## 3 Results and Discussion  
### Method Performance--Matrix and Analyte Extension  
The US-DLLME-GC-MS method was previously developed in our lab to determine MDA and ACRL in beverages [22]. Herein, we studied the method's applicability to four additional carbonyl compounds (FCHO, ACE, MGO, and DMGO) in adult formula. We used a standard adult formula as a blank sample to ensure the method's suitability for the simultaneous extraction of these six analytes from the adult formula. First, a kinetic study was performed in triplicate to assess the formation of carbonyl-DNPH derivatives (Figure 1). MDA and ACRL reached equilibrium after 5 min of ultrasound treatment, FCHO after 10 min, while ACE, MGO, and DMGO required 20 min. To ensure all analytes reached equilibrium and were successfully extracted as their corresponding DNPH derivatives, we set the ultrasound incubation time to 20 min. The analytical performance of the method was evaluated under these conditions (Table 1). The method's specificity is based on specific ions for each analyte and internal standards eluting at specific retention times previously described in this manuscript (Section 2.5). Figure 2 confirms the absence of interfering signals in the retention time region for all analytes and the internal standards. The LOD ranged from 8 to 89 ng/mL, and the LOQ ranged from 0.061 to 0.671 ug/mL. Standard addition calibrations showed excellent linearity, with determination coefficients of \(\geq\)0.9990. The method's precision, assessed at concentrations of 0.7 ug/mL (QC1), 1.0 ug/mL (QC2), and 2.0 ug/mL (QC3), demonstrated good intraday precision with relative standard deviations (RSD) between 1.3% and 5.8%, and interday precision with RSD between 0.9% and 4.6%. Similarly, method accuracy at the same concentrations showed recoveries ranging from 98.0% to 102.6%. Table S2 compares the analytical performance of this method with other methods for detecting carbonyl compounds in infant formula, milk, and dairy products. Single HPLC-UV determinations of MDA have been reported, using either the TBARS test or as a TBA-MDA derivative after liquid-liquid extraction or dilution. Despite being feasible and reproducible, with acceptable RSD values, these methods exhibit poorer linearity and lower recovery rates than those in this study. Similarly, single UV determinations of FCHO in milk have shown higher LOD and LOQ values than those reported here. Single MDA determination in infant formula using HPLC-MS without derivatization has been reported to have significantly lower detection limits than other HPLC methods. When paired with derivatization, HPLC-MS methods achieve even further reductions in detection limits, as reported for MGO and DA determination in baby food by [PERSON] & [PERSON] (2014), and in milk and dairy products reported by [PERSON] et al., 2022 [PERSON] et al. (2022, 2024). In a previous study, we identified five carbonyl compounds, including MDA, ACRL, MGO, and DA, in infant formula using Gas-Diffusion Microextraction (GDME) with o-PDA derivatization via HPLC-UV; this method, though accurate and precise, had higher detection limits [PERSON] et al. (2022). Additionally, we previously reported the development of an HS-SPME-GC-MS method for seven carbonyl and dicarbonyl compounds (including FCHO, ACE, MGO, DA, and MDA) as PFPH derivatives from infant formula, achieving detection limits similar to or slightly lower than those reported here. However, this method had higher RSD values [PERSON] et al. (2022). Figure 1: Kinetic analysis of DNPH derivatization and extraction of carbonyl compounds from adult formula using US-DLLME-GC-MS.