Oktawia Borecka1,2, Lesley E Rhodes3,4, Ann R Webb5, John J Dutton6, William D Fraser6,7. 1. Department of Earth and Environmental Sciences, Faculty of Science and Engineering, University of Manchester, Manchester, M13 9PL, UK. octavia.borecka@gmail.com. 2. Division of Musculoskeletal and Dermatological Sciences, Faculty of Medicine Biology and Health, School of Biological Sciences, University of Manchester, Manchester, M13 9PL, UK. octavia.borecka@gmail.com. 3. Division of Musculoskeletal and Dermatological Sciences, Faculty of Medicine Biology and Health, School of Biological Sciences, University of Manchester, Manchester, M13 9PL, UK. 4. Photobiology Unit, Dermatology Research Centre, Salford Royal NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, M6 8HD, UK. 5. Department of Earth and Environmental Sciences, Faculty of Science and Engineering, University of Manchester, Manchester, M13 9PL, UK. 6. Bioanalytical Facility, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK. 7. Departments of Clinical Biochemistry and Endocrinology, Norfolk and Norwich University Hospital, Norwich, NR4 7UY, UK.
Abstract
BACKGROUND: 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. OBJECTIVES: To develop and validate a reliable method to measure 7DHC concentration in skin. METHODS: 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. RESULTS: 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%. CONCLUSIONS: 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.
BACKGROUND: 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. OBJECTIVES: To develop and validate a reliable method to measure 7DHC concentration in skin. METHODS: 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. RESULTS: 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%. CONCLUSIONS: 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.
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