{"id":43384,"date":"2020-10-13T01:00:00","date_gmt":"2020-10-13T01:00:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/lc-ms-ms-analysis-of-urinary-free-cortisol-and-cortisone-without-matrix-interferences-wrong\/"},"modified":"2026-03-17T20:40:07","modified_gmt":"2026-03-17T20:40:07","slug":"lc-ms-ms-analysis-of-urinary-free-cortisol-and-cortisone-without-matrix-interferences-wrong","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/es\/articles\/cfss2824\/lc-ms-ms-analysis-of-urinary-free-cortisol-and-cortisone-without-matrix-interferences-wrong","title":{"rendered":"LC-MS\/MS Analysis of Urinary Free Cortisol and Cortisone without Matrix Interferences"},"content":{"rendered":"\n<ul class=\"wp-block-list\">\n<li>Raptor Biphenyl column provides reliable separation of analytes from endogenous matrix interferences.<\/li>\n\n\n\n<li>Heightened selectivity for compounds that are hard to resolve on C18 and other phenyl chemistries enables accurate results.<\/li>\n\n\n\n<li>Fast 3-minute analysis allows high sample throughput.<\/li>\n<\/ul>\n\n\n\n<p>Cortisol is a steroid hormone synthesized from cholesterol by a multienzyme cascade in the adrenal glands. It is the main glucocorticoid in humans and plays a critical role in glucose metabolism and immune response regulation. Cortisone, a downstream metabolite of cortisol, is measured in conjunction with cortisol to help diagnose various adrenocortical disorders, particularly hypercortisolism (Cushing syndrome). Urinary free cortisol and cortisone levels correlate well with bioactive plasma concentrations and, thus, are important biomarkers. LC-MS\/MS is the preferred screening technique for these compounds because it is highly selective and helps eliminate analytical interferences that can affect immunoassay-based methods.<\/p>\n\n\n\n<p>Due to the structural similarities between cortisol and cortisone and the potential for matrix effects from urine components, appropriate column selectivity is critical for obtaining accurate results. In the LC-MS\/MS analysis of urinary free cortisol and cortisone developed here, both compounds were completely resolved from endogenous matrix interferences using a highly selective Raptor Biphenyl column. Chromatographic separation was essential in this case because some of the matrix compounds are isobaric to cortisol and cannot be distinguished by the mass spectrometer. Using a Raptor Biphenyl column and the conditions shown here, baseline separation was obtained in a 3-minute analysis, which allows labs to quickly generate more accurate quantitative data for clinical diagnosis.<\/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_cf0697.png\" alt=\"Cortisol and Cortisone in Extracted Female Human Urine on Raptor Biphenyl by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0697<\/p><div class=\"chromatogram-peaks\"><h4>Peaks<\/h4><table class=\"peaks col-lg-6 col-12\">\n<thead><tr><th><\/th><th style=\"text-align: left;width: 75px\">Peaks<\/th><th style=\"text-align: center;width: 75px\">t<sub>R<\/sub> (min)<\/th><th style=\"text-align: center;width: 75px\">Precursor Ion<\/th><th style=\"text-align: center;width: 75px\">Product Ion<\/th><th style=\"text-align: center;width: 75px\">Product Ion<\/th><\/tr><\/thead>\n<tbody><tr><td class=\"num\">1.<\/td><td class=\"cmpd\">Cortisol-d4<\/td><td class=\"oth\">0.75<\/td><td class=\"oth\">367.4<\/td><td class=\"oth\">121.0<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Endogenous cortisol\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/50-23-7\/Endogenous cortisol\" target=\"_blank\" rel=\"noopener\">Endogenous cortisol<\/a><\/td><td class=\"oth\">0.76<\/td><td class=\"oth\">363.3<\/td><td class=\"oth\">120.9<\/td><td class=\"oth\">91.1<\/td><\/tr>\n<tr><td class=\"num\">3.<\/td><td class=\"cmpd\">Cortisone-d8<\/td><td class=\"oth\">0.88<\/td><td class=\"oth\">369.4<\/td><td class=\"oth\">167.9<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<tr><td class=\"num\">4.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Endogenous cortisone\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/53-06-5\/Endogenous cortisone\" target=\"_blank\" rel=\"noopener\">Endogenous cortisone<\/a><\/td><td class=\"oth\">0.89<\/td><td class=\"oth\">361.3<\/td><td class=\"oth\">163.2<\/td><td class=\"oth\">91.0<\/td><\/tr>\n<\/tbody><\/table><div class=\"peaknotes\">*Isobaric matrix peaks<\/div><\/div><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 Biphenyl  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9309A52?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0697\" rel=\"noopener\">cat.# 9309A52<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Dimensions:<\/th><td>50 mm x 2.1 mm ID<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Particle Size:<\/th><td>2.7 \u00b5m<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Pore Size:<\/th><td>90 \u00c5<\/td><\/tr><tr><td><\/td><tr><th class=\"sub conditions_header\" scope=\"row\">Guard Column:<\/th><td>Raptor Biphenyl EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 \u00b5m (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9309A0252?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0697\" rel=\"noopener\">cat.# 9309A0252<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>40 \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>Mobile phase A<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Conc.:<\/th><td>  Calculated concentration is 105.1 ng\/mL and 135.9 ng\/mL for cortisol and cortisone, respectively<\/td><\/tr><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Inj. Vol.:<\/th><td>10 \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>Water, 0.1% formic acid <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>Acetonitrile, 0.1% formic acid <\/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>70<\/td><td>30<\/td><\/tr><tr><td>1.00<\/td><td>0.6<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>1.01<\/td><td>0.6<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>1.50<\/td><td>0.6<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>1.51<\/td><td>0.6<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>3.00<\/td><td>0.6<\/td><td>70<\/td><td>30<\/td><\/tr><\/tbody><\/table><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Max Pressure:<\/th><td>400 bar<\/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>UHPLC<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Sample Preparation<\/th><td>1. Centrifuge female human urine for 5 min at 4500 rpm, 10 \u00b0C.<br \/>2. Aliquot 380 \u00b5L supernatant. Add 20 \u00b5L each of internal standard solution (1 \u00b5g\/mL in methanol). <br \/>3. Load 200 \u03bcL of sample on to ISOLUTE SLE+ 200 \u00b5L supported liquid extraction plate (part# 820-0200-P01). <br \/>4. Apply a pulse of vacuum to initiate flow.<br \/>5. Wait 5 min for sample to completely absorb.<br \/>6. Extract samples with 1 mL of MTBE. Allow solvent to flow for 5 min under gravity. Apply vacuum for 10-30 sec to complete elution.<br \/>7. Evaporate extracts to dryness under a stream of nitrogen.<br \/>8. Reconstitute in 200 \u00b5L mobile phase A prior to analysis.<br \/>9. Vortex to mix.<br \/><\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0697.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>In general, LC-MS\/MS analysis of urinary free cortisol and cortisone can provide better specificity than immunoassay-based methods. However, it is critical that the analytical column has the proper selectivity for chromatographically separating cortisol from isobaric matrix components. In the method presented here, complete separation of the target analytes and matrix interferences was obtained in a 3-minute analysis on a Raptor Biphenyl column.<\/p>\n","protected":false},"author":11,"featured_media":90000,"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":[2391,2373,2399,2374,2375],"resource-technique":[2299,2301,2302],"hf_cat_post":[650,2624],"ppma_author":[414],"class_list":["post-43384","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","product-library-liquid-reference-standards","product-library-reference-standard-products","product-library-sample-preparation-products","resource-technique-liquid-chromatography","resource-technique-mass-spectrometry-ms","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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