{"id":39956,"date":"2020-10-15T14:30:00","date_gmt":"2020-10-15T14:30:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/rapid-analysis-of-17-bile-acids-in-human-plasma-by-lc-msms\/"},"modified":"2026-02-09T21:51:16","modified_gmt":"2026-02-09T21:51:16","slug":"rapid-analysis-of-17-bile-acids-in-human-plasma-by-lc-msms","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/ja\/application-notes\/cfan2911\/rapid-analysis-of-17-bile-acids-in-human-plasma-by-lc-msms","title":{"rendered":"Rapid Analysis of 17 Bile Acids in Human Plasma by LC-MS\/MS"},"content":{"rendered":"\n<h2 class=\"wp-block-heading\">Abstract<\/h2>\n\n\n\n<p>The quantitative analysis of bile acids in plasma is critical for diagnosing many structural liver diseases. Accurate reporting can be difficult because of analyte characteristics, matrix effects, and other factors. In this work, we describe a robust, selective LC-MS\/MS method for the analysis of 17 bile acids in under 10 minutes, including three isomer groups, with special attention given to a matrix interference observed in routine human plasma analysis.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Introduction<\/h2>\n\n\n\n<p>Bile acids are a group of major catabolic products of cholesterol, and they are critical signaling molecules that regulate cholesterol and glucose. The analysis of bile acids in human plasma is an important diagnostic tool as bile acids are biomarkers of liver disease and are also used as indicators of potentially harmful side effects of new drugs. There are two main types of bile acids based upon their functional groups: unconjugated (or free) and conjugated, primarily with glycine- or taurine-based residues (Figure 1, Table I). Quantitation of bile acids in matrix can be very challenging due to several factors. These include structural similarities, varying polarity and stereochemistry, the presence of isomers, limited fragmentation of unconjugated bile acids in a mass spectrometer, high endogenous levels, and matrix effects caused by phospholipids or triglycerides.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 1:<\/strong> Bile Acids Base Chemical Structure\u00a0<br><\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<style>.kb-image39956_f9b5f5-b9 .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<figure class=\"wp-block-kadence-image kb-image39956_f9b5f5-b9 size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"1800\" height=\"560\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_01.jpg\" alt=\"\" class=\"kb-img wp-image-18724\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_01.jpg 1800w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_01-300x93.jpg 300w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_01-1024x319.jpg 1024w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_01-768x239.jpg 768w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_01-1536x478.jpg 1536w\" sizes=\"auto, (max-width: 1800px) 100vw, 1800px\" \/><\/figure>\n\n<\/div><\/div><\/div>\n\n\n<p><strong>Table I:<\/strong> R Group Identifications for Figure 1<\/p>\n\n\n<figure class=\"wp-block-table\">\n<table>\n<tbody>\n<tr style=\"background-color: #272a59; color: #ffffff;\">\n<td><strong>Compound Name<\/strong><\/td>\n<td><strong>R<\/strong><\/td>\n<td><strong>R<sub>1<\/sub><\/strong><\/td>\n<td><strong>R<sub>2<\/sub><\/strong><\/td>\n<td><strong>R<sub>3<\/sub><\/strong><\/td>\n<td><strong>R<sub>4<\/sub><\/strong><\/td>\n<td><strong>R<sub>5<\/sub><\/strong><\/td>\n<\/tr>\n<tr>\n<td>Ursodeoxycholic acid (UDCA)<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>OH<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>Hyodeoxycholic acid (HDCA)<\/td>\n<td>OH<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr>\n<td>Cholic acid (CA)<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>OH<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>Chenodeoxycholic acid (CDCA)<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>OH<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr>\n<td>Deoxycholic acid (DCA)<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>Dehydrolithocholic acid (DHLCA)<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>=O<\/td>\n<\/tr>\n<tr>\n<td>Lithocholic acid (LCA)<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>Glycoursodeoxycholic acid (GUDCA)<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>NHCH<sub>2<\/sub>CO<sub>2<\/sub>H<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr>\n<td>Glycocholic acid (GCA)<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>NHCH<sub>2<\/sub>CO<sub>2<\/sub>H<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>Glycochenodeoxycholic acid (GCDCA)<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>NHCH<sub>2<\/sub>CO<sub>2<\/sub>H<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr>\n<td>Glycodeoxycholic acid (GDCA)<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>NHCH<sub>2<\/sub>CO<sub>2<\/sub>H<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>Glycolithocholic acid (GLCA)<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>NHCH<sub>2<\/sub>CO<sub>2<\/sub>H<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr>\n<td>Tauroursodeoxycholic acid (TUDCA)<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>NHCH<sub>2<\/sub>CH<sub>2<\/sub>SO<sub>3<\/sub>H<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>Taurocholic acid (TCA)<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>NHCH<sub>2<\/sub>CH<sub>2<\/sub>SO<sub>3<\/sub>H<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr>\n<td>Taurochenodeoxycholic acid (TCDCA)<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>NHCH<sub>2<\/sub>CH<sub>2<\/sub>SO<sub>3<\/sub>H<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>Taurodeoxycholic acid (TDCA)<\/td>\n<td>H<\/td>\n<td>OH<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>NHCH<sub>2<\/sub>CH<sub>2<\/sub>SO<sub>3<\/sub>H<\/td>\n<td>OH<\/td>\n<\/tr>\n<tr>\n<td>Taurolithocholic acid (TLCA)<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>H<\/td>\n<td>NHCH<sub>2<\/sub>CH<sub>2<\/sub>SO<sub>3<\/sub>H<\/td>\n<td>OH<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/figure>\n\n\n<p>As mentioned above, matrix effects are a commonly encountered issue in bile acids analysis where interferences with the analytes of interest are caused by the sample matrix or other substances that are added to the sample. Matrix interferences may present as a partially or fully coeluting peak with one of the analytes or internal standards, or as a reduction in the MS signal, which may impact the limit of detection and method accuracy and precision for a particular analyte(s). As a result, matrix interferences can cause issues with identification and quantitation of the analytes of interest. Pinpointing the source of matrix interferences can be difficult as they can be caused by many factors. Patient medications and lifestyle\/diet factors; sample preservatives or anticoagulants; contamination during sample processing; or compounds endogenous to the matrix all could result in matrix interference. Because matrix interferences can have such an impact on the performance of an LC-MS\/MS assay, it is extremely important that any potential interferences are identified and explored during method development and validation.