{"id":76459,"date":"2016-06-30T00:00:00","date_gmt":"2016-06-30T00:00:00","guid":{"rendered":"https:\/\/discover.restek.com\/?p=76459"},"modified":"2025-12-26T20:57:52","modified_gmt":"2025-12-26T20:57:52","slug":"simple-analysis-of-bile-acids-by-gc-ms","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/zh-hans\/blogs-de\/gnbl5536\/simple-analysis-of-bile-acids-by-gc-ms","title":{"rendered":"Simple Analysis of Bile Acids by GC-MS"},"content":{"rendered":"\n<p>Bile acids are a group of steroidal acids with carboxyl and hydroxyl groups on the side. They are the major metabolic products of cholesterol. Bile acids play important roles as biomarkers for early diagnosis and therapeutic monitoring of many diseases, especially liver and intestinal diseases. For instance, high levels of intestinal bile acids, in particular, deoxcholic acid, is an indication of colon carcinogenesis.[1]&nbsp;Below are structures of some of the most common bile acids.<\/p>\n\n\n<style>.kb-image76459_7b09a7-34 .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<figure class=\"wp-block-kadence-image kb-image76459_7b09a7-34 size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"1417\" height=\"1081\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-01.png\" alt=\"\" class=\"kb-img wp-image-78605\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-01.png 1417w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-01-300x229.png 300w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-01-1024x781.png 1024w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-01-768x586.png 768w\" sizes=\"auto, (max-width: 1417px) 100vw, 1417px\" \/><\/figure>\n\n\n\n<p><br>LC-MS and LC-MS\/MS are commonly used for bile acid analysis in biological fluids without derivatization.\u00a0Disadvantages of LC-based methods include relatively high carry-over on LC columns and low peak capacity.<\/p>\n\n\n\n<p>GC is a robust, simple and inexpensive lab tool for volatile or semi-volatile compound analysis. The only drawback of GC versus HPLC for bile acid analysis is&nbsp;polar functional groups (carboxyl and hydroxyl) need to be derivatized into GC-detectable forms. Trimethylsilylating reagents, such as N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA), are commonly used for derivatization of both hydroxyl and carboxyl groups. Choosing the right reagents can be tricky. Kumar et al.[2]&nbsp;compared four types of derivatization mixtures: MSTFA:NH<sub>4<\/sub>I:DTE (500:4:2, 40 lL, 60 C, 30 min), MSTFA:TMSI:TMCS (100:2:5, 40 lL, 60 C, 30 min), MSTFA:TMCS (100:1, 40 lL, 60 C, 30 min), and BSTFA:TMCS (100:1, 40 lL, 60 C,30 min). Test results showed that MSTFA:NH<sub>4<\/sub>I:DTE (500:4:2, v\/w\/w) has the highest efficiency for bile acid derivatization. However, the mixture must be freshly prepared and thus is not convenient for fast analysis. Fortunately, my colleague Amanda Rigdon developed a one-step method for&nbsp;cannabinoid acids derivatization&nbsp;using&nbsp;BSTFA+1% TMCS, and the reaction&nbsp;is only 30 min.<\/p>\n\n\n\n<p>Here, the same derivatization method was applied to bile acids, and the preliminary results are very promising. Basically, this reagent simply replaces the hydrogen in the \u2013OH or \u2013COOH groups with a trimethylsilyl group (see the picture below). The procedure is&nbsp;simple and straightforward.<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li>Place 100 \u00b5L of bile acid standards (~100 ppm) into an\u00a0interlock vial<\/li>\n\n\n\n<li>Evaporate the solvent under nitrogen<\/li>\n\n\n\n<li>Add 50 \u00b5L of ethyl acetate and 50 \u00b5L of\u00a0BSTFA+1% TMCS<\/li>\n\n\n\n<li>Incubate for 30 min at 70 \u00b0C<\/li>\n\n\n\n<li>Cool down and dilute with ethyl acetate if necessary<\/li>\n<\/ul>\n\n\n<style>.kb-image76459_fba385-20 .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<figure class=\"wp-block-kadence-image kb-image76459_fba385-20 size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"1364\" height=\"443\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-02.png\" alt=\"\" class=\"kb-img wp-image-78599\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-02.png 1364w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-02-300x97.png 300w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-02-1024x333.png 1024w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-02-768x249.png 768w\" sizes=\"auto, (max-width: 1364px) 100vw, 1364px\" \/><\/figure>\n\n\n\n<p>The derivatized bile acid standards were analyzed by GC-MS under full scan mode using an&nbsp;Rxi-5ms&nbsp;30 m \u00d7 0.25 mm \u00d7 0.25 \u00b5m column. The chromatogram below shows good separation of closely related bile acids (isomers and enantiomers), such as HDCA and UDCA.<\/p>\n\n\n<style>.kb-image76459_134952-9b .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<figure class=\"wp-block-kadence-image kb-image76459_134952-9b size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"1252\" height=\"870\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-03.png\" alt=\"\" class=\"kb-img wp-image-78593\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-03.png 1252w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-03-300x208.png 300w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-03-1024x712.png 1024w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-03-768x534.png 768w\" sizes=\"auto, (max-width: 1252px) 100vw, 1252px\" \/><\/figure>\n\n\n\n<p>The reaction and GC conditions (reagent\/bile acid amount ratio, reaction temperature, time, column dimensions, etc.) can be further optimized. Hopefully, this preliminary work could open the door for the fast GC analysis of bile acids. Keep in mind there are some limitations on conjugated bile acids.