{"id":43386,"date":"2020-10-13T00:00:00","date_gmt":"2020-10-13T00:00:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/simplified-methylmalonic-acid-analysis-without-derivatization-in-human-plasma-by-lc-msms\/"},"modified":"2025-12-10T13:52:56","modified_gmt":"2025-12-10T13:52:56","slug":"simplified-methylmalonic-acid-analysis-without-derivatization-in-human-plasma-by-lc-msms","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/es\/articoli\/cfss2829\/simplified-methylmalonic-acid-analysis-without-derivatization-in-human-plasma-by-lc-msms","title":{"rendered":"Simplified Methylmalonic Acid Analysis without Derivatization in Human Plasma by LC-MS\/MS"},"content":{"rendered":"\n<ul class=\"wp-block-list\">\n<li>Baseline separation of isomers ensures accurate reporting.<\/li>\n\n\n\n<li>Simple protein crash sample preparation, no derivatization required.<\/li>\n\n\n\n<li>Fast 5-minute analysis is suitable for high-throughput labs.<\/li>\n<\/ul>\n\n\n\n<p>Vitamin B<sub>12<\/sub>&nbsp;plays an essential role in metabolic energy production, and deficiency can be difficult to diagnose without testing as it can manifest through a wide variety of symptoms. In clinical testing of plasma samples, elevated levels of methylmalonic acid (MMA) can be used to diagnose functional vitamin B<sub>12<\/sub>&nbsp;deficiency as well as methylmalonic acidemia, an inherited metabolic disorder. Methylmalonic acid determination is a very sensitive test and is more specific than a homocysteine test, but it typically requires extensive sample pre-treatment using liquid-liquid extraction, derivatization, solvent evaporation, and\/or SPE. Additionally, chromatographic resolution can be difficult to achieve between methylmalonic acid and its naturally occurring isomer, succinic acid.<\/p>\n\n\n\n<p>To overcome these obstacles, we developed a simplified method for methylmalonic acid analysis. The approach shown here uses a protein crash sample preparation and does not require derivatization. By eliminating derivatization, considerable time and resource savings are realized and a significant source of variation is removed. For analysis, a direct injection of the supernatant is made onto a Force C18 column. LC-MS\/MS results show that methylmalonic acid is clearly resolved from succinic acid, which eliminates potential isobaric interference and makes peak identification and quantitation straightforward. In addition, the 5-minute total chromatographic analysis time makes this method suitable as a high-throughput assay for the clinical diagnosis of vitamin B<sub>12<\/sub>&nbsp;deficiency and methylmalonic acidemia.<\/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_cf0718.png\" alt=\"Methylmalonic Acid in Human Plasma on Force C18 by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0718<\/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\"><a class=\"cmpd_link\" title=\"View compound information for Succinic acid\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/110-15-6\/Succinic acid\" target=\"_blank\" rel=\"noopener\">Succinic acid<\/a><\/td><td class=\"oth\">1.68<\/td><td class=\"oth\">117.3<\/td><td class=\"oth\">73.1<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\">Methyl-D3-malonic acid<\/td><td class=\"oth\">2.02<\/td><td class=\"oth\">120.2<\/td><td class=\"oth\">76.2<\/td><\/tr>\n<tr><td class=\"num\">3.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Methylmalonic acid\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/516-05-2\/Methylmalonic acid\" target=\"_blank\" rel=\"noopener\">Methylmalonic acid<\/a><\/td><td class=\"oth\">2.03<\/td><td class=\"oth\">117.3<\/td><td class=\"oth\">73.1<\/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>Force C18   (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/963431E?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0718\" rel=\"noopener\">cat.# 963431E<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Dimensions:<\/th><td>100 mm x 3.0 mm ID<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Particle Size:<\/th><td>3 \u00b5m<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Pore Size:<\/th><td>100 \u00c5<\/td><\/tr><tr><td><\/td><tr><th class=\"sub conditions_header\" scope=\"row\">Guard Column:<\/th><td>Force C18 EXP guard column cartridge 3 mm ID, (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/963450253?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0718\" rel=\"noopener\">cat.# 963450253<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>35 \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\">Conc.:<\/th><td>  13 ng\/mL Methylmalonic acid (endogenous) in double charcoal stripped human plasma<\/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>0.5% Formic acid in water <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>0.5% Formic acid in 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>0.7<\/td><td>95<\/td><td>5<\/td><\/tr><tr><td>0.50<\/td><td>0.7<\/td><td>95<\/td><td>5<\/td><\/tr><tr><td>3.00<\/td><td>0.7<\/td><td>5<\/td><td>95<\/td><\/tr><tr><td>3.01<\/td><td>0.7<\/td><td>95<\/td><td>5<\/td><\/tr><tr><td>5.00<\/td><td>0.7<\/td><td>95<\/td><td>5<\/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>HPLC<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Sample Preparation<\/th><td>100 \u03bcL of sample (double charcoal stripped human plasma containing 13 ng\/mL of endogenous methylmalonic acid) was aliquoted for extraction. 5 \u03bcL of internal standard (2500 ng\/mL MMA-D3 in water) was added to the sample. The sample was precipitated using 300 \u03bcL of 0.5% formic acid in methanol followed by a 10 second vortex at 3000 rpm. The sample was then centrifuged at 4000 rpm for 10 minutes at 10 \u00b0C. 250 \u03bcL of the supernatant was filtered using a Thomson SINGLE StEP standard filter vial (PVDF, 0.2 \u03bcm, Restek cat.# 25895) prior to analysis.<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Notes<\/th><td>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_cf0718.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:20px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n<figure class=\"wp-block-table\">\n<table style=\"background-color: #e4f7fa; border-style: none;\">\n<tbody>\n<tr>\n<td style=\"padding: 20px;\">\n<h4 style=\"color: #02366d;\">Want even better performance when analyzing MMA and other metal-sensitive compounds?<\/h4>\n<p>Learn more at <a href=\"https:\/\/www.restek.com\/articles\/accurately-analyze-metal-sensitive-compounds-with-resteks-new-inert-lc-columns\" target=\"_blank\" rel=\"noopener\">www.restek.com\/inert<\/a><\/p>\n<\/td>\n<td style=\"vertical-align: middle; padding: 20px;\"><img decoding=\"async\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/teaser-product-lc-columns-inert-lc-02.png\" alt=\"inert lc column\" title=\"-\"><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/figure>\n\n\n<div style=\"height:100px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n","protected":false},"excerpt":{"rendered":"<p>Methylmalonic acid analysis is an important clinical test for diagnosing vitamin B12 deficiency as well as methylmalonic acidemia. Methods typically require extensive sample preparation, which involves significant time and expense. The LC-MS\/MS method developed here offers a much simpler alternative, providing complete chromatographic resolution of isobaric analytes from the direct injection of a protein crash sample supernatant onto a Force C18 column.<\/p>\n","protected":false},"author":11,"featured_media":6493,"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],"hf_cat_post":[650,2624],"ppma_author":[414],"class_list":["post-43386","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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