and contains a low amount of fat (3 g of fat per 100 g) compared to red meat which contains more than 5 g per 100 g. Chicken meat is a staple in the local diet, and analysis and monitoring of pesticide residues in this food product are necessary to ensure the safety of consumers. Every year, the European Food Safety Authority (EFSA) provides an annual report on the number of samples tested for the presence of pesticides in Europe (19). The report covers the type of food tested, the frequency of occurrence of pesticides in food, and the type and number of pesticides detected. Safety concerns have led to the establishment of maximum residue limits (MRLs) for pesticide residues in different food test matrix categories. Maximum residue levels are based on a risk assessment derived from pesticide residue data from food monitoring and food consumption data. Values of MRLs are applied to 315 fresh products and to the same products after processing, adjusted to content for dilution or concentration during the process. The general recommended value for the maximum residue level of pesticide contamination is 0.01 mg/kg and covers types of food for which values of pesticide contamination are not specifically defined (10).

The low limits of detection required by regulatory inspections and the complex nature of the matrix being tested for the presence of pesticides require efficient sample preparation and identification of traces of pesticides at the level of maximum residue values (12). The development of a multi-residue method is difficult, because pesticides have different polarities, solubility, volatility and pKa values (4). Over time, the amount of pesticides present can decrease because of their physical/chemical properties and environmental factors. The stability of pesticides can be influenced by the type of matrix, i.e. its composition, the presence of organic matter, pH influence and water content (3). Analysis of pesticide residues in fatty foods is a major challenge for analytical chemists, mostly due to the presence of water, fatty acids and cholesterol that make determination difficult. Pesticide residues can be analysed using gas chromatography with electron capture detection (GC/ECD) and gas (GC/MS) or liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) (13, 17). Most of the published multi-year studies for the analysis of pesticides in food of animal origin were carried out using gas chromatography/flame ionization detection (GC/FID) and the determination of persistent organic pollutants, i.e. organochlorine pesticides (2), but the scope of recent studies where, in addition to organochlorine pesticides, organophosphate pesticides, pyrethroids and carbamates are monitored, is proving to be more and more justified (23).

In the last few years, many studies have been published with the aim of developing sensitive and accurate methods for the determination of pesticides in animal matrices (28, 29). There is no unique standard preparation method using the quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction procedure for matrices such as meat, fish and other fatty samples, but in scientific works and EFSA validations there is an increasing number of modified methods that include existing QuEChERS preparations for plant products (1, 4). Standard procedures for extraction of food of animal origin require the use of large quantities of reagents and solvents, many of which are toxic. They have several stages besides filtering, high-volume quantitative transfers and need the use of suitable laboratory dishes, evaporation and condensation steps, as well as annealing and activation of florisil for column cleanup. This complexity is highly relevant because every extra step makes the process more difficult and increases the risk of random and systematic mistakes (26).

The QuEChERS sample preparation method is a fast and modifiable method that was introduced in 2003 for rapid and efficient analysis of pesticide residues in products of plant origin by Anastassiades and Lehotay (1), and has been succesfully modified and improved by many other authors (21, 26). Thus, this method of dispersive solid phase extraction and cleanup has become the primary one in advanced analysis and simultaneous determination of a large number of pesticides, offering the possibility of modifying the method and adjusting the preparation technique for testing specific targeted pesticides. After two decades, this modern method of sample preparation has become the basic method and most rational choice. The use of QuEChERS for the preparation of samples for pesticide analysis has expanded over the years to also include fatty foods as possible samples (27), i.e. products of animal origin (meat, eggs, milk, etc.). QuEChERS has replaced the European standard method certified by the European Committee for Standardization (EN1528) in many laboratories because of the short duration of QuEChERS analyses and the method’s use of less of particular expensive solvents. New sorbent materials with different types of adsorbent and different packages and ratios of composition quantities have become the basis of a new technology for testing the presence of pesticides. The components of dispersive solid phase consist of magnesium sulphate, C18 silica, primary-secondary amine (PSA), graphite carbon black, Florisil, Z sep sorbent and a specially designed enhanced matrix removal lipid. The analysis of pesticides in food requires sample preparation, where precise separation and detection is important. The preparation techniques for analysing pesticides from the sample are: liquid-liquid extraction, liquid-liquid micro extraction, solid-phase extraction (SPE), solid-phase micro extraction and gel-permeable chromatography. Solid-phase extraction is currently the best represented and best used extraction technique (29). The first step in pesticide analysis is the choice of method and the second step is the validation of the precise method and optimisation of the technique on the instrument on which the determination is performed.

