Which Column is Best for the Analysis of Methylmalonic Acid (MMA)?

If you search Restek’s website for methylmalonic acid (MMA), you will find two different solutions for this analysis in plasma. One using a Force C18 column (https://www.restek.com/articles/evaluation-of-a-simple-method-for-methylmalonic-acid-analysis-in-human-plasma-by-lc-msms) and one using a Raptor Polar X column (https://www.restek.com/articles/method-for-the-simultaneous-analysis-of-methionine-pathway-metabolites-and-methylmalonic-acid). You might be wondering why this is the case.

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 µm Raptor Polar X column with an accompanying Raptor EXP Polar X guard were used for this method.

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 µL 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.

Figure 1: Separation of Succinic Acid and Methylmalonic Acid on a Force C18 Column 100 x 3 mm, 3 µm.

Column:	Force C18 100 x 3mm, 3 µm (Cat. #963431E)
Mobile Phase A:	0.5% Formic Acid in Water
Mobile Phase B:	0.5% Formic Acid in Methanol
Injection Volume:	5 µL
Concentration:	100 ng/mL
Flow:	0.7 mL/min
Gradient:	Time (min) %B 0.00 5 0.50 60 3.00 75 3.01 100 5.00 STOP
Column Temp.:	35°C

 

Analyte	Retention Time (min)	Peak Height (cps)
Succinic Acid	1.67	34500
Methylmalonic Acid	2.03	840000

 

 

Figure 2: Separation of Succinic Acid and Methylmalonic Acid on a Raptor Polar X Column 50 x 2.1 mm, 2.7 µm.

Column:	Raptor Polar X 50 x 2.1 mm, 2.7 µm (Cat. #9311A52)
Mobile Phase A:	0.5% Formic Acid in Water
Mobile Phase B:	Acetonitrile
Injection Volume:	5 µL
Concentration:	100 ng/mL
Flow:	0.6 mL/min
Gradient:	Time (min) %B 0.00 85 1.00 55 3.00 10 3.01 85 4.00 STOP
Column Temp.:	40°C

 

Analyte	Retention Time (min)	Peak Height (cps)
Succinic Acid	1.35	60100
Methylmalonic Acid	2.95	1600000

 

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.

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! 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.