We would like to thank EAN University and the University of La Laguna.

1. Preparation of the stock and working solutions
NOTE: For safety reasons, it is advisable to wear nitrile gloves, a laboratory coat, and safety glasses throughout the protocol.
<ol>
	<li>Prepare individual stock solutions of each of the 45 commercial pesticide standards (see Table of Materials) at approximately 1,000 mg/L in acetonitrile in 10 mL volumetric flasks.</li>
	<li>Combine the above individual stock solutions to prepare a 400 mg/L stock solution in acetonitrile in a 25 mL volumetric flask. 
	NOTE: This mixed solution will be utilized to prepare the working solutions for recovery and calibration experiments.</li>
	<li>Prepare stock solutions of atrazine-d5 and triphenyl phosphate (TPP) at concentrations of 750 mg/L and 1,050 mg/L, respectively, in acetonitrile in 10 mL volumetric flasks. Utilize atrazine-d5 as a procedural internal standard (P-IS) and TPP as an injection internal standard (I-IS). 
	NOTE: The ideal scenario would involve the utilization of an isotopically labeled internal standard for each specific target analyte.</li>
	<li>Prepare stock recovery solutions in acetonitrile containing 0.05% (v/v) of formic acid (to prevent degradation) in 10 mL volumetric flasks to yield 10, 100, and 400 &#181;g/kg sample equivalents for the pesticides and 200 &#181;g/kg for the P-IS separately. Store these solutions in amber glass vials in the dark at &#8722;20 &#176;C.</li>
	<li>Prepare calibration solutions of the pesticides and P-IS together in acetonitrile with 0.05% (v/v) of formic acid in 10 mL volumetric flasks to yield 5, 10, 25, 75, 200, 400, and 600 &#181;g/kg, and 200 ng/ng, respectively, and store them in amber glass vials in the darkness at &#8722;20 &#176;C. 
	NOTE: The same solutions may be utilized throughout the experimental work but storing them under the specified conditions immediately after each use is essential.</li>
	<li>Prepare a mixture of analyte protectants containing 100 g/L of 3-ethoxy-1,2-propanediol, 10 g/L of L-gulonic acid &#947;-lactone, 10 g/L of D-sorbitol, and 5 g/L of shikimic acid in a 4/1 (v/v) ratio of acetonitrile to water with 0.5% (v/v) of formic acid. 
	NOTE: This mixture of analyte protectants is to be added just before the injection to mitigate ME.</li>
</ol>
2. Sample collection
<ol>
	<li>Collect samples from three avocado species (e.g., Criollo, Hass, and Lorena) available at supermarkets. Ensure that each sample weighs approximately 1 kg, which is enough for conducting all the subsequent studies and aligns with Directive 2002/63/CE21. 
	NOTE: Organic samples were preferentially selected to minimize the likelihood of the presence of pesticide residues.</li>
	<li>Transport the collected avocado samples to the laboratory, and individually homogenize them without the pipe using a chopper (see Table of Materials). Store the homogenized samples in amber glass containers at 4 &#176;C until analysis. 
	NOTE: The same avocado samples will be used throughout the entire study. Therefore, it is crucial to store them under the specified conditions immediately after each use.</li>
</ol>
3. Sample preparation utilizing the QuEChERS method with ammonium formate
NOTE: Figure 1 illustrates a schematic representation of the QuEChERS method with ammonium formate.
<ol>
	<li>Weigh 5 g of each avocado sample in a 50 mL centrifuge tube (see Table of Materials).</li>
	<li>Add 50 &#181;L of the P-IS solution to yield a concentration of 200 &#181;g/kg. For recovery assessment, also add the pesticide solutions prepared in step 1.4 to yield concentrations of 10, 100, and 400 &#181;g/kg (n = 5 each).</li>
	<li>Vortex the tube for 30 s to ensure thorough integration of the spike into the sample.</li>
	<li>Add 10 mL of acetonitrile to the centrifuge tube. Shake the tube at 70 rpm for 5 min.</li>
	<li>Add 2.5 g of ammonium formate, shake the tube again at 70 rpm for 5 min, and subsequently centrifuge it at 1,800 &#215; g for 5 min.</li>
	<li>To a 2 mL centrifuge tube containing 150 mg of anhydrous MgSO4, 50 mg of primary-secondary amine (PSA), 50 mg of octadecylsilane (C18), 10 mg of graphitized carbon black (GCB), and 60 mg of a zirconium oxide-based sorbent Z-Sep+, add 1 mL of the extract for purification utilizing dispersive-solid phase extraction (d-SPE). Vortex the tube for 30 s and centrifuge it at 1,800 &#215; g for 5 min.</li>
	<li>Transfer 200 &#181;L of the extract to an autosampler vial, add 20 &#181;L of the analyte protectant solution prepared in step 1.6, and include 50 &#181;L of the TPP solution.</li>
	<li>Perform instrumental analysis using a GC-MS/MS system (see section 4).</li>
	<li>Perform matrix-matched calibration following the same procedure as described above, using blank extracts, except, during the d-SPE step (step 3.6), clean 5 mL of the supernatant in 15 mL tubes. Add the spike and P-IS solutions at step 3.7. Add the calibration standard solutions to the autosampler vials to yield 5, 10, 25, 50, 100, 200, 400, and 600 &#181;g/kg, along with the TPP, resulting in a final volume of 270 &#181;L. 
	NOTE: Overall, be sure to construct matrix-matched calibration curves for each avocado variety plus the acetonitrile-only calibrations.</li>
</ol>
4. Instrumental analysis using GC-MS/MS
<ol>
	<li>Conduct the analyses employing a GC-MS/MS system with a triple quadrupole (TQ) equipped with an electron ionization interface (&#8722;70 eV) and an autosampler (see Table of Materials).</li>
	<li>Employ an MS GC column (silica Bond of 30 m length, 0.25 mm inner diameter, 0.25 &#181;m film thickness) along with ultrahigh purity Helium as the carrier gas at a constant flow rate of 1.2 mL/min.</li>
	<li>Verify the following parameters before proceeding with the equipment operation:
	<ol>
		<li>Ensure that the gas pressures are correct: Helium at 140 psi and Argon at 65 psi.</li>
		<li>Check the condition of the rotary pump oil to ensure that it is clear and at the appropriate level.</li>
		<li>Ensure that the injection syringe does not have any obstructions from previous injections.</li>
		<li>Confirm that the wash vials contain a sufficient volume of each solvent.</li>
		<li>Check that the consumables counter (septum, liner) has not reached its limit.</li>
	</ol>
	</li>
	<li>Turn on the main GC switch located on the front panel and turn on the MS switch located at the back.</li>
	<li>Open the GCMS Real Time Analysis software that controls all the parameters of the GC-MS/MS system. 
	NOTE: The instrument system includes the GCMS Real Time Analysis software by default.</li>
	<li>Click on Vacuum Control | Advanced | Rotary Pump 1 to initiate the vacuum system. 
	NOTE: In this window, monitor the pressure to determine the optimal vacuum values, which should be lower than 9.0 Pa. It will take approximately 12 h.</li>
	<li>Click on Start to turn on turbo molecular pump 1 and turbo molecular pump 2.</li>
	<li>Click on Start for the Ion Source Heater option. 
	NOTE: After a recommended time of 1 h, check the system&#39;s vacuum to confirm that the recommended value is lower than 1.6e-3 Pa.</li>
	<li>Set the MS interface temperature to 250 &#176;C and the ion source temperature to 300 &#176;C.</li>
	<li>Maintain the GC oven at an initial temperature of 50 &#176;C for 1 min, then ramp it up to 180 &#176;C at a rate of 25 &#176;C/min. Subsequently, increase the temperature to 230 &#176;C at 5 &#176;C/min and then to 290 &#176;C at 25 &#176;C/min. Finally, keep the temperature constant at 290 &#176;C for 6 min. The total analysis time is 24.6 min.</li>
	<li>Click on Close once all these systems are turned on.</li>
	<li>Click on the Tuning option from the analysis software and click on Peak Monitor View to perform an initial verification of the mass spectrometer conditions. 
	&#8203;NOTE: If necessary, perform autotuning.</li>
	<li>Click on Acquisition, and from the displayed window, click on Download Initial Parameters. Verify that the equipment is ready GC and ready MS.</li>
</ol>
5. Data acquisition
<ol>
	<li>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.</li>
	<li>Click on Batch Start and let the injection process commence.</li>
	<li>Perform the injection at 250 &#176;C in the splitless mode, maintaining an injection volume of 1 &#181;L. After 1 min following the injection, open the split. 
	NOTE: Between injections, be sure to clean the 10 &#181;L syringe with methanol, ethyl acetate, and acetonitrile, using a single rinse with each solvent. All the injections are performed in triplicate.</li>
	<li>Analyze the analytes using the multiple reaction monitoring (MRM) mode, which is the standard mode employed in MS/MS systems with a TQ. 
	NOTE: Table 1 provides the retention times (in min) and the quantifier and qualifier transitions for the multiclass pesticides, P-IS, and I-IS. The quantitative analysis relies on the ratio of the peak area of the quantitation ion to the P-IS ion. The I-IS is employed for quality control during injection. Supplementary File 1 contains chromatograms for all the 45 analyzed pesticides.</li>
	<li>Open the Postrun Analysis software for data analysis. 
	NOTE: The instrument system includes the GCMS Postrun Analysis software by default.</li>
	<li>Click on the injection to be analyzed, navigate through the table containing the analytes, and select the peak of interest.</li>
	<li>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.</li>
</ol>

