{"id":43383,"date":"2020-10-13T00:00:00","date_gmt":"2020-10-13T00:00:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/fast-accurate-lc-msms-method-for-folate-deficiency-biomarkers-in-plasma\/"},"modified":"2025-12-08T14:29:56","modified_gmt":"2025-12-08T14:29:56","slug":"fast-accurate-lc-msms-method-for-folate-deficiency-biomarkers-in-plasma","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/de\/articoli\/cfss2789\/fast-accurate-lc-msms-method-for-folate-deficiency-biomarkers-in-plasma","title":{"rendered":"Fast, Accurate LC-MS\/MS Method for Folate Deficiency Biomarkers in Plasma"},"content":{"rendered":"\n<ul class=\"wp-block-list\">\n<li>Strong retention on a Raptor HILIC-Si column prevents matrix interference.<\/li>\n\n\n\n<li>Complete separation from phospholipids ensures accurate results in a quick, 5-minute analysis.<\/li>\n\n\n\n<li>Divert matrix to waste to keep your MS source clean and reduce downtime for maintenance.<\/li>\n<\/ul>\n\n\n\n<p>Folate deficiency is considered a risk factor for a wide range of human health problems, including neural tube defects in newborns, cardiovascular diseases, Alzheimer\u2019s disease, and certain forms of cancer. Plasma levels of folic acid and its metabolites (5-formyl tetrahydrofolate and 5-methyltetrahydrofolic acid) are used as biomarkers to diagnose folate deficiency. However, LC-MS\/MS methods for folate deficiency biomarkers in plasma can be very challenging because these small, polar compounds are not retained well on traditional reversed-phase LC columns. Retention can be improved using a HILIC method, but in this case, the column must provide strong enough retention to prevent coelution with the phospholipid components in the plasma sample matrix. Although a good sample preparation protocol will help, 100% removal of phospholipids is very difficult, and even low levels of phospholipids can interfere with target analytes, compromise quantitation, and contaminate the MS source.<\/p>\n\n\n\n<p>Using a HILIC approach with a Raptor HILIC-Si column is a much better alternative for LC-MS\/MS methods for folate deficiency biomarkers in plasma because you can quickly and completely separate the matrix interferences from the target analytes. The increased retention obtained on a Raptor HILIC-Si column ensures good separation of folate deficiency biomarkers from phospholipids and allows labs to accurately quantitate these important compounds, even at just 25\u201350 ng\/mL, with no ion suppression from matrix interferences. In addition, more resolution between analytes and matrix components lets you divert matrix to waste, which keeps your MS cleaner longer. The LC-MS\/MS method for folate deficiency biomarkers in plasma shown here allows folic acid, 5-formyl tetrahydrofolate, and 5-methyltetrahydrofolic acid to be accurately analyzed with no matrix interference in a fast, 5-minute analysis.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<div class=\"wp-block-custom-chromatogram-article\"><div class=\"wp-block-custom-chromatogram-article\"><div class=\"chromatogram-image wide-image\"><img decoding=\"async\" src=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0698.png\" alt=\"Folate Deficiency Biomarkers in Human Plasma on Raptor HILIC-Si by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0698<\/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\">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\">5-Formyl tetrahydrofolate<\/td><td class=\"oth\">2.23<\/td><td class=\"oth\">50<\/td><td class=\"oth\">474.2<\/td><td class=\"oth\">327.0<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Folic acid\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/59-30-3\/Folic acid\" target=\"_blank\" rel=\"noopener\">Folic acid<\/a><\/td><td class=\"oth\">2.23<\/td><td class=\"oth\">25<\/td><td class=\"oth\">442.2<\/td><td class=\"oth\">295.0<\/td><\/tr>\n<tr><td class=\"num\">3.<\/td><td class=\"cmpd\">5-Methyltetrahydrofolic acid<\/td><td class=\"oth\">2.25<\/td><td class=\"oth\">25<\/td><td class=\"oth\">460.3<\/td><td class=\"oth\">313.0<\/td><\/tr>\n<\/tbody><\/table><\/div><div class=\"chromatogram-conditions\"><h4>Conditions<\/h4><div class=\"conditions-container container-fluid\"><div class=\"row\"><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Column<\/th><td>Raptor HILIC-Si  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9310A5E?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0698\" rel=\"noopener\">cat.