<\/p>\n\n\n\n<p>As many method developers know, it can be difficult to address all the potential matrix interferences that may be present in authentic patient samples when using a surrogate matrix during routine validation. Several years ago, Restek released an application note for the analysis of 17 bile acids in plasma by LC-MS\/MS. Utilizing a Raptor C18 column, this method effectively separated 17 bile acids, including three sets of isomers, in an 8.5-minute runtime. &nbsp;When further studies were performed with this method, it was discovered that a matrix interference present in clinical patient samples was partially coeluting with one of the deuterated bile acid internal standards, resulting in inconsistent internal standard response. To address this issue, a new analytical method was developed herein. The objectives of the revised method were to resolve the matrix interference while still achieving respectable separation of the 17 target bile acids, including the three sets of isomers, for subsequent qualification and quantification LC-MS\/MS studies.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Original Bile Acids Method and Matrix Interference<\/h3>\n\n\n\n<p>The original application was developed on a Raptor C18 column, and the method parameters are shown in Figure 2.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 2:<\/strong> 17 Bile Acids in Human Plasma on Raptor C18 (Original Method)<\/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 wide-image\"><img decoding=\"async\" src=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0711.png\" alt=\"Bile Acids in Human Plasma on Raptor C18 (1.8 \u03bcm) by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0711<\/p><div class=\"chromatogram-peaks\"><h4>Peaks<\/h4><table class=\"peaks col-lg-6 col-12 peak-50\">\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\">Conc.<br \/>(ng\/mL)<\/th><th style=\"text-align: center;width: 75px\">Precursor 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\">Glycoursodeoxycholic acid (GUDCA)<\/td><td class=\"oth\">1.45<\/td><td class=\"oth\">540<\/td><td class=\"oth\">448.4<\/td><td class=\"oth\">74.1<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Tauroursodeoxycholic acid (TUDCA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/14605-22-2 \/Tauroursodeoxycholic acid\" target=\"_blank\" rel=\"noopener\">Tauroursodeoxycholic acid (TUDCA)<\/a><\/td><td class=\"oth\">1.59<\/td><td class=\"oth\">1,080<\/td><td class=\"oth\">498.4<\/td><td class=\"oth\">80.1<\/td><\/tr>\n<tr><td class=\"num\">3.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Glycocholic acid (GCA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/475-31-0\/Glycocholic acid\" target=\"_blank\" rel=\"noopener\">Glycocholic acid (GCA)<\/a><\/td><td class=\"oth\">2.01<\/td><td class=\"oth\">540<\/td><td class=\"oth\">464.3<\/td><td class=\"oth\">74.2<\/td><\/tr>\n<tr><td class=\"num\">4.<\/td><td class=\"cmpd\">Taurocholic acid (TCA)<\/td><td class=\"oth\">2.16<\/td><td class=\"oth\">1,080<\/td><td class=\"oth\">514.4<\/td><td class=\"oth\">80.0<\/td><\/tr>\n<tr><td class=\"num\">5.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Ursodeoxycholic acid (UDCA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/128-13-2\/Ursodeoxycholic acid\" target=\"_blank\" rel=\"noopener\">Ursodeoxycholic acid (UDCA)<\/a><\/td><td class=\"oth\">2.25<\/td><td class=\"oth\">360<\/td><td class=\"oth\">391.4<\/td><td class=\"oth\">391.4<\/td><\/tr>\n<tr><td class=\"num\">6.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Hyodeoxycholic acid (HDCA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/83-49-8\/Hyodeoxycholic acid\" target=\"_blank\" rel=\"noopener\">Hyodeoxycholic acid (HDCA)<\/a><\/td><td class=\"oth\">2.56<\/td><td class=\"oth\">360<\/td><td class=\"oth\">391.4<\/td><td class=\"oth\">391.4<\/td><\/tr>\n<tr><td class=\"num\">7.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Cholic acid (CA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/81-25-4\/Cholic acid\" target=\"_blank\" rel=\"noopener\">Cholic acid (CA)<\/a><\/td><td class=\"oth\">2.74<\/td><td class=\"oth\">90<\/td><td class=\"oth\">407.3<\/td><td class=\"oth\">407.2<\/td><\/tr>\n<tr><td class=\"num\">8.<\/td><td class=\"cmpd\">Glycochenodeoxycholic acid (GCDCA)<\/td><td class=\"oth\">3.29<\/td><td class=\"oth\">540<\/td><td class=\"oth\">448.4<\/td><td class=\"oth\">74.1<\/td><\/tr>\n<\/tbody><\/table>\n<table class=\"peaks col-lg-6 col-12 peak-50\"><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\">Conc.<br \/>(ng\/mL)<\/th><th style=\"text-align: center;width: 75px\">Precursor Ion<\/th><th style=\"text-align: center;width: 75px\">Product Ion<\/th><\/tr><\/thead><tbody>\n<tr><td class=\"num\">9.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Taurochenodeoxycholic acid (TCDCA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/516-35-8 \/Taurochenodeoxycholic acid\" target=\"_blank\" rel=\"noopener\">Taurochenodeoxycholic acid (TCDCA)<\/a><\/td><td class=\"oth\">3.42<\/td><td class=\"oth\">1,080<\/td><td class=\"oth\">498.4<\/td><td class=\"oth\">80.1<\/td><\/tr>\n<tr><td class=\"num\">10.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Glycodeoxycholic acid (GDCA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/360-65-6\/Glycodeoxycholic acid\" target=\"_blank\" rel=\"noopener\">Glycodeoxycholic acid (GDCA)<\/a><\/td><td class=\"oth\">3.54<\/td><td class=\"oth\">540<\/td><td class=\"oth\">448.4<\/td><td class=\"oth\">74.1<\/td><\/tr>\n<tr><td class=\"num\">11.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Taurodeoxycholic acid (TDCA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/516-50-7 \/Taurodeoxycholic acid\" target=\"_blank\" rel=\"noopener\">Taurodeoxycholic acid (TDCA)<\/a><\/td><td class=\"oth\">3.65<\/td><td class=\"oth\">1,080<\/td><td class=\"oth\">498.4<\/td><td class=\"oth\">80.1<\/td><\/tr>\n<tr><td class=\"num\">12.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Chenodeoxycholic acid (CDCA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/474-25-9\/Chenodeoxycholic acid\" target=\"_blank\" rel=\"noopener\">Chenodeoxycholic acid (CDCA)<\/a><\/td><td class=\"oth\">4.20<\/td><td class=\"oth\">360<\/td><td class=\"oth\">391.4<\/td><td class=\"oth\">391.4<\/td><\/tr>\n<tr><td class=\"num\">13.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Deoxycholic acid (DCA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/83-44-3\/Deoxycholic acid\" target=\"_blank\" rel=\"noopener\">Deoxycholic acid (DCA)<\/a><\/td><td class=\"oth\">4.40<\/td><td class=\"oth\">90<\/td><td class=\"oth\">391.4<\/td><td class=\"oth\">391.4<\/td><\/tr>\n<tr><td class=\"num\">14.<\/td><td class=\"cmpd\">Glycolithocholic acid (GLCA)<\/td><td class=\"oth\">4.57<\/td><td class=\"oth\">540<\/td><td class=\"oth\">432.3<\/td><td class=\"oth\">74.0<\/td><\/tr>\n<tr><td class=\"num\">15.<\/td><td class=\"cmpd\">Taurolithocholic acid (TLCA)<\/td><td class=\"oth\">4.69<\/td><td class=\"oth\">1,080<\/td><td class=\"oth\">482.4<\/td><td class=\"oth\">80.0<\/td><\/tr>\n<tr><td class=\"num\">16.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Dehydrolithocholic acid (DHLCA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/1553-56-6\/Dehydrolithocholic acid\" target=\"_blank\" rel=\"noopener\">Dehydrolithocholic acid (DHLCA)<\/a><\/td><td class=\"oth\">5.62<\/td><td class=\"oth\">90<\/td><td class=\"oth\">373.3<\/td><td class=\"oth\">373.3<\/td><\/tr>\n<tr><td class=\"num\">17.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Lithocholic acid (LCA)\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/434-13-9\/Lithocholic acid\" target=\"_blank\" rel=\"noopener\">Lithocholic acid (LCA)<\/a><\/td><td class=\"oth\">5.76<\/td><td class=\"oth\">180<\/td><td class=\"oth\">375.5<\/td><td class=\"oth\">375.3<\/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 C18  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9304252?