{"id":43339,"date":"2020-10-07T14:30:00","date_gmt":"2020-10-07T14:30:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/fast-accurate-lc-msms-analysis-of-the-c3-epimer-of-25-hydroxyvitamin-d-on-raptor-fluorophenyl-columns\/"},"modified":"2026-03-17T17:19:36","modified_gmt":"2026-03-17T17:19:36","slug":"fast-accurate-lc-msms-analysis-of-the-c3-epimer-of-25-hydroxyvitamin-d-on-raptor-fluorophenyl-columns","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/zh-hans\/articles\/bass2523\/fast-accurate-lc-msms-analysis-of-the-c3-epimer-of-25-hydroxyvitamin-d-on-raptor-fluorophenyl-columns","title":{"rendered":"Fast, Accurate LC-MS\/MS Analysis of the C3 Epimer of 25-Hydroxyvitamin D on Raptor FluoroPhenyl Columns"},"content":{"rendered":"\n<ul class=\"wp-block-list\">\n<li>5-minute analysis time allows more samples per day.<\/li>\n\n\n\n<li>Complete separation of vitamin D epimers ensures accurate results.<\/li>\n\n\n\n<li>Long-lasting, rugged Raptor FluoroPhenyl LC columns reduce cost per sample.<\/li>\n<\/ul>\n\n\n\n<p>Vitamin D analysis has increased dramatically in clinical practice due to its association with multiple human diseases including cancer, rheumatoid arthritis, and diabetes. It also has an important role in calcium and phosphorous absorption in relation to bone strength, so there is growing concern about the prevalence of vitamin D deficiency worldwide. Accurate LC analysis of vitamin D epimers is critical for quantitative determination of the bioactive forms of vitamin D.<\/p>\n\n\n\n<p>Vitamin D exists in two forms, vitamin D2 and vitamin D3, which undergo metabolism to form 25-hydroxyvitamins D2 and D3, respectively. These metabolites are used as biomarkers to assess vitamin D status; however, both have epimeric forms (3-epi-25-hydroxyvitamins D2 and D3) that contribute significantly to the total 25-hydroxyvitamin D2 and D3 concentrations. Studies have shown that these vitamin D epimers have much lower bioactivity than the primary metabolites; therefore, they must be separated in order to prevent overestimating vitamin D levels.<\/p>\n\n\n\n<p>LC-MS\/MS is a common technique for vitamin D analysis; however, because the biomarkers and their epimers are isobaric, chromatographic separation is essential for accurate quantitation. Many published methods employ a C18 column, but its utility is limited by incomplete resolution of the biomarkers and their epimers as well as by analysis times of 10 minutes or more. The Raptor FluoroPhenyl column is a better choice as it provides alternate selectivity to a C18 column and completely separates the isobaric epimers and biomarkers in just 5 minutes. As shown below, Raptor FluoroPhenyl columns improve LC analysis of vitamin D epimers, reliably providing accurate measurement of vitamin D content for disease and deficiency investigation and monitoring.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<div class=\"wp-block-custom-chromatogram-article\"><div class=\"wp-block-custom-chromatogram-article\"><div class=\"chromatogram-image regular-image\"><img decoding=\"async\" src=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0644.png\" alt=\"25-Hydroxyvitamin D2\/D3 and 3-Epi-25-Hydroxyvitamin D2\/D3 on Raptor FluoroPhenyl by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0644<\/p><div class=\"chromatogram-conditions\"><h4>Conditions<\/h4><div class=\"conditions-container container-fluid\"><div class=\"row\"><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Column<\/th><td>Raptor FluoroPhenyl  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9319A1E?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0644\" rel=\"noopener\">cat.