Table 1. MS/MS parameters for 39 pesticides in MRM mode.
METHOD VALIDATION AND CALCULATION OF MEASUREMENT UNCERTAINTY FOR PESTICIDE RESIDUE ANALYSIS IN FRESH RED PEPPER
G. ÖZTÜRK and Y. YAKAR
Food Engineering Department, Faculty of Engineering, Harran University, 63100, Sanliurfa, Turkey
yasinyakar@harran.edu.tr
Cite this article as:
Ozturk, G., Yakar, Y. (2022) “Method validation and calculation of measurement uncertainty for pesticide residue analysis in fresh red pepper”, Latin American Applied Research 52(3), pp 181-186.
Abstract-- A multiple analysis method based on QuEChERS (fast, easy, inexpensive, effective, robust and safe) extraction method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for 39 selected pesticides in fresh red pepper. This method has been validated according to the Sante guide published by the European Union Reference Laboratories (EURLs). The linearity study was conducted in the range of 1-80 µg/kg and in seven different concentrations. The correlation coefficients of the calibration curves were (R2)> 0.992. Quantification limits (LOQs) ranged from 4 to 19 μg/kg. The recovery values were between 70.84% and 108.0%. The repeatability values (RSDr), which were between 0.99% and 17.5% in terms of in-laboratory reproducibility values (RSDWR), were determined to be between 0.98% and 19.57%. Repeatability and trueness parameters were used to calculate measurement uncertainty. The extended uncertainty values were calculated as 20.88% to 49.94%. The method was successfully used to analyze 22 fresh red pepper samples. Nine different pesticide residues were identified in 12 samples. The amount of carbendazim in a sample was found to be above the legal limits of the EU and Turkey. A residue of carbofuran, which is banned in Turkey, was found in one sample.
Keywords-- Validation, measurement uncertainty, pesticide, pepper, LC-MS/MS
Pepper (Capsicum annum L.) is a plant rich in bioactive components, including ascorbic acid, carotenoids, tocopherols, flavonoids, and capsaicin. It has been consumed fresh and as a spice since ancient times (Liu et al., 2016; Rani et al., 2019; Song et al., 2019).
In 2019, 38,027,164 tons of fresh peppers were produced worldwide. Turkey produced 2,625,669 tons, which was 6.9% of the worldwide production, meaning that Turkey was the third largest producer after China and Mexico (FAOSTAT, 2021).
In Turkey, red pepper is produced in the Mediterranean, Aegean, Marmara, and Southeastern Anatolia regions (Beyaz et al., 2009). Although it is often consumed fresh, pepper is also consumed as a canned food, in pepper paste, as a pickle, and in hot sauce, chili pepper and ground pepper (Doymaz and Pala, 2002; Arslan and Özcan, 2011).
Pesticides are chemicals that enable agricultural yield to be increased by minimizing the losses caused by insects, pests and fungi (Esturk et al., 2014; Golge et al., 2018; Alcântara et al., 2019; Kumar et al., 2019; Bhandari et al., 2019; Ons et al., 2020; Tudi et al., 2021). Their use in agriculture has become necessary to meet the increasing need for food resulting from the rapid increase in the world population. However, if pesticides are not used correctly, they can have detrimental effects on humans, the environment, and non-target species. They can cause significant health problems for consumers, especially because of their subacute and chronic toxicity (Golge and Kabak, 2015; Liu et al., 2016; Zamora-Sequeira et al., 2019; Narenderan et al., 2020). For this reason, controlling the use of pesticides in agricultural production, monitoring the residue levels in fruits and vegetables, and ensuring that their use is legally regulated are all very important for human health.
Many extraction methods have been developed to determine the presence of pesticide residues. These include solid-phase extraction (SPE), solid-phase microextraction (SPME), accelerated solvent extraction (ASE), supercritical fluid extraction (SFE), matrix solid-phase dispersion (MSPD), microwave-assisted extraction (MAE), membrane extraction, gel permeation chromatography (GPC), and the QuEChERS method (Zhu et al., 2014; Golge et al., 2018; Velkoska-Markovska et al., 2018; Hepsağ, 2019).
The QuEChERS method, which was developed by Anastassiades et al. (2003), has become a widely used technique in recent years due to its applicability to a wide variety of pesticides (Alcântara et al., 2019; Rizzetti et al., 2016; Tartaglia et al., 2020).
This study aimed to carry out validation studies for 39 pesticides in fresh red pepper using QuEChERS extraction method and the LC-MS/MS technique, and to calculate the measurement uncertainty.
Certified pesticide standards ranging from 95-99% were obtained from Dr. Ehrenstorfer (Augsburg, Germany). Single stock standard solutions were prepared as 1000 mg/L in acetone and stored at -18 ºC. These solutions were mixed to obtain a multi-standard solution of 10 mg/L and stored at -18 ºC. Multi-standard working solutions were prepared daily by diluting the multi-standard solution with pesticide-free pepper extract.