Analyzing Pesticides in Avocado Using the QuEChERS Method and CG-MS/MS

<ol>
	<li>Raposo, F., Barcel&#243;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+strategies+of+matrix+effects+using+chromatography-mass+spectrometry:+An+overview+from+research+versus+regulatory+viewpoints.">Challenges and strategies of matrix effects using chromatography-mass spectrometry: An overview from research versus regulatory viewpoints.</a> Trends Analyt Chem. 134, 116068 (2021).</li><li>Rahman, M. M., Abd El-Aty, A. M., Shim, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+enhancement+effect:+a+blessing+or+a+curse+for+gas+chromatography?-A+review.">Matrix enhancement effect: a blessing or a curse for gas chromatography?-A review.</a> Anal Chim Acta. 801, 14-21 (2013).</li><li>Poole, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-induced+response+enhancement+in+pesticide+residue+analysis+by+gas+chromatography.">Matrix-induced response enhancement in pesticide residue analysis by gas chromatography.</a> J Chromatogr A. 1158 (1-2), 241-250 (2007).</li><li>Anastassiades, M., Ma&#353;tovsk&#225;, K., Lehotay, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+analyte+protectants+to+improve+gas+chromatographic+analysis+of+pesticides.">Evaluation of analyte protectants to improve gas chromatographic analysis of pesticides.</a> J Chromatogr A. 1015 (1-2), 163-184 (2003).</li><li>Trufelli, H., Palma, P., Famiglini, G., Cappiello, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+matrix+effects+in+liquid+chromatography-mass+spectrometry.">An overview of matrix effects in liquid chromatography-mass spectrometry.</a> Mass Spectrom Reviews. 30 (3), 491-509 (2011).</li><li>European Commission SANTE/11312/2021. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+document+on+analytical+quality+control+and+method+validation+procedures+for+pesticide+residues+analysis+in+food+and+feed.">Guidance document on analytical quality control and method validation procedures for pesticide residues analysis in food and feed.</a> European Commission. , (2021).</li><li>Kwon, H., Lehotay, S. J., Geis-Asteggiante, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+of+matrix+effects+in+liquid+and+gas+chromatography-mass+spectrometry+analysis+of+pesticide+residues+after+QuEChERS+sample+preparation+of+different+food+crops.">Variability of matrix effects in liquid and gas chromatography-mass spectrometry analysis of pesticide residues after QuEChERS sample preparation of different food crops.</a> J Chromatogr A. 1270, 235-245 (2012).</li><li>Lehotay, S. J., Ma&#353;tovsk&#225;, K., Yun, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+two+fast+and+easy+methods+for+pesticide+residue+analysis+in+fatty+food+matrixes.">Evaluation of two fast and easy methods for pesticide residue analysis in fatty food matrixes.</a> J AOAC Int. 88 (2), 630-638 (2005).</li><li>Gonz&#225;lez-Curbelo, M. &. #. 1. 9. 3. ;., Gonz&#225;lez-S&#225;lamo, J., Varela-Mart&#237;nez, D. A., Hern&#225;ndez-Borges, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+pesticide+residues+in+pollen+and+dairy+products.">Analysis of pesticide residues in pollen and dairy products.</a> Sustainable Agriculture Reviews 47. 47, 47-89 (2020).</li><li>Madej, K., Kalenik, T. K., Piekoszewski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation+and+determination+of+pesticides+in+fat-containing+foods.">Sample preparation and determination of pesticides in fat-containing foods.</a> Food Chem. 269, 527-541 (2018).</li><li>Yanty, N. A. M., Marikkar, J. M. N., Long, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+varietal+differences+on+composition+and+thermal+characteristics+of+avocado+oil.">Effect of varietal differences on composition and thermal characteristics of avocado oil.</a> J Am Oil Chem Soc. 88, 1997-2003 (2011).</li><li>Pano-Farias, N. S., Ceballos-Maga&#241;a, S. G., Muniz-Valencia, R., Gonzalez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+assessment+of+matrix+effect+and+uncertainty+of+a+gas+chromatography+coupled+to+mass+spectrometry+method+for+pesticides+in+papaya+and+avocado+samples.">Validation and assessment of matrix effect and uncertainty of a gas chromatography coupled to mass spectrometry method for pesticides in papaya and avocado samples.</a> J Food Drug Anal. 25 (3), 501-509 (2017).</li><li>Pano-Farias, N. S., Ceballos-Maga&#241;a, S. G., Jurado, J. M., Aguayo-Villarreal, I. A., Mu&#241;iz-Valencia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+method+for+pesticides+in+avocado+and+papaya+by+means+of+ultra-high+performance+liquid+chromatography+coupled+to+a+triple+quadrupole+mass+detector:+Validation+and+uncertainty+assessment.">Analytical method for pesticides in avocado and papaya by means of ultra-high performance liquid chromatography coupled to a triple quadrupole mass detector: Validation and uncertainty assessment.</a> J Food Sci. 83 (8), 2265-2272 (2018).</li><li>Pano-Farias, N. S., Ceballos-Maga&#241;a, S. G., Gonzalez, J., Jurado, J. M., Mu&#241;iz-Valencia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercritical+fluid+chromatography+with+photodiode+array+detection+for+pesticide+analysis+in+papaya+and+avocado+samples.">Supercritical fluid chromatography with photodiode array detection for pesticide analysis in papaya and avocado samples.</a> J Sep Sci. 38 (7), 1240-1247 (2015).</li><li>Lozano, A., Rajski, &. #. 3. 2. 1. ;., Ucl&#233;s, S., Belmonte-Valles, N., Mezcua, M., Fern&#225;ndez-Alba, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+zirconium+dioxide-based+sorbents+to+decrease+the+matrix+effect+in+avocado+and+almond+multiresidue+pesticide+analysis+followed+by+gas+chromatography+tandem+mass+spectrometry.">Evaluation of zirconium dioxide-based sorbents to decrease the matrix effect in avocado and almond multiresidue pesticide analysis followed by gas chromatography tandem mass spectrometry.</a> Talanta. 118, 68-83 (2014).</li><li>Han, L., Matarrita, J., Sapozhnikova, Y., Lehotay, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+recent+product+to+remove+lipids+and+other+matrix+co-extractives+in+the+analysis+of+pesticide+residues+and+environmental+contaminants+in+foods.">Evaluation of a recent product to remove lipids and other matrix co-extractives in the analysis of pesticide residues and environmental contaminants in foods.