To begin the sample preparation using the QuEChERS method, weigh five grams of an avocado sample of interest in a 50 milliliter centrifuge tube. Add 50 microliters of the procedural internal standard, or P-IS solution to the tube. Add 10 milliliters of aceto nitrile to the centrifuge tube and shake the tube at 70 RPM for five minutes.
Then, add 2.5 grams of ammonium formate to it, and shake the tube again at 70 RPM for five minutes. Subsequently, centrifuge the tube at 1, 800 G for five minutes. Next, in a two milliliter centrifuge tube, take the appropriate amounts of the reagents displayed on the screen.
Then, add one milliliter of the extract into the tube for purification using dispersive solid phase extraction. Vortex the tube for 30 seconds before centrifuging it at 1, 800 G for five minutes. Transfer 200 microliters of the purified extract to an autosampler vial, and add 20 microliters of the analyte protectant solution, followed by adding 50 microliters of a pre-prepared triphenyl phosphate solution.
Finally, perform instrumental analysis using a GC-MS/MS system. Conduct the analyses using GC-MS/MS system with a triple quadrupole equipped with an electron ionization interface of 70 electron volts in an autosampler. Ensure that the gas pressures are correct, helium at 140 PSI, and argon at 65 PSI.
Turn on the main GC switch located on the front panel, and turn on the MS switch located at the back. Open the GC-MS real-time analysis software that controls all the parameters of the GC-MS/MS system. Click on Vacuum Control, then Advanced, followed by Rotary Pump 1 to initiate the vacuum system.
Click on Start to turn on the turbomolecular pumps one and two. Then, click on the Start option for the ion source heater. Set the MS interface temperature to 250 degrees Celsius and the ion source temperature to 300 degrees Celsius.
Maintain the GC oven at an initial temperature of 50 degrees Celsius for one minute, then ramp it up to 180 degrees Celsius at a rate of 25 degrees Celsius per minute. Subsequently, increase the temperature to 230 degrees Celsius at five degrees Celsius per minute, and then to 290 degrees Celsius at 25 degrees Celsius per minute. Then, keep the temperature constant at 290 degrees Celsius for six minutes.
The total analysis time is 24.6 minutes. Once all these systems are turned on, click on Close. Then, click on the Tuning option from the Analysis Software and select Peak Monitor View to perform an initial verification of the mass spectrometer conditions.
Click on Acquisition, and from the displayed window, click on Download Initial Parameters. Verify that the equipment is ready GC and ready MS.For data acquisition, click on New Batch File from the software, and create a sequence containing information such as sample name, sample ID, method file, data file, injection volume, and tuning file. Add rows as necessary, and click on Save.
Click on Batch Start, and let the injection process commence. Perform the injection at 250 degrees Celsius in the split list mode, maintaining an injection volume of one microliters. After one minute following the injection, open the split.
Analyze the analytes using the Multiple Reaction Monitoring, or MRM mode, which is the standard mode employed in MS/MS systems with a triple quadrupole. Open the post-run analysis software for data analysis. Click on the injection to be analyzed, navigate through the table containing the analytes, and select the peak of interest.
Click on the peak or the region of interest to visualize the chromatogram, review the peak integrations, and if necessary, perform manual integration. Verify the areas of all analytes to perform the necessary calculations and method evaluation. Generation of matrix-matched calibration curves revealed a relatively consistent matrix effect of less than 20%classified as a soft ME for most pesticide combinations.
The results in the displayed figure demonstrated the count of pesticides whose average recovery percentages were within the acceptable range of 70 to 120%for each type of avocado. A significant proportion of the tested pesticides exhibited recovery percentages falling within the specific range, with relative standard deviation values below 20%