Acetonitrile (MeCN), Methanol (MeOH), and glacial acetic acid (AcOH) which were HPLC pure were obtained from Merck (Darmstadt, Germany). The anhydrous magnesium sulfate and anhydrous sodium acetate were purchased from Merck, while primary secondary amine (PSA) and graphitized carbon black were purchased from Supelco (Bellefonte, PA, USA). Ultra-pure water was freshly produced using a Milli-Q water treatment system (Bedford, MA, USA).
The fresh red pepper samples used in the validation study were free of pesticides. They were obtained from a grocery store selling organic products in the Sanliurfa province of Turkey. 1 kg each of the 22 samples was obtained from different markets and district markets in September 2019. The peppers were stored in a freezer at -18 ºC before being ground in a homogenizer and analyzed.
The red pepper samples were extracted using the QuEChERS method described by Anastassiades et al. (Anastassiades et al., 2003). 15 g of the homogenized sample was weighed into a 50 ml propylene tube. 15 ml of MeCN-AcOH (99:1, v/v). 6 g of anhydrous MgSO4, and 1.5 g of anhydrous sodium acetate was the added. After the cap of the tube was closed, it was first shaken for 1 minute and then centrifuged at 5000 rpm for 5 minutes. 4 ml of the upper phase was then transferred to a 15 ml propylene tube containing 600 mg of anhydrous MgSO4 and 200 mg PSA. Shaking and centrifugation were repeated. The vial was taken from the upper phase and kept at -18 ° C until analyzed.
Pesticide analyses were conducted using Shimadzu-8030 liquid chromatography-mass spectrophotometer (LC-MS/MS), which can operate in positive and negative mode and is equipped with an electro spray ionization source (ESI). Quantitative analysis of pesticides was carried out using the multiple reaction monitoring (MRM) method. Capillary voltage was 4500 V, the Desolvation Line (DL) temperature was 250 °C, the interface temperature was 350 °C, the heat block temperature was 400 °C, the nebulizing gas flow was 3 L/min, and the drying gas flow was 15 L/min. A list of the MRM mode transitions and parameters of the 39 pesticides is given in Table 1.
Table 1. MS/MS parameters for 39 pesticides in MRM mode.
An Ods-4 C18 column was used for chromatographic separation. Mobile phase (A) was prepared as water containing 10 mm ammonium acetate and was followed by mobile phase (B) MeOH. The mobile phase flow rate was set to 0.3 ml/min. A gradient flow program was applied to the column. In the mobile phase (B) the solution was passed through the column to 95% in 3.5 minutes. Then the flow continued in the same way for 2 minutes. The mobile phase B was reduced to 5% and streaming continued for 5 minutes. The injection volume was set to 5 μL.
In the validation studies, the "Analytical Quality Control and Validation Procedures Guide for Pesticide Residues and Analysis in Food and Feed" published by the European Union Reference Laboratories (EURLs) was used as a reference. The validation studies were carried out based on the parameters of linearity, matrix effect, quantification limit (LOQ), specificity, trueness (systematic error), and precision (repeatability and reproducibility within the laboratory) (SANTE, 2017).
The linearity study was carried out in seven different concentrations (1, 2, 5, 10, 20, 40, and 80 µg/kg) and in three replicates. It was ensured that the deviation between the actual concentration and the calculated concentration was not more than ± 20%.
The "Matrix-matched" calibration method was used to balance the matrix effect. The LOQ study was performed at the level of 5 µg/kg and 10 replicates. LOQ values were obtained by taking 10 times the calculated standard deviation. LOQs were calculated with recovery between 70% and 120% and repeatability RSD below 20% ( IUPAC Technical Report, 2002).
In the specificity study, it was noted that there was a maximum difference of ± 0.1 minutes between the retention time of the analytic calibration for the analysis sought in the sample extract.
Trueness, the averages of the recovery studies performed during the
repeatability and in-laboratory reproducibility control studies were checked
for compliance
Table 2. Validation and measurement uncertainty data of 39 pesticides in fresh red pepper samples.
Table 2. (Continue)
within the range of 70-120%. Compliance with RSD ≤ 20% for repeatability (RSDr) and in-laboratory reproducibility values was checked.
Quantification uncertainty was estimated according to the procedures recommended in the EURACHEM/ CITAC Guide CG4 (IUPAC Technical Report, 2002). Standard uncertainties from reproducibility and trueness parameters were used for each pesticide to calculate measurement uncertainty. The Combined Standard Uncertainty (Uc) values were calculated. Then, the expanded uncertainty (U') values were obtained by multiplying these values with the coverage factor (k = 2) at a 95% confidence level.
The results of the validation studies are given in Table 2. In the linearity study, it was determined that the deviation between the actual concentrations and the calculated concentrations were less than ± 20%, and the correlation coefficients (R2) were between 0.992 and 0.999. The LOQ values were calculated as 4 µg/kg and 19 µg/kg. The trueness check was done by calculating the recovery values. Recovery values were obtained from 71.06% to 101.71 at the 10 µg/kg level and from 70.84% to 108.00% at the 50 µg/kg level.
A repeatability check was done for the recovery study. It was determined that the % RSDr values calculated at the 10 µg/kg level were between 1.20 and 17.50, and the values at the 50 µg/kg level were between 0.99 and 12.66.
A reproducibility control was also carried out through the recovery study. It was observed that the % RSDWR values calculated at the 10 µg/kg level were between 3.32 and 19.57, and the values at the 50 µg/kg level were bet-ween 0.98 and 12.44. Reproducibility and trueness parameters were used to calculate the measurement uncertainty. The expanded uncertainty values were found to be between 20.88 and 49.94.
In a study where a total of 725 cucumber and pepper samples from three different provinces of Turkey wereanalyzed by LC-MS/MS for 170 different pesticide residues, the LOQs were 0.003 and 0.016 mg/kg, the recovery rates were between 80.5% and 118.2%, and the RSDs were found to be between 0.7 and 19%. The expanded measurement uncertainty for pesticides ranged from 10.4% to 42.4% (Golge et al., 2018).
Liu et al. (2016) used GC-MS and a modified QuEChERS method to identify 29 pesticide residues in green, red, and dried red peppers in their 2016 study. Ca-libration curves for 29 pesticides were run at a concentration of 0.1–10 μg/mL and the R2 values were found to be> 0.998. The LOD values were determined to be 0.006-0.06 mg/kg for green pepper, 0.005-0.039 mg/kg for red pepper and 0.014-0.25 mg/kg for dried red pepper. The average recoveries ranged from 70.1% to 110%, with RSD values <13%.
Morales et al. (2011) conducted a study in 2011 to detect 16 pesticides in pepper samples using an LC-MS/MS device and the QuEChERS extraction method. They validated the method according to the SANCO European Guidelines. Under optimized conditions, they found recoveries (RSD ≤ 20%) in the range of 70-110%. They obtained a linear equation for each analyte in the concentration range of 10 to 100 µg/kg, and found the correlation coefficients to be > 0.997.
Zhang et al. (2016) used GC-MS in their 2016 study and modified the QuEChERS method to determine eight pyrethroid pesticide residues in green, red, and dried chili peppers. Calibration curves for pyrethroid pesticides showed good linearity in the range 0.05-20 μg/mL, and they found R2 values of > 0.997. They found the LOQ values of eight pyrethroids to be 0.004-0.04 mg/kg for green and red peppers, and 0.04-0.5 mg/kg for dried red peppers. They determined that the average recoveries varied between 79.0 and 104% and the RSDs were>11%. They found that some commercial samples contained residual pyrethroid pesticides below legal limits.
Zhu et al. (2014) modified the QuEChERS method to determine 227 pesticides in pepper samples with LC-MS/MS. They determined r2 values to be > 0.99, LOQ values to be between 0.13 and 13.51 µg/kg, and LOD values to be between 0.04 and 4.05 µg/kg for 227 pesticides.
The fresh red pepper samples were obtained from various markets in Sanliurfa province of Turkey. Analysis of 22 red pepper samples found no residue in 10 samples. Nine different pesticide residues were detected in 12 samples. Acetamiprid (13.4-638.7 µg/kg) was found in 10 samples, azoxystrobin (18.6 µg/kg) in one sample, boscalid in two samples (19.9-24.4 µg/kg), carbofuran in one sample. (111.2 µg kg--1), imidachloropride in five samples (32.7-133.5 µg/kg), pyridine in four samples, pyriproxyfen in five samples (9.9-223.7 µg/kg), residues of spiromesifen in one sample (144.2 µg/kg) and tebuconazole (44.5-190.3 µg/kg) in four samples. Acetamiprid residue detected in one sample exceeded the MRL. In one sample, the prohibited carbofuran residue was detected.
This study conducted validation studies for 39 pesticides in fresh red pepper using the LC-MS/MS technique and the QuEChERS extraction method, and calculated measurement uncertainties. In the validation studies, the linearity, matrix effect, quantification limit (LOQ), trueness, repeatability (RSDr), reproducibility (RSDwR) parameters specified in the SANTE guideline were used. The results obtained met the conditions specified in the manual. The quantification uncertainty was also calculated for each of the 39 pesticides. The quantification uncertainty values were found to be less than 50%. In addition, at least one pesticide residue was detected in 12 of the 22 red pepper samples analyzed. It was observed that the acetamiprid residue detected in one sample was above the MRL. In another sample, carbofuran residue, which is banned in Turkey, was detected.
ACKNOWLEDGMENTS
This study was supported by the Harran University Scientific Research Institutions (HUBAK Project No: 14009).
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Received: August 5, 2021
Sent to Subject Editor: September 16, 2021
Accepted: December 16, 2021
Recommended by Subject Editor Ardson Vianna Jr.