{"id":43352,"date":"2020-10-12T00:00:00","date_gmt":"2020-10-12T00:00:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/evaluation-of-a-simple-method-for-methylmalonic-acid-analysis-in-human-plasma-by-lc-msms\/"},"modified":"2025-12-11T17:04:42","modified_gmt":"2025-12-11T17:04:42","slug":"evaluation-of-a-simple-method-for-methylmalonic-acid-analysis-in-human-plasma-by-lc-msms","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/zh-hans\/application-notes-ja\/cfan3010\/evaluation-of-a-simple-method-for-methylmalonic-acid-analysis-in-human-plasma-by-lc-msms","title":{"rendered":"Evaluation of a Simple Method for Methylmalonic Acid Analysis in Human Plasma by LC-MS\/MS"},"content":{"rendered":"\n<h2 class=\"wp-block-heading\">Abstract<\/h2>\n\n\n\n<p>A new LC-MS\/MS method for methylmalonic acid (MMA) analysis was developed that provides complete chromatographic resolution from isobaric succinic acid in plasma samples. Excellent chromatographic results were obtained from the direct injection of a protein crash sample supernatant onto a Force C18 column, providing a much simpler sample preparation compared to typical methods.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Introduction<\/h2>\n\n\n\n<p>Vitamin B12 deficiency can manifest clinically in a wide variety of physical and behavioral signs and symptoms, but methylmalonic acid (MMA) can be used as a specific biomarker for diagnosis. In the metabolic cycle for energy production, vitamin B12 promotes the conversion of methylmalonyl CoA to succinyl CoA. If there is not enough B12 available, blood levels of MMA begin to rise. High levels of MMA can also result from the metabolic disorder methylmalonic acidemia, which causes the inability to properly digest specific fats and proteins. Therefore, elevated levels of MMA can be used to diagnose functional vitamin B12 deficiency as well as methylmalonic acidemia.<\/p>\n\n\n\n<p>The MMA test typically requires extensive sample pre-treatment incorporating liquid-liquid extraction, derivatization, solvent evaporation, and\/or SPE. Additionally, chromatographic resolution can be difficult to achieve between MMA and its naturally occurring isomer, succinic acid. Herein, we present a simple sample preparation method\u2014without derivatization\u2014that allows for the direct injection of protein crash supernatant while still maintaining resolution between MMA and succinic acid in a 5-minute cycle time.<\/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>\u00a0Structures of Methylmalonic Acid and Succinic Acid.<\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<style>.kb-image43352_e74b42-06 .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<div class=\"wp-block-kadence-image kb-image43352_e74b42-06\"><figure class=\"aligncenter size-medium_large\"><img loading=\"lazy\" decoding=\"async\" width=\"768\" height=\"243\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN3010-01-768x243.jpg\" alt=\"\" class=\"kb-img wp-image-18982\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN3010-01-768x243.jpg 768w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN3010-01-300x95.jpg 300w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN3010-01-1024x324.jpg 1024w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN3010-01-1536x486.jpg 1536w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN3010-01.jpg 1800w\" sizes=\"auto, (max-width: 768px) 100vw, 768px\" \/><\/figure><\/div>\n\n<\/div><\/div><\/div>\n\n\n<div style=\"height:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\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<\/h3>\n\n\n\n<p>Double charcoal stripped human plasma contains endogenous levels of methylmalonic acid; therefore, a surrogate matrix was created by fortifying SeraFlx BIOMATRIX (Cerilliant) at 1000 ng\/mL with succinic acid. The surrogate matrix was then spiked with MMA to prepare calibration standards and QC samples. The calibration range was 10\u2013500 ng\/mL and surrogate matrix QC samples were prepared at 10, 20, 150, and 400 ng\/mL.<\/p>\n\n\n\n<p>The amount of endogenous methylmalonic acid in the double charcoal stripped human plasma was determined to be 13 ng\/mL using the calibration standards prepared in the surrogate matrix. Additional QC samples were prepared in double charcoal stripped human plasma by fortifying at the same levels as the surrogate matrix and then quantitatively accounting for the additional endogenous MMA level. The QC samples in human plasma contained combined (endogenous and fortified) MMA levels of 23, 33, 163, and 413 ng\/mL.<\/p>\n\n\n\n<p>Internal standard MMA-D3 was prepared at 2,500 ng\/mL in water; 5 \u00b5L was then added to each sample, and samples were vortexed prior to protein precipitation.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Sample Preparation<\/h3>\n\n\n\n<p>Protein precipitation was performed by aliquoting 300 \u00b5L of 0.5% formic acid in methanol into 100 \u00b5L of sample. The sample was then vortexed for 10 seconds at 3000 rpm followed by centrifugation at 4000 rpm for 10 minutes at 10 \u00b0C. The supernatant (250 \u00b5L) was then filtered using a Thomson SINGLE StEP standard filter vial (0.2 \u00b5m PVDF membrane, Restek (cat.