# 9310A5E<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Dimensions:<\/th><td>50 mm x 3.0 mm ID<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Particle Size:<\/th><td>2.7 \u00b5m<\/td><\/tr><tr><td><\/td><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>30 \u00b0C<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Standard\/Sample<\/th><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Diluent:<\/th><td>20 mM Ammonium acetate in acetonitrile:water (80:20) containing 10 mg\/mL 2-mercaptoethanol<\/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>50:50 Water:acetonitrile, 20 mM ammonium acetate <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>20:80 Water:acetonitrile, 20 mM ammonium acetate <\/td><\/tr><tr><td><\/td><td><table class=\"cgram_ramp\"><thead><tr><th>Time (min)<\/th><th>Flow (mL\/min)<\/th><th>%B<\/th><\/tr><\/thead><tbody><tr><td>0.00<\/td><td>0.5<\/td><td>100<\/td><\/tr><tr><td>3.00<\/td><td>0.5<\/td><td>0<\/td><\/tr><tr><td>3.20<\/td><td>0.5<\/td><td>0<\/td><\/tr><tr><td>3.21<\/td><td>0.5<\/td><td>100<\/td><\/tr><tr><td>5.21<\/td><td>0.5<\/td><td>100<\/td><\/tr><\/tbody><\/table><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Max Pressure:<\/th><td>344 bar<\/td><\/tr><\/table><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Detector<\/th><td>MS\/MS<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Ion Mode:<\/th><td>ESI+ <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Mode:<\/th><td>MRM <\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Instrument<\/th><td>UHPLC<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Sample Preparation<\/th><td>1. Aliquot 380 \u03bcL of human plasma (K2EDTA, 2x charcoal stripped) containing 100 \u03bcg\/mL 2-mercaptoethanol and add 20 \u03bcL fortification solution. <br \/>2. Vortex for 2 min and centrifuge for 2 min at 4000 rpm.<br \/>3. Condition EVOLUTE EXPRESS WAX 30 mg SPE plate (Biotage 604-0030-PX01) with 1 mL methanol and then equilibrate with 1 mL 2% formic acid in water. Apply vacuum to dry the plate completely.<br \/>4. Load 400 \u03bcL of sample onto the plate and apply vacuum to initiate the flow.<br \/>5. Wash the plate with 1 mL water. Apply vacuum to dry the plate completely.<br \/>6. Elute samples with 300 \u03bcL 5% ammonium hydroxide in methanol containing 10 mg\/mL 2-mercaptoethanol. Apply vacuum for elution.<br \/>7. Evaporate extracts to dryness under nitrogen at 30 \u00b0C.<br \/>8. Reconstitute in 200 \u03bcL mobile phase B containing 10 mg\/mL 2-mercaptoethanol.<br \/><br \/>Note: The whole sample preparation process was performed under dim light.<br \/><\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Notes<\/th><td>Endogenous peaks for phospholipids and lysophospholipids are displayed because they are common matrix interferences and are known to suppress ionization efficiency.<\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0698.pdf\" target=\"_blank\" rel=\"noopener noreferrer\"><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" width=\"18\" height=\"18\" viewBox=\"0 0 18 18\"><g data-name=\"Group 2996\"><path data-name=\"Rectangle 1246\" d=\"M0 0h18v18H0z\" style=\"fill: none;\"><\/path><\/g><g data-name=\"Group 2997\"><path data-name=\"Path 729\" d=\"M13.412 11.4v2.017H5.345V11.4H4v2.017a1.349 1.349 0 0 0 1.345 1.345h8.068a1.349 1.349 0 0 0 1.345-1.345V11.4zm-.672-2.694-.948-.948-1.741 1.735V4H8.706v5.493L6.965 7.758l-.948.948 3.361 3.361z\" transform=\"translate(-.437 -.414)\" style=\"fill: rgb(13, 123, 196);\"><\/path><\/g><\/svg>Download PDF<\/a><\/div><\/div><\/div>\n<\/div><\/div><\/div>\n\n\n<div style=\"height:100px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n","protected":false},"excerpt":{"rendered":"<p>LC-MS\/MS methods for folate deficiency biomarkers in plasma are complicated by the presence of matrix phospholipids and lysophospholipids. These interferences are problematic with reversed-phase LC because they can suppress response, reduce accuracy, and contaminate the MS source. Using a Raptor HILIC-Si column and the HILIC method shown here, folate deficiency biomarkers are completely separated from matrix interferences, allowing accurate determinations to be made in a fast, 5-minute analysis.<\/p>\n","protected":false},"author":11,"featured_media":6487,"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":[788],"tags":[],"industries-application":[2245,2246],"post-badge":[],"resource-type":[2768],"product-library":[2535,2517],"resource-technique":[2334,2364],"ppma_author":[414],"class_list":["post-43383","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-articoli","industries-application-clinico","industries-application-diagnostica-clinica","resource-type-featured-application","product-library-colonne-lc","product-library-prodotti-per-cromatografia-liquida","resource-technique-cromatografia-liquida-lc","resource-technique-ms-ms-it"],"acf":[],"taxonomy_info":{"category":[{"value":788,"label":"Articoli"}],"industries-application":[{"value":2245,"label":"Clinico"},{"value":2246,"label":"Diagnostica 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