Analyses of residues from plant protection substances have been carried out centrally at two investigative offices in Baden-Württemberg since 1 January, 2002. The Chemisches und Veterinäruntersuchungsamt (CVUA) Stuttgart conducts analyses on foods of plant origin and CVUA Freiburg is responsible for foods of animal origin. The number of different pesticidal substances on the world market is growing constantly. About 1,000 substances are routinely analyzed as part of the food monitoring program at CVUA Stuttgart.
In order to ensure the likelihood of detecting an ever growing number of greatly diverse pesticide substances with a broad spectrum of chemical properties, existing analytical methods must be constantly optimized for new substances and new methods must be developed. Given the low concentrations of residues in foods, their analysis is very challenging. Much knowledge is required for the ever-constant development of new analytical methods, and the most modern of analytical instruments are absolutely indispensable. Only with this degree of expertise and equipment can a thorough and precise determination of pesticide residues be guaranteed and consumer protection be effectively carried out.
Most laboratories make use of so-called multi-methods, e.g. QuEChERS and QuPPe, for processing the samples to be analyzed for the presence of pesticide residues. A very large number of substances can be hereby captured in a single procedural step and prepared for the actual measurement. Very different methods of measurement come into play here, each of which is matched to specific pesticide substances.
In summary, the routine analysis of samples for pesticide residues and contaminants conducted at CVUA Stuttgart are currently carried out in the following steps:
The sample description involves checking the amount of sample, the batch unit, the condition of the samples, and whether the information which is legally required, such as country of origin or use-by date, has been provided.
The quantified residues should represent not only those in the individual sample being analyzed, but those of the whole production batch. A minimum amount of sample must be available for the analyses, to ensure statistical certainty (as a rule, 1 kg and/or 10 sample pieces). A basic requirement is that all parts of the sample must be from the same batch. These and further requirements for the sampling are defined in Directive 2002/63/EC (Community methods of sampling for the official control of pesticide residues in and on products of plant and animal origin).
Assessing the condition of the samples involves careful observation, testing for any odors, cutting the sample into pieces and tasting them. The state of the sample is recorded in writing and photographed.
Special focus is given to sensory anomalies, such as mold, decay, rancid smells or taste. In individual cases, for example when an infestation of insects is found, the sample is reported to the authorities, without any further analysis.
Likewise, a sample will not be further analyzed for pesticide residues if it is visibly damaged to a large extent, because the spoiling process destroys the pesticide residues. The metabolic processes of mildew, e.g., can degrade pesticide substances, so any analytical results would have little meaning here.
The country of origin is especially important for the judgment of possible pesticide residue amounts, because every EU member state approves the pesticide substances, based on the local prevailing climatic conditions and crops grown. Guidelines regarding the application of pesticides and their active substances may differ from those of other member states of the EU. Samples from Germany are also checked for compliance with national guidelines, in addition to those established for the EU.
Products labeled as of German origin are sometimes found to have actually come from a third country, deduced from the type of pesticide residues detected. In some cases these false claims were discovered as a result of cooperation with local official food inspectors.
Especially for pre-packaged products that are packed in the absence of consumers and need not be consumed immediately, there are several mandatory details whose presence on the sample label must be verified. These are defined in Regulation (EU) No 1169/2011 on the provision of food information to consumers.
Samples to be analyzed for pesticides are not washed beforehand, because this reduces the amount of residues on the sample. Moreover, particular parts of some food samples are not analyzed, in accordance with specifications in the pesticide residue maximum level regulation (Reg. (EC) 396/2005). For example, the leaves of strawberries and the stems of grapes are not analyzed with the fruit.
In order to ensure that the analytical results are representative, it is crucial not only to have a suitable number of samples, but also to conduct the comminution and finally the homogenization in a proper and careful manner.
While finely ground wheat products usually need no further comminution, stone fruit processing, for example, requires removing the seed before analysis and then weighing the seed to re-include it in the calculation of the pesticide amount.
Long, thin vegetables, on the other hand, are cut into five, equally long pieces, whereby only the two ends and the middle piece are used for the homogenization. Larger products such as apples are cut into four sections, and the two opposite pieces are further processed for the analysis (see also Illustrations 1a–1e). These recommendations were developed by Gesellschaft Deutscher Chemiker and have since been used by CVUA Stuttgart.
Illustrations 1a–1e: Comminution of Samples; Sectioning Techniques
After the coarsely chopped sample pieces are frozen overnight, they can be milled in their frozen state in a mixer with dry ice into a finely granulated, homogenous mass (cryogenic milling).
Depending on the type of sample, 1 g to 10 g of this homogenous mass is then weighed in a suitable extraction vessel for the processing of the sample (see also Illustrations 2a–2c).
Illustrations 2a–2c: Weighing of Homogenous Sample Mass in the Extraction Vessel
A major advance in modern pesticide residue analysis is that, for over 99 % of the residues to be analyzed, only two different methods of processing have to be performed. The so-called multi-methods make it possible to process a large number of substances in a significantly short time and with a minimum of material expenditure. Low or medium polar substances are processed using the QuEChERS method, whereas highly polar substances are processed with the QuPPe method. Both methods were developed by an employee of CVUA Stuttgart and are now used in laboratories around the world.