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0711\" rel=\"noopener\">cat.# 9304252<\/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>1.8 \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_CF0711\" rel=\"noopener\">cat.# 25810<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>60 \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>70:30 Water:methanol<\/td><\/tr><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Inj. Vol.:<\/th><td>3 \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 acetate in water <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>50:50 Acetonitrile: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>0.5<\/td><td>65<\/td><td>35<\/td><\/tr><tr><td>2.00<\/td><td>0.5<\/td><td>60<\/td><td>40<\/td><\/tr><tr><td>2.50<\/td><td>0.5<\/td><td>55<\/td><td>45<\/td><\/tr><tr><td>3.50<\/td><td>0.5<\/td><td>50<\/td><td>50<\/td><\/tr><tr><td>4.60<\/td><td>0.5<\/td><td>45<\/td><td>55<\/td><\/tr><tr><td>5.70<\/td><td>0.5<\/td><td>20<\/td><td>80<\/td><\/tr><tr><td>5.90<\/td><td>0.8*<\/td><td>5<\/td><td>95<\/td><\/tr><tr><td>6.50<\/td><td>0.8*<\/td><td>5<\/td><td>95<\/td><\/tr><tr><td>6.51<\/td><td>0.5<\/td><td>65<\/td><td>35<\/td><\/tr><tr><td>8.50<\/td><td>0.5<\/td><td>65<\/td><td>35<\/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\">Notes<\/th><td>*The flow rate was increased to 0.8 mL\/min to more thoroughly flush phospholipids from the analytical column, thereby reducing matrix effects.<br \/><br \/>Want even better performance when analyzing metal-sensitive compounds? Check out Inert LC columns at <a href=\"https:\/\/www.restek.com\/inert\" target=\"_blank\" rel=\"noopener\">www.restek.com\/inert<\/a>.<\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0711.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<p><\/p>\n\n\n\n<p>When patient samples were later analyzed with the original method described in Figure 2, a matrix interference was found to be partially coeluting with D<sub>4 <\/sub>UDCA, the deuterated internal standard used for quantitation of UDCA. The matrix interference present when analyzed with the original method is shown in Figure 3.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 3:<\/strong> Matrix Interference Partially Coeluting with D<sub>4 <\/sub>UDCA on Raptor C18 (Original Method)<\/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 wide-image\"><img decoding=\"async\" src=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0818.png\" alt=\"Plasma Matrix Interference Co-Eluting with UDCA-D&lt;sub&gt;4&lt;\/sub&gt; in Patient Sample on Raptor C18\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0818<\/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><\/tr><\/thead>\n<tbody><tr><td class=\"num\">1.<\/td><td class=\"cmpd\">Matrix interference<\/td><td class=\"oth\">2.25<\/td><td class=\"oth\">&#8211;<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\">D<sub>4<\/sub> UDCA<\/td><td class=\"oth\">2.35<\/td><td class=\"oth\">395.40<\/td><td class=\"oth\">395.40<\/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 C18  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9304252?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0818\" rel=\"noopener\">cat.# 9304252<\/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>1.8 \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_CF0818\" rel=\"noopener\">cat.# 25810<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>60 \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>65:35 Water:methanol <\/td><\/tr><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Inj. Vol.:<\/th><td>3 \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 acetate in water, pH unadjusted <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>Methanol:acetonitrile (v\/v, 50:50) <\/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>0.5<\/td><td>65<\/td><td>35<\/td><\/tr><tr><td>2.00<\/td><td>0.5<\/td><td>60<\/td><td>40<\/td><\/tr><tr><td>2.50<\/td><td>0.5<\/td><td>55<\/td><td>45<\/td><\/tr><tr><td>3.50<\/td><td>0.5<\/td><td>50<\/td><td>50<\/td><\/tr><tr><td>4.60<\/td><td>0.5<\/td><td>45<\/td><td>55<\/td><\/tr><tr><td>5.70<\/td><td>0.5<\/td><td>20<\/td><td>80<\/td><\/tr><tr><td>5.90<\/td><td>0.8*<\/td><td>5<\/td><td>95<\/td><\/tr><tr><td>6.50<\/td><td>0.8*<\/td><td>5<\/td><td>95<\/td><\/tr><tr><td>6.51<\/td><td>0.5<\/td><td>65<\/td><td>35<\/td><\/tr><tr><td>8.50<\/td><td>0.5<\/td><td>65<\/td><td>35<\/td><\/tr><\/tbody><\/table><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Max Pressure:<\/th><td>275 bar<\/td><\/tr><\/table><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Detector<\/th><td>Shimadzu LCMS-8045 in ESI- mode<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Instrument<\/th><td>Shimadzu Nexera X2<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Sample Preparation<\/th><td>A 50 \u03bcL aliquot of patient sample was added to a microcentrifuge tube. Fifty microliters of internal standards was added to the tube and vortexed. The samples were protein precipitated using 800 \u03bcL of ice-cold acetonitrile. After vortexing and centrifugation at 4200 rpm for 10 min, the supernatant was transferred to a new vial and dried down at 60 \u00b0C under nitrogen. All samples were reconstituted in 200 \u03bcL of 35% methanol in water.<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Notes<\/th><td>*The flow rate was increased to 0.8 mL\/min to more thoroughly flush phospholipids from the analytical column, thereby reducing matrix effects.<br \/><br \/>The flow was diverted to waste before 1 minute and after 6 minutes to protect the mass spectrometer.<\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0818.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<p><\/p>\n\n\n\n<p>In some cases of matrix interferences, the simplest option to remove the interference is to choose an alternative product ion that is not affected. Because unconjugated bile acids exhibit limited fragmentation in a mass spectrometer, D<sub>4 <\/sub>UDCA uses the parent mass as both the precursor and product ions, meaning that choosing a different product ion is not an option for this compound.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Method Requirements<\/h3>\n\n\n\n<p>The primary requirement of the redeveloped method was to chromatographically separate the matrix interference from D<sub>4 <\/sub>UDCA. Additionally, the analyte list contained three sets of isomeric compounds: taurine conjugated isomers (TUDCA, TCDCA, TDCA); glycine conjugated isomers (GUDCA, GCDCA, GDCA); and unconjugated isomers (UDCA, HDCA, CDCA, and DCA). The method was also required to adequately resolve the isomers within these sets.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Method Optimization<\/h3>\n\n\n\n<p>A Raptor Inert ARC-18 100 x 2.1 mm, 2.7 \u00b5m analytical column (cat.# 9314A12-T) was selected for development of the new method. A Raptor Inert ARC-18 5 x 2.1 mm, 2.7 \u00b5m EXP guard column (cat.# 9314A0252-T) was also installed to protect the analytical column. The elution gradient, column temperature, flow rate, and injection volume were optimized to provide adequate resolution of all analytes and the matrix interference.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Experimental<\/h2>\n\n\n\n<h3 class=\"wp-block-heading\">Calibration Standards and Quality Control Samples&nbsp;<\/h3>\n\n\n\n<p>Bile acid standard mixtures were obtained from Cambridge Isotope Laboratories. The stable, isotope-labeled bile acids mixes (unconjugated, cat.# <a href=\"https:\/\/isotope.com\/metabolomics-mixes-and-kits\/bile-acid-standard-mix-1-unconjugated-msk-ba1-1\" target=\"_blank\" rel=\"noreferrer noopener\">MSK-BA1<\/a>; conjugated, cat.