# 25895).<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><tbody><tr><td>Analytical column:<\/td><td colspan=\"2\">Force C18 3 \u00b5m, 100 mm x 3.0 mm (cat.# 963431E)<\/td><\/tr><tr><td>Guard column:<\/td><td colspan=\"2\">Force C18 EXP guard column cartridge (cat.# 963450253)<\/td><\/tr><tr><td>Mobile phase A:<\/td><td colspan=\"2\">Water, 0.5% formic acid<\/td><\/tr><tr><td>Mobile phase B:<\/td><td colspan=\"2\">Methanol, 0.5% formic acid<\/td><\/tr><tr><td>Gradient<\/td><td>Time (min)<\/td><td>%B<\/td><\/tr><tr><td>&nbsp;<\/td><td>0.00<\/td><td>5<\/td><\/tr><tr><td>&nbsp;<\/td><td>0.50<\/td><td>5<\/td><\/tr><tr><td>&nbsp;<\/td><td>3.00<\/td><td>95<\/td><\/tr><tr><td>&nbsp;<\/td><td>3.01<\/td><td>5<\/td><\/tr><tr><td>&nbsp;<\/td><td>5.00<\/td><td>5<\/td><\/tr><tr><td>Flow rate:<\/td><td colspan=\"2\">0.7 mL\/min<\/td><\/tr><tr><td>Injection volume:<\/td><td colspan=\"2\">3 \u00b5L<\/td><\/tr><tr><td>Column temp.:<\/td><td colspan=\"2\">35 \u00b0C<\/td><\/tr><tr><td>Sample temp.:&nbsp;<\/td><td colspan=\"2\">10 \u00b0C<\/td><\/tr><tr><td>Ion mode:<\/td><td colspan=\"2\">Negative ESI<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<p><strong>Table I<\/strong>: Analyte Transitions for Methylmalonic Acid Analysis.<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><tbody><tr><td class=\"has-text-align-center\" data-align=\"center\"><strong>Compound<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\"><strong>Precursor Ion<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\"><strong>Product Ion<\/strong><\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\">MMA<\/td><td class=\"has-text-align-center\" data-align=\"center\">117.3<\/td><td class=\"has-text-align-center\" data-align=\"center\">73.1<\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\">MMA-D<sub>3<\/sub><\/td><td class=\"has-text-align-center\" data-align=\"center\">120.2<\/td><td class=\"has-text-align-center\" data-align=\"center\">76.2<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<h2 class=\"wp-block-heading\">Results and Discussion<\/h2>\n\n\n\n<h3 class=\"wp-block-heading\">Sample Preparation Evaluation<\/h3>\n\n\n\n<p>To ensure that the supernatant was compatible with direct analysis, an initial solvent evaluation was conducted. Trichloroacetic acid (TCA) was initially investigated for use as the precipitating solvent. While using a small volume of 40% TCA (w\/v) (25 \u00b5L per 100 \u00b5L of sample) effectively precipitated proteins, it resulted in no signal for methylmalonic acid, despite dilution with 300 \u00b5L of 0.5% formic acid in water prior to analysis (Figure 2). It is speculated that the TCA present in the final extract is not only responsible for the improved peak shape of succinic acid, but also for ion suppression that resulted in reduced sensitivity for MMA. Precipitation with 0.5% formic acid in methanol greatly increased sensitivity compared to TCA when evaluating the same sample (Figure 3). The methanol-based supernatant was compatible with direct analysis providing a sample volume of 3 \u00b5L or less was injected in order to avoid solvent effects.<\/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>\u00a040% Trichloroacetic Acid (w\/v) Protein Precipitation.<\/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_cf0719.png\" alt=\"Methylmalonic Acid in Human Plasma (Trichloroacetic Acid Preparation)\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0719<\/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\">1.54<\/td><td class=\"oth\">117.3<\/td><td class=\"oth\">73.1<\/td><\/tr>\n<\/tbody><\/table><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0719.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 3:<\/strong>\u00a00.5% Formic Acid in Methanol (v\/v) Protein Precipitation.<\/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_cf0720.png\" alt=\"Methylmalonic Acid in Human Plasma (Formic Acid Preparation)\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0720<\/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\">1.60<\/td><td class=\"oth\">117.3<\/td><td class=\"oth\">73.1<\/td><\/tr>\n<tr><td class=\"num\">2.<\/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\">1.96<\/td><td class=\"oth\">117.3<\/td><td class=\"oth\">73.1<\/td><\/tr>\n<\/tbody><\/table><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0720.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:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\">Chromatographic Performance<\/h3>\n\n\n\n<p>A fast, 5-minute analysis of methylmalonic acid (Figure 4) was obtained from the direct injection of the supernatant. MMA was clearly resolved (USP resolution of 3.0) from the isobaric interference, succinic acid, which made peak identification and quantitation easy. While analytical columns containing superficially porous particles can offer a speed advantage for some applications, a fully porous particle Force C18 column was used here because the additional surface area provides the retention required to chromatographically separate MMA from succinic acid.