Standardization of an analytical method to quantify ochratoxin A in green coffee beans by high performance liquid chromatography

Nowadays, Brazil is the largest producer and exporter of coffee, also the second largest consumer of the beverage. The importance of ensuring food safety for consumers has influenced research to improve and monitor the final product quality. Coffee is a product that presents a high risk of fungal contamination, which can result in the presence of mycotoxins and poses risks to human and animal health. Therefore, this study aimed to standardize a chromatographic method to test and quantify ochratoxin A in 13 samples of green coffee beans. The green coffee beans were stored in sheds without temperature or humidity control. Samples were ground, and the analyte was extracted by a 3% methanol:sodium bicarbonate (1:2 v/v) solution. Ochratoxin A was quantified in a high performance liquid chromatograph. The method was validated by testing the selectivity, linearity, accuracy, limits of detection and quantification. The method presented robustness to the tested parameters and among the analyzed samples. Ochratoxin A was detected above the limit established by the legislation (75.19 µg kg-1) only in one sample. Overall, the storage of green coffee beans in these sheds was adequate, since 12 samples had a low content of ochratoxin A and they were within the limit established by legislation. Therefore, food safety was guaranteed without any severe mycotoxin contamination.


Abstract
Nowadays, Brazil is the largest producer and exporter of coffee, also the second largest consumer of the beverage. The importance of ensuring food safety for consumers has influenced research to improve and monitor the final product quality. Coffee is a product that presents a high risk of fungal contamination, which can result in the presence of mycotoxins and poses risks to human and animal health. Therefore, this study aimed to standardize a chromatographic method to test and quantify ochratoxin A in 13 samples of green coffee beans. The green coffee beans were stored in sheds without temperature or humidity control.
Samples were ground, and the analyte was extracted by a 3% methanol:sodium bicarbonate (1:2 v/v) solution. Ochratoxin A was quantified in a high performance liquid chromatograph.
The method was validated by testing the selectivity, linearity, accuracy, limits of detection and quantification. The method presented robustness to the tested parameters and among the analyzed samples. Ochratoxin A was detected above the limit established by the legislation (75.19 µg kg-1) only in one sample. Overall, the storage of green coffee beans in these sheds was adequate, since 12 samples had a low content of ochratoxin A and they were within the limit established by legislation. Therefore, food safety was guaranteed without any severe mycotoxin contamination.

Introduction
Coffee is one of the most consumed beverages in the world, and the drink has its guaranteed space on the consumers' table in Brazil. According to the Ministério da Agricultura, Pecuária e Abastecimento (MAPA), coffee arrived in Brazil in 1727 brought from the French Guiana. The climatic conditions and geographical relief were favorable for coffee cultivation, which spread it throughout the country and resulted in a predominant production for years in the Vale do Paraíba region.
Brazil is considered the largest coffee producer and exporter and the second largest coffee consumer in the word, moving US$5.2 billion in 2017. Nowadays, the concern with food quality is not only focused on the required prerequisites such as appearance, taste, aroma, texture, nutritional value and safety, but also chemical and microbiological contaminations. Food contamination caused by fungi results in major economic losses, a waste of raw material and changes in the product quality and safety, directly impacting the agricultural and food industries (Das et al., 2015). Even more, some of these filamentous fungi can produce mycotoxins that pose a serious risk to human and animal health, which can cause diseases or even lead to death (Bennet & Klich, 2003).
Mycotoxin production depends on fungal growth, so it can be produced in the field, before or during harvesting, or during storage. Mycotoxins can remain in the food even after the fungi that produce it has been eliminated. This can be explained for its thermal stability, resistance to heat treatments, or dehydrations processes (Bennet & Klich, 2003;Iamanaka & Taniwaki, 2010).
Ochratoxin A is a mycotoxin classified by the International Agency for Research on Cancer (IARC) as a group 2B human carcinogen. It can cause development of kidney disease, which is considered a contributing factor to a chronic disease in which the kidneys decrease in size and weight with diffuse fibrosis and urinary tract tumors (Iarc, 1993). Ochratoxin A is also related to nephrotoxic properties in animals. In addition, it behaves as a hepatoxin, Research, Society and Development, v. 9, n. 8, e39985070, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5070 5 immunosuppressive, teratogenic and carcinogenic substance. Studies indicate that this toxin induces cancer in liver, kidneys, mammary glands and testicles of animals (Jeleń & Grabarkiewicz-Szczȩsna, 2005;Amezqueta et al., 2012).
Ochratoxin A can be found in oats, barley, rye, wheat, coffee beans, nuts, dried peanuts, beans, spices, dried fruits and wine (Amezqueta et al., 2012). The Agência Nacional de Vigilância Sanitária (ANVISA) assigns maximum limits for mycotoxins in food. This regulation is applied to companies that import, produce, distribute and marked food products.
Verification of ochratoxin A in food can be performed by several chromatographic methods, such as thin-layer chromatography, gas chromatography coupled to mass spectrometry, high performance liquid chromatography coupled to a mass spectrometry and mass spectrometry coupled to inductive plasma; and by enzymatic immunological assays, such as ELISA (EFSA, 2004;Soleimany & Abas, 2012;Prelle et al., 2013;Pereira & Cunha 2014). However, the most common is high performance liquid chromatography combined with immunoaffinity columns and florescence detectors. Studies show that this technique has been used successfully to analyze the mycotoxin presence in food matrices (Prelle et al., 2013;Yashin et al., 2017).
The sample complexity requires a prior treatment, comprising several steps, before the analyte is determined. Most analytical methods used to analyze ochratoxin A has several steps in common, such as sampling, homogenization, extraction, separation / purification and detection. However, these steps can be considered as a source of variation of the results (Prelle et al., 2013;Pereira & Cunha 2014).
In order to be considered appropriate and in compliance, the analytical technique needs to provide safe results and quantify low concentration of compounds in a reliable and reproducible manner. The performance criteria established for the method must include all parameters, such as the limit of detection, coefficient of variation, coefficient of repeatability, reproducibility and the percentage of recovery. Using these parameters, laboratories would be free to adopt the most appropriate analytical method for their facilities (Codex, 2019). Thus, the standardization of a chromatographic method allows the reliability and reproducibility of results. Therefore, this study aimed to standardize a chromatographic method using HPLC and a fluoresce detector to quantify ochratoxin A in 13 samples of green coffee beans stored in sheds for 24 months.

