|= Emerging. More than 5 years before clinical availability. (9.82%)|
|= Expected to be clinically available in 1 to 4 years. (12.95%)|
|= Clinically available now. (22.77%)|
Authors: Trelle, Morten Beck
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.
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.
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
- 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.
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.
References & Acknowledgements:
IP Royalty: no
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