<\/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>\u00a050 ng\/mL MMA in SeraFlx BIOMATRIX.<\/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_cf0717.png\" alt=\"Methylmalonic Acid in SeraFlx BIOMATRIX on Force C18 by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0717<\/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\">1.69<\/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-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0717.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:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\">Linearity<\/h3>\n\n\n\n<p>Using linear 1\/x weighted regression, the method showed good linearity for MMA across a range from 10\u2013500 ng\/mL with r<sup>2<\/sup>&nbsp;values of at least 0.999 in all three accuracy and precision experiments.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Accuracy and Precision<\/h3>\n\n\n\n<p>Precision and accuracy analyses were performed on three different days. The method accuracy was demonstrated to be within 6.00% of the nominal concentration for all QC levels. The %RSD was 5.19%\u20136.50% and 7.67% for intra- and interday, respectively, at the LLOQ. The %RSD was 0.866%\u20136.24% and 1.97%\u20134.99% for intra- and inter-run, respectively, across the low, mid, and high QC levels, indicating good method precision (Table II).<\/p>\n\n\n\n<p><strong>Table II<\/strong>: Interday Accuracy and Precision of SeraFlx BIOMATRIX QC Samples.<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><tbody><tr><td class=\"has-text-align-center\" data-align=\"center\" rowspan=\"2\"><strong>Analyte<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\" colspan=\"3\"><strong>QC LLOQ (10 ng\/mL)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\" colspan=\"3\"><strong>QC Low (20 ng\/mL)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\" colspan=\"3\"><strong>QC Mid (150 ng\/mL)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\" colspan=\"3\"><strong>QC High (400 ng\/mL)<\/strong><\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\"><strong>Avg. Conc. (ng\/mL)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\"><strong>Avg. Accuracy (%)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\"><strong>Precision (%RSD)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\"><strong>Avg. Conc. (ng\/mL)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\"><strong>Avg. Accuracy (%)<\/strong><\/td><td><strong>Precision (%RSD)<\/strong><\/td><td><strong>Avg. Conc. (ng\/mL)<\/strong><\/td><td><strong>Avg. Accuracy (%)<\/strong><\/td><td><strong>Precision (%RSD)<\/strong><\/td><td><strong>Avg. Conc. (ng\/mL)<\/strong><\/td><td><strong>Avg. Accuracy (%)<\/strong><\/td><td><strong>Precision (%RSD)<\/strong><\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\">MMA<\/td><td class=\"has-text-align-center\" data-align=\"center\">9.40<\/td><td class=\"has-text-align-center\" data-align=\"center\">9.40<\/td><td class=\"has-text-align-center\" data-align=\"center\">7.67<\/td><td class=\"has-text-align-center\" data-align=\"center\">19.0<\/td><td>95.1<\/td><td>4.99<\/td><td>152<\/td><td>101<\/td><td>4.84<\/td><td>404<\/td><td>101<\/td><td>1.97<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<h3 class=\"wp-block-heading\">Matrix Surrogate Evaluation<\/h3>\n\n\n\n<p>Because endogenous methylmalonic acid was found in double charcoal stripped human plasma (Figure 5), an evaluation of the use of a surrogate matrix for preparing calibration curves and QC samples was performed. SeraFlx BIOMATRIX has physical and chemical characteristics similar to human serum\/plasma and tested negative for the presence of MMA. A calibration curve prepared in SeraFlx BIOMATRIX was used to quantitate the endogenous level of MMA in the double charcoal stripped human plasma. A concentration of 13 ng\/mL was determined. The plasma was then spiked with additional MMA at concentrations corresponding to the LLOQ and low, mid, and high QC levels. The method accuracy was demonstrated to be within 7.00% of the nominal concentration for all QC levels. The method precision was 6.77%, which is within the acceptance criteria, for all QC levels, indicating that SeraFlx BIOMATRIX was an appropriate matrix surrogate for the analysis of MMA in human plasma (Table III). The suitability of this surrogate matrix for human plasma can improve lab efficiency by eliminating the need to prescreen control material for detectable levels of MMA.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 5:<\/strong>\u00a0Endogenous MMA (13 ng\/mL) in Double Charcoal Stripped Plasma.<\/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-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:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p><strong>Table III<\/strong>: Inter-Day Accuracy and Precision of Human Plasma QC Samples.