Simple click chemistry-based derivatization to quantify endogenous formaldehyde in milk using ultra-high-performance liquid chromatography/tandem mass spectrometry in selected reaction monitoring mode.

Abstract

Formaldehyde (FA) exposure via environmental pollution or through the food chain poses a serious threat to human health, especially in developing countries like India. Although the addition of FA to food is proscribed, it is often illegally added to foods such as milk to increase the shelf-life. There are challenges in differentiating the endogenous FA content in milk from externally added FA. We have developed a simple method using ultra-high-performance liquid chromatography/tandem mass spectrometry in selected reaction monitoring mode (UHPLC/MS/SRM) for the absolute quantification of endogenous FA in milk. The steps include fat removal, protein precipitation using acid, and spiking with labelled FA (FA*), followed by simple click chemistry-based derivatization using Girard P reagent (GP) and final analysis. A standard curve with FA* was constructed and used for the calculation of endogenous FA in milk. The optimal conditions for the derivatization reaction using 500 μL of milk were: GP, 50 μg; temperature, 37°C; time, 60 min; and 0.1% HCl. The validation parameters such as accuracy (95.84 to 99.73%), precision (2.84 to 8.02%) and spiked recovery (>95%) are within the FDA guidelines. This method is highly sensitive [limit of detection (LOD) of 1 ng/mL] with a dynamic range of 3.12 to 200 ng/mL. The endogenous FA level in pasteurized cow milk is 70 ng/mL (n = 60). The FA content in raw milk samples from cow, goat and buffalo (each n = 10) varied from 134 to 255 ng/mL. This method is precise and sufficiently sensitive to quantify endogenous FA in milk samples using a minimal sample volume. As it involves simple sample preparation steps, it can be used routinely to quantify endogenous FA.