These steps are preparatory work for every comprehensive monitoring programme conducted to verify compliance with legislation; such programmes are essential for realistic assessments of exposure to dietary chemicals. The use of a modified QuEChERS method for monitoring different matrices marks significant progress in assessing food safety, but validated QuEChERS methods need to be confirmed through proficiency testing (PT) activities and accreditation under ISO/IEC 17025. The aim of the study is primarily to summarise an instance of progress in research on gas chromatographical quantification of pesticide residues in chicken fillets and to confirm the importance of multi-class analysis. The validated method was applied to test pesticides in conventional and organic muscle chicken breast fillets.

Validation is always a balance between skills and technical possibilities. Validation using QuEChERS preparations avoids the use of large amounts of expensive organic solvents with the help of extremely powerful and highly sophisticated techniques. The validation guideline (SANTE/11312/2021) (9) prescribed by European regulations precisely defines the conditions for linearity, correlation coefficient, recovery, repeatability, accuracy and relative standard deviations. Following the steps prescribed by the guide allows the laboratories to have free choice of methods, which is beneficial for the continuous evaluation of the analytical methods. Laboratories performing analyses of pesticide residues also tend to work under a quality system and standard ISO/IEC 17025. In this study, LOD and LOQ values ranged 0.9–1.5 µg/kg and 3.0–4.9 µg/kg, respectively. The LOQ values of all studied pesticides were below their respective MRLs, which confirms that the validated method has satisfactory sensitivity. During the validation of the method, there were no deviations in recovery values within the range of 70–120%, and all obtained repeatability RSD values were less than 20% (Table 1). Acceptable recoveries were obtained for the tested pesticides. Satisfactory sensitivity was achieved and the correlation coefficients of all tested pesticides of the constructed calibration curves were greater than 0.99 (Table S1). When optimising the extraction procedure, solvents such as acetonitrile, methanol, acetone and ethyl acetate can be used as mixtures with water and/or additives for pH value adjustment (11, 16, 28). Therefore, differents amount of extraction agent can be used for the same mass of sample; freezing or ultrasound can also be effective techniques (25, 28, 29). Rutkowska et al. (25) used 20 mL of acetonitrile and 10 mL of water, which was cold in order to reduce the endothermic reaction of magnesium sulphate and protect heat-sensitive pesticides such as captan, folpet, tolylfluanid, and dichlofluanid, when analysing 10g of commercial crop seed for residues. The composition of the matrix and target pesticides must be considered so that the preparation phase gives a satisfactory recovery and the influence of the matrix effect is minimised. The matrix effect is the significant influence of interfering substances from the sample matrix that is important to consider in both liquid and gas chromatography. The QuEChERS method grants the possibility of easy modification and adjustment of the preparation procedure for better reliability of testing specific or required pesticides in the tested matrix. Some modifications of the original QuEChERS method have been carried out to adapt it better to the matrix analysed and the characteristics of the analysed pesticides. By choosing appropriate sorbents for sample preparation, the influence of the matrix can be significantly reduced. Oliveira et al. (22) conducted a mixture design experiment on trahira (Hoplias marabaricus) samples to find an adequate combination of C18, PSA and Z-Sep+ with which to extract pyrethroids. The same experiment also investigated temperature treatment in sample preparation and achieved good results when a freezing time was chosen of 12 hours (overnight).