# <a href=\"https:\/\/isotope.com\/metabolomics-mixes-and-kits\/bile-acid-standard-mix-2-conjugated-msk-ba2-1\" target=\"_blank\" rel=\"noreferrer noopener\">MSK-BA2<\/a>) were used as internal standards. D<sub>4 <\/sub>hyodeoxycholic acid&nbsp;(cat.# <a href=\"https:\/\/isotope.com\/hyodeoxycholic-acid-hdca-2-2-4-4-d4-dlm-11626-pk\" target=\"_blank\" rel=\"noreferrer noopener\">DLM-11626-0<\/a>) was also obtained from Cambridge Isotope Laboratories as a separate standard. Unlabeled bile acids mixes (unconjugated, cat.# <a href=\"https:\/\/isotope.com\/metabolomics-mixes-and-kits\/bile-acid-standard-mix-1-unconjugated-unlabeled-msk-ba1-us-1\" target=\"_blank\" rel=\"noreferrer noopener\">MSK-BA1-US<\/a>; conjugated, cat.# <a href=\"https:\/\/isotope.com\/metabolomics-mixes-and-kits\/bile-acid-standard-mix-2-conjugated-unlabeled-msk-ba2-us-1\" target=\"_blank\" rel=\"noreferrer noopener\">MSK-BA2-US<\/a>) were used as primary reference standards. Reference standards for hyodeoxycholic acid (HDCA) and dehydrolithocholic acid (DHLCA) were obtained separately.&nbsp;<br>Calibration standards and fortified QC samples were prepared at the concentrations shown in Table II. An internal standard working solution comprising the 17 isotopically labeled bile acids was prepared to a concentration of 2 \u00b5M.&nbsp;<\/p>\n\n\n\n<p><strong>Table II:<\/strong> Calibration Standard and Quality Control Sample Concentrations in Plasma (\u00b5M) for All Analytes&nbsp;<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table><tbody><tr><td><strong>Cal F<\/strong><\/td><td><strong>Cal E<\/strong><\/td><td><strong>Cal D<\/strong><\/td><td><strong>Cal C<\/strong><\/td><td><strong>Cal B<\/strong><\/td><td><strong>Cal A<\/strong><\/td><td><strong>LLOQ<\/strong><\/td><td><strong>Low QC<\/strong><\/td><td><strong>Medium QC<\/strong><\/td><td><strong>High QC<\/strong><\/td><\/tr><tr><td>0.05<\/td><td>0.1<\/td><td>0.2<\/td><td>0.5<\/td><td>1<\/td><td>5<\/td><td>0.05<\/td><td>0.1<\/td><td>0.5<\/td><td>1<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<h3 class=\"wp-block-heading\">Sample Preparation<\/h3>\n\n\n\n<p>For control samples, a 90 \u00b5L aliquot of 2x charcoal-stripped plasma (K<sub>2<\/sub>\u2219EDTA) was spiked with 10 \u00b5L of calibrator\/QC material and vortexed. Internal standards (10 \u00b5L) were added and vortexed. Samples were protein precipitated using 400 \u00b5L of ice-cold acetonitrile. After vortexing and centrifugation at 4200 rpm for 15 minutes, the supernatant was transferred to a glass test tube and dried down under nitrogen. All samples were reconstituted in 200 \u00b5L of 60:40 water:mobile phase B (MPB). MPB was 50:50 methanol:acetonitrile (v\/v).&nbsp;<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Instrument Conditions<\/h3>\n\n\n\n<p>LC-MS\/MS analysis of bile acids in human plasma was performed on a Shimadzu Nexera UHPLC with a Shimadzu LCMS-8045 MS\/MS in negative ESI mode. Instrument conditions and analyte transitions for the new method are provided in Figure 4.&nbsp;<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Results and Discussion<\/h2>\n\n\n\n<h3 class=\"wp-block-heading\">Chromatographic Performance<\/h3>\n\n\n\n<p>The 17 bile acids, including three sets of isomers, were well separated over 9.5 minutes (total run time). The matrix interference was also appreciably separated from D<sub>4<\/sub> UDCA (Figure 5).<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 4:<\/strong> Bile Acids with Isomer Separation in Human Plasma on Raptor Inert ARC-18 (New Method)<\/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_cf0817.png\" alt=\"Bile Acids with Isomer Separation in Human Plasma on Raptor Inert ARC-18 by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0817<\/p><div class=\"chromatogram-peaks\"><h4>Peaks<\/h4><table class=\"peaks col-lg-6 col-12 peak-50\">\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><\/tr><\/thead>\n<tbody><tr><td class=\"num\">1.<\/td><td class=\"cmpd\">GUDCA<\/td><td class=\"oth\">1.32<\/td><td class=\"oth\">448.40<\/td><td class=\"oth\">74.15<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for TUDCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/14605-22-2 \/TUDCA\" target=\"_blank\" rel=\"noopener\">TUDCA<\/a><\/td><td class=\"oth\">1.40<\/td><td class=\"oth\">498.10<\/td><td class=\"oth\">80.05<\/td><\/tr>\n<tr><td class=\"num\">3.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for GCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/475-31-0\/GCA\" target=\"_blank\" rel=\"noopener\">GCA<\/a><\/td><td class=\"oth\">1.78<\/td><td class=\"oth\">464.10<\/td><td class=\"oth\">74.15<\/td><\/tr>\n<tr><td class=\"num\">4.<\/td><td class=\"cmpd\">TCA<\/td><td class=\"oth\">1.80<\/td><td class=\"oth\">514.20<\/td><td class=\"oth\">80.05<\/td><\/tr>\n<tr><td class=\"num\">5.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for UDCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/128-13-2\/UDCA\" target=\"_blank\" rel=\"noopener\">UDCA<\/a><\/td><td class=\"oth\">2.63<\/td><td class=\"oth\">391.50<\/td><td class=\"oth\">391.50<\/td><\/tr>\n<tr><td class=\"num\">6.<\/td><td class=\"cmpd\">GCDCA<\/td><td class=\"oth\">2.66<\/td><td class=\"oth\">448.40<\/td><td class=\"oth\">74.15<\/td><\/tr>\n<tr><td class=\"num\">7.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for TCDCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/516-35-8 \/TCDCA\" target=\"_blank\" rel=\"noopener\">TCDCA<\/a><\/td><td class=\"oth\">2.66<\/td><td class=\"oth\">498.10<\/td><td class=\"oth\">80.05<\/td><\/tr>\n<tr><td class=\"num\">8.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for GDCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/360-65-6\/GDCA\" target=\"_blank\" rel=\"noopener\">GDCA<\/a><\/td><td class=\"oth\">2.88<\/td><td class=\"oth\">448.40<\/td><td class=\"oth\">74.15<\/td><\/tr>\n<\/tbody><\/table>\n<table class=\"peaks col-lg-6 col-12 peak-50\"><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><\/tr><\/thead><tbody>\n<tr><td class=\"num\">9.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for TDCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/516-50-7 \/TDCA\" target=\"_blank\" rel=\"noopener\">TDCA<\/a><\/td><td class=\"oth\">2.88<\/td><td class=\"oth\">498.10<\/td><td class=\"oth\">80.05<\/td><\/tr>\n<tr><td class=\"num\">10.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for CA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/81-25-4\/CA\" target=\"_blank\" rel=\"noopener\">CA<\/a><\/td><td class=\"oth\">2.92<\/td><td class=\"oth\">407.20<\/td><td class=\"oth\">407.20<\/td><\/tr>\n<tr><td class=\"num\">11.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for HDCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/83-49-8\/HDCA\" target=\"_blank\" rel=\"noopener\">HDCA<\/a><\/td><td class=\"oth\">2.99<\/td><td class=\"oth\">391.50<\/td><td class=\"oth\">391.50<\/td><\/tr>\n<tr><td class=\"num\">12.<\/td><td class=\"cmpd\">TLCA<\/td><td class=\"oth\">3.87<\/td><td class=\"oth\">482.10<\/td><td class=\"oth\">80.05<\/td><\/tr>\n<tr><td class=\"num\">13.<\/td><td class=\"cmpd\">GLCA<\/td><td class=\"oth\">4.00<\/td><td class=\"oth\">432.20<\/td><td class=\"oth\">74.15<\/td><\/tr>\n<tr><td class=\"num\">14.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for CDCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/474-25-9\/CDCA\" target=\"_blank\" rel=\"noopener\">CDCA<\/a><\/td><td class=\"oth\">4.51<\/td><td class=\"oth\">391.50<\/td><td class=\"oth\">391.50<\/td><\/tr>\n<tr><td class=\"num\">15.