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

Análise de pesticidas em abacate usando o método QuEChERS e 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.

Preparação de amostras de abacate usando o método QuEChERS com formato de amônio para análise de pesticidas

Determinação de 45 pesticidas em variedades de abacate pelo método QuEChERS e cromatografia gasosa - espectrometria de massas em tandem

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.

Análise GC-MS/MS e aquisição de dados para avaliação de pesticidas em amostras de abacate preparadas pelo método QuEChERS

Realizar as análises usando um sistema GC-MS/MS com um quadrupolo triplo equipado com uma interface de ionização de elétrons de 70 elétron-volts em um amostrador automático. Certifique-se de que as pressões do gás estejam corretas. Hélio a 140 PSI e argônio a 65 PSI.Ligue o interruptor GC principal localizado no painel frontal. E ligue o interruptor MS localizado na parte traseira. Abra o software de análise em tempo real GCMS que controla todos os parâmetros do sistema GC-MS/MS.Clique em controle de vácuo. Em seguida, avançou seguido pela bomba rotativa um para iniciar o sistema de vácuo. Clique em iniciar para ligar as bombas turbomoleculares um e dois.Em seguida, clique na opção de início do aquecedor de fonte de íons. Defina a temperatura da interface MS para 250 graus Celsius e a temperatura da fonte de íons para 300 graus Celsius. Mantenha o forno GC a uma temperatura inicial de 50 graus Celsius por um minuto.Em seguida, aumente para 180 graus Celsius a uma taxa de 25 graus Celsius por minuto. Posteriormente, aumente a temperatura para 230 graus Celsius a cinco graus Celsius por minuto e depois para 290 graus Celsius a 25 graus Celsius por minuto. Em seguida, mantenha a temperatura constante em 290 graus Celsius por seis minutos.O tempo total de análise é de 24,6 minutos. Depois que todos esses sistemas estiverem ligados, clique em fechar. Em seguida, clique na opção de ajuste do software de análise e selecione a visualização do monitor de pico para realizar uma verificação inicial das condições do espectrômetro de massa.Clique em Aquisição e, na janela exibida, clique em Baixar parâmetros iniciais. Verifique se o equipamento está pronto GC e pronto MS. Para aquisição de dados, clique no novo arquivo em lote do software e crie uma sequência contendo informações como nome da amostra, ID da amostra, arquivo de método, arquivo de dados, volume de injeção e arquivo de ajuste. Adicione linhas conforme necessário e clique em salvar.Clique em Batch Start e deixe o processo de injeção começar. Realize a injeção a 250 graus Celsius no modo de lista dividida, mantendo um volume de injeção de um microlitro. Após um minuto após a injeção, abra a divisão.Analise os analitos usando o monitoramento de reação múltipla, ou modo MRM, que é o modo padrão empregado em sistemas MS/MS com um triplo quadrupolo. Abra o software de análise pós-execução para análise de dados. Clique na injeção a ser analisada.Navegue pela tabela que contém os analitos e selecione o pico de interesse. Clique no pico ou na região de interesse para visualizar o cromatograma. Revise as integrações de pico e, se necessário, execute a integração manual.Verificar as áreas de todas as substâncias a analisar para efectuar os cálculos necessários e a avaliação do método. A geração de curvas de calibração pareadas com a matriz revelou um efeito de matriz relativamente consistente de menos de 20% classificado como EM suave para a maioria das combinações de pesticidas. Os resultados na figura apresentada demonstraram a contagem de pesticidas cujas porcentagens médias de recuperação estavam dentro da faixa aceitável de 70 a 120% para cada tipo de abacate.Uma proporção significativa dos pesticidas testados apresentou percentuais de recuperação dentro dessa faixa específica, com valores de desvio padrão relativo abaixo de 20%

Research

JoVE Journal

Chemistry

GC-MS/MS Analysis and Data Acquisition for Assessing Pesticides in Avocado Samples Prepared by the QuEChERS Method

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 ...

Química