{"id":51858,"date":"2024-01-09T00:00:00","date_gmt":"2024-01-09T00:00:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/which-column-is-best-for-the-analysis-of-methylmalonic-acid-mma\/"},"modified":"2025-12-15T23:36:53","modified_gmt":"2025-12-15T23:36:53","slug":"which-column-is-best-for-the-analysis-of-methylmalonic-acid-mma","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/ja\/blogs\/gnbl4867\/which-column-is-best-for-the-analysis-of-methylmalonic-acid-mma","title":{"rendered":"Which Column is Best for the Analysis of Methylmalonic Acid (MMA)?"},"content":{"rendered":"\n

If you search Restek\u2019s website for methylmalonic acid (MMA), you will find two different solutions for this analysis in plasma. One using a Force C18 column (Evaluation of a Simple Method for Methylmalonic Acid Analysis in Human Plasma by LC-MS\/MS<\/a>) and one using a Raptor Polar X column (Method for the Simultaneous Analysis of Methionine Pathway Metabolites and Methylmalonic Acid<\/a>). You might be wondering why this is the case.<\/p>\n\n\n\n

For starters, MMA is an important biomarker for a number of cobalamin disorders, however it is not the only biomarker that is looked at for this type of diagnosis. While MMA is analyzed in our Force C18 method, homocysteine, methionine, cystathionine, and cysteine are also important analytes to look at when trying to diagnose cobalamin disorders. In order to provide a more efficient method for our customers, we added these analytes to one method for a more comprehensive test of cobalamin disorders. This method includes a simple sample preparation without derivatization, as well as a rapid 4-minute LC-MS\/MS analysis. A 50 x 2.1 mm, 2.7 \u00b5m Raptor Polar X column with an accompanying Raptor EXP Polar X guard were used for this method.<\/p>\n\n\n\n

In addition to the method being more comprehensive on Polar X, the conditions in which these compounds are analyzed enables increased sensitivity of the analytes. While the Force C18 method is effective at retaining MMA and separating its natural occurring isomer, succinic acid, the method utilizes 0.5% formic acid (v\/v) modified mobile phases in order to get MMA in its neutral form so that it can be retained on a traditional reversed-phase column. However, high levels of formic acid are known to suppress analyte response in ESI- mode. By utilizing Polar X, which features a combined HILIC and ion-exchange retention mechanism, retention of MMA can be achieved while in its ionized form which means much less formic acid is needed in the mobile phase resulting in increased sensitivity. In the examples below, both methods used a 5 \u00b5L injection of a 100 ng\/mL sample. You can see that the sensitivity is improved using the Polar X column. This is especially important when working with very low limits of detection and limits of quantitation.<\/p>\n\n\n