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for DCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/83-44-3\/DCA\" target=\"_blank\" rel=\"noopener\">DCA<\/a><\/td><td class=\"oth\">4.68<\/td><td class=\"oth\">391.50<\/td><td class=\"oth\">391.50<\/td><\/tr>\n<tr><td class=\"num\">16.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for LCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/434-13-9\/LCA\" target=\"_blank\" rel=\"noopener\">LCA<\/a><\/td><td class=\"oth\">6.59<\/td><td class=\"oth\">375.40<\/td><td class=\"oth\">375.40<\/td><\/tr>\n<tr><td class=\"num\">17.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for DHLCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/1553-56-6\/DHLCA\" target=\"_blank\" rel=\"noopener\">DHLCA<\/a><\/td><td class=\"oth\">6.68<\/td><td class=\"oth\">373.20<\/td><td class=\"oth\">373.20<\/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 Inert ARC-18  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9314A12-T?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0817\" rel=\"noopener\">cat.# 9314A12-T<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Dimensions:<\/th><td>100 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 Inert ARC-18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 \u00b5m (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9314A0252-T?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0817\" rel=\"noopener\">cat.# 9314A0252-T<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>50 \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>60:40 Water:mobile phase B<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Conc.:<\/th><td>5 \u00b5M <\/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>5 mM Ammonium acetate in water, pH unadjusted <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>Methanol:acetonitrile (v\/v, 50:50) <\/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>0.5<\/td><td>60<\/td><td>40<\/td><\/tr><tr><td>6.00<\/td><td>0.5<\/td><td>30<\/td><td>70<\/td><\/tr><tr><td>7.00<\/td><td>0.5<\/td><td>20<\/td><td>80<\/td><\/tr><tr><td>7.50<\/td><td>0.8*<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>8.10<\/td><td>0.8*<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>8.20<\/td><td>0.5<\/td><td>60<\/td><td>40<\/td><\/tr><tr><td>9.50<\/td><td>0.5<\/td><td>60<\/td><td>40<\/td><\/tr><\/tbody><\/table><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Max Pressure:<\/th><td>365 bar<\/td><\/tr><\/table><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Detector<\/th><td>Shimadzu LCMS-8045 in ESI- mode<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Instrument<\/th><td>Shimadzu Nexera X2<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Sample Preparation<\/th><td>For control samples, a 90 \u00b5L aliquot of 2x charcoal-stripped plasma (K<sub>2<\/sub>EDTA) was added to a microcentrifuge tube and spiked with 10 \u00b5L of calibrator\/QC material and vortexed. Ten microliters of internal standards was added and vortexed. Samples were protein precipitated using 400 \u00b5L of ice-cold acetonitrile. After vortexing and centrifugation at 4200 rpm for 15 minutes, the supernatant was transferred to a glass test tube and dried down under nitrogen. All samples were reconstituted in 200 \u00b5L of 60:40 water:mobile phase B. The sample was transferred to a clean 2 mL screw-thread vial (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/21143?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0817\" rel=\"noopener\">cat.# 21143<\/a>) with a glass insert (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/21776?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0817\" rel=\"noopener\">cat.# 21776<\/a>) and capped with short-cap, screw-vial closures (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/24498?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0817\" rel=\"noopener\">cat.# 24498<\/a>).<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Notes<\/th><td>*The flow rate was increased to 0.8 mL\/min to more thoroughly flush phospholipids from the analytical column, thereby reducing matrix effects.<br \/><br \/>The flow was diverted to waste before 1 minute and after 7 minutes to protect the mass spectrometer. <\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0817.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<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 5:<\/strong> Plasma Matrix Interference Separated from D<sub>4 <\/sub>UDCA on Raptor Inert ARC-18<\/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 wide-image\"><img decoding=\"async\" src=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0819.png\" alt=\"Plasma Matrix Interference Separated from UDCA-D&lt;sub&gt;4&lt;\/sub&gt; in Patient Sample on Raptor Inert ARC-18\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0819<\/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><\/tr><\/thead>\n<tbody><tr><td class=\"num\">1.<\/td><td class=\"cmpd\">Matrix interference<\/td><td class=\"oth\">2.11<\/td><td class=\"oth\">&#8211;<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\">Matrix interference<\/td><td class=\"oth\">2.41<\/td><td class=\"oth\">&#8211;<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<tr><td class=\"num\">3.<\/td><td class=\"cmpd\">D<sub>4<\/sub> UDCA<\/td><td class=\"oth\">2.67<\/td><td class=\"oth\">395.40<\/td><td class=\"oth\">395.40<\/td><\/tr>\n<tr><td class=\"num\">4.<\/td><td class=\"cmpd\">D<sub>4<\/sub> HDCA<\/td><td class=\"oth\">3.02<\/td><td class=\"oth\">395.40<\/td><td class=\"oth\">395.40<\/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 Inert ARC-18  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9314A12-T?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0819\" rel=\"noopener\">cat.# 9314A12-T<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Dimensions:<\/th><td>100 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 Inert ARC-18 EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 \u00b5m (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9314A0252-T?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0819\" rel=\"noopener\">cat.# 9314A0252-T<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>50 \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>60:40 Water:MPB <\/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>5 mM ammonium acetate in water, pH unadjusted <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>Methanol:acetonitrile (v\/v, 50:50) <\/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>0.5<\/td><td>60<\/td><td>40<\/td><\/tr><tr><td>6.00<\/td><td>0.5<\/td><td>30<\/td><td>70<\/td><\/tr><tr><td>7.00<\/td><td>0.5<\/td><td>20<\/td><td>80<\/td><\/tr><tr><td>7.50<\/td><td>0.8*<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>8.10<\/td><td>0.8*<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>8.20<\/td><td>0.5<\/td><td>60<\/td><td>40<\/td><\/tr><tr><td>9.50<\/td><td>0.5<\/td><td>60<\/td><td>40<\/td><\/tr><\/tbody><\/table><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Max Pressure:<\/th><td>365 bar<\/td><\/tr><\/table><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Detector<\/th><td>Shimadzu LCMS-8045 in ESI- mode<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Instrument<\/th><td>Shimadzu Nexera X2<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Sample Preparation<\/th><td>For control samples, a 100 \u00b5L aliquot of patient sample was added to a microcentrifuge tube. Ten microliters of internal standards was added and vortexed. Samples were protein precipitated using 400 \u00b5L of ice-cold acetonitrile. After vortexing and centrifugation at 4200 rpm for 15 minutes, the supernatant was transferred to a glass test tube and dried down under nitrogen. All samples were reconstituted in 200 \u00b5L of 60:40 water:MPB. The sample was transferred to a clean 2 mL screw-thread vial (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/21143?