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Not AvailableIn this study, sterols in milk fat of cow, goat, bufalo and camel were analyzed for their variability across diferent season and species. A GC–MS-SIM (gas chromatography- mass spectrometry-single ion monitoring mode) method was developed for accurate determination of sterols through the selection of correct set of target and reference ions in diferent time windows. The simple and rapid method enabled accurate analysis of sterols without subjecting them to derivatization. The sterols characterized in milk fat included cholesterol, lathosterol, desmosterol, lanosterol and dihydrolanosterol. Species compared to season had a pronounced efect on variability of sterols. Among the diferent species, goat milk contained the highest cholesterol level (325.18 mg 100 g−1 fat) while minor sterols were most abundant in bufalo milk (20.62 mg 100 g−1 fat). The content of minor sterols: lanosterol, lathosterol, desmosterol and dihydrolanosterol in milk of diferent animals varied from 5.94 to 9.11, 3.01 to 7.50, 0.60 to 2.77 & 0.57 to 1.25 mg 100 g−1 fat respectively. Chemometric statistical approaches consisting of principal component analysis and hierarchical cluster analysis (PCA and HCA) were used to process the data. PCA was a suitable approach to fnd out the similarity and diferences between diferent species and accounted for 96.6% of total variability in data. However, the variation based on season could not be explained well by PCA. HCAcould suitably classify the milch animals into four clusters based on sterol contentNot Availabl