Mindy Gao (Presenter)
Thermo Fisher Scientific
Bio: Over 15 years experience of LC/MS in pharmaceutical industry and biomarker, clinical applications
Authorship: Mindy Gao
Thermo Fisher Scientific, CA, USA
| Short Abstract Methylmalonic acid (MMA) is polar molecule that poses challenges for the development of quantitative LC-MS methods. Two analytical approaches have been implemented: analysis of MMA in negative ionization mode and analysis of derivatized MMA in positive ionization mode. The product of the derivatization reaction with n-butanol shows improved reverse phase chromatographic retention and higher ionization efficiency than underivatized MMA, making this method a preferred quantitative solution. The derivatization reaction parameters described in the literature resulted in variable, up to 100 fold, reaction efficiency. Several reaction parameters were investigated and optimized to ensure reproducible and efficient butylation reaction of MMA. |
Long Abstract
Introduction
Methylmalonic acid (MMA) is small polar molecule that poses challenges for the development of quantitative LC-MS methods. Two analytical approaches have been described in the literature: analysis of methylmalonic acid in negative ionization mode and analysis of derivatized MMA in positive ionization mode. The product of the derivatization reaction with n-butanol shows improved retention to reverse phase chromatographic column and higher ionization efficiency than underivatized MMA, making this method a preferred solution for quantitative analysis. The derivatization reaction parameters described in the literature resulted in variable, up to 100 fold, reaction efficiency demonstrated by large variability in internal standard signal. Several reaction parameters were investigated and optimized to ensure reproducible and efficient butylation reaction of MMA.
Methods
The sample preparation method included protein precipitation followed by derivatization reaction, evaporation and reconstitution.
The chromatographic separation was performed using a 2.8-minute isocratic LC method. Methylmalonic acid and its deuterated analog (internal standard) were detected on a hybrid quadrupole-orbitrap mass spectrometer using APCI ionization probe. The mass spectrometry method collected MS/MS spectra for each analyte in a PRM experiment at a resolution of 70K.
The following derivatization reaction parameters were optimized: reaction time, temperature, n-butanol additives, and other additives which could catalyze the reaction.
Results
We found that reaction temperature does not have significant effect on reaction efficacy and reproducibility so we recommend that reaction is carried on at room temperature. We also found that extending the reaction time up to 1 hour did not improve reaction performance. The main reaction parameter found to play significant role in reaction efficacy and reproducibility was an additive. Addition of appropriate salt increased reaction efficacy and significantly improved reaction reproducibility, resulting in reproducible internal standard signal (within 10% difference). Implementation of new derivatization reaction parameters allowed us to develop a quantitative method with a limit of quantitation of of 25 nM, precision and accuracy within 10%, and negligible matrix effects.
For in vitro diagnostic use.
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