The goal of this project was to develop a fit-for purpose assay that could be used for the quantification of 4 lead drug candidates that were all structural analogs across 3 matrices. A fifth analog was used as the internal standard. Successful development would conclude in the qualification of each assay over a linear range of 5.0 – 5,000 ng/mL using a 0.025 mL aliquot of either rat, dog or monkey plasma.
Protein precipitation extraction
After mass spectrometer optimization, the first day of method evaluation was as follows:
Aliquot 25µL plasma ISWS 25µL 500ng/mL in 50/50 ACN/H2O PPT 400µL acetonitrile Mix 5 min multi-vortex Centrifuge 5 min 3000 rpm, ambient temp Transfer 100µL supernatant reconstitute 200µL 75/25 ACN/H2O vortex 1 min medium speed
Mobile phase A: 0.1% Formic Acid (aq)
Mobile phase B: 0.1% Formic Acid in ACN
Column: Waters Xbridge C18
The first extraction was a simple protein precipitation with a range of 5.0 to 5,000 ng/mL. However quantification of all the analytes with one method was not possible due to quadratic and divergent behavior observed in the calibrators presumably caused by matrix effects. In order to minimize these effects a liquid-liquid extraction was attempted. Both MTBE and MTBE/Hexane mixtures were evaluated as extraction solvents using acidic, neutral, and basic conditions. Observed recoveries were low at34%. The next strategy to minimize matrix effects was to return to the original extraction and investigate new chromatography. Because of the known challenges that exist with matrix effects, especially when using an analog internal standard, one of the first steps to troubleshooting chromatography is to change column chemistry and/ or mobile phase.
Monitoring of the endogenous phospholipids in the sample extracts allowed us to determine where they were eluting and that they were co-eluting with the analyte of concern but not the internal standard. Since the matrix effects would likely impact the analyte and internal standard differently, it was decided to change the chromatography and mobile phase to try and have the analyte and internal standard co-elute so any matrix effects will ideally impact both compounds equally.
New chromatographic conditions:
MPA: 0.1% Formic Acid (aq)
MPB: 0.1% Formic Acid in Methanol
Column: Raptor Biphenyl
The new conditions caused the analyte of interest and the internal standard to co-elute, and it changed the elution order of the compounds. After some difficulty with accurate and precise quantification early on the method development, changing the chromatography helped tremendously. This project involved the quantification of four structurally similar compounds in three different matrices. The beginning stages of method development were done in rat plasma and with one compound at a time. The four compounds shared an analog internal standard which made it difficult to reduce matrix effects in all compounds simultaneously.
The final assay allowed each analyte to be quantified using the same extraction, chromatography, and mass spec platform. This “drop-in” method approach allows for the rapid analysis of samples by eliminating the lag time incurred by system, equipment, and reagents changes. The method was qualified for 4 analytes within +/- 15% accuracy and precision results.