<\/p>\n\n\n\n<p>GC analysis time too long? EZGC can help! Here is one of the modulation solutions using shorter column. The total analysis time is less than 10 min.<\/p>\n\n\n<style>.kb-image76459_36d902-4d .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<figure class=\"wp-block-kadence-image kb-image76459_36d902-4d size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"1367\" height=\"549\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-04.png\" alt=\"\" class=\"kb-img wp-image-78587\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-04.png 1367w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-04-300x120.png 300w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-04-1024x411.png 1024w, https:\/\/discover.restek.com\/wp-content\/uploads\/blog-simple-analysis-of-bile-acids-by-gc-ms-04-768x308.png 768w\" sizes=\"auto, (max-width: 1367px) 100vw, 1367px\" \/><\/figure>\n\n\n\n<p><br role=\"presentation\" data-uw-rm-sr=\"\"><\/p>\n\n\n\n<p>References<\/p>\n\n\n\n<p>[1] (a) Jensen, R. G.; Clark, R. M.; deJong, F. A.; Hamosh, M.; Liao, T. H.; Mehta, N. R., The lipolytic triad: human lingual, breast milk, and pancreatic lipases: physiological implications of their characteristics in digestion of dietary fats.&nbsp;<em>Journal of pediatric gastroenterology and nutrition&nbsp;<\/em><strong>1982,<\/strong>&nbsp;<em>1<\/em>&nbsp;(2), 243-55; (b) Reddy, B. S., Diet and excretion of bile acids.&nbsp;<em>Cancer research&nbsp;<\/em><strong>1981,<\/strong>&nbsp;<em>41<\/em>&nbsp;(9 Pt 2), 3766-8.<\/p>\n\n\n\n<p>[2] Kumar, B. S.; Chung, B. C.; Lee, Y. J.; Yi, H. J.; Lee, B. H.; Jung, B. H., Gas chromatography-mass spectrometry-based simultaneous quantitative analytical method for urinary oxysterols and bile acids in rats.&nbsp;<em>Anal. Biochem.&nbsp;<\/em><strong>2011,<\/strong>&nbsp;<em>408<\/em>&nbsp;(2), 242-52.<\/p>\n\n\n\n<p>&nbsp;<\/p>\n\n\n\n<p>&#8220;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Bile acids are a group of steroidal acids with carboxyl and hydroxyl groups on the side. They are the major metabolic products of cholesterol. Bile acids play important roles as biomarkers for early diagnosis and therapeutic monitoring of many diseases,&#8230;<\/p>\n","protected":false},"author":3,"featured_media":0,"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":[792],"tags":[],"industries-application":[],"post-badge":[],"resource-type":[],"product-library":[],"resource-technique":[],"hf_cat_post":[623],"ppma_author":[587],"class_list":["post-76459","post","type-post","status-publish","format-standard","hentry","category-blogs-de"],"acf":[],"taxonomy_info":{"category":[{"value":792,"label":"Blogs"}]},"featured_image_src_large":false,"author_info":{"display_name":"Audrey Crater","author_link":"https:\/\/discover.restek.com\/zh-hans\/author\/audrey-craterrestek-com\/"},"comment_info":0,"category_info":[{"term_id":792,"name":"Blogs","slug":"blogs-de","term_group":0,"term_taxonomy_id":792,"taxonomy":"category","description":"","parent":0,"count":442,"filter":"raw","cat_ID":792,"category_count":442,"category_description":"","cat_name":"Blogs","category_nicename":"blogs-de","category_parent":0}],"tag_info":false,"authors":[{"term_id":587,"user_id":0,"is_guest":1,"slug":"dan-li","display_name":"Dan Li","avatar_url":"https:\/\/secure.gravatar.com\/avatar\/?s=96&d=mm&r=g","0":null,"1":"","2":"","3":"","4":"","5":"","6":"","7":"","8":""}],"_links":{"self":[{"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/posts\/76459","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/comments?post=76459"}],"version-history":[{"count":1,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/posts\/76459\/revisions"}],"predecessor-version":[{"id":78612,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/posts\/76459\/revisions\/78612"}],"wp:attachment":[{"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/media?parent=76459"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/categories?post=76459"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/tags?post=76459"},{"taxonomy":"industries-application","embeddable":true,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/industries-application?post=76459"},{"taxonomy":"post-badge","embeddable":true,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/post-badge?post=76459"},{"taxonomy":"resource-type","embeddable":true,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/resource-type?post=76459"},{"taxonomy":"product-library","embeddable":true,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/product-library?post=76459"},{"taxonomy":"resource-technique","embeddable":true,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/resource-technique?post=76459"},{"taxonomy":"hf_cat_post","embeddable":true,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/hf_cat_post?post=76459"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/discover.restek.com\/zh-hans\/wp-json\/wp\/v2\/ppma_author?post=76459"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}