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1%, respectively, for all target analytes. Limits of quantification were below 3.03 μg/L for all compounds. The methodology was then validated and applied for the evaluation of human plasma samples in order to demonstrate its applicability to the analysis of vitamin D analogues in biological samples. Samples of five individuals were analyzed. Results show that linoleate-D3, vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 24,25-dihydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 could be detected in most samples, while the two latter also