Figure 1:<\/strong> Separation of Succinic Acid and Methylmalonic Acid on a Force C18 Column 100 x 3 mm, 3 \u00b5m<\/div>
\n\n<\/colgroup>\n\n\n\n\n\n\n\n\n
Column:<\/span><\/td>\nForce C18 100 x 3mm, 3 µm (Cat. # 963431E)<\/td>\n<\/tr>\n
Mobile Phase A:<\/span><\/td>\n0.5% Formic Acid in Water<\/td>\n<\/tr>\n
Mobile Phase B:<\/span><\/td>\n0.5% Formic Acid in Methanol<\/td>\n<\/tr>\n
Injection Volume:<\/span><\/td>\n5 µL<\/td>\n<\/tr>\n
Concentration:<\/span><\/td>\n100 ng\/mL<\/td>\n<\/tr>\n
Flow:<\/span><\/td>\n0.7 mL\/min<\/td>\n<\/tr>\n
Gradient:<\/span><\/td>\n\n<\/colgroup>\n\n\n\n\n\n\n\n\n
Time (min)<\/strong><\/td>\n%B<\/strong><\/td>\n<\/tr>\n
0.00<\/td>\n5<\/td>\n<\/tr>\n
0.50<\/td>\n60<\/td>\n<\/tr>\n
3.00<\/td>\n75<\/td>\n<\/tr>\n
3.01<\/td>\n100<\/td>\n<\/tr>\n
5.00<\/td>\nSTOP<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/td>\n<\/tr>\n
Column Temp.:<\/span><\/td>\n35°C<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n<\/colgroup>\n\n\n\n\n
Analyte<\/span><\/td>\nRetention Time (min)<\/td>\nPeak Height (cps)<\/td>\n<\/tr>\n
Succinic Acid<\/span><\/td>\n1.67<\/td>\n34500<\/td>\n<\/tr>\n
Methylmalonic Acid<\/span><\/td>\n2.03<\/td>\n840000<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/figure>\n\n<\/div><\/div><\/div>\n\n\n
<\/div>\n\n\n
Figure 2:<\/strong> Separation of Succinic Acid and Methylmalonic Acid on a Raptor Polar X Column 50 x 2.1 mm, 2.7 \u00b5m<\/div>
\n\n<\/colgroup>\n\n\n\n\n\n\n\n\n
Column:<\/span><\/td>\nRaptor Polar X 50 x 2.1 mm, 2.7 µm (Cat. # 9311A52)<\/td>\n<\/tr>\n
Mobile Phase A:<\/span><\/td>\n0.5% Formic Acid in Water<\/td>\n<\/tr>\n
Mobile Phase B:<\/span><\/td>\nAcetonitrile<\/td>\n<\/tr>\n
Injection Volume:<\/span><\/td>\n5 µL<\/td>\n<\/tr>\n
Concentration:<\/span><\/td>\n100 ng\/mL<\/td>\n<\/tr>\n
Flow:<\/span><\/td>\n0.6 mL\/min<\/td>\n<\/tr>\n
Gradient:<\/span><\/td>\n\n<\/colgroup>\n\n\n\n\n\n\n\n\n
Time (min)<\/td>\n%B<\/td>\n<\/tr>\n
0.00<\/td>\n85<\/td>\n<\/tr>\n
1.00<\/td>\n55<\/td>\n<\/tr>\n
3.00<\/td>\n10<\/td>\n<\/tr>\n
3.01<\/td>\n85<\/td>\n<\/tr>\n
4.00<\/td>\nSTOP<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/td>\n<\/tr>\n
Column Temp.:<\/span><\/td>\n40°C<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n<\/colgroup>\n\n\n\n\n
Analyte<\/span><\/td>\nRetention Time (min)<\/td>\nPeak Height (cps)<\/td>\n<\/tr>\n
Succinic Acid<\/span><\/td>\n1.35<\/td>\n60100<\/td>\n<\/tr>\n
Methylmalonic Acid<\/span><\/td>\n2.95<\/td>\n1600000<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/figure>\n\n<\/div><\/div><\/div>\n\n\n
<\/div>\n\n\n\n

Additionally, the new method on Polar X achieves better resolution of MMA and its naturally occurring isomer, succinic acid. In the original method, these two analytes were separated, but it is clear in the chromatograms above that there is increased resolution between these analytes using Polar X.<\/p>\n\n\n\n

One further consideration for the analysis of MMA is the use of an inert LC column. Because of the chelating properties of MMA, the use of traditional stainless steel column hardware can affect sensitivity and peak shape. When using an inert column for this analysis, peak shape and sensitivity were greatly improved for MMA. An example showing the use of a Force Inert C18 can be found in Figure 3 below.<\/p>\n\n\n

Figure 3: <\/strong>Recovery Comparison of MMA on Traditional vs. Inert LC Column Hardware<\/div>
\n\n
\"\"<\/figure>\n\n<\/div><\/div><\/div>\n\n\n
<\/div>\n\n\n\n

Overall, we wanted to create a new method that included more relevant analytes in order to create a more efficient and comprehensive method for our customers. However, if you are only analyzing MMA and feel more comfortable using traditional reversed-phase chromatography, our original method on the Force C18 column still works great! You also have the option of using a Force Inert C18 to improve sensitivity. If you are looking at additional analytes, or looking for better sensitivity or resolution between analytes, the method on the Polar X column may be a better solution for you. If you are interested in trying an inert column, contact your local sales representative<\/a>!<\/p>\n\n\n\n

