High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is a powerful method for the microanalysis of compounds in biological samples. Compared with gas chromatography-mass spectrometry (GC-MS), this method is more broadly applicable to various compounds and usually does not require a derivatization step before analysis. However, when neutral sterols are analyzed, the sensitivities of usual HPLC-MS/MS method are not superior to those of GC-MS because the sterols are relatively resistant to ionization. In this review, we introduce the recent development of HPLC-MS/MS analysis for the quantification of non-cholesterol sterols. By adding an effective derivatization step to the conventional procedure, sterol analysis by HPLC-MS/MS surpassed that obtained by GC-MS in sensitivity. In addition, sufficient specificity of this method was achieved by selected reaction monitoring (SRM) and thorough chromatographic separation of each sterol.
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