Validation and Monitoring of Carminic Acid using HPLC-DAD and LC-MS/MS in Processed Foods

Carmine and cochineal extract are vibrant red colored pigments derived from cochineal insects, containing carminic acid as their primary component. These colorants are commonly used in widely consumed food products including candies and fish cakes. Carminic acid has recently been associated with allergic reactions linked to specific proteins. This study aimed to develop and authenticate a method for quantifying carminic acid using highperformance liquid chromatography with a diode array detector (HPLC-DAD) and a C18 UG120 column. Conditions included a water-trifluoroacetic acid (TFA) 0.1% (A) and acetonitrile-TFA 0.1% (B) mobile phase, with a flow rate of 1.0 mL/min and a column temperature of 30oC. Calibration curves (0.2-50 mg/L) show good linearity (r2≥0.9999). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) facilitates qualitative analysis, with a 0.05 mg/kg limit of detection (LOD) and a 0.15 mg/kg limit of quantification (LOQ). Intra-day and inter-day measurements exhibited accuracy (87.3-97.1%, recovery) and precision (0.48-8.90%, relative standard deviation, RSD. Measurement uncertainty was also estimated. The developed method is applicable for the effective monitoring of carmine and cochineal extracts in diverse food types, this is crucial for understanding and mitigating the potential health concerns associated with carminic acid.