Pesticidal substances themselves, however, differ greatly from each other in terms of their chemistry. There are residues that degrade very quickly in a sour extract with low pH values, but remain stable over a longer period in a neutral or lightly alkaline extract with high pH values. The pH value of the processing method must therefore be adjusted, to make sure that a sample extract can be used for the measurement of a specific pesticide substance. For sour samples such as lemons or pomegranates, something alkaline such as sodium hydroxide will be given. This ensures that the pH value stays in an acceptable range and the pesticide residues can be measured without any losses.
The sample matrix in which the pesticide residues should be detected also has a large influence on the quality of the analytical results. In order to accurately analyze the dark green extract in herbs, for example, the green-colored „chlorophyll“ must be removed from the extract (see also Illustration 3). For this purpose, a special carbon powder (activated carbon) is added, which the color binds itself to. The extract is then put in a centrifuge, where the -carbon-bound chlorophyll can be separated from the cleaned extract.
Illustration 3: Colored QuEChERS-Extracts
These adjustments for the two processing methods, depending on the type of sample and specific residues, are necessary, but require little effort. The analyses and evaluation of the complex results that follow, however, require additional experience and much know-how.
Following the sample preparation the chemically diverse pesticide substances are grouped and analyzed using several different measuring instruments and specific investigatory methods. Using different instruments and measuring methods ensures reliable identification and preliminary quantification of the pesticide residues in the samples. If positive findings are made, either of quantities that are close to the legal limit values or of unauthorized substances, a follow-up analysis is conducted. This involves more precise quantification by means of a more sophisticated procedure.
Two main chromatographic separation procedures are used for the separation of pesticide residues from each other and from parts of the sample (investigatory matrix) that have gotten into the extract: gas (GC) and liquid chromatography (LC). In both cases, there is an immobile separating substance (solid particles in the case of LC and a liquid film in the case of GC) in which the separation occurs (separation phase). In addition, a further phase is needed to transport the molecules and substances. A liquid is used for the liquid chromatography, and a gas for the gas chromatography.
The simplest model of a liquid chromatography is represented by paper chromatography. The paper is the solid separation phase, while water or a solvent can be used as the „transporter“. The various colors contained in a black (or other colored) felt-tip marker can thus be separated (see Illustration 4).
Illustration 4: Paper Chromatography for a Black Felt-tip Marker Using Methanol
It is easy to imagine that using thicker or rougher paper could lead to different results. That is why we also use different solid phases in the lab, specially adjusted for the different substance groups. Temperature, flow velocity, and the combination of solvents also influence the result of the separation, and are thus optimized for each method.
Directly after the substances are separated they are then identified by means of various detection systems. In most cases the system consists of two linked mass spectrometers (MS/MS), but single mass spectrometers (MS) and time-of-flight (TOF) mass spectrometers are also used for this purpose.
Mass spectrometers function as mass filters. Simply put, molecules are separated in an MS according to their mass. The strength of the signal can also be measured, based on the number of molecules measured, upon which an initial quantification of the analyzed substances can be made. In an MS/MS system the molecules are separated in the first MS, fragmented into smaller pieces, and then measured in the second MS. Since the molecule fragments are often very specific to the original substance from which they came, the MS/MS system offers a more reliable identification of substances.
After measuring the extracts, each pesticide residue is evaluated individually, with help from calibration standards and an evaluation program. If a positive finding is made, the substance will be evaluated as to whether the amount lies near or even above the legal limit. If so, the pesticide amount must be checked again using a more sophisticated procedure (precise quantification). To ensure accuracy, the sample must be processed two more times (double determination).
Carrying out more precise quantification measures of pesticide residues involves special analytical procedures that cannot be used for the first round of analysis because they are too time-consuming and costly. The double determination confirms the initially determined amount with its twice repeated, more precise analysis. This is especially necessary when a pesticide amount was found to be near or even above the limit value in the first analysis.
The most reliable and commonly used procedure used for the precise determination of residues, especially for problematic matrices, is the standard addition. Various amounts of the pesticide substance (the analyte) are added to the sample.
In addition, special internal standards (ISTD) are used, preferably those that have the same chemical properties as the analyte (e.g. with isotope labeled standards). The calculation is made using the area ratio of the analyte and internal standard. The results are very exact and can stand up to every test.
The evaluation of residue findings must consider an analytical margin of error, in accordance with EU Document No. SANTE/11813/2017. For samples of plant origin this margin is 50 %, calculated from a large number of EU proficiency tests of pesticide residues.
From the viewpoint of the official laboratory, a sample where e.g. the legal limit of 0.01 mg/kg applies is normally only considered to be in violation with the law when the analytical result was at least 0.021 mg/kg (measured value minus 50 %). An exceedance of the maximum level with an amount less than 0.021 mg/kg cannot be supposed with absolute certainty (95 % confidence interval). On the other hand, this means that the importers, producers and suppliers should make sure that the maximum levels found by their own monitoring analyses are not close to the legal limits. To ensure that goods are in compliance with food law requirements at a level of 95 % confidence, the residue amounts in the sample should lie well under the maximum level (measured value plus 50 %), thus giving room for measurement uncertainty.