{"id":43388,"date":"2020-10-13T00:00:00","date_gmt":"2020-10-13T00:00:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/fast-simple-lc-msms-analysis-of-creatine-and-creatinine\/"},"modified":"2025-12-08T14:37:21","modified_gmt":"2025-12-08T14:37:21","slug":"fast-simple-lc-msms-analysis-of-creatine-and-creatinine","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/es\/articles\/cfss2937\/fast-simple-lc-msms-analysis-of-creatine-and-creatinine","title":{"rendered":"Fast, Simple LC-MS\/MS Analysis of Creatine and Creatinine"},"content":{"rendered":"\n<ul class=\"wp-block-list\">\n<li>Simple isocratic method, no complex mobile phases.<\/li>\n\n\n\n<li>Fast, 1.5 min analysis without additional equilibration time.<\/li>\n\n\n\n<li>Highly reproducible results.<\/li>\n<\/ul>\n\n\n\n<p>Creatine is a compound that is made primarily in the liver and then transported to muscles where it is used as an energy source. Once in the muscle, some of the creatine is spontaneously converted to creatinine. It is important to monitor creatine and creatinine because these compounds can be used as biomarkers of renal, liver, and heart health. Historically, colorimetric and enzymatic assays have been used, but LC-MS\/MS is a better technique because it reduces interferences. Although LC-MS\/MS analysis of creatine and creatinine using an alkyl-bonded (e.g., C18) column is the usual approach, these highly polar analytes are very difficult to retain and separate from each other using a reversed-phase column. When a reversed-phase method is employed, complex ion-pairing reagents (e.g., tetrabutylammonium hydroxide) are generally required to increase analyte hydrophobicity.<\/p>\n\n\n\n<p>Using HILIC, ion-exchange, or other newer LC phases can be a better alternative to reversed-phase methods because these phases employ different mechanisms of interaction. Some non-C18 columns do provide increased retention, but analysis times can be long (e.g., 10 min) and may require the use of complex mobile phases and gradients. Labs needing faster LC-MS\/MS analysis of creatine and creatinine should consider adopting the HILIC method shown here. Good separations in both plasma and urine samples were obtained in just 1.5 min using a Raptor HILIC-Si (2.7 \u03bcm, 50 mm \u00d7 2.1 mm) column and a simple, isocratic mobile phase. Results during in-house testing were highly reproducible, making this fast, simple LC-MS\/MS analysis a good procedure for high-throughput labs.<\/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\n<p class=\"has-medium-font-size\"><strong>Human Plasma<\/strong><\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article\"><div class=\"wp-block-custom-chromatogram-article\"><div class=\"chromatogram-image wide-image\"><img decoding=\"async\" src=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0722.png\" alt=\"Creatine and Creatinine in Human Plasma on Raptor HILIC-Si by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0722<\/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\"><a class=\"cmpd_link\" title=\"View compound information for Creatinine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/60-27-5\/Creatinine\" target=\"_blank\" rel=\"noopener\">Creatinine<\/a><\/td><td class=\"oth\">0.537<\/td><td class=\"oth\">114.0<\/td><td class=\"oth\">44.3<\/td><td class=\"oth\">86.0<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Creatine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/57-00-1\/Creatine\" target=\"_blank\" rel=\"noopener\">Creatine<\/a><\/td><td class=\"oth\">1.232<\/td><td class=\"oth\">132.1<\/td><td class=\"oth\">43.3<\/td><td class=\"oth\">90.2<\/td><\/tr>\n<\/tbody><\/table><div class=\"peaknotes\"> <\/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 HILIC-Si  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9310A52?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0722\" rel=\"noopener\">cat.# 9310A52<\/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>UltraShield UHPLC precolumn filter 0.2 \u00b5m (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/25810?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0722\" rel=\"noopener\">cat.