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0819\" rel=\"noopener\">cat.# 21143<\/a>) with a glass insert (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/21776?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0819\" rel=\"noopener\">cat.# 21776<\/a>) and capped with short-cap, screw-vial closures (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/24498?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0819\" rel=\"noopener\">cat.# 24498<\/a>).<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Notes<\/th><td>The flow rate was increased to 0.8 mL\/min to more thoroughly flush phospholipids from the analytical column, thereby reducing matrix effects.<br \/><br \/>The flow was diverted to waste before 1 minute and after 7 minutes to protect the mass spectrometer. <\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0819.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<p><\/p>\n\n\n\n<p>The new method developed here utilized a Raptor ARC-18 column, whereas the original method used a Raptor C18 column. Both the Raptor ARC-18 and Raptor C18 columns are C18 stationary phases and show similar selectivity for the three sets of isomers, allowing for adequate separation of these analytes. The difference between these columns is that the Raptor C18 is a traditional C18 ligand containing methyl side chains with an end-capped surface, while the Raptor ARC-18 is a sterically protected C18 ligand containing isobutyl side chains (Figure 6). Stationary phase differences, such as end capping and carbon load, can influence the retention and selectivity of a stationary phase. In this instance, the difference in selectivity allowed the Raptor ARC-18 column to resolve all analytes as well as the matrix interference, which was not possible on the Raptor C18 column.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 6:<\/strong> Retention Properties of Raptor C18 (Left) and Raptor ARC-18 (Right)<br><\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<style>.kb-image39956_cf73bc-b1 .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<div class=\"wp-block-kadence-image kb-image39956_cf73bc-b1\"><figure class=\"aligncenter size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"1800\" height=\"459\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_06.jpg\" alt=\"\" class=\"kb-img wp-image-18730\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_06.jpg 1800w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_06-300x77.jpg 300w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_06-1024x261.jpg 1024w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_06-768x196.jpg 768w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure_article_CFAN2911b_06-1536x392.jpg 1536w\" sizes=\"auto, (max-width: 1800px) 100vw, 1800px\" \/><\/figure><\/div>\n\n<\/div><\/div><\/div>\n\n\n<p>For this revised method, a column with inert hardware was chosen over one with the traditional stainless-steel hardware. These columns have a premium inert coating applied to the stainless-steel surface of the LC column that is designed to reduce nonspecific binding of chelating analytes. As bile acids are acidic in nature, some chelation with metal surfaces may occur, resulting in undesirable peak shapes and loss of sensitivity. When analyzed with an inert column, lower detection limits may be achieved for this group of analytes. In Figure 7 below, the performance of a select number of bile acids are compared when analyzed on an inert versus an untreated (non-inert) column. &nbsp;<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 7:<\/strong> Comparison of Peak Response for Bile Acids on Raptor Inert ARC-18 and Raptor ARC-18 Columns<\/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_cf0820.png\" alt=\"Comparison of Bile Acids on Raptor Inert ARC-18 and Raptor ARC-18\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0820<\/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\">Precursor 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 TUDCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/14605-22-2 \/TUDCA\" target=\"_blank\" rel=\"noopener\">TUDCA<\/a><\/td><td class=\"oth\">498.10<\/td><td class=\"oth\">80.05<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for GCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/475-31-0\/GCA\" target=\"_blank\" rel=\"noopener\">GCA<\/a><\/td><td class=\"oth\">464.10<\/td><td class=\"oth\">74.15<\/td><\/tr>\n<tr><td class=\"num\">3.<\/td><td class=\"cmpd\">TCA<\/td><td class=\"oth\">514.20<\/td><td class=\"oth\">80.05<\/td><\/tr>\n<tr><td class=\"num\">4.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for TCDCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/516-35-8 \/TCDCA\" target=\"_blank\" rel=\"noopener\">TCDCA<\/a><\/td><td class=\"oth\">498.10<\/td><td class=\"oth\">80.05<\/td><\/tr>\n<tr><td class=\"num\">5.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for TDCA\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/516-50-7 \/TDCA\" target=\"_blank\" rel=\"noopener\">TDCA<\/a><\/td><td class=\"oth\">498.10<\/td><td class=\"oth\">80.05<\/td><\/tr>\n<tr><td class=\"num\">6.<\/td><td class=\"cmpd\">TLCA<\/td><td class=\"oth\">482.10<\/td><td class=\"oth\">80.05<\/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>See notes.<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Dimensions:<\/th><td>100 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\">Temp.:<\/th><td>50 \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>60:40 Water:mobile phase B<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Conc.:<\/th><td>0.25 \u03bcM <\/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>5 mM ammonium acetate in water, pH unadjusted <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>Methanol:acetonitrile (v\/v, 50:50) <\/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>0.5<\/td><td>60<\/td><td>40<\/td><\/tr><tr><td>6.00<\/td><td>0.5<\/td><td>30<\/td><td>70<\/td><\/tr><tr><td>7.00<\/td><td>0.5<\/td><td>20<\/td><td>80<\/td><\/tr><tr><td>7.50<\/td><td>0.8*<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>8.10<\/td><td>0.8*<\/td><td>0<\/td><td>100<\/td><\/tr><tr><td>8.20<\/td><td>0.5<\/td><td>60<\/td><td>40<\/td><\/tr><tr><td>9.50<\/td><td>0.5<\/td><td>60<\/td><td>40<\/td><\/tr><\/tbody><\/table><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Max Pressure:<\/th><td>300 bar<\/td><\/tr><\/table><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Detector<\/th><td>Shimadzu 8045-LCMS in ESI- mode<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Instrument<\/th><td>Shimadzu Nexera X2<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Sample Preparation<\/th><td>For control samples, a 100 \u00b5L aliquot of patient sample was added to a microcentrifuge tube. Ten microliters of internal standards were added and vortexed. Samples were protein precipitated using 400 \u00b5L of ice-cold acetonitrile. After vortexing and centrifugation at 4200 rpm for 15 minutes, the supernatant was transferred to a glass test tube and dried down under nitrogen. All samples were reconstituted in 200 \u00b5L of 60:40 water:mobile phase B. The sample was transferred to a clean 2 mL screw-thread vial (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/21143?