<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><tbody><tr><td class=\"has-text-align-center\" data-align=\"center\" rowspan=\"2\"><strong>Analyte<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\" colspan=\"3\"><strong>QC LLOQ (23 ng\/mL)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\" colspan=\"3\"><strong>QC Low (33 ng\/mL)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\" colspan=\"3\"><strong>QC Mid (163 ng\/mL)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\" colspan=\"3\"><strong>QC High (413 ng\/mL)<\/strong><\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\"><strong>Avg. Conc. (ng\/mL)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\"><strong>Avg. Accuracy (%)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\"><strong>Precision (%RSD)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\"><strong>Avg. Conc. (ng\/mL)<\/strong><\/td><td class=\"has-text-align-center\" data-align=\"center\"><strong>Avg. Accuracy (%)<\/strong><\/td><td><strong>Precision (%RSD)<\/strong><\/td><td><strong>Avg. Conc. (ng\/mL)<\/strong><\/td><td><strong>Avg. Accuracy (%)<\/strong><\/td><td><strong>Precision (%RSD)<\/strong><\/td><td><strong>Avg. Conc. (ng\/mL)<\/strong><\/td><td><strong>Avg. Accuracy (%)<\/strong><\/td><td><strong>Precision (%RSD)<\/strong><\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\">MMA<\/td><td class=\"has-text-align-center\" data-align=\"center\">24.0<\/td><td class=\"has-text-align-center\" data-align=\"center\">104<\/td><td class=\"has-text-align-center\" data-align=\"center\">4.16<\/td><td class=\"has-text-align-center\" data-align=\"center\">34.2<\/td><td>104<\/td><td>6.77<\/td><td>175<\/td><td>107<\/td><td>1.53<\/td><td>421<\/td><td>102<\/td><td>1.34<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<h2 class=\"wp-block-heading\">Conclusion<\/h2>\n\n\n\n<p>It was demonstrated that a simple, cost-effective sample preparation method can be used as an alternative to the tedious methods traditionally used for the analysis of MMA. The direct analysis of supernatant reduces costs associated with training, consumables, and extraction time. A Force C18 analytical column is able to successfully separate the naturally occurring isomer of MMA, succinic acid, with good resolution and a fast cycle time of only 5 minutes. SeraFlx BIOMATRIX was found to be a suitable matrix surrogate for the analysis of human plasma, eliminating the need to prescreen control material. With a fast, simple sample preparation procedure and 5 minutes of chromatographic analysis time, the established method provides a high-throughput assay for the clinical diagnosis of vitamin B12 deficiency and methylmalonic acidemia.<\/p>\n\n\n\n<div style=\"height:20px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\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-1489b0bd8c7fa6a213f7f4f1cf134d8f\" style=\"color:#02366d\">Want even better performance when analyzing mycotoxins and other metal-sensitive compounds?<\/h3>\n\n\n\n<p>Learn more at&nbsp;<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<\/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:20px\" 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\/963431E\">                                Catalog No. 963431E                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Force C18, 3 \u00b5m, 100 x 3.0 mm LC Column<\/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\/963431E\">\u67e5\u770b\u4ea7\u54c1<\/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\/963450253\">                                Catalog No. 963450253                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Force C18 Guard Cartridge, 5 x 3.0 mm EXP, 3-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\/963450253\">\u67e5\u770b\u4ea7\u54c1<\/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>A new LC-MS\/MS method for methylmalonic acid (MMA) analysis was developed that provides complete chromatographic resolution from isobaric succinic acid in plasma samples. Excellent chromatographic results were obtained from the direct injection of a protein crash sample supernatant onto a Force C18 column, providing a much simpler sample preparation compared to typical methods.<\/p>\n","protected":false},"author":3,"featured_media":6367,"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":[785],"tags":[],"industries-application":[2266,2267],"post-badge":[],"resource-type":[],"product-library":[2571,2572,2553],"resource-technique":[2343,2365],"ppma_author":[702,586],"class_list":["post-43352","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-application-notes-ja","industries-application-clinical-ja","industries-application-clinical-diagnostics-ja","product-library-lc-columns-ja","product-library-lc-ms-supplies-ja","product-library-liquid-chromatography-products-ja","resource-technique-liquid-chromatography-ja","resource-technique-ms-ms-ja"],"acf":[],"taxonomy_info":{"category":[{"value":785,"label":"\u30a2\u30d7\u30ea\u30b1\u30fc\u30b7\u30e7\u30f3"}],"industries-application":[{"value":2266,"label":"\u81e8\u5e8a"},{"value":2267,"label":"\u81e8\u5e8a\u691c\u67fb"}],"product-library":[{"value":2571,"label":"LC 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