A newly developed and validated LC-MS/MS method for measuring 7-dehydrocholesterol (7DHC) concentration in human...

UVB absorption by 7-dehydrocholesterol (7DHC) in the skin triggers the production of vitamin D and its metabolites, which maintain calcium homeostasis. Detection and measurement of 7DHC in skin using modern liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques have been lacking, yet there is need for such a technique to provide more information on 7DHC concentration and its UVB responses in human skin.

To develop and validate a reliable method to measure 7DHC concentration in skin.

Human skin punch biopsies of 5 mm diameter obtained through the Manchester Skin Health Biobank were utilised. 7DHC was extracted with ethyl acetate:methanol 1:1 (v/v) and derivatised using 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), to allow for improved ionisation of 7DHC through Electrospray Ionisation Mass Spectrometry (ESI-MS). Solid supported liquid extraction (SLE) was also employed to allow the removal of larger lipids from 7DHC and minimise potential matrix effects.

The LC-MS/MS assay satisfied International Council for Harmonisation research standards for method validation. Calibration curve was linear with a typical r² of 0.997, coefficient of variation was 11.1% and 4.32% for inter-assay and intra-assay imprecision, respectively. Lower limit of quantification was 1.6 µg/g and upper limit of quantification was 100 µg/g, SLE recovery of 7DHC was on average 91.4%.

We have developed a robust, precise and accurate assay for the detection and quantification of 7DHC in small samples of human skin (0.2 cm² surface area). This novel method of extraction and quantification will be valuable to future vitamin D photobiology research.