</a> J Chromatogr A. 1449, 17-29 (2016).</li><li>Chamkasem, N., Ollis, L. W., Harmon, T., Lee, S., Mercer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+136+pesticides+in+avocado+using+a+modified+QuEChERS+method+with+LC-MS/MS+and+GC-MS/MS.">Analysis of 136 pesticides in avocado using a modified QuEChERS method with LC-MS/MS and GC-MS/MS.</a> J Agric Food Chem. 61 (10), 2315-2329 (2013).</li><li>Rajski, &. #. 3. 2. 1. ;., Lozano, A., Ucl&#233;s, A., Ferrer, C., Fern&#225;ndez-Alba, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+pesticide+residues+in+high+oil+vegetal+commodities+by+using+various+multi-residue+methods+and+clean-ups+followed+by+liquid+chromatography+tandem+mass+spectrometry.">Determination of pesticide residues in high oil vegetal commodities by using various multi-residue methods and clean-ups followed by liquid chromatography tandem mass spectrometry.</a> J Chromatogr A. 1304, 109-120 (2013).</li><li>Hern&#225;ndez-Borges, J., Ravelo-P&#233;rez, L. M., Hern&#225;ndez-Su&#225;rez, E. M., Carnero, A., Rodr&#237;guez-Delgado, M. &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+abamectin+residues+in+avocados+by+high-performance+liquid+chromatography+with+fluorescence+detection.">Analysis of abamectin residues in avocados by high-performance liquid chromatography with fluorescence detection.</a> J Chromatogr A. 1165 (1-2), 52-57 (2007).</li><li>Moreno, J. F., Li&#233;banas, F. A., Frenich, A. G., Vidal, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+different+sample+treatments+for+determining+pesticide+residues+in+fat+vegetable+matrices+like+avocado+by+low-pressure+gas+chromatography-tandem+mass+spectrometry.">Evaluation of different sample treatments for determining pesticide residues in fat vegetable matrices like avocado by low-pressure gas chromatography-tandem mass spectrometry.</a> J Chromatogr A. 1111 (1), 97-105 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commission+Directive+2002/63/EC+of+11+July+2002+establishing+Community+methods+of+sampling+for+the+official+control+of+pesticide+residues+in+and+on+products+of+plant+and+animal+origin+and+repealing+Directive+79/700/EEC.">Commission Directive 2002/63/EC of 11 July 2002 establishing Community methods of sampling for the official control of pesticide residues in and on products of plant and animal origin and repealing Directive 79/700/EEC.</a> Official Journal of the European Union. L187, 30-43 (2002).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=European+Regulation,+396/2005,+Regulation+(EC)+NO+396/2005+of+the+European+Parliament+and+of+the+Council+of+23+February+2005+on+maximum+residue+levels+of+pesticides+in+or+on+food+and+feed+of+plant+and+animal+origin+and+amending+Council+Directive+91/414/EEC.">European Regulation, 396/2005, Regulation (EC) NO 396/2005 of the European Parliament and of the Council of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC.</a> Official Journal of the European Union. L70, 1-16 (2005).</li><li>Gonz&#225;lez-Curbelo, M. &. #. 1. 9. 3. ;., Lehotay, S. J., Hern&#225;ndez-Borges, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+modified+QuEChERS+version+for+high-throughput+analysis+of+a+wide+range+of+pesticides+in+foods.">Validation of a modified QuEChERS version for high-throughput analysis of a wide range of pesticides in foods.</a> Abstracts of Papers of the American Chemical Society. 246, (2013).</li><li>Gonz&#225;lez-Curbelo, M. &. #. 1. 9. 3. ;., Lehotay, S. J., Hern&#225;ndez-Borges, J., Rodr&#237;guez Delgado, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonium+formate+buffer+in+QuEChERS+for+high+throughput+analysis+of+pesticides+in+food+by+fast,+low-pressure+GC-MS/MS+and+LC-MS/MS.">Ammonium formate buffer in QuEChERS for high throughput analysis of pesticides in food by fast, low-pressure GC-MS/MS and LC-MS/MS.</a> Abstracts of Papers of the American Chemical Society. 248, (2014).</li><li>Gonz&#225;lez-Curbelo, M. &. #. 1. 9. 3. ;., Lehotay, S. J., Hern&#225;ndez-Borges, J., Rodr&#237;guez-Delgado, M. &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+ammonium+formate+in+QuEChERS+for+high-throughput+analysis+of+pesticides+in+food+by+fast,+low-pressure+gas+chromatography+and+liquid+chromatography+tandem+mass+spectrometry.">Use of ammonium formate in QuEChERS for high-throughput analysis of pesticides in food by fast, low-pressure gas chromatography and liquid chromatography tandem mass spectrometry.</a> J Chromatogr A. 1358, 75-84 (2014).</li><li>Varela-Mart&#237;nez, D. A., Gonz&#225;lez-S&#225;lamo, J., Gonz&#225;lez-Curbelo, M. &. #. 1. 9. 3. ;., Quick Hern&#225;ndez-Borges, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quick,+easy,+cheap,+effective,+rugged,+and+safe+(QuEChERS)+extraction.">Quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction.</a> Handbooks in Separation Science. , 399-437 (2020).</li><li>Gonz&#225;lez-Curbelo, M. &. #. 1. 9. 3. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+organochlorine+pesticides+in+a+soil+sample+by+a+modified+QuEChERS+approach+using+ammonium+formate.">Analysis of organochlorine pesticides in a soil sample by a modified QuEChERS approach using ammonium formate.</a> J Vis Exp. 191, e64901 (2023).</li><li>Gonz&#225;lez-Curbelo, M. &. #. 1. 9. 3. ;., Dionis-Delgado, S., Asensio-Ramos, M., Hern&#225;ndez-Borges, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pesticide+analysis+in+toasted+barley+and+chickpea+flours.">Pesticide analysis in toasted barley and chickpea flours.</a> J Sep Sci. 35 (2), 299-307 (2012).</li><li>Varela-Mart&#237;nez, D. A., Gonz&#225;lez-Curbelo, M. &. #. 1. 9. 3. ;., Gonz&#225;lez-S&#225;lamo, J., Hern&#225;ndez-Borges, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+analysis+of+pesticides+in+minor+tropical+fruits+from+Colombia.">High-throughput analysis of pesticides in minor tropical fruits from Colombia.</a> Food Chem. 280, 221-230 (2019).</li><li>Li, L., Li, W., Qin, D. M., Jiang, S. R., Liu, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+graphitized+carbon+black+to+the+QuEChERS+method+for+pesticide+multiresidue+analysis+in+spinach.">Application of graphitized carbon black to the QuEChERS method for pesticide multiresidue analysis in spinach.</a> J AOAC Int. 92 (2), 538-547 (2009).</li></ol>

