Combining a liquid-liquid extraction with successive air assisted liquid-liquid microextraction for the analysis of phytosterols present in animal based butter and oil samples.

In this study, an elevated temperature liquid-liquid extraction combined method with successive air-assisted liquid-liquid microextraction has been proposed for the extraction of four phytosterols in cow milk butter and animal oil samples prior to gas chromatography-flame ionization detector. The method is started by combining a few grams of the melted butter or oil samples with ethanol. The mixture is vortexed and placed into a water-bath adjusted at 50 ⁰C. After a few minutes, the mixture is allowed to cool at room temperature. In this step, the butter or oil is become stiff and ethanol is collected on top of the sample. The separated ethanol phase is collected and mixed with deionized water to obtain a homogenous solution. After that, a few microliters of ethyl methyl ammonium chloride: pivalic acid deep eutectic solvent is added into the solution and the mixture was pulled into a glass test tube and pushed back to the tube for five times. After centrifugation, whole of the collected phase at the bottom of tube was withdrawn and transferred into a microtube and contacted with sodium hydroxide solution. The mixture is withdrawn and released to the tube 2 times to remove the extracted fatty acids. The validation data verified that high enrichment factors (385-450) and extraction recoveries (77-90%), low limits of quantification (2.6-5.2 ng g) and detection (0.73-1.5 ng g), and satisfactory relative standard deviations (≤ 9.3%) can be obtained with this method. At last, the developed method was successfully used for the analysis of phytosterols in various butter and oil samples marketed in Tabriz, Iran.