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0820\" rel=\"noopener\">cat.# 21143<\/a>) with a glass insert (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/21776?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0820\" rel=\"noopener\">cat.# 21776<\/a>) and capped with short-cap, screw-vial closures (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/24498?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0820\" rel=\"noopener\">cat.# 24498<\/a>).<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Notes<\/th><td>*The flow rate was increased to 0.8 mL\/min to more thoroughly flush phospholipids from the analytical column, thereby reducing matrix effects.<br \/><br \/>The flow was diverted to waste before 1 minute and after 7 minutes to protect the mass spectrometer.<br \/><br \/>Columns are:<br \/>\u2022 Raptor Inert ARC-18 (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9314A12-T?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0820\" rel=\"noopener\">cat.# 9314A12-T<\/a>)<br \/>\u2022 Raptor ARC-18 (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9314A12?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0820\" rel=\"noopener\">cat.# 9314A12<\/a>)<br \/><\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0820.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<p><\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Linearity<\/h3>\n\n\n\n<p>Method linearity was verified by analysis of a six-point calibration curve (n=3). A 1\/x weighted linear regression was used for all compounds. Representative calibration curves are shown in Figure 8. The method has a dynamic range of 0.05\u20135 \u00b5M for all analytes and excellent correlation coefficients (&gt;0.99). The calibration range was selected to represent typical bile acids concentrations in human plasma samples.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 8:<\/strong> Representative Calibration Curves<\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<style>.kb-image39956_740d7a-31 .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<div class=\"wp-block-kadence-image kb-image39956_740d7a-31\"><figure class=\"aligncenter size-large\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"671\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-cfan2911b-08-1024x671.jpg\" alt=\"\" class=\"kb-img wp-image-18952\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-cfan2911b-08-1024x671.jpg 1024w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-cfan2911b-08-300x197.jpg 300w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-cfan2911b-08-768x503.jpg 768w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-cfan2911b-08-1536x1007.jpg 1536w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-cfan2911b-08.jpg 1800w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><\/figure><\/div>\n\n<\/div><\/div><\/div>\n\n\n<p><\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Accuracy and Precision<\/h3>\n\n\n\n<p>Accuracy and precision were assessed at four different concentrations (LLOQ, low QC, medium QC, high QC; see Table II for details) and evaluated both intraday and as an average of three days (n=9). Blank matrix samples were extracted and analyzed to confirm that endogenous levels of bile acids in the charcoal stripped plasma were below the detection limit. Method accuracy was assessed as the percentage of the measured concentration relative to the fortified concentration. The interday precision of the method was assessed using percent relative standard deviation (%RSD). These results, shown in Table III, demonstrate that the method is accurate and precise over the range studied for the quantitative analysis of bile acids in human plasma.<\/p>\n\n\n\n<p><strong>Table III:<\/strong> Method Accuracy and Precision Results in Plasma (Interday)<\/p>\n\n\n<figure class=\"wp-block-table\">\n<table>\n<tbody>\n<tr style=\"background-color: #272a59; color: #ffffff;\">\n<td><strong>Analyte<\/strong><\/td>\n<td colspan=\"2\"><strong>LLOQ (0.05 \u00b5M)<\/strong><\/td>\n<td colspan=\"2\"><strong>Low QC (0.1 \u00b5M)<\/strong><\/td>\n<td colspan=\"2\"><strong>Medium QC (0.5 \u00b5M)<\/strong><\/td>\n<td colspan=\"2\"><strong>High QC (1 \u00b5M)<\/strong><\/td>\n<\/tr>\n<tr style=\"background-color: #272a59; color: #ffffff;\">\n<td>\u00a0<\/td>\n<td><strong>%Recovery<\/strong><\/td>\n<td><strong>%RSD<\/strong><\/td>\n<td><strong>%Recovery<\/strong><\/td>\n<td><strong>%RSD<\/strong><\/td>\n<td><strong>%Recovery<\/strong><\/td>\n<td><strong>%RSD<\/strong><\/td>\n<td><strong>%Recovery<\/strong><\/td>\n<td><strong>%RSD<\/strong><\/td>\n<\/tr>\n<tr>\n<td>GUDCA<\/td>\n<td>81.6%<\/td>\n<td>7.77%<\/td>\n<td>91.8%<\/td>\n<td>7.75%<\/td>\n<td>94.3%<\/td>\n<td>5.73%<\/td>\n<td>95.2%<\/td>\n<td>6.31%<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>GCA<\/td>\n<td>84.4%<\/td>\n<td>2.84%<\/td>\n<td>90.1%<\/td>\n<td>9.36%<\/td>\n<td>93.8%<\/td>\n<td>7.66%<\/td>\n<td>95.1%<\/td>\n<td>5.55%<\/td>\n<\/tr>\n<tr>\n<td>TCA<\/td>\n<td>87.8%<\/td>\n<td>6.20%<\/td>\n<td>88.1%<\/td>\n<td>9.20%<\/td>\n<td>91.9%<\/td>\n<td>7.20%<\/td>\n<td>93.3%<\/td>\n<td>8.58%<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>UDCA<\/td>\n<td>81.3%<\/td>\n<td>4.21%<\/td>\n<td>92.0%<\/td>\n<td>9.50%<\/td>\n<td>94.2%<\/td>\n<td>5.25%<\/td>\n<td>95.0%<\/td>\n<td>6.21%<\/td>\n<\/tr>\n<tr>\n<td>GCDCA<\/td>\n<td>87.3%<\/td>\n<td>2.94%<\/td>\n<td>92.2%<\/td>\n<td>8.71%<\/td>\n<td>93.9%<\/td>\n<td>5.80%<\/td>\n<td>85.1%<\/td>\n<td>7.16%<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>TCDCA<\/td>\n<td>88.4%<\/td>\n<td>6.45%<\/td>\n<td>84.4%<\/td>\n<td>10.08%<\/td>\n<td>94.6%<\/td>\n<td>5.82%<\/td>\n<td>93.0%<\/td>\n<td>8.59%<\/td>\n<\/tr>\n<tr>\n<td>CA<\/td>\n<td>84.7%<\/td>\n<td>6.83%<\/td>\n<td>92.0%<\/td>\n<td>10.22%<\/td>\n<td>94.3%<\/td>\n<td>6.92%<\/td>\n<td>95.3%<\/td>\n<td>6.18%<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>GDCA<\/td>\n<td>87.6%<\/td>\n<td>9.33%<\/td>\n<td>90.6%<\/td>\n<td>11.74%<\/td>\n<td>93.7%<\/td>\n<td>7.80%<\/td>\n<td>94.1%<\/td>\n<td>6.99%<\/td>\n<\/tr>\n<tr>\n<td>HDCA<\/td>\n<td>84.9%<\/td>\n<td>7.73%<\/td>\n<td>88.7%<\/td>\n<td>11.71%<\/td>\n<td>92.5%<\/td>\n<td>8.07%<\/td>\n<td>94.7%<\/td>\n<td>7.58%<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>TDCA<\/td>\n<td>86.7%<\/td>\n<td>9.66%<\/td>\n<td>88.0%<\/td>\n<td>10.61%<\/td>\n<td>94.8%<\/td>\n<td>3.48%<\/td>\n<td>95.4%<\/td>\n<td>6.41%<\/td>\n<\/tr>\n<tr>\n<td>TUDCA<\/td>\n<td>82.2%<\/td>\n<td>7.47%<\/td>\n<td>91.0%<\/td>\n<td>9.94%<\/td>\n<td>94.1%<\/td>\n<td>6.43%<\/td>\n<td>92.3%<\/td>\n<td>9.07%<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>TLCA<\/td>\n<td>86.4%<\/td>\n<td>7.02%<\/td>\n<td>87.8%<\/td>\n<td>10.38%<\/td>\n<td>93.3%<\/td>\n<td>8.51%<\/td>\n<td>95.6%<\/td>\n<td>5.90%<\/td>\n<\/tr>\n<tr>\n<td>GLCA<\/td>\n<td>82.9%<\/td>\n<td>5.61%<\/td>\n<td>91.8%<\/td>\n<td>7.66%<\/td>\n<td>93.9%<\/td>\n<td>6.29%<\/td>\n<td>93.6%<\/td>\n<td>7.76%<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>CDCA<\/td>\n<td>84.4%<\/td>\n<td>8.25%<\/td>\n<td>93.6%<\/td>\n<td>5.88%<\/td>\n<td>95.7%<\/td>\n<td>4.83%<\/td>\n<td>95.7%<\/td>\n<td>5.91%<\/td>\n<\/tr>\n<tr>\n<td>DCA<\/td>\n<td>87.1%<\/td>\n<td>5.54%<\/td>\n<td>89.0%<\/td>\n<td>9.77%<\/td>\n<td>94.3%<\/td>\n<td>6.40%<\/td>\n<td>95.7%<\/td>\n<td>6.03%<\/td>\n<\/tr>\n<tr style=\"background-color: #ced4d9;\">\n<td>LCA<\/td>\n<td>83.3%<\/td>\n<td>2.84%<\/td>\n<td>92.1%<\/td>\n<td>7.97%<\/td>\n<td>93.4%<\/td>\n<td>6.75%<\/td>\n<td>95.2%<\/td>\n<td>6.36%<\/td>\n<\/tr>\n<tr>\n<td>DHLCA<\/td>\n<td>83.6%<\/td>\n<td>3.72%<\/td>\n<td>91.1%<\/td>\n<td>8.62%<\/td>\n<td>94.4%<\/td>\n<td>5.28%<\/td>\n<td>95.1%<\/td>\n<td>6.19%<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/figure>\n\n\n<h2 class=\"wp-block-heading\">Conclusions<\/h2>\n\n\n\n<p>A robust, selective LC-MS\/MS method was developed and verified for the analysis of 17 bile acids in human plasma using a Raptor Inert ARC-18 column. The method was paired with a simple protein precipitation sample preparation step. Standard curves demonstrated excellent linearity and precision\/accuracy over routine intraday and interday analyses.