The authors have no conflicts of interest to disclose.

The primary limitation associated with matrix-matched calibration arises from the use of blank samples as calibration standards. This leads to an augmented number of samples to be processed for analysis and an increased injection of matrix components in each analytical sequence, potentially leading to higher instrument maintenance demands. Nonetheless, this strategy is more suitable than standard addition, which would generate a much larger number of samples to be injected due to the need to perform a calibration curve f...

In chemical analysis, the matrix effect (ME) can be defined in various ways, but a widely accepted general definition is as follows: it refers to the change in the signal, particularly a change in the slope of the calibration curve when the sample matrix or portion of it is present during the analysis of a specific analyte. As a critical aspect, ME necessitates thorough investigation during the validation process of any analytical method, as it directly affects the accuracy of quantitative measurement for the target analytes1. Ideally, a sample pretreatment procedure should be selective enough to avoid extracting any components from the sample matrix. However, despite significant efforts, many of these matrix components still end up in the final determination systems in most cases. Consequently, such matrix components often compromise the recovery and precision values, introduce additional noise, and escalate the overall cost and labor involved in the method.
In gas chromatography (GC), ME arises due to the presence of active sites within the GC system, which interact with the target analytes through various mechanisms. On the one hand, the matrix constituents block or mask these active sites that would otherwise interact with the target analytes, resulting in frequent signal enhancement2. On the other hand, active sites that remain unobstructed may cause peak tailing or analyte decomposition due to strong interactions, leading to a negative ME. However, this can offer potential benefits in certain cases2. It is crucial to emphasize that achieving complete inertness in a GC system is exceedingly challenging, despite using highly inert components and proper maintenance. With continuous use, the accumulation of matrix components in the GC system becomes more pronounced, causing an increased ME. Nowadays, it is widely recognized that analytes containing oxygen, nitrogen, phosphorus, sulfur, and similar elements, exhibit a greater ME as they readily interact with these active sites. Conversely, highly stable compounds such as hydrocarbons or organohalogens do not undergo such interactions and do not show observable ME during analysis2,3.
Overall, ME cannot be fully eliminated, leading to the development of several strategies for compensation or correction when complete removal of matrix components is not feasible. Among these strategies, the utilization of deuterated internal standards (ISs), analyte protectants, matrix-matched calibration, the standard addition method, or the modification of injection techniques have been documented in scientific literature1,2,4,5. The SANTE/11312/2021 guidelines have also recommended these strategies6.
Regarding the application of matrix-matched calibration to compensate for MEs, sample sequences in practical situations encompass diverse types of foods or various samples from the same commodity. In this case, the assumption is made that employing any sample from the same commodity will effectively compensate for ME in all samples. However, there is a lack of sufficient studies in the existing literature that specifically investigate this issue7.
The multiresidue determination of pesticides in matrices containing an appreciable percentage of fat and pigments constitutes a challenging task. The considerable amount of coextracted material can significantly affect the extraction efficiency and interfere with the subsequent chromatographic determination, potentially damaging the column, source, and detector, and resulting in significant MEs8,9,10. Consequently, the analysis of pesticides at trace levels in such matrices necessitates a significant reduction of matrix components before analysis while ensuring high recovery values7. Obtaining high recovery values is crucial to ensure that pesticide analyses remain reliable, accurate, and compliant with regulatory standards. This is vital for ensuring food safety, environmental protection, and informed decision-making in agriculture and related fields.
Avocado is a fruit of high commercial value cultivated in tropical and Mediterranean climates worldwide and widely consumed both in its regions of origin and in the numerous export markets. From the analytical point of view, avocado is a complex matrix containing a significant number of fatty acids (i.e., oleic, palmitic, and linoleic), similar to nuts, a significant pigment content, as in green leaves, as well as sugars and organic acids, similar to those found in other fruits11. Due to its fatty nature, special attention must be given when employing any analytical method for analysis. While pesticide residue analysis has been conducted on avocados using GC-MS in some instances8,12,13,14,15,16,17,18,19,20, it has been relatively less frequent compared to other matrices. In most cases, a version of the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method has been applied8,12,13,14,15,16,17,18. None of these studies have investigated the consistency of MEs among different avocado varieties.
Therefore, the aim of this work was to study the consistency of MEs and recovery values for 45 representative pesticides across different varieties of avocado (i.e., Criollo, Hass, and Lorena) using the QuEChERS method with ammonium formate and GC-MS/MS. To the best of our knowledge, this is the first time that this type of study has been conducted on such fatty matrix samples.

