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A longer 50 min C30 chromatographic assay did not result in an expected improvement in the chromatographic separation of overlapping isomer peaks compared to the 30 min method but did result in loss of accuracy of peak picking algorithms. We recommend the 30 min C30 assay with scheduled MS/MS acquisition as an efficient tool to analyse complex biological matrices and to annotate lipid species based on MS/MS data.7-Dehydrocholesterol is an essential biomarker of Smith-Lemli-Opitz syndrome, a congenital autosomal recessive disorder. This s