Plant Samples
Thirteen samples of green coffee beans, Coffee arabica variety, were collected in 2016. The samples were stored in sheds without control of temperature or humidity for 24 months. The samples were obtained from the cities of Bambui, Doresopolis, Piumhi and São Roque in the state of Minas Gerais, Brazil.

Ochratoxin A extraction
The green coffee beans were ground and sieved to get a 200 mesh size. Ochratoxin A was extracted by stirring 25.00 g of sample for 10 min in 200 mL of 3% methanol:sodium bicarbonate solution (1:1, v/v) at 254 rpm. Subsequently, 10 mL of the filtrate was diluted 10 times in a phosphate-buffered saline solution (PBS) and passed through an immunoaffinity column (OchratestTM -Vicam, Watertown. USA) at a flow rate of 2-3 mL per min. Prior to the full passage of the sample, the column was washed with 10 mL of ultra-pure water at 5 ml per min. Then, the excess liquid was removed by allowing air passage in the column. Elution was carried out with 4 mL of methanol HPLC grade left in contact with the column for 3 min.
Finally, the eluate was evaporated in a water bath at 80 °C and resuspended in 400 µL of mobile phase.

Chromatographic validation
Selectivity, linearity, limits of detection (LD), limits of quantification (LQ) and accuracy were used to validate the chromatographic method and ensure the good analytical quality of the results (Brasil, 2003;Ribani et al., 2004;Harris, 2008).
Selectivity was assessed by comparing the analytical curve of the sample with and without the addition of the standard. Linearity, obtained by external standardization, was determined by correlating the signal (peak area) and the concentration of the compound, and expressed by the equation of the analytical curve and its respective coefficient of determination(r2). The limits of detection (LD) and quantification (LQ) were determined from the parameters of the analytical curve. Both limits were calculated using the mathematical equations: LD= 3.3 x s/S and LQ= 10 x s/S, where is the estimate standard deviation of the response and S is the angular coefficient of the analytical curve. Last, accuracy was tested by recovery assays using three randomly selected samples that were fortified with ochratoxin A at 3 different concentrations. The recovery was determined considering the relationship between the amount of present addicted analyte in the sample and the response using the following mathematical correlation: Recovery (%) = [(observed concentration) / (expected concentration) x 100 (Brasil, 2003;Ribani et al., 2004;Harris, 2008).

Statistical analysis
Ochratoxin A values were submitted to an analysis of variance, and means were compared by the Tukey test at a 5% probability level. The experimental design was completely randomized (DRC) and Sisvar software was used (Ferreira, 2011).