<table><tbody><tr><td>3-Ethoxy-1,2-propanediol</td><td>Sigma Aldrich</td><td>260428-1G</td><td></td></tr><tr><td>Acetonitrile</td><td>Merk</td><td>1006652500</td><td></td></tr><tr><td>Ammonium formate</td><td>Sigma Aldrich</td><td>156264-1KG</td><td></td></tr><tr><td>AOAC 20i/s autosampler</td><td>Shimadzu</td><td>221-723115-58</td><td></td></tr><tr><td>Automatic shaker MX-T6-PRO</td><td>SCILOGEX</td><td>8.23222E+11</td><td></td></tr><tr><td>Balance</td><td>OHAUS</td><td>PA224</td><td></td></tr><tr><td>Centrifuge tubes, 15 mL</td><td>Nest</td><td>601002</td><td></td></tr><tr><td>Centrifuge tubes, 2 mL</td><td>Eppendorf</td><td>4610-1815</td><td></td></tr><tr><td>Centrifuge tubes, 50 mL</td><td>Nest</td><td>602002</td><td></td></tr><tr><td>Centrifuge Z206A</td><td>MERMLE</td><td>6019500118</td><td></td></tr><tr><td>Choper 2L</td><td>Oster</td><td>2114111</td><td></td></tr><tr><td>Column SH-Rxi-5sil MS, 30 m x 0.25 mm, 0.25 &amp;micro;m</td><td>Shimadzu</td><td>221-75954-30</td><td>MS GC column&amp;nbsp;</td></tr><tr><td>Dispensette 5-50 mL</td><td>BRAND</td><td>4600361</td><td></td></tr><tr><td>DSC-18</td><td>Sigma Aldrich</td><td>52600-U</td><td></td></tr><tr><td>D-Sorbitol</td><td>Sigma Aldrich</td><td>240850-5G</td><td></td></tr><tr><td>Ethyl acetate</td><td>Merk</td><td>1313181212</td><td></td></tr><tr><td>GCMS-TQ8040&amp;nbsp;</td><td>Shimadzu</td><td>211552</td><td></td></tr><tr><td>Graphitized carbon black</td><td>Sigma Aldrich</td><td>57210-U</td><td></td></tr><tr><td>Injection syringe</td><td>Shimadzu</td><td>LC2213461800</td><td></td></tr><tr><td>L-Gulonic acid &amp;gamma;-lactone</td><td>Sigma Aldrich</td><td>310301-5G</td><td></td></tr><tr><td>Linner splitless</td><td>Shimadzu</td><td>221-4887-02</td><td></td></tr><tr><td>Magnesium sulfate anhydrus</td><td>Sigma Aldrich</td><td>M7506-2KG</td><td></td></tr><tr><td>Methanol</td><td>Panreac</td><td>131091.12.12</td><td></td></tr><tr><td>Milli-Q ultrapure (type 1) water</td><td>Millipore</td><td>F4H4783518</td><td></td></tr><tr><td>Pipette tips 10 - 100 &amp;micro;L</td><td>Biologix</td><td>200010</td><td></td></tr><tr><td>Pipette tips 100 - 1000 &amp;micro;L</td><td>Brand</td><td>541287</td><td></td></tr><tr><td>Pipette tips 20 - 200 &amp;micro;L</td><td>Brand</td><td>732028</td><td></td></tr><tr><td>Pipettes Pasteur</td><td>NORMAX</td><td>5426023</td><td></td></tr><tr><td>Pippette Transferpette S variabel 10 - 100 &amp;micro;L</td><td>BRAND</td><td>704774</td><td></td></tr><tr><td>Pippette Transferpette S variabel 100 - 1000 &amp;micro;L</td><td>BRAND</td><td>704780</td><td></td></tr><tr><td>Pippette Transferpette S variabel 20 - 200 &amp;micro;L</td><td>SCILOGEX</td><td>7.12111E+11</td><td></td></tr><tr><td>Primary-secondary amine</td><td>Sigma Aldrich</td><td>52738-U</td><td></td></tr><tr><td>Shikimic acid</td><td>Sigma Aldrich</td><td>S5375-1G</td><td></td></tr><tr><td>Syringe Filter PTFE/L 25 mm, 0.45 &amp;micro;m</td><td>NORMAX</td><td>FE2545I</td><td></td></tr><tr><td>Triphenyl phosphate (QC)</td><td>Sigma Aldrich</td><td>241288-50G</td><td></td></tr><tr><td>Vials with fused-in insert</td><td>Sigma Aldrich</td><td>29398-U</td><td></td></tr><tr><td>Z-SEP+</td><td>Sigma Aldrich</td><td>55299-U</td><td>zirconium oxide-based sorbent</td></tr><tr><td>&lt;strong&gt;Pesticides&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;CAS registry number&lt;/strong&gt;</td></tr><tr><td>4,4&amp;acute;-DDD</td><td>Sigma Aldrich</td><td>35486-250MG</td><td>72-54-8</td></tr><tr><td>4,4&amp;acute;-DDE</td><td>Sigma Aldrich</td><td>35487-100MG</td><td>72-55-9</td></tr><tr><td>4,4&amp;acute;-DDT</td><td>Sigma Aldrich</td><td>31041-100MG</td><td>50-29-3</td></tr><tr><td>Alachlor</td><td>Sigma Aldrich</td><td>45316-250MG</td><td>15972-60-8</td></tr><tr><td>Aldrin</td><td>Sigma