Analysis of oxysterols and vitamin D metabolites in mouse brain and cell line samples by ultra-high-performance liquid chromatography-atmospheric pressure photoionization–mass spectrometry

• We report two sensitive and fast methods for the analysis of biological samples.
• 25-Hydroxyvitamin D3 was identified in mouse brain samples for the first time.
• Oxidative stressed cell line samples were compared to non-stressed control samples.
• The role of oxysterols in the formation of α-syn aggregates cannot be ruled out.

We have developed an ultra-high-performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometric (UHPLC-APPI–MS/MS) method for the simultaneous quantitative analyses of several oxysterols and vitamin D metabolites in mouse brain and cell line samples. An UHPLC-APPI-high resolution mass spectrometric (UHPLC-APPI-HRMS) method that uses a quadrupole-time of flight mass spectrometer was also developed for confirmatory analysis and for the identification of non-targeted oxysterols. Both methods showed good quantitative performance. Furthermore, APPI provides high ionization efficiency for determining oxysterols and vitamin D related compounds without the time consuming derivatization step needed in the conventionally used electrospray ionization method to achieve acceptable sensitivity. Several oxysterols were quantified in mouse brain and cell line samples. Additionally, 25-hydroxyvitamin D3 was detected in mouse brain samples for the first time.