gc-ms/ms analysis and data acquisition for assessing pesticides in avocado samples prepared by the quechers method

Analysere plantevernmidler i avokado ved hjelp av QuEChERS-metoden og CG-MS/MS

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.

Avokadoprøveforberedelse ved bruk av QuEChERS-metoden med ammoniumformiat for plantevernmiddelanalyse

Bestemmelse av 45 plantevernmidler i avokadosorter ved QuEChERS-metoden og gasskromatografi-tandem massespektrometri

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 auto-sampler 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.

GC-MS/MS-analyse og datainnsamling for vurdering av plantevernmidler i avokadoprøver utarbeidet ved QuEChERS-metoden

Gjennomføre analysene ved hjelp av et GC-MS/MS-system med en trippel kvadrupol utstyrt med et elektronioniseringsgrensesnitt på 70 elektronvolt i en autosampler. Sørg for at gasstrykket er riktig. Helium ved 140 PSI og argon ved 65 PSI.Slå på hoved-GC-bryteren på frontpanelet. Og slå på MS-bryteren på baksiden. Åpne GCMS-programvaren for sanntidsanalyse som styrer alle parameterne til GC-MS/MS-systemet.Klikk på vakuumkontroll. Deretter avansert etterfulgt av roterende pumpe en for å starte vakuumsystemet. Klikk på start for å slå på de turbomolekylære pumpene en og to.Klikk deretter på startalternativet for ionekildevarmeren. Sett MS-grensesnitttemperaturen til 250 grader Celsius, og ionekildetemperaturen til 300 grader Celsius. Hold GC-ovnen på en starttemperatur på 50 grader Celsius i ett minutt.Øk den deretter opp til 180 grader Celsius med en hastighet på 25 grader Celsius per minutt. Deretter øker du temperaturen til 230 grader Celsius ved fem grader Celsius per minutt, og deretter til 290 grader Celsius ved 25 grader Celsius per minutt. Hold deretter temperaturen konstant på 290 grader Celsius i seks minutter.Den totale analysetiden er 24,6 minutter. Når alle disse systemene er slått på, klikker du på lukk. Klikk deretter på innstillingsalternativet fra analyseprogramvaren og velg toppmonitorvisning for å utføre en innledende verifisering av massespektrometerforholdene.Klikk på Anskaffelse, og fra det viste vinduet klikker du på Last ned innledende parametere. Kontroller at utstyret er klart GC og klart MS. For datainnsamling, klikk på ny batch file fra programvaren og lag en sekvens som inneholder informasjon som sample navn, sample ID, metode file, data file, injeksjonsvolum og tuning file. Legg til rader etter behov og klikk på lagre.Klikk på Batch Start og la injeksjonsprosessen starte. Utfør injeksjonen ved 250 grader Celsius i delt listemodus, og hold et injeksjonsvolum på én mikroliter. Etter ett minutt etter injeksjonen åpnes splitten.Analyser analyttene ved hjelp av multippel reaksjonsovervåking, eller MRM-modus, som er standardmodusen som brukes i MS/MS-systemer med en trippel kvadrupol. Åpne analyseprogramvaren etter kjøring for dataanalyse. Klikk på injeksjonen som skal analyseres.Naviger gjennom tabellen som inneholder analyttene og velg toppen av interesse. Klikk på toppen eller området av interesse for å visualisere kromatogrammet. Se gjennom toppintegrasjonene, og utfør om nødvendig manuell integrasjon.Verifiser områdene til alle analytter for å utføre nødvendige beregninger og metodeevaluering. Generering av matrisematchede kalibreringskurver avslørte en relativt konsistent matriseeffekt på mindre enn 20 % klassifisert som en myk ME for de fleste plantevernmiddelkombinasjoner. Resultatene i den viste figuren viste antallet plantevernmidler hvis gjennomsnittlige utvinningsprosent var innenfor det akseptable området på 70 til 120 % for hver type avokado.En betydelig andel av de testede plantevernmidlene viste gjenvinningsprosenter som falt innenfor dette spesifikke området, med relative standardavviksverdier under 20 %