Aldrich</td><td>36666-25MG</td><td>309-00-2</td></tr><tr><td>Atrazine</td><td>Sigma Aldrich</td><td>45330-250MG-R</td><td>1912-24-9</td></tr><tr><td>Atrazine-d5 (IS)</td><td>Sigma Aldrich</td><td>34053-10MG-R</td><td>163165-75-1</td></tr><tr><td>Buprofezin</td><td>Sigma Aldrich</td><td>37886-100MG</td><td>69327-76-0</td></tr><tr><td>Carbofuran</td><td>Sigma Aldrich</td><td>32056-250-MG</td><td>1563-66-2</td></tr><tr><td>Chlorpropham</td><td>Sigma Aldrich</td><td>45393-250MG</td><td>101-21-3</td></tr><tr><td>Chlorpyrifos</td><td>Sigma Aldrich</td><td>45395-100MG</td><td>2921-88-2</td></tr><tr><td>Chlorpyrifos-methyl</td><td>Sigma Aldrich</td><td>45396-250MG</td><td>5598-13-0</td></tr><tr><td>Deltamethrin</td><td>Sigma Aldrich</td><td>45423-250MG</td><td>52918-63-5</td></tr><tr><td>Dichloran</td><td>Sigma Aldrich</td><td>45435-250MG</td><td>99-30-9</td></tr><tr><td>Dichlorvos</td><td>Sigma Aldrich</td><td>45441-250MG</td><td>62-73-7</td></tr><tr><td>Dieldrin</td><td>Sigma Aldrich</td><td>33491-100MG-R</td><td>60-57-1</td></tr><tr><td>Diphenylamine</td><td>Sigma Aldrich</td><td>45456-250MG</td><td>122-39--4</td></tr><tr><td>Endosulfan A</td><td>Sigma Aldrich</td><td>32015-250MG</td><td>115-29-7</td></tr><tr><td>Endrin</td><td>Sigma Aldrich</td><td>32014-250MG</td><td>72-20-8</td></tr><tr><td>EPN</td><td>Sigma Aldrich</td><td>36503-100MG</td><td>2104-64-5</td></tr><tr><td>Esfenvalerate</td><td>Sigma Aldrich</td><td>46277-100MG</td><td>66230-04-4</td></tr><tr><td>Ethion</td><td>Sigma Aldrich</td><td>45477-250MG</td><td>563-12-2</td></tr><tr><td>Fenamiphos</td><td>Sigma Aldrich</td><td>45483-250MG</td><td>22224-92-6</td></tr><tr><td>Fenitrothion</td><td>Sigma Aldrich</td><td>45487-250MG</td><td>122-14-5</td></tr><tr><td>Fenthion</td><td>Sigma Aldrich</td><td>36552-250MG</td><td>55-38-9</td></tr><tr><td>Fenvalerate</td><td>Sigma Aldrich</td><td>45495-250MG</td><td>51630-58-1</td></tr><tr><td>HCB</td><td>Sigma Aldrich</td><td>45522-250MG</td><td>118-74-1</td></tr><tr><td>Iprodione</td><td>Sigma Aldrich</td><td>36132-100MG</td><td>36734-19-7</td></tr><tr><td>Lindane</td><td>Sigma Aldrich</td><td>45548-250MG</td><td>58-89-9</td></tr><tr><td>Malathion</td><td>Sigma Aldrich</td><td>36143-100MG</td><td>121-75-5</td></tr><tr><td>Metalaxyl</td><td>Sigma Aldrich</td><td>32012-100MG</td><td>57837-19-1</td></tr><tr><td>Methidathion</td><td>Sigma Aldrich</td><td>36158-100MG</td><td>950-37-8</td></tr><tr><td>Myclobutanil</td><td>Sigma Aldrich</td><td>34360-100MG</td><td>88671-89-0</td></tr><tr><td>Oxyfluorfen</td><td>Sigma Aldrich</td><td>35031-100MG</td><td>42874-03-3</td></tr><tr><td>Parathion-methyl</td><td>Sigma Aldrich</td><td>36187-100MG</td><td>298-00-0</td></tr><tr><td>Penconazol</td><td>Sigma Aldrich</td><td>36189-100MG</td><td>66246-88-6</td></tr><tr><td>Pirimiphos-methyl</td><td>Sigma Aldrich</td><td>32058-250MG</td><td>29232-93-7</td></tr><tr><td>Propiconazole</td><td>Sigma Aldrich</td><td>45642-250MG</td><td>60207-90-1</td></tr><tr><td>Propoxur</td><td>Sigma Aldrich</td><td>45644-250MG</td><td>114-26-1</td></tr><tr><td>Propyzamide</td><td>Sigma Aldrich</td><td>45645-250MG</td><td>23850-58-5</td></tr><tr><td>Pyriproxifen</td><td>Sigma Aldrich</td><td>34174-100MG</td><td>95737-68-1</td></tr><tr><td>Tolclofos-methyl</td><td>Sigma Aldrich</td><td>31209-250MG</td><td>5701804-9</td></tr><tr><td>Triadimefon</td><td>Sigma Aldrich</td><td>45693-250MG</td><td>43121-43-3</td></tr><tr><td>Triflumizole</td><td>Sigma Aldrich</td><td>32611-100MG</td><td>68694-11-1</td></tr><tr><td>&amp;alpha;-HCH</td><td>Sigma Aldrich</td><td>33377-50MG</td><td>319-86-8</td></tr><tr><td>&amp;beta;-HCH</td><td>Sigma Aldrich</td><td>33376-100MG</td><td>319-85-7</td></tr></tbody></table>

determination of 45 pesticides in avocado varieties by the quechers method and gas chromatography-tandem mass spectrometry

