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Title:   Author  item
                                            Pedersen, Theresaitem
                                            Keyes, Williamitem
                                            Shahab-Ferdows, Settiitem
                                            Allen, Lindsay

                                                         - *A*item
                                            Newman, John
  Submitted to: Journal of Chromatography
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 3/21/2011
                                    Publication  Date: 5/16/2011
                                    Citation: Pedersen, T.L., Keyes, W.R., Shahab-Ferdows, S., Allen, L.H., Newman, J.W. 2011. An Optimized Method for Measuring Methylmalonic Acid in Low Volumes of Serum Using UPLC-MS/MS. Journal of Chromatography. B. 879, 1502-6.Interpretive Summary: Methylmalonic acid (MMA) is a metabolic intermediate transformed to succinic acid (SA) by a vitamin B12-dependent catalytic step, and is broadly used as a clinical biomarker of functional vitamin B12 status.  However, reported methods use between 100 and 1000_L of serum or plasma making them sub-optimal for sample-limited studies, including those with neonates and infants. LC–MS/MS based protocols to measure MMA as n-butyl esters in the presence of tri-deuterated MMA (MMA-d3) were modified for use with 25_L of human serum by scaling down sample processing volumes and analysis by UPLC–MS/MS. Plasma-based calibration solutions were found to be unnecessary, and chromatographic resolution and peak shape of SA and MMA was optimized in <4 min with isocratic 53:47 methanol/1.67mM (pH 6.5) ammonium formate. Additionally, 1-cyclohexyl-urido-3-dodecanoic acid (CUDA) was included as internal standard allowing direct assessment of MMA recovery. Sample concentrations in the low normal range produced a signal:noise of >100:1.MMAintra- and inter-assay variability was under 10%. MMA-d3 surrogate recovery averaged 93±14%. MMA stability exceeded three years in frozen samples and was unaffected by up to five freeze/thaw cycles. In conclusion, we report that methylmalonic acid can be measured with 25_L of serum using water based standards. The assay signal:noise per concentration indicates that the method could perform as implemented with as little as 5_L of serum. The reported method is applicable for studies of functional B12 status in sample limited experiments including investigations of nutritional status in neonates and in studies where low normal MMA levels are expected.
                                Technical Abstract: Background: Methylmalonic acid (MMA) is a metabolic intermediate which is transformed to succinic acid (SA) by a vitamin B12-dependent catalytic step.  MMA is broadly used as a clinical biomarker of functional vitamin B12 status.  However, currently validated protocols use between 100 -1000 μL of serum or plasma making them less than ideal for sample-limited studies, including those with neonates and infants.  

Methods:  Current analytical protocols were modified to measure MMA in 25 μL of human serum as n-butyl esters by scaling down sample processing volumes, with analysis by UPLC MS/MS. Calibration solutions were prepared in water, as opposed to dialyzed plasma. A complete baseline resolution of SA and MMA was achieved in <4min with isocratic 53:47 methanol/1.67mM (pH6.5) ammonium formate. Additionally, tri deuterated MMA (MMA-d3) and 1 cyclohexyl-urido-3-dodecanoic acid (CUDA) were included as internal standards.  

Results:  Sample concentrations in the low normal range produced a signal:noise of >100:1. MMA intra- and inter-assay variability was under 10%. MMA-d3 surrogate recovery averaged 93 ± 14%. MMA stability exceeded three years in frozen samples and was unaffected by up to five freeze/thaw cycles.   

Conclusions:  Methylmalonic acid can be measured with 25 μL of serum using water based standards.  The assay signal:noise per concentration indicates that the method could perform with as little as 5 μL of serum. The reported method is applicable for clinical nutrition studies of functional B12 status in sample limited experiments and can be directly transferred to high throughput robotic liquid handlers.


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