Development of Homogeneous Liquid-Liquid Extraction in Combination with Dispersive Liquid-Liquid Microextraction Based on Deep Eutectic Solvents for the Extraction and Assessment of Phytosterols in Animal Cream Samples using Gas Chromatography Equipped with Flame Ionization Detector

Background and Objectives: Food quality and safety control is an important part of the public health assessment of food adulteration has been a key issue in recent years. Cream is a product containing milk fat and is one of the most expensive and widely consumed edible fats, which is often targeted for cheating by mixing various vegetable oils with natural milk fats. A way to detect this type of fraud is to assess phytosterols in this dairy product. Materials & Methods: In this study, homogeneous liquid-liquid extraction method combined with dispersive liquid-liquid microextraction method was used for the extraction of stigmasterol, brassicaestrol, compesterol and β-sitestrol from animal cream samples. Samples were analyzed using gas chromatography-flame ionization detection method. To achieve high efficiency, effects of various factors in each extraction step, including type and volume of the extraction solvent, volume of the separating agent, speed and duration of centrifugation in the homogeneous liquid-liquid extraction step were investigated and optimized. Moreover, type and volume of the extraction solvent and salting effects in the dispersive liquid-liquid microextraction step were investigated and optimized as well. Results: The suggested method was successfully carried on on cream samples. Limit of detection and limit of quantification of the method were in ranges of 0.92–2.1 and 2.1–6.4 ng/g, respectively. Method included a proper precision with relative standard deviation of less than ≤ 8.8 for intra and inter-day precisions. Results showed that the total phytosterol content in 21 samples (70%) was more than 3%, contrary to the legal limits by the Institute of Standards and Industrial Research of Iran. Conclusion: Results of this study have verified that the suggested method is simple and reliable, including short analysis time and high ability to extract and measure phytosterols from animal cream samples.