Deuterium or 13C-labeled internal standards for quantification of methylmalonic acid in serum? Morten Trelle (Presenter) Department of Clinical Biochemistry, OUH Svendborg

Authors: Trelle, Morten Beck
Department of Clinical Biochemistry, OUH Svendborg Hospital, Baagøes Alle 15, DK-5700 Svendborg, Denmark

In an LCMS-based quantification method for methylmalonic acid (MMA) in human serum, we have observed decreasing levels of MMA in batches of 81 replicates of a patient serum pool despite the use of methyl-d3-malonic acid (d3-MMA) as an internal standard. Clearly, either the sample preparation or the LCMS analysis was not robust. In the end, an exchange of d3-MMA for 13C4-Methylmalonic acid (13C4-MMA) solved the problem. As d3-MMA display slightly reduced retention time compared to MMA and 13C4-MMA, we suggest that the drift in quantified MMA was the result of slightly different matrix effects on MMA and d3-MMA.

Problem

Decreasing levels of MMA in batches of 81 replicates of a patient serum pool despite the use of methyl-d3-malonic acid (d3-MMA) as an internal standard.

Method Information

Sample preparation: Protein precipitation; 100 µL serum plus 300 µL acetonitril with 0.4% formic acid (FA) and internal standard (d3-MMA or 13C4-MMA). Mixed and filtered on Agilent Captive ND 96 well plates, flow-through evaporated under nitrogen and reconstituted in 5% methanol with 0,4% FA.

LC-MS/MS system: Transcend TLX1 HPLC system coupled to TSQ Quantum Ultra (Thermo-Fisher Scientific).

Mobile phase A: 0.1% FA in MilliQ water

Mobile phase B: 0.1% FA in Methanol LC-MS grade

7 min gradient: 0-2.0 min increase from 5% to 30% B, 2-2.5 min increase to 100% B, 2.5-3 min at 100% B, 3-7 min at 5%B, flow 0,5 mL/min

Column: Phenomenex Gemini C18, 3 µm, 110 Å, 100 x 3 mm

Column oven: 30°C

Injection volume: 20 µL

Troubleshooting Steps

- Considered whether the problem was related to sample preparation or LCMS analysis. Thought it was most likely related to the LCMS analysis.

- Exchanged d3-MMA with 13C4-MMA as internal standard.

- This solved the problem.

Outcome

The problem of unstable quantification of MMA during a batch of 81 replicates of a serum pool was solved by replacement of d3-MMA with 13C4-MMA as internal standard. D3-MMA exhibits a slightly reduced retention time compared to 13C4-MMA and MMA, which may result in a slightly different matrix effect and in the end unstable and unreliable quantification of MMA. This observation suggests that 13C-labeled internal standards, if available, should be preferred over deuterium-labeled internal standards when new quantitative analyses are being developed.