# 25810<\/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>20:80 Water:acetonitrile<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Conc.:<\/th><td>Endogenous levels <\/td><\/tr><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Inj. Vol.:<\/th><td>0.2 \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>5 mM Ammonium formate in 20:80 water:acetonitrile <\/td><\/tr><tr><td><\/td><td><table class=\"cgram_ramp\"><thead><tr><th>Time (min)<\/th><th>Flow (mL\/min)<\/th><th>%A<\/th><\/tr><\/thead><tbody><tr><td>0.00<\/td><td>0.5<\/td><td>100<\/td><\/tr><tr><td>1.5<\/td><td>Stop<\/td><td><\/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>UHPLC<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Sample Preparation<\/th><td>Endogenous levels of creatinine and creatine in human plasma were determined using a single protein precipitation step followed by LC-MS\/MS analysis. A 50 \u03bcL aliquot of human plasma (K2EDTA) was mixed with 950 \u03bcL acetonitrile. After vortexing and centrifuging at 4300 rpm for 10 min, 200 \u03bcL of the supernatant was transferred to a new vial and mixed with  50 \u03bcL of water. Centrifugation was performed again before injection. <br \/><\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Notes<\/th><td>Endogenous peaks for phospholipids and lysophospholipids are displayed because they are common matrix interferences and are known to suppress ionization efficiency.<\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0722.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\n\n<p class=\"has-medium-font-size\"><strong>Human Urine<\/strong><\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article\"><div class=\"wp-block-custom-chromatogram-article\"><div class=\"chromatogram-image wide-image\"><img decoding=\"async\" src=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0723.png\" alt=\"Creatine and Creatinine in Human Urine on Raptor HILIC-Si by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0723<\/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\"><a class=\"cmpd_link\" title=\"View compound information for Creatinine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/60-27-5\/Creatinine\" target=\"_blank\" rel=\"noopener\">Creatinine<\/a><\/td><td class=\"oth\">0.537<\/td><td class=\"oth\">114.0<\/td><td class=\"oth\">44.3<\/td><td class=\"oth\">86.0<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Creatine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/57-00-1\/Creatine\" target=\"_blank\" rel=\"noopener\">Creatine<\/a><\/td><td class=\"oth\">1.240<\/td><td class=\"oth\">132.1<\/td><td class=\"oth\">43.3<\/td><td class=\"oth\">90.2<\/td><\/tr>\n<\/tbody><\/table><\/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 HILIC-Si  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9310A52?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0723\" rel=\"noopener\">cat.# 9310A52<\/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>UltraShield UHPLC precolumn filter 0.2 \u00b5m (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/25810?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0723\" rel=\"noopener\">cat.# 25810<\/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>20:80 Water:acetonitrile<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Conc.:<\/th><td>  Endogenous levels<\/td><\/tr><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Inj. Vol.:<\/th><td>0.2 \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>5 mM Ammonium formate in 20:80 water:acetonitrile <\/td><\/tr><tr><td><\/td><td><table class=\"cgram_ramp\"><thead><tr><th>Time (min)<\/th><th>Flow (mL\/min)<\/th><th>%A<\/th><\/tr><\/thead><tbody><tr><td>0.00<\/td><td>0.5<\/td><td>100<\/td><\/tr><tr><td>1.5<\/td><td>Stop<\/td><td><\/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>UHPLC<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Sample Preparation<\/th><td>Endogenous levels of creatinine and creatine in human urine were determined using a simple dilute-and-shoot method. A 50 \u03bcL aliquot of human urine was mixed with 950 \u03bcL acetonitrile. After vortexing and centrifuging at 4300 rpm for 10 min, 10 \u03bcL of the supernatant was added to the 1490 \u03bcL of 80%  acetonitrile in water. Centrifugation was performed again before injection. <\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0723.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>A rapid, robust method was established for the analysis of creatine and creatinine in human plasma and urine. An efficient separation was achieved in less than 1.5 min using a Raptor HILIC-Si column and a simple isocratic mobile phase.<\/p>\n","protected":false},"author":11,"featured_media":6505,"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":[2223,2224],"post-badge":[],"resource-type":[2623],"product-library":[2499,2481],"resource-technique":[2355,2326,2363],"hf_cat_post":[650,2624],"ppma_author":[414],"class_list":["post-43388","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-articles","industries-application-klinisch","industries-application-klinische-diagnostik","resource-type-featured-application","product-library-lc-saulen","product-library-flussigchromatographie-produkte","resource-technique-hilic-de","resource-technique-flussigchromatografie","resource-technique-ms-ms-de"],"acf":[],"taxonomy_info":{"category":[{"value":7,"label":"Articles"}],"industries-application":[{"value":2223,"label":"Klinisch"},{"value":2224,"label":"Klinische 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