Methylmalonic acid analysis without a drying step

The Diagnotix MMA Direct Injection Kit introduces a new LC-MS/MS workflow for the analysis of methylmalonic acid (MMA) in plasma and serum. The method combines a simple aqueous sample preparation with direct injection while maintaining reversed phase chromatography on a C18 column.

By eliminating the evaporation step typically required in MMA workflows, the method enables straightforward automation while retaining the robustness and stability associated with reversed phase LC-MS/MS separations.

This approach allows laboratories to integrate MMA analysis easily within existing LC-MS/MS workflows that already rely on reversed phase chromatography.

Conventional MMA LC-MS/MS workflows

Most MMA LC-MS/MS methods rely on protein precipitation using methanol or acetonitrile during sample preparation. This results in a supernatant containing a high percentage of organic solvent.

Under these conditions, highly polar compounds such as methylmalonic acid and succinic acid are poorly retained on reversed phase columns. As a result, laboratories typically use one of two strategies.

Evaporation and reconstitution

One common approach is to evaporate the methanol or acetonitrile and reconstitute the sample in water.

Advantage:
This enables the use of reversed phase chromatography, allowing robust chromatographic separation between methylmalonic acid and succinic acid.

Disadvantage:
The evaporation step introduces an additional manual step and makes full automation more difficult.

See Diagnotix' first Methylmalonic in serum/plasma LC-MS/MS kit

Direct injection with alternative chromatography

A second strategy is to inject the supernatant directly using alternative chromatographic techniques such as HILIC or cation-exchange chromatography.

Advantage:
The drying step can be avoided and sample preparation can be automated more easily.

Disadvantages:
These chromatographic systems are generally more sensitive to variations in sample composition. For example, differences in salt concentration between samples can influence retention behaviour and peak shape. As a result, chromatographic performance may vary during a sample batch. In addition, these techniques often require dedicated columns and mobile phases that differ from the reversed phase conditions used for many other LC-MS/MS assays.

The Diagnotix Direct Injection RP approach

The Diagnotix MMA Direct Injection Kit combines the advantages of both strategies.

The workflow uses simple aqueous sample preparation followed by direct injection, while maintaining reversed phase chromatography on a C18 column. This allows laboratories to avoid the drying step while retaining the robustness and reproducibility of reversed phase LC-MS/MS separations.

As a result, methylmalonic acid and succinic acid can be separated reliably without introducing alternative chromatographic systems.

Designed for routine LC-MS/MS laboratories

Many LC-MS/MS laboratories run multiple assays on the same instrument, often using reversed phase chromatography with similar mobile phases and column chemistries.

The Diagnotix MMA Direct Injection method fits naturally within these environments. Because the assay uses chromatographic conditions comparable to other reversed phase LC-MS/MS methods, it can be integrated easily into existing analytical workflows.

This simplifies method switching, troubleshooting and system optimisation when multiple assays are performed on the same LC-MS/MS instrument.