Pesticide residues screening in wine by mass spectrometry

Recently, a study (from PAN Europe) covered 40 bottles of wine – 34 conventional and six organic ones – purchased inside the EU. According to the results, the 34 bottles of conventional wine together contained 148 pesticide residues. All 34 bottles contained from one to ten pesticides, bringing the average per bottle to more than four. Of the six bottles of organic wine tested, one sample contained a low concentration of a possibly carcinogenic pesticide. According to PAN Europe, the “contamination of wines is a direct result of over-reliance on pesticides in grape production”. This study, between others, to prove the importance of develop methods sensivity and confident for pesticide detection in wine. A multiresidue method was developed for the determination ca of 250 pesticide residues in wine using Quechers extraction, gas chromatography-tandem mass spectrometry (GC-MS-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). The method was validated with the evaluation of follow parameters: Linearity, Precision, Accuracy, Matrix effect, Limit of detection and Limit of Quantification. The method was approved and was able to quantify pesticide residues in more than 60 samples of wine.


Introduction
In this paper, we report a rapid modified QuEChERS method for multiresidue analysis for ca of 300 pesticides in wine with good selectivity, sensitivity, and cost effectiveness.In order to demonstrate the suitability of the method for routine regulatory purposes, the method was validated and the statistical parameters are discussed.

Reagents and standards
The certificated analytical standards were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Alemanha).All the solvents and chemicals used in the study were of HPLC grade.

Instrumentation
Ultra high-performance liquid chromatography UHPLC-MS/MS (liquid chromatography, Shimadzu and MSMS, API 4000 Applied Biosystems) equipped with Phenomenex Kinetex column (50 mm × 2.1 mm, 2.6 µm) was used for quantification of pesticides.The flow rate was 250 µL/min, the column temperature 30 • C, and the injection volume 5 µL.A binary gradient of 0.1% HCOOH and HCOONH 5 mM in water (A) and 0.1% HCOOH and HCOONH 5 mM in CHOH (B) was employed.The mobile-phase gradient was programmed as follows: 0 min, 20% B; 10 min, 80% B; 10.01 min, 90% B; 13 min, 90% B; 13.01 min, 20% B; and 16 min, 20% B. Mass spectral analyses were operated in the positive ion mode using a ESI interface.The electrospray ionization (ESI) needle spray voltage was 5000.The heated capillary was 500 • C. Collision Gas Pressure was 5.The pesticides were detected in MS/MS conditions, programming the chromatographic run in MRM mode (multiple reaction monitoring).

Specificity
The specificity of the analytical method for pesticides detection was confirmed by obtaining positive results from honey containing the analyte, coupled with negative results from samples which do not contain it (negative controls).The matrix effect was assessed by preparing pesticide standards in blank matrix extracted from untreated honey.The matrix extracts were analyzed before spiking to confirm the absence of the test pesticides in them.

Linearity
The quantification of pesticide was based on a six-point matrix-matched calibration graph by plotting the detector response against concentration of the calibration standards within the range 2.5-50 µg/L making three replicates for each concentration.A linear regression of six calibration points for each component was used to determine the relationship with the analyte concentrations calculated for each component on the basis of their occurrence in the reference material.

Limit of Detection (LOD) and Limit of Quantification (LOQ)
The LOD and LOQ were determined by signal-to-noise approach.

Method Accuracy (Recovery) and Precision (Repeatability)
Method recovery studies were performed at two spiking concentration levels (10 µg/kg and 50 µg/kg).

Determination of uncertainties
Combined uncertainty in estimation was determined for all the pesticides at the two fortification levels studied (10 and 50 µg/kg) as the statistical procedure of the EURACHEM/CITAC Guide CG 4.

Specificity
The specificity of the method toward the studied analytes was good.No interferences due to matrixes were found.Hence, no further time-consuming concentration/cleanup pretreatments were required.

Linearity
The analytes showed linear behavior in the studied concentration range of 2,5-50 µg/L.The correlation coefficient (r 2 ) was found to be ≥ 0.99 for all pesticides.

LOD and LOQ
LOD and LOQ were estimated as the lowest concentrations of pesticide injected that yielded a signal/noise ratio of 3 and 10, respectively.The LOQs attained in the proposed method fit with maximum residue limits (MRLs) for grape.The limits were reported in Table 1.

Recovery and precision
The single-step extraction method adopted for wine samples provided satisfactory recovery.These data are in agreement with the criteria of document no.SANCO/12495/2011, that recommend general recovery limits of 70-120% within laboratory repeatability ≤ 20% [18].Therefore, the method could be considered sufficiently accurate and precise for the purpose.

Uncertainty of measurement
The study of uncertainty was performed at 2 concentration levels (10 and 50 µg/kg), identifying and studying the most important parameters that determined the uncertainty of the analytical method.The parameters selected were point calibration, standard solution, weigh, volume, and precision; their contributions to method uncertainty were calculated as indicated in the experimental section.The different contributions of uncertainty for each concentration level, together with the relative combined standard uncertainty, are shown in Table 1.

Wine samples analysis
Sixty wine samples were analyzed between 2012 and 2015.The samples were from different wineries in the state of Rio Grande do Sul, Brazil.About 300 pesticides were analyzed and 30 were detected.40 samples were determined at least one pesticide.

Conclusion
Based on these results, the methodology for the determination of pesticides in wine, described in this report, are validated and approved for execution.

Table 1 .
Results for LOQ, Recovery, Precision and Uncertainty of Measurement.