Rhys Jones (Presenter)
Biotage GB Limited
Authorship: Rhys Jones1, Lee Williams1, Alan Edgington1, Adam Senior1, Helen Lodder1, Geoff Davies1, Steve Jordan1, Claire Desbrow1, Paul Roberts1, Victor Vandell2 & Elena Gairloch2.
1Biotage GB Limited, Distribution Way, Dyffryn Business Park, Cardiff, CF82 7TS, UK.
| Short Abstract This poster summarises various sample preparation strategies for the extraction of methylmalonic acid from serum prior to LC-MS/MS analysis. A range of extraction techniques were evaluated: protein precipitation, phospholipid depletion, supported liquid extraction and solid phase extraction using both silica and polymer-based mixed-mode anion exchange chemistries. Analysis was performed using an ACQUITY IClass UPLC interfaced to a Xevo TQS triple quadrupole mass spectrometer via electrospray ionization operating in multiple reaction monitoring mode. Method performance was evaluated for evaporative effects, assay recovery and ion suppression effects using post column infusion experiments. Phospholipid removal was also determined. Full results will be presented. |
Long Abstract
Introduction
The screening for elevated levels of methylmalonic Acid (MMA) in serum is commonly used as a clinical diagnostic indicator of Cobalamin (Vitamin B12) deficiency in humans. MMA is commonly analyzed using LC-MS/MS with or without prior derivatization.This poster summarises various sample preparation strategies for the extraction of MMA from serum without the necessity for derivatization, prior to LC-MS/MS analysis. A range of techniques of varying complexity were evaluated: protein precipitation, phospholipid depletion, supported liquid extraction and solid phase extraction using both silica and polymer-based mixed-mode anion exchange chemistries. Method performance was evaluated for evaporative effects, assay recovery and ion suppression effects using post column infusion experiments. Phospholipid removal was also determined, because of their well characterised potential for interference.
Methodology
MMA was spiked into charcoal stripped human serum at various concentrations. 100 µL of serum was extracted for each sample preparation technique. MMA-13C4 radiolabelled internal standard was spiked at 100 ng/mL.
A solvent first protein crash methodology was employed using both ISOLUTE® PPT+ (protein precipitation) and ISOLUTE® PLD+ (phospholipid depletion) 96-well plates in conjunction with vortex mixing. Initial method development involved optimization of organic solvent composition and crash ratio for efficient precipitation of proteins, phospholipid removal and maximum analyte recovery. Supported liquid extraction was performed using ISOLUTE SLE+ 200 µL capacity plates. Sample pH control and extraction solvent was investigated. Various forms of SPE: Silica and polymer-based mixed-mode strong anion exchange (ISOLUTE® SAX and EVOLUTE® EXPRESS AX respectively); and polymer-based mixed-mode weak anion exchange (EVOLUTE EXPRESS WAX) were evaluated. Extraction strategies focused on sample pre-treatment conditions, pH control and elution optimization.
Extracts were evaporated at 40 °C and reconstituted in 100 µL of 0.4% formic acid in water prior to LC-MS/MS analysis. Samples were analyzed using a Waters ACQUITY IClass UPLC coupled to a XEVO TQS triple quadrupole mass spectrometer. Electrospray ionization was utilized and run in negative ion, whilst acquiring data in multiple reaction monitoring mode.
Results
MMA is a polar, low molecular weight dicarboxylic acid having an isobaric species in the form of succinic acid (SA). These properties can result in issues with analysis in terms of volatility during evaporation, chromatography, MS sensitivity, incompatibility of mobile phases with ionization mode and low molecular weight fragmentation.
Initial method development efforts focussed on analyte losses during the evaporation process. When drying under neutral mobile phase conditions substantial losses were observed. Acidic conditions yielded far higher recoveries. Another option to avoid evaporative losses was the addition of a small volume (2 µL) of glycol as a “keeper solvent”.
Chromatographic separation of MMA and isobaric SA was achieved using a mobile phase of 0.4% formic acid in both water and methanol with a Gemini 3 µm C18 (100 x 3 mm id) column. Although acidic mobile phases are not optimal for negative ionization we were unable to find a suitable alternative.
The ISOLUTE PPT+ and PLD+ extraction approaches required precipitation with acidified ACN for optimal performance. ISOLUTE SLE+ required pre-treatment with formic acid followed by extraction with MTBE. Both silica and polymer-based SPE performed best when using water based extraction protocol in the absence of ionic strength or pH control, with elution using 2% formic acid in acetonitrile. Weak anion exchange SPE required pH control to 5 during the procedure and could be eluted in either acidic or basic solvent due to neutralization of the sorbent or analyte for this mechanism. Ultimately we were were able to achieve high reproducible recoveries greater than 85 % with corresponding RSDs below 10 % for all sample preparation techniques.
Phospholipid MRM testing (+ ion mode) of 16 of the most abundant PLs previously identified through full scan, SIM and pre-cursor ion scanning demonstrated excellent removal for all techniques apart from the ISOLUTE PPT+ protein precipitation plate. This is simply a plate for removal of proteins from sample matrices and does not remove PLs to any significant degree.
Post column infusion experiments to highlight regions of suppression in the chromatogram for each technique demonstrated no appreciable effect for the ISOLUTE SLE+, ISOLUTE SAX, EVOLUTE EXPRESS AX and WAX approaches, with only a subtle effect on the ISOLUTE PLD+ and PPT+ approaches. Subtle differences in the baseline were observed for each approach in the early part of the chromatogram.
Calibration curves were constructed using spiked serum. Good linearity was observed from 10 to 2000 ng/mL, demonstrating coefficients of determination (r2) greater than 0.999 for all sample preparation techniques. Signal to noise ratios at 10 ng/mL were demonstrated to be greater than 10:1 for all methods.
Conclusion
This poster demonstrates the applicability of various 96-well plate based extraction strategies for the extraction of MMA from serum.
Each sample preparation technique offered differing degrees of complexity during processing. Subtle differences in baseline noise were observed between the protocols which ultimately affected sensitivity. All approaches apart from protein precipitation performed well in terms of high reproducible recoveries, low suppression effects with respect to phospholipids and excellent extract cleanliness resulting in the ability to reach LLOQs at 10 ng/mL or below.
References & Acknowledgements:
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IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |