In Situ Real-Time Tracing of Organophosphorus Pesticides in Apples by Solid-Phase Microextraction with Developed Sampling-Rate Calibration

An in situ tracing study based on solid-phase microextraction (SPME) was conducted to investigate the uptake and elimination of organophosphorus pesticides in apples. A matrix-compatible polydimethylsiloxane/poly(styrene-co-divinylbenzene)/polydimethylsiloxane fiber was produced to meet the needs of in situ sampling. The fiber had high extraction ability, good sensitivity and accuracy with respect to the analytes in apple pulp, and could be used 85 times. Although the sampling rate was changing over time, quantification was still achieved by the sampling rate calibration method. Some factors that affect its applicability were studied. The limits of detection were 0.18 ng/g for diazinon and 0.20 ng/g for chlorpyrifos, rather lower than the maximum residue limits of the National Food Safety Standard of China (GB 2763-2016) and the European Commission (Reg.(EU) No 834/2013, 2018/686). The accuracy of in situ SPME quantification was verified by comparing with the results obtained by the traditional liquid–liquid extraction method. In this work, the in situ sampling method is developed using apples, diazinon, and chlorpyrifos as a model system; however, this method can be used for in vivo analysis of fruits and vegetables for nutrition and safety monitoring.


Supplementary Materials
.Rinsing and washing conditions tested in this study.

Preparation of SPME Fibers
Sylgard 184 PDMS prepolymer and curing agent were mixed thoroughly at a ratio of 10:1, according to the manufacturer's manual, in a polypropylene centrifuge tube, and subjected to centrifugation for 8 min at 12000 rpm for degassing. A portion of 45 mg of P(St-co-DVB) microspheres was mixed into 700 mg of the resulted PDMS solution to obtain uniform PDMS/P(St-co-DVB) mixture. A corroded stainless steel wire was dipped vertically into the mixture and subsequently pulled out at a slow rate, and a PDMS/P(St-co-DVB) coating was formed on the surface of the wire end (about 1.5 cm). The coated fiber was placed in a drying oven at 150 °C for 2 min to cure the coating. For each fiber, the coating/curing process was repeated to obtain appropriate thickness. The PDMS overcoated PDMS/P(St-co-DVB) fibers were prepared according to the previous study by Souza Silva [13]. PDMS fibers without P(St-co-DVB) microspheres were also prepared for daily quality control (QC) tests. The coatings with thickness of 59 μm and length of 1.5 cm were selected in this study.
Prior to use, the fibers were conditioned in a GC injection port under inert gas flow (N2) at 250 °C for 2 h, and then 280 °C for 1 h. During the usage, the coatings were inspected using an optical microscope.

SPME Procedure
After chopping and removing the cores, the apple was homogenized along with 2-fold amount of deionized water using a high speed disperser. The processed sample was placed in amber glass bottles and stored in a freezer at 4 °C until analysis.
DI-SPME was carried out in 10-mL vials sealed with Teflon-coated septum sealed steel caps. After being spiked with pesticides, the apple pulp was agitated for one hour at 600 rpm and 30 °C before further pretreatment in order to allow for the sufficient binding of the analytes to the matrix. Each portion of 8 g of apple pulp was weighed in a 10-mL vial. A 15-min pre-extraction incubation of the sample was performed in the agitation unit at 600 rpm and 25 °C, followed by 30 min of extraction at the same temperature and stirring rate. Following extraction, the fiber was rinsed in a rinsing solvent, desorbed for 10 min at 280 °C, and then washed with a washing solvent. After each sample, a KimWipe tissue soaked with the washing solvent was used manually to gently clean the fiber surface of any attached debris. All experiments were performed in triplicate.
For the evaluation of DI-SPME/GC method, the extraction temperature was 30 °C, the extraction time was 50 min, the stirring rate was 1200 rpm and the desorption time was 5 min. Other conditions were the same as above.In this part, all experiments were performed in quintuplicate.
The stability of the instrumental response was monitored by QC tests, which were conducted by a home-made PDMS fiber in 8 mL of spiked deionized water at the beginning of each working day.
Duringin situ sampling the fiber only went to a depth of 2 cm in the apple, and the sample for LLE also took the similar part of apple.
Briefly, a portion of 20 g of chopped apple sample was homogenized along with 50 mL of acetonitrile using a high speed disperser, and then the mixture was vigorously shaken for 5 min and filtrated by suction filtration. The filtrate with the volume of 40-50 mL was collected in a 100-mL polypropylene centrifugal tube. Subsequent phase separation was facilitated by adding 6 g of sodium chloride and shaking for 1 min. After centrifugation for 15 min at 4000 rpm, 10 mL of acetonitrile solution from the upper layer was suctioned out and put into a 50-mL polypropylene centrifugal tube and then evaporated to near dryness in water bath under 80 °C with a steam of nitrogen. Less than 2 mL of methanol was applied to flush the tube two times, and the solutions were totally transferred