<\/div>\n\n\n
\n

Products Mentioned<\/h3>\n
\n
\n
\n
\n Catalog No. 963431E <\/a> <\/div>\n
\n
Force C18, 3 \u00b5m, 100 x 3.0 mm LC\u30ab\u30e9\u30e0<\/div>\n <\/div>\n
\n \u88fd\u54c1\u60c5\u5831\u3092\u898b\u308b<\/a>\n <\/div>\n <\/div>\n
\n
\n Catalog No. 9311A52 <\/a> <\/div>\n
\n
Raptor Polar X, 2.7 \u00b5m, 50 x 2.1 mm LC\u30ab\u30e9\u30e0<\/div>\n <\/div>\n
\n \u88fd\u54c1\u60c5\u5831\u3092\u898b\u308b<\/a>\n <\/div>\n <\/div>\n <\/div>\n <\/div>\n \n\n\n
<\/div>\n","protected":false},"excerpt":{"rendered":"

If you search Restek’s website for methylmalonic acid (MMA), you will find two different solutions for this analysis in plasma. One uses a Force C18 LC column and another a Raptor Polar X column. Why is this the case?<\/p>\n","protected":false},"author":24,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_kadence_starter_templates_imported_post":false,"_kad_post_transparent":"","_kad_post_title":"","_kad_post_layout":"","_kad_post_sidebar_id":"","_kad_post_content_style":"","_kad_post_vertical_padding":"","_kad_post_feature":"","_kad_post_feature_position":"","_kad_post_header":false,"_kad_post_footer":false,"footnotes":""},"categories":[9],"tags":[],"industries-application":[2153,2154,2155],"post-badge":[],"resource-type":[],"product-library":[],"resource-technique":[2299,2302],"hf_cat_post":[623],"ppma_author":[444],"class_list":["post-51858","post","type-post","status-publish","format-standard","hentry","category-blogs","industries-application-clinical","industries-application-clinical-diagnostics","industries-application-clinical-research","resource-technique-liquid-chromatography","resource-technique-ms-ms"],"acf":[],"taxonomy_info":{"category":[{"value":9,"label":"Blogs"}],"industries-application":[{"value":2153,"label":"Clinical"},{"value":2154,"label":"Clinical Diagnostics"},{"value":2155,"label":"Clinical Research"}],"resource-technique":[{"value":2299,"label":"Liquid Chromatography"},{"value":2302,"label":"MS\/MS"}]},"featured_image_src_large":false,"author_info":{"display_name":"Samantha Herbick","author_link":"https:\/\/discover.restek.com\/ja\/author\/samantha-herbick\/"},"comment_info":0,"category_info":[{"term_id":9,"name":"Blogs","slug":"blogs","term_group":0,"term_taxonomy_id":9,"taxonomy":"category","description":"","parent":0,"count":442,"filter":"raw","cat_ID":9,"category_count":442,"category_description":"","cat_name":"Blogs","category_nicename":"blogs","category_parent":0}],"tag_info":false,"authors":[{"term_id":444,"user_id":24,"is_guest":0,"slug":"samantha-herbick","display_name":"Samantha Herbick","avatar_url":{"url":"https:\/\/discover.restek.com\/wp-content\/uploads\/people-herbick-samantha.jpg","url2x":"https:\/\/discover.restek.com\/wp-content\/uploads\/people-herbick-samantha.jpg"},"0":null,"1":"","2":"","3":"","4":"","5":"","6":"","7":"","8":""}],"_links":{"self":[{"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/posts\/51858","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/users\/24"}],"replies":[{"embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/comments?post=51858"}],"version-history":[{"count":12,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/posts\/51858\/revisions"}],"predecessor-version":[{"id":74519,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/posts\/51858\/revisions\/74519"}],"wp:attachment":[{"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/media?parent=51858"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/categories?post=51858"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/tags?post=51858"},{"taxonomy":"industries-application","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/industries-application?post=51858"},{"taxonomy":"post-badge","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/post-badge?post=51858"},{"taxonomy":"resource-type","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/resource-type?post=51858"},{"taxonomy":"product-library","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/product-library?post=51858"},{"taxonomy":"resource-technique","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/resource-technique?post=51858"},{"taxonomy":"hf_cat_post","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/hf_cat_post?post=51858"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/ppma_author?post=51858"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}