Gas chromatography (GC) tandem mass spectrometry (MS/MS) stands as a preeminent analytical instrument extensively employed for the surveillance of pesticide residues in food. Nevertheless, these methods are vulnerable to matrix effects (MEs), which can potentially affect accurate quantification depending on the specific combination of analyte and matrix. Among the various strategies to mitigate MEs, matrix-matched calibration represents the prevailing approach in pesticide residue applications due to its cost-effectiveness and straightforward implementation. In this study, a total of 45 representative pesticides were analyzed in three different varieties of avocado (i.e., Criollo, Hass, and Lorena) using the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method with ammonium formate and GC-MS/MS. 
For this purpose, 5 g of the avocado sample was extracted with 10 mL of acetonitrile, and then 2.5 g of ammonium formate was added to induce phase separation. Subsequently, the supernatant underwent a cleanup process via dispersive solid-phase extraction employing 150 mg of anhydrous MgSO4, 50 mg of primary-secondary amine, 50 mg of octadecylsilane, 10 mg of graphitized carbon black, and 60 mg of a zirconium oxide-based sorbent (Z-Sep+). The GC-MS/MS analysis was successfully performed in less than 25 min. Rigorous validation experiments were carried out to assess the performance of the method. The examination of a matrix-matched calibration curve for each variety of avocado revealed that the ME remained relatively consistent and less than 20% (considered as a soft ME) for most pesticide/variety combinations. Furthermore, the method&#180;s limits of quantification were lower than 5 &#181;g/kg for all three varieties. Finally, the recovery values for most pesticides fell within the acceptable range of 70-120%, with relative standard deviation values below 20%.

Comprehensive validation of the analytical method was conducted according to SANTE/11312/2021 guidelines6, encompassing assessments of linearity, ME, recovery, and repeatability.
For the linearity assessment, matrix-matched calibration curves were constructed using spiked blank samples at multiple concentration levels (ranging from 5 to 600 &#181;g/kg). The determination coefficients (R2) for most of the selected pesticides were found to be higher than or equ...

Read this Scientific Article Protocol about Author Spotlight: A Matrix-Matched Calibration Approach for Mitigating Matrix Effects in Avocado Pesticide Residue Analysis at JoVE.com

Avocado Sample Preparation Using the QuEChERS Method with Ammonium Formate for Pesticide Analysis

The present protocol describes the analysis of multiclass pesticide residues in avocado varieties using the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method with ammonium formate, followed by gas chromatography-tandem mass spectrometry.

Determination of 45 Pesticides in Avocado Varieties by the QuEChERS Method and Gas Chromatography-Tandem Mass Spectrometry

Gas chromatography (GC) tandem mass spectrometry (MS/MS) stands as a preeminent analytical instrument extensively employed for the surveillance of ...

determination-45-pesticides-avocado-varieties-quechers-method-gas

Research

JoVE Journal

Chemistry

581 Views.  Universidad EAN. Om te beginnen met de monstervoorbereiding met behulp van de QuEChERS-methode, weegt u vijf gram van een avocadomonster af in een centrifugebuis van 50 milliliter. Voeg 50 microliter van de procedurele interne standaard of P-IS-oplossing toe aan de buis. Voeg 10 milliliter acetonitril toe aan de centrifugebuis en schud de buis vijf minuten op 70 RPM.Voeg er vervolgens 2,5 gram ammoniumformiaat aan toe en schud de buis opnieuw vijf minuten op 70 RPM. Centrifugeer vervolgens de buis gedu...

Video: Voorbereiding van avocadomonsters met behulp van de QuEChERS-methode met ammoniumformiaat voor analyse van pesticiden

Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Biochemistry

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

Determination of Carbonyl Functional Groups in Bio-oils by Potentiometric Titration: The Faix Method

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls cause an increase in viscosity (often referred to as &#39;aging&#39;) during storage of bio-oils. As such, carbonyl content has previously been used as a method of tracking bio-oil aging and condensation reactions with less variability than viscosity measurements. Additionally, carbonyls are also responsible for coke formation in bio-oil upgrading processes. Given the importance of carbonyls in bio-oils, accurate analytical methods for their quantification are very important for the bio-oil community. Potentiometric titration methods based on carbonyl oximation have long been used for the determination of carbonyl content in pyrolysis bio-oils. Here, we present a modification of the traditional carbonyl oximation procedures that results in less reaction time, smaller sample size, higher precision, and more accurate carbonyl determinations. While traditional carbonyl oximation methods occur at room temperature, the Faix method presented here occurs at an elevated temperature of 80 &#176;C.

Here we present a potentiometric titration technique for accurately quantifying carbonyl compounds in pyrolysis bio-oils.

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls ...

Determination of the Absorption, Translocation, and Distribution of Imidacloprid in Wheat

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. Imidacloprid, the most widely used insecticide worldwide, is a representative first-generation neonicotinoid and is used in pest control for crops, vegetables, and fruit trees. With such a broad application of imidacloprid, its residue in crops has attracted increasing scrutiny. In the present study, 15 wheat seedlings were placed in a culture medium containing 0.5 mg/L or 5 mg/L imidacloprid for hydroculture. The content of imidacloprid in the wheat roots and leaves was determined after 1 day, 2 days, and 3 days of hydroculture to explore the migration and distribution of imidacloprid in wheat. The results showed that imidacloprid was detected both in the roots and leaves of the wheat plant, and the content of imidacloprid in the roots was higher than that in the leaves. Furthermore, the imidacloprid concentration in the wheat increased with increasing exposure time. After 3 days of exposure, the roots and leaves of the wheat in the 0.5 mg/L treatment group contained 4.55 mg/kg &plusmn; 1.45 mg/kg and 1.30 mg/kg &plusmn; 0.08 mg/kg imidacloprid, respectively, while the roots and leaves of the 5 mg/L treatment group contained 42.5 mg/kg &plusmn; 0.62 mg/kg and 8.71 mg/kg &plusmn; 0.14 mg/kg imidacloprid, respectively. The results from the present study allow for a better understanding of pesticide residues in crops and provide a data reference for the environmental risk assessment of pesticides.