<\/p>\n\n\n\n<p>This work highlights the importance of alternative column chemistries for the resolution of isomers and matrix interferences. The Raptor C18 and Raptor Inert ARC-18 are both C18-based stationary phases, but each have a unique selectivity resulting from differences, such as end capping and carbon load. In this study, this difference in selectivity allowed for a critical matrix interference to be resolved while still maintaining the separation of the isomers. In addition, the use of an inert LC column in this work resulted in increased peak responses. The improved analyte sensitivity this afforded, makes it well suited for future applications of experimental test samples, as would be the case in microbiome research.<\/p>\n\n\n\n<p><em>This method has been developed for research use only; it is not suitable for use in diagnostic procedures without further evaluation.<\/em><\/p>\n\n\n\n<div class=\"wp-block-columns has-background is-layout-flex wp-container-core-columns-is-layout-9d6595d7 wp-block-columns-is-layout-flex\" style=\"background-color:#e4f7fa\">\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:66.66%\">\n<h3 class=\"wp-block-heading has-text-color has-link-color wp-elements-4b996d52b2cd7337d1a86cbbb02ffac0\" style=\"color:#02366d\">Want even better performance when analyzing bile acids and other metal-sensitive compounds?<\/h3>\n\n\n\n<p>Learn more at\u00a0<a href=\"https:\/\/discover.restek.com\/GNSS4155\/accurately-analyze-metal-sensitive-compounds-with-resteks-new-inert-lc-columns\/\" data-type=\"post\" data-id=\"45251\">www.restek.com\/inert<\/a><\/p>\n<\/div>\n\n\n\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:33.33%\"><div class=\"wp-block-image\">\n<figure class=\"alignright size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"400\" height=\"123\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/teaser-product-lc-columns-inert-lc-02.png\" alt=\"\" class=\"wp-image-29764\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/teaser-product-lc-columns-inert-lc-02.png 400w, https:\/\/discover.restek.com\/wp-content\/uploads\/teaser-product-lc-columns-inert-lc-02-300x92.png 300w\" sizes=\"auto, (max-width: 400px) 100vw, 400px\" \/><\/figure>\n<\/div><\/div>\n<\/div>\n\n\n\n<div style=\"height:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n        <div class=\"cpb\">\n            <h3 class=\"cpb-heading\">Products Mentioned<\/h3>\n            <hr class=\"cpb-heading-underline\" \/>\n            <div class=\"cpb-list\">\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9304252\">                                Catalog No. 9304252                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Raptor C18, 1.8 \u00b5m, 50 x 2.1 mm HPLC\u30ab\u30e9\u30e0<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9304252\">\u88fd\u54c1\u60c5\u5831\u3092\u898b\u308b<\/a>\n                                                    <\/div>\n                    <\/div>\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9314A12-T\">                                Catalog No. 9314A12-T                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Raptor Inert ARC-18, 2.7 \u00b5m, 100 x 2.1 mm HPLC\u30ab\u30e9\u30e0<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9314A12-T\">\u88fd\u54c1\u60c5\u5831\u3092\u898b\u308b<\/a>\n                                                    <\/div>\n                    <\/div>\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9314A0252-T\">                                Catalog No. 9314A0252-T                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Raptor Inert ARC-18 EXP\u30ac\u30fc\u30c9\u30ab\u30e9\u30e0\u30ab\u30fc\u30c8\u30ea\u30c3\u30b8\uff5c\u30b3\u30a2\u30b7\u30a7\u30eb\uff5c\u30a4\u30ca\u30fc\u30c8\u30ab\u30e9\u30e0\uff5c2.7 \u00b5m\uff5c5 x 2.1 mm\uff5cHPLC\u30fbUHPLC\u5bfe\u5fdc<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9314A0252-T\">\u88fd\u54c1\u60c5\u5831\u3092\u898b\u308b<\/a>\n                                                    <\/div>\n                    <\/div>\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/21143\">                                Catalog No. 21143                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Short-Cap Vial with Grad Marking Spot, 9-425 Screw-Thread, 2.0 mL, 9 mm, 12 x 32 (vial only), Amber, 1000-pk.<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/21143\">\u88fd\u54c1\u60c5\u5831\u3092\u898b\u308b<\/a>\n                                                    <\/div>\n                    <\/div>\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/21776\">                                Catalog No. 21776                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Vial Inserts, Glass, Big Mouth w\/Bottom Spring, 250 \u00b5L, 100-pk.<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/21776\">\u88fd\u54c1\u60c5\u5831\u3092\u898b\u308b<\/a>\n                                                    <\/div>\n                    <\/div>\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/24498\">                                Catalog No. 24498                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Short Screw Cap, Polypropylene, Screw-Thread, PTFE\/Silicone\/PTFE Septa, Blue, Preassembled, 2.0 mL, 9 mm, 1000-pk.<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/24498\">\u88fd\u54c1\u60c5\u5831\u3092\u898b\u308b<\/a>\n                                                    <\/div>\n                    <\/div>\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/25810\">                                Catalog No. 25810                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">UltraShield UHPLC PreColumn Filter, 0.2 \u00b5m Frit, 5-pk.<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/25810\">\u88fd\u54c1\u60c5\u5831\u3092\u898b\u308b<\/a>\n                                                    <\/div>\n                    <\/div>\n                            <\/div>\n        <\/div>\n        \n\n\n<div style=\"height:100px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n","protected":false},"excerpt":{"rendered":"<p>In this study, 17 bile acids (including three isomer groups) and a critical matrix interference were accurately quantified using a 10-min LC-MS\/MS method with a dynamic range of 0.05\u20135 \u00b5M.<\/p>\n","protected":false},"author":13,"featured_media":6355,"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":[13],"tags":[],"industries-application":[2153,2154],"post-badge":[],"resource-type":[],"product-library":[2391,2373],"resource-technique":[2299,2301,2302],"hf_cat_post":[651],"ppma_author":[432,656],"class_list":["post-39956","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-application-notes","industries-application-clinical","industries-application-clinical-diagnostics","product-library-lc-columns","product-library-liquid-chromatography-products","resource-technique-liquid-chromatography","resource-technique-mass-spectrometry-ms","resource-technique-ms-ms"],"acf":[],"taxonomy_info":{"category":[{"value":13,"label":"Application 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