Breastfeeding is the main and most complete form of nutrition for babies according to the World Health Organization (WHO) [1]. However, many children do not have access to breastfeeding due to mother's biological factors, such as mothers who are HIV positive or who use medication/drugs that may harm the baby; as well as biochemical factors related to the baby, such as the inability to digest breast milk [2,3]. In these cases, one of the alternatives is the use of breast milk substitutes, such as infant formula (IF) .

IFs are mainly produced with cow's milk adding vegetable oils, carbohydrates and enriched with vitamins and minerals. The development and improvement of IF increased over the last years. Currently, there are IF based on soy milk, goat's milk, lactose-free, gluten-free and with partially and extensively hydrolysed protein, enabling a correct and healthy diet for babies with the most diverse nutritional requirements [4,5].

IFs can contain toxic chemical substances, such as pesticides and mycotoxins, resulting from the contamination of raw materials and inputs used in the production chain, which can cause adverse effects to infants [6]. Thus, it is extremely important to mitigate and monitor the exposure of babies and children to contaminants from food.

Pesticides are chemical or biological substances used for agricultural processing around the world, for the purpose of controlling pests and diseases and preventing food losses [7]. Brazil is one of the countries with the highest consumption of pesticides in the world, with a rising trend reported in recent years [8].

Mycotoxins are secondary metabolites produced by fungi, mainly Aspergillus sp., Penicillium sp. and Fusarium sp, which are associated with a variety of diseases that cause mutagenicity, teratogenicity and carcinogenicity [9]. Aflatoxins produced by fungi Aspergillus sp. are classified as carcinogenic to humans (group 1) by the International Agency for Research on Cancer (IARC), being the most widespread in Brazil [10].

Brazil does not have regulations for pesticide residues in IF. However, the European Community sets a maximum residue limit (MRL) of 0.01 mg/kg for pesticides in infant foods [11]. With regard to mycotoxins, the Brazilian Health Regulatory Agency (ANVISA) established the maximum tolerated limits of 1 µg/kg for aflatoxin B1, B2, G1 and G2 in IF and follow-up IF for infants and toddlers [12]. According to the Regulation (EU) 2023/915, there are no established limits for aflatoxin in infant foods [13].

Glyphosate and metabolites, organochlorine pesticides, organophosphorus pesticides, and pyrethroids levels were reported in IF from Brazil [14,15], India [16], Turkey [17], and Romania [18], [19], [20], and the QuEChERS (quick, easy, cheap, effective, rugged, and safe) methodology was used in the most of these studies.

The QuEChERS approach was originally developed to analyse pesticides in fruits and vegetables [21], but recently it has been used to determine different classes of contaminants in foodstuff, including mycotoxins, and their metabolites, especially in IF [19,20,22]. Gas chromatography coupled to mass spectrometry (GC-MS) was applied to analyse pesticides in IF [18,23]. However, studies related to mycotoxins by GC-MS have only been conducted in cereal-based products specifically targeted at infants [24]. On the other hand, liquid chromatography coupled to mass spectrometry (LC-MS/MS) is the most frequently used techniques, improving the quantitative determination of pesticides and mycotoxins [25], [26], [27], [28], [29], [30]. However, LC-high-resolution mass spectrometry (HRMS) was also applied to determine compounds belonging to different classes in IF [14,19].

High-resolution mass spectrometers (HRMS) have been employed in non-target screening methods owing to their ability to discern peaks with minimal mass differences, along with exceptional mass accuracy, resolution, and broad mass ranges [31]. This capability makes them well-suited for the concurrent detection of numerous substances as performed in suspect screening and non-target analysis. For this, Quadrupole Time of Flight (QToF) and Orbitrap mass spectrometers were the most used techniques coupled to LC or GC [32], [33], [34], [35]. In this context, the aim of the present study was the development and validation of an analytical method to determine 19 pesticides, and 4 aflatoxins (G1, G2, B1, B2) in 30 different samples of IF marketed in Brazil. For this, the method consists of an efficient, simple, and fast analytical approach to ensure good recovery, minimize potential matrix effects, and adhere as much as possible to the principles of green chemistry. On the other hand, the tentative identification and confirmation of other contaminants in infant formulas were carried out using two analytical methods - suspect screening and unknown analysis - by UHPLC-Q-Orbitrap-MS.

The most common pesticides and mycotoxins found in vegetables used in the formulation of infant formulas, such as corn, sunflower, rice, palm, and coconut, were selected according to reports of the Brazilian PARA program (Pesticide Residue Analysis Program in Food) developed by ANVISA. Since many formulas are milk-based, the pesticides reported by PARA in the grass consumed by cows were also included in this selection [36]. The samples were extracted using a QuEChERS-based protocol extraction and ultra-high performance LC-triple quadrupole MS/MS (UHPLC-QqQ-MS/MS). Up to our knowledge, this is the first report on these pesticides and mycotoxins in a multiclass method residue in Brazilian IF.