Presented here is a protocol for the determination of the absorption, translocation, and distribution of imidacloprid in wheat under hydroponic conditions using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The results showed that imidacloprid can be absorbed by wheat, and imidacloprid was detected in both the wheat roots and leaves.

Neonicotinoids, a class of insecticides, are widely used because of their novel modes of action, high insecticidal activity, and strong root uptake. ...

Environment

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars with enhanced organoleptic characteristics and thus, to increase consumer acceptance. High-throughput metabolomic platforms have been recently developed to quantify a wide range of metabolites in different plant tissues, including key compounds responsible for fruit taste and aroma quality (volatilomics). A method using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is described here for the identification and quantification of VOCs emitted by ripe blackcurrant fruits, a berry highly appreciated for its flavor and health benefits.
Ripe fruits of blackcurrant plants (Ribes nigrum) were harvested and directly frozen in liquid nitrogen. After tissue homogenization to produce a fine powder, samples were thawed and immediately mixed with sodium chloride solution. Following centrifugation, the supernatant was transferred into a headspace glass vial containing sodium chloride. VOCs were then extracted using a solid-phase microextraction (SPME) fiber and a gas chromatograph coupled to an ion trap mass spectrometer. Volatile quantification was performed on the resulting ion chromatograms by integrating peak area, using a specific m/z ion for each VOC. Correct VOC annotation was confirmed by comparing retention times and mass spectra of pure commercial standards run under the same conditions as the samples. More than 60 VOCs were identified in ripe blackcurrant fruits grown in contrasting European locations. Among the identified VOCs, key aroma compounds, such as terpenoids and C6 volatiles, can be used as biomarkers for blackcurrant fruit quality. In addition, advantages and disadvantages of the method are discussed, including prospective improvements. Furthermore, the use of controls for batch correction and minimization of drift intensity have been emphasized.

A headspace solid-phase microextraction-gas-chromatography platform is described here for fast, reliable, and semi-automated volatile identification and quantification in ripe blackcurrant fruits. This technique can be used to increase knowledge about fruit aroma and to select cultivars with enhanced flavor for the purpose of breeding.

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...

Analysis of Organochlorine Pesticides in a Soil Sample by a Modified QuEChERS Approach Using Ammonium Formate

Currently, the QuEChERS method represents the most widely used sample preparation protocol worldwide for analyzing pesticide residues in a broad variety of matrices both in official and non-official laboratories. The QuEChERS method using ammonium formate has previously proven to be advantageous compared to the original and the two official versions. On the one hand, the simple addition of 0.5 g of ammonium formate per gram of sample is sufficient to induce phase separation and achieve good analytical performance. On the other hand, ammonium formate reduces the need for maintenance in routine analyses. Here, a modified QuEChERS method using ammonium formate was applied for the simultaneous analysis of organochlorine pesticide (OCP) residues in agricultural soil. Specifically, 10 g of the sample was hydrated with 10 mL of water and then extracted with 10 mL of acetonitrile. Next, phase separation was carried out using 5 g of ammonium formate. After centrifugation, the supernatant was subjected to a dispersive solid-phase extraction clean-up step with anhydrous magnesium sulfate, primary-secondary amine, and octadecylsilane. Gas chromatography-mass spectrometry was used as the analytical technique. The QuEChERS method using ammonium formate is demonstrated as a successful alternative for extracting OCP residues from a soil sample.

The present protocol describes the utilization of ammonium formate for phase partitioning in QuEChERS, together with gas chromatography-mass spectrometry, to successfully determine organochlorine pesticide residues in a soil sample.

Currently, the QuEChERS method represents the most widely used sample preparation protocol worldwide for analyzing pesticide residues in a broad variety ...

Evaluation of Wild Peanut Species for Resistance to Aflatoxin Accumulation

Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (Arachis spp.) Seeds

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate economically important agricultural commodities, including peanuts, posing a high risk to human and animal health. Due to the narrow genetic base, peanut cultivars demonstrate limited resistance to fungal pathogens. Therefore, numerous wild peanut species with tolerance to Aspergillus have received substantial consideration by scientists as sources of disease resistance.
Exploring plant germplasm for resistance to aflatoxins is difficult since aflatoxin accumulation does not follow a normal distribution, which dictates the need for the analyses of thousands of single peanut seeds. Sufficiently hydrated peanut (Arachis spp.) seeds, when infected by Aspergillus species, are capable of producing biologically active stilbenes&#160;(stilbenoids) that are considered defensive phytoalexins. Peanut stilbenes inhibit fungal development and aflatoxin production. Therefore, it is crucial to analyze the same seeds for peanut stilbenoids to explain the nature of seed resistance/susceptibility to the Aspergillus invasion. None of the published methods offer single-seed analyses for aflatoxins and/or stilbene phytoalexins.
We attempted to fulfill the demand for such a method that is environment-friendly, uses inexpensive consumables, and is sensitive and selective. In addition, the method is non-destructive since it uses only half of the seed and leaves the other half containing the embryonic axis intact. Such a technique allows germination and growth of the peanut plant to full maturity from the same seed used for the aflatoxin and stilbenoid analysis. The integrated part of this method, the manual challenging of the seeds with Aspergillus, is a limiting step that requires more time and labor compared to other steps in the method. The method has been used for the exploration of wild Arachis germplasm to identify species resistant to Aspergillus and to determine and characterize novel sources of genetic resistance to this fungal pathogen.

Author Spotlight: Quantification of Aflatoxins and Phytoalexins in Peanut Seeds to Identify Genetic Resistance Against Aspergillus

We demonstrate a medium-throughput method for quantification of aflatoxins and stilbenoid phytoalexins in single peanut seeds using ultra performance liquid chromatography. This method was specifically developed for the analyses of wild Arachis species challenged by the aflatoxigenic Aspergillus species.

Determination of 45 Pesticides in Avocado Varieties by the QuEChERS Method and Gas Chromatography-Tandem Mass Spectrometry

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