# 9319A1E<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Dimensions:<\/th><td>100 mm x 3 mm ID<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Particle Size:<\/th><td>2.7 \u00b5m<\/td><\/tr><tr><td><\/td><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>30 \u00b0C<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Standard\/Sample<\/th><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Diluent:<\/th><td>Water:methanol (50:50)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Conc.:<\/th><td>25-50 ng\/mL<\/td><\/tr><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Inj. Vol.:<\/th><td>5 \u00b5L <\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Mobile Phase<\/th><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">A:<\/th><td>0.1% Formic acid in water <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>Methanol <\/td><\/tr><tr><td><\/td><td><table class=\"cgram_ramp\"><thead><tr><th>Time (min)<\/th><th>Flow (mL\/min)<\/th><th>%A<\/th><th>%B<\/th><\/tr><\/thead><tbody><tr><td>0.00<\/td><td>0.6<\/td><td>25<\/td><td>75<\/td><\/tr><tr><td>4.00<\/td><td>0.6<\/td><td>15<\/td><td>85<\/td><\/tr><tr><td>4.10<\/td><td>0.6<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>5.00<\/td><td>0.6<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>5.01<\/td><td>0.6<\/td><td>25<\/td><td>75<\/td><\/tr><tr><td>7.00<\/td><td>0.6<\/td><td>25<\/td><td>75<\/td><\/tr><\/tbody><\/table><\/td><\/tr><\/table><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Detector<\/th><td>MS\/MS<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Ion Mode:<\/th><td>ESI+ <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Mode:<\/th><td>MRM <\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Instrument<\/th><td>HPLC<\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-related-skus\"><h4>Related SKUs<\/h4><ul><li>9319A1E<\/li><\/ul><\/div><div class=\"chromatogram-related-searches\"><h4>Related Searches<\/h4><ul><li>epimer<\/li><li>vitamin D<\/li><li>vitamin D epimer<\/li><li>vitamin D metabolite<\/li><\/ul><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0644.pdf\" target=\"_blank\" rel=\"noopener noreferrer\"><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" width=\"18\" height=\"18\" viewBox=\"0 0 18 18\"><g data-name=\"Group 2996\"><path data-name=\"Rectangle 1246\" d=\"M0 0h18v18H0z\" style=\"fill: none;\"><\/path><\/g><g data-name=\"Group 2997\"><path data-name=\"Path 729\" d=\"M13.412 11.4v2.017H5.345V11.4H4v2.017a1.349 1.349 0 0 0 1.345 1.345h8.068a1.349 1.349 0 0 0 1.345-1.345V11.4zm-.672-2.694-.948-.948-1.741 1.735V4H8.706v5.493L6.965 7.758l-.948.948 3.361 3.361z\" transform=\"translate(-.437 -.414)\" style=\"fill: rgb(13, 123, 196);\"><\/path><\/g><\/svg>Download PDF<\/a><\/div><\/div><\/div>\n<\/div><\/div><\/div>\n\n\n<div style=\"height:100px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n","protected":false},"excerpt":{"rendered":"<p>Raptor FluoroPhenyl columns can provide greater selectivity and retention for analytes that are not easily separated using a C18 column. For example, the epimeric forms of vitamin D2 and D3 25-hydroxy metabolites are important diagnostic markers for vitamin D status, but these compounds cannot be distinguished on a C18 column. The new Raptor FluoroPhenyl column provides ample chromatographic resolution for these isobaric epimers, which possess unequal bioactivities, so accurate results are generated and thus the proper diagnosis can be made.<\/p>\n","protected":false},"author":11,"featured_media":89994,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_kadence_starter_templates_imported_post":false,"_kad_post_transparent":"","_kad_post_title":"","_kad_post_layout":"","_kad_post_sidebar_id":"","_kad_post_content_style":"","_kad_post_vertical_padding":"","_kad_post_feature":"","_kad_post_feature_position":"","_kad_post_header":false,"_kad_post_footer":false,"footnotes":""},"categories":[7],"tags":[],"industries-application":[2153,2154],"post-badge":[],"resource-type":[2623],"product-library":[2391,2373],"resource-technique":[2299,2302],"hf_cat_post":[650,2624],"ppma_author":[414],"class_list":["post-43339","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-articles","industries-application-clinical","industries-application-clinical-diagnostics","resource-type-featured-application","product-library-lc-columns","product-library-liquid-chromatography-products","resource-technique-liquid-chromatography","resource-technique-ms-ms"],"acf":[],"taxonomy_info":{"category":[{"value":7,"label":"Articles"}],"industries-application":[{"value":2153,"label":"Clinical"},{"value":2154,"label":"Clinical 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