Emanuela Gionfriddo1, 2, Dominika Gruszecka, Xiujuan Li1, 3, Janusz Pawliszyn1
1Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada, 2Department of Chemistry and Biochemistry, The University of Toledo, Toledo OH, 43606, USA, 3Key Laboratory of Environment Correlative Dietology (Ministry of Education), College of Food Science & Technology, Huazhong Agricultural University, Wuhan, China
A matrix-compatible direct-immersion solid-phase microextraction (SPME) fiber, named PDMS/DVB/PDMS or SPME-OC Fiber, was used for the determination of pesticides in soy milk via direct immersion. Combined with gas chromatography-mass spectrometry, it eliminated the need for extensive sample pre-treatment procedures. To extend the lifetime of the SPME device, rapid pre- and post-desorption cleaning steps were implemented. This method allowed to achieve limits of quantitation (1–2.5 μg/kg) for the targeted analytes that were below the Maximum Residue Levels (MRLs) mandated for soy-based products.1,2
Soy-based products are a category of nutraceuticals extensively used worldwide for their health benefits and also as a more sustainable alternative to dairy products. Raw soy grains are the starting material for all soy- based products and are often exposed to agrochemicals from agricultural and post-harvesting practices. It is important to monitor the level of pesticide residues in soy derivatives to ensure their compliance with tolerance limits set by various regulatory agencies across the world. Soy milk, being a stable emulsion of oils, water, and proteins, is a challenging sample to treat for the extraction of pesticides residues at ultra-trace levels. To propose an automated and sensitive method, solid phase microextraction (SPME) was considered as an extraction technique in this work. This is because of SPME’s ability to provide an automated analytical workflow and pre-concentration to achieve limits of quantitation for the targeted pesticides at low part-per-billion levels.3 Moreover, the use of a matrix- compatible SPME fiber enabled direct immersion extraction from soymilk, improving the recovery of pesticides with good water solubility.
Table 1 describes the final optimized method.
Calibration was performed via matrix-matched calibration, spiking the analytes of interest and three deuterated internal standards: diazinon-D10, malathion-D6, and thiabendazole-D4. The soymilk samples were purchased at local grocery stores and were refrigerated until analyzed.
The SPME procedure necessitated the optimization of fiber washing after the extraction (rinsing) and desorption (washing), in order to prolong its lifetime. And previous studies demonstrated that this optimization needed to be performed based on the type of food matrix analyzed and the targeted analytes.4-7 Several rinsing and washing solutions were tested (Table 2).
The best cleaning method involved a rinsing step in water:acetone (9:1 v/v) for 10 s and 1 min washing in water:acetone (1:1 v/v), in combination with a 1:1 dilution of the soy milk sample with ultra pure water prior to SPME. This method allowed for 120 consecutive extractions with an average signal variation of +/- 25% and % RSD of less than 15%. Furthermore, the matrix modifiers were optimized for enhanced extraction of hydrophobic analytes. Salting out effects were investigated by varying the ionic strength of the solution, by adding sodium chloride within a range from 5 to 20% to the soy milk/water mixture (1:1, w:w). However, due to no significant improvement noticed in the recovery of the analytes, the addition of salt was discarded for further optimization. An alternative strategy to improve recovery is the addition of organic modifiers. For aqueous samples, optimal recoveries are obtained keeping the content of the organic solvent below 1%. But for complex samples containing matrix constituents that can bind the analytes, the addition of organic modifiers is useful to shift the binding equilibrium toward the free, unbound form thus improving recovery by SPME. In this work, four organic solvents were considered, namely, acetonitrile, acetone, methanol, and ethanol. Each solvent was added at concentrations of 10%, 20%, 30%, and 50% (v:v) to the samples. Solvent concentrations above 50% induced congealing of the soy milk, thus were not further tested. The results showed that the addition of a solution containing 30:70 acetone:water (v:v) to the soy milk sample (dilution ratio 1:1) allowed the best recovery of the targeted analytes. Further, other parameters were finely tuned to optimize both the extraction and desorption process (Table 1).
A matrix-matched calibration approach was used by spiking pesticide-free soymilk samples with all analytes in a concentration range of 1-1000 µg/kg; with the exception of phosalone which was spiked at 2.5-1000 µg/kg. Calculations were performed using linear regression for each of the targeted analytes, except phosalone, which required a 1/x2 weight. The accuracy and precision of the method were assessed at three concentration levels of 15, 75, and 200 µg/kg in quadruplicate measurements over three days. Limits of quantitation (LOQs) were determined at the lowest concentration level with an RSD of below 20%, and accuracy within 30% of the nominal concentration. LOQs ranged between 1 and 2.5 µg/kg. The LOQs achieved by this method allowed the detection of the targeted pesticides below the recommended limits set for soy products by the European Commission6 and Office of the Federal Register7 for the USA Market. A summary of the figures of merit for this work is provided in Table 3.
The validated method was further used for the analysis of different brands of soy milk samples obtained from local grocery stores. The results in Table 4, show the occurrence of several targeted pesticides, up to 118.9 µg/kg, in two different commercial brands of soy milk.
A new method for the analysis of pesticides in soymilk was optimized and validated using a matrix-compatible SPME fiber. This DI-SPME-GC-MS method was able to quantitatively monitor the presence of pesticides with LOQs of 1-2.5 µg/kg, with a completely automated workflow including rinsing and washing of the SPME fiber. The excellent robustness of the SPME matrix compatible fiber enabled its use of up to 120 extraction/desorption cycles. For our complete SPME portfolio or to download the guide "SPME for GC - Getting Started with Solid Phase Microextraction" visit us at SigmaAldrich.com/SPME