This MMA workflow is designed for routine LC-MS/MS laboratories looking to reduce complexity without compromising analytical performance.

• Reliable results through stable and reproducible chromatography
• Simplified workflow with minimal sample preparation and no evaporation step
• Seamless integration into existing LC-MS/MS systems using standard reversed phase conditions

To understand how this workflow simplifies MMA analysis, it is important to consider the trade-offs between existing MMA LC-MS/MS approaches. This requirement for reliable chromatographic separation directly impacts both sample preparation and chromatographic strategy, leading to different analytical approaches in routine laboratories.

A well-known analytical challenge in methylmalonic acid analysis is the presence of succinic acid. Methylmalonic acid and succinic acid are isobaric compounds, meaning they produce identical precursor and fragment ions in the mass spectrometer. As a result, chromatographic separation is essential for reliable MMA analysis.

In practice the challenge is further complicated by the fact that succinic acid is usually present at significantly higher concentrations than methylmalonic acid. Without sufficient chromatographic resolution, the much larger succinate peak may interfere with MMA detection.

Reversed phase (RP) chromatography is the most widely used chromatographic technique for the analysis of small organic molecules by LC-MS/MS. For this reason, RP chromatography is the standard separation technique in many clinical chemistry laboratories, therapeutic drug monitoring (TDM) workflows and toxicology assays.

A key reason for this widespread use is the robustness and reproducibility of RP separations. Retention times and peak shapes typically remain stable from injection to injection and from batch to batch, even during large analytical runs. Typical stationary phases include C18, C8, biphenyl, F5 and phenyl-hexyl columns. Mobile phases commonly consist of water combined with methanol or acetonitrile, often with a volatile buffer.

Because these chromatographic conditions are widely used across LC-MS/MS assays, RP methods integrate easily on instruments that already run multiple routine analyses.

Traditional MMA sample preparation often relies on protein precipitation with methanol or acetonitrile, a widely used technique in LC-MS/MS sample preparation.

This results in a supernatant with a high organic content (methanol or acetonitrile). Under these conditions, highly polar compounds such as methylmalonic acid and succinic acid are poorly retained on reversed phase columns, making chromatographic separation difficult.

For this reason, most laboratories adopt one of two analytical strategies.

1. Evaporation and reconstitution

The first strategy involves evaporating the methanol or acetonitrile followed by reconstitution of the sample in water.

Advantage: Reversed phase chromatography becomes possible, allowing robust chromatographic separation between methylmalonic acid and succinic acid.
Disadvantage: The evaporation step introduces an additional handling step and complicates automation. This approach is used in the standard Diagnotix MMA method (1000 KIT M MMA).

2. Direct injection using alternative chromatography

The second strategy avoids the drying step by using direct injection combined with alternative chromatographic techniques such as HILIC or cation-exchange chromatography.

Advantage: Sample preparation can be automated and the evaporation step is avoided.
Disadvantages: These chromatographic systems are generally more sensitive to variations in sample composition. For example, differences in salt concentration between samples may influence retention behaviour and peak shape. As a result, retention times and chromatographic performance may change during a sample batch. In addition, these methods require dedicated columns and mobile phases that differ from the reversed phase conditions used in many other LC-MS/MS assays.

The new Diagnotix MMA Direct Injection method combines the advantages of both strategies while avoiding their main limitations.

The workflow uses a simple aqueous sample preparation that allows direct injection of the supernatant after protein precipitation into the LC-MS/MS system while maintaining reversed phase chromatography on a C18 column.

This enables reliable chromatographic separation between methylmalonic acid and succinic acid without introducing a drying step, while maintaining reversed phase chromatography.

Reversed phase direct injection MMA analysis

The Diagnotix MMA Direct Injection method (1000DI KIT M MMA) uses reversed phase chromatography, allowing direct injection while maintaining compatibility with other reversed phase LC-MS/MS methods running on the same instrument.

Because the method uses mobile phases and column chemistries comparable to other RP workflows, laboratories can integrate the assay easily within existing LC-MS/MS setups. This also simplifies troubleshooting, system optimisation and switching between analytical methods.

This method is designed to reduce complexity in routine MMA analysis while maintaining the performance and predictability expected from standard reversed phase LC-MS/MS workflows.

Consistent chromatographic behaviour is essential for reliable MMA quantification in routine laboratory settings.

• Reliable and stable chromatography
• Consistent performance between runs
• Reproducible results across sample batches
• Robust separation of MMA and succinic acid

Reducing manual steps improves efficiency and supports automation in high-throughput environments.

• Simple and fast sample preparation
• No evaporation step required
• Easy to automate
• Reduced hands-on time

Using standard reversed phase conditions ensures compatibility with routine workflows and simplifies day-to-day operation.

• Column lifetime comparable to standard reversed phase methods
• Standard RP conditions using conventional column chemistry and mobile phases
• Easier method switching on shared LC-MS/MS systems
• Familiar conditions that simplify troubleshooting and system optimisation

Transparency in LC-MS/MS methods

Diagnotix believes that transparency in analytical methods is important for laboratories working with LC-MS/MS. In routine laboratories, multiple assays often run on the same instrument and analysts need to understand how a method behaves within their LC-MS/MS system.

For this reason, Diagnotix does not aim to provide completely closed “black-box” solutions with fixed chromatographic configurations. Instead, transparency about the chromatographic principle, column chemistry and mobile phases allows laboratories to understand how the method works in practice.

This becomes particularly important during troubleshooting, system optimisation or when switching between different LC-MS/MS methods on the same instrument.

A practical consideration when using commercial kits

When implementing an MMA method without a drying step, it can be useful to check which chromatographic technique is used in the assay.

When a workflow includes an evaporation and reconstitution step, reversed phase chromatography is typically used. However, MMA methods that avoid the drying step often rely on alternative chromatographic techniques such as HILIC or ion-exchange chromatography.

Laboratories using commercial or “black-box” kits may therefore wish to check the instructions for use to see which chromatographic principle is applied. If the chromatographic technique is not explicitly mentioned, it may be useful to ask the supplier whether the method uses reversed phase chromatography or an alternative separation technique.