Method validation
The reliability of an analytical method is of considerable importance to quantify compounds, which through a robust method maintain the reproducibility and quality of results. The standardization of a method is a way to perform an analysis and publish its results with confidence and responsibility (Ribani et al., 2004).
It was possible to observe the ochratoxin A separation and its chromatographic profile from a standard solution of the analyte under study (Figure 1). The average retention time for ochratoxin A was 8.0 ± 0.1 min. Other authors reported values of 8.5 min (Medina, 2015), 5 min (Galarce-Bastos et al., 2014), 7.2 min (Casal et al., 2014) and 11.2 min (Benites et al., 2017), lower and even higher values than the one found in this study. However, it is noteworthy that the equipment and chromatographic conditions were different.  Through the construction of the analytical curve, it was possible to test the linearity of the method by estimating the coefficient of determination referring to the equation of the line (y = 3796,59x -3149,52). The coefficient of determination was 0.999, which shows the strong linear correlation between the concentration of the analyzed compound and the peak area, a value above 0.99 is recommended (Brasil, 2003). Also, this coefficient of determination is higher than those reported by Benites et al. (2017)  The LD and LQ were estimated using the parameters from the constructed analytical curve, which were 0.24 and 0.8 μg kg-1, respectively. These values are similar to those reported by Benites et al. (2017), being 0.266 and 0.5 μg kg-1for LD and LQ, respectively. LD and LQ were considerably low, showing a good sensitivity of the method for the analysis of ochratoxin A in coffee samples. Ribani et al. (2004) attribute the small differences in the parameters to the various chromatographic conditions, such as apparatus and methodologies.
The accuracy was evaluated through recovery assays to ensure that any matrix interferences did not influence the ochratoxin A detection. The recovery ranged from 80 to 90% for the three concentrations in the tested samples. Based on the acceptable limits for recovery (70 to 120%), it can be observed that the method presented a good recovery for the analyzed compound and the partial values and average values were within the acceptable limit range. For more complex samples, this acceptable limit range can be from 50 to 120%. Research, Society and Development, v. 9, n. 8, e39985070, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5070 Medina (2015) found recovery values between 78 and 94% in tests done at 5 concentration levels, while Benites et al. (2017) reported values between 95.5 and 109.8%.
Through the validation analysis it is possible to notice that the method presented selectivity, linearity, limits of detection and quantification, recovery and robustness, which generated reproducible and reliable results in the quantification of ochratoxin A.

Ochratoxin A quantification in green coffee beans
The quantification results of ochratoxin A in 13 samples of stored green coffee beans are shown in Table 1. Ochratoxin A concentration ranged from 0.69 to 75.19 μg kg-1, where sample 4 ( Figure 3) presented a value considerable superior to the limit allowed by legislation for roasted and green coffee beans (10 μg kg-1).  to 40 °C. Hence, these attributes make fungi capable of causing spoilage in food with different levels of humidity and climatic conditions (Medina, 2015).
Water activity and temperature are the factors that most affect fungal growth, sporulation and toxin production (Magan et al., 2011). In general, high humidity (20 -25%), high relative humidity (70 -90%) and warm temperatures (22 -30 °C) increase fungal proliferation and mycotoxin production (Vanesa and Ana, 2013 the year, these favorable conditions for the production of ochratoxin A can be considered a problem in the southern region of Minas Gerais because in the harvest months there is a considerable increase in the region's temperature and humidity, which may have been a factor for the ochratoxin A detected in the coffee beans that were analyzed. Passamani et al. (2014) evaluated the production of ochratoxin A by Aspergillus niger and A. carbonarius at different pH, temperatures and water activity. The authors reported an ochratoxin A production of 7 µg g-1 and 1 µg g-1, respectively for each fungi, at a water activity of 0.99, pH 5.35 and 15 °C. These values were higher than those found in our study, but it was expected since the mentioned study established the most favorable conditions for the fungi to produce ochratoxin A. Batista and Chaulfoun. (2007) quantified ochratoxin A in samples from different coffee fractions that were dried under different conditions (soil, asphalt and cement).
Ochratoxin A ranged from 0.1 to 5.0 µg kg-1 of coffee regardless the drying conditions.
These values were below those found in this study and within the limit required by legislation. Barcelo et al. (2017) analyzed the contamination of ochratoxin A in roasted coffee, green coffee beans with and without defects. The concentrations of ochratoxin A ranged from 17.14 to 21.02 µg kg-1 for the defective green coffee beans while the green coffee beans without defects did not present ochratoxin A. After roasting the ochratoxin A ranged from 5.57 to 6.79 µg kg-1, resulting in a reduction of approximately 70% of the contamination. The authors concluded that the roasting process is a practice that decreases contaminants in coffee beans, especially ochratoxin A.

Conclusion
All coffee samples presented ochratoxin A contamination, but only one of them showed a concentration above the limit allowed by legislation. Therefore, the adoption of good agricultural practices during all coffee processing can significantly influence the risk of contamination by microorganisms, consequently the production of mycotoxins.
Considering the validation results, the method performed in this study for the identification and quantification of ochratoxin A was adequate for the analysis of green coffee beans.
As coffee is one of the most consumed beverages in the world, the need to assess contaminants is important for the consumer's safety. The method to identify ochratoxin A needs to be efficient, robust, and reproducible in many parts of the world because in each Research, Society andDevelopment, v. 9, n. 8, e39985070, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5070 13 country a different form of post-harvest is employed, which can contribute to the proliferation of toxin-producing fungi.