Sixty percent of the tested pesticides had a matrix effect value lower than 5%, 40% of them had a matrix effect value between 5 and 10%, and only pyridaben had a higher value of matrix effect, of 12.59%. The quantification was carried out in the matrix and the mean value of recovery was taken into account when quantifying the results. By examining the samples, the presence of pesticide residues was determined in four tested samples of chicken breast fillet. In 96.0% of the tested samples, none of the 56 tested multi-class pesticides were detected, while pesticide residues were detected in four samples, albeit at levels below the maximum residue levels (MRLs) values set by European regulation in 2005 (8, 10). This regulation was relevant to the present research in Belgrade because European Union legislation has been adopted and implemented in Serbian regulations regarding the use of pesticides and the presence of residues in food. In one sample from domestic producers, cypermethrin was detected in the amount of 11.5 µg/kg, which is less than the MRL value of 100 µg/kg (or 0.1 mg/kg). Also, in one sample from domestic producers, the persistent organochlorine pesticide endosulfan sulphate was detected in an amount of 4.9 µg/kg, while the MRL value is 50 µg/kg (0.05 mg/kg). Permethrin was detected in one sample from domestic producers in the amount of 15.2 µg/kg (MRL = 50 µg/kg) and endosulfan alpha was quantified in the same sample in the amount of 5.4 µg/kg (EU MRL value is 50 µg/kg). In a fourth sample, which was imported, the persistent pesticide p,p´-DDT was detected in an amount of 6 µg/kg, significantly less than the amount prescribed by law for the sum of all metabolites of DDT of 1,000 µg/kg. In our investigation, in addition to p,p’-DDT, the presence of the metabolite p,p’-DDE was confirmed in the amount of 3.2 µg/kg, but since the value is lower than the LOQ, it is not shown in Table 2. European Commission Regulation (EC) 2023/163 prescribes MRLs for DDT and its metabolites in various meat products at a maximum of 1,000 mg/kg. Dichlorodiphenyltrichloroethane is an active substance known as a persistent organic pollutant, and despite the cessation of the use of DDT in the European Union, it can still be detected at very low levels in some plant and animal products because it persists long in the environment. Based on the most recent data from 2016 to 2020, which show that DDT residues occur in wild boar products at levels higher than the current MRL, the Commission has established values for other farmed terrestrial animals at 1 mg/kg. For the pesticides for which MRLs are not prescribed, a default MRL of 0.01 mg/kg applies, and this is the reason for the quantification limits set in the SANTE guidelines, which must be achieved during method validation. Also, when using a lower calibration range from 5 to 50 µg/kg, validation can achieve lower LOD and LOQ values. Such lower values were also achieved by Kartalović et al. (14), whose LODs ranged from 0.27 to 1.51 µg/kg and LOQ values from 1.10 to 5.20 µg/kg. Using the conversion factor, there was a change in the value when calculating the total amount of endosulfan, whereby the amount of endosulfan sulfate (from 4.9 µg/kg) expressed per endosulfan was reduced to 4.7 µg/kg. All detected values are significantly lower than the EU MRL values and do not pose a risk to human or animal health. In the samples produced by organic production, none of the tested pesticides was detected. The organic samples had no traces of pesticides present. Good agricultural and animal husbandry practices led to chicken breast fillets farmed organically being pesticide residue free, which satisfies a requirement of organic production regulations. Above all, surveillance should not be omitted, because the EFSA recommended focusing monitoring activities on commodities that frequently contain pesticide residues or that have the potential to result in a significant short-term intake. The results obtained are in accordance with those of a study by Lee et al. (18), which examined 29 pesticides and their metabolites in 70 commercial chicken products from Korea. In their results, difenoconazole and propiconazole were detected in two samples and the concentrations were 11 μg/kg and 5 μg/kg. While they detected pesticides different to those found in our study, the detected amounts were in the same range as the one obtained by testing in our work. The presence of difenoconazole, imidacloprid and fipronil and its metabolites was confirmed by Wang et al. (28) examining chicken faeces using LC/MS/MS, which is important for validation and monitoring. Meat and meat products have also been evaluated in the neighboring Republic of Bosnia and Herzegovina: smoked pork products were examined and their concentrations of α-HCH, lindane, PCB 28, PCB 52 and PCB 153 were quantified. The detected values were in the range of 5.1 ± 1.1 µg/kg for α-HCH and up to 22.11 ± 7.12 µg/kg for lindane, but the concentrations varied depending on the way the product was treated (14). Examinations of fish from the Serbian section of the Danube river are published more frequently in scientific literature. For example, the presence of p,p’-DDE and p,p’-DDD was confirmed in silver bream and barbel in the amount of 0.84 to 9.06 µg/g of fat (7). Considerable quantities of organochlorine pesticides in Serbia were quantified as metabolites of DDT, aldrin, lindane and endrin-ketone in amounts from 1.1 to 137.9 µg/kg in wild animals (24). The presence of pesticide residues in wild animals is explained by their presence in regions with intensive agricultural production and waste. However, there are no published articles on the topic of pesticides in chicken fillets in Serbia. In that regard, this study is the first of its kind.

The study presents a multi-class method for monitoring and controlling the presence of pesticide residues in chicken breast fillets. Sample preparation methods were validated and optimised for the analysis of pesticide residues in the chicken fillets using GC/MS for quantification. The determination of pesticides in chicken meat is necessary for ensuring that human exposure to possible contamination is minimal and is an important tool for the determination of environmental contamination by pesticide residues. Analysis of pesticides in fatty food such as poultry is a difficult and demanding laboratory task. Extraction and preparation of samples with satisfactory recovery is an important aspect of pesticide analysis which was achieved in this study. Quantification using sensitive instruments and the newest techniques, when reliable, is an important method for monitoring and screening of poultry meat. QuEChERS methods and means of monitoring which are suitable for determining traces of residues have led to modernisation and have shed more light on the actual levels of pesticide residues in food consumed on a daily basis. The development of fast, safe and efficient methods of preparation has made it possible to determine different classes of compounds.