Bioanalysis of Small Molecule Pharmaceuticals: Simultaneous Determination in Biological Fluid Samples from Multiple Species by the Management of Matrix Effects

Gray, Nicholas (2011) Bioanalysis of Small Molecule Pharmaceuticals: Simultaneous Determination in Biological Fluid Samples from Multiple Species by the Management of Matrix Effects. Doctoral thesis, Northumbria University.

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Abstract

A strategy is described for method evaluation to manage the influence of endogenous compounds to induce bias in pharmaceutical quantification from blood samples of different animal species resulting from ionisation matrix effects. The approach introduces the possibility of simultaneous cross animal species calibration for ethical reasons using a simple indicative test, which also rapidly demonstrates the utility of a test assay in other matrices.

A quantitative Turboflow LC-MS/MS assay was evaluated using samples prepared in species matched controlled matrices with deuterated internal standardisation. The method was subsequently tested to assess the analysis of samples from multiple animal species using calibration samples from a single matrix origin. The object of this was to enable the substitution of analytical control samples in rodent plasma, reducing the plasma volume required, therefore the number of rodents used indirectly to support development studies. The method was unsuccessful due to concentration bias in identically prepared test samples. The bias origin was investigated using a fixed ratio solution of the analyte and its deuterated analogue with matrix test aliquots. The investigation identified non equivalent relative ionisation efficiency between compounds. The ratio method is proposed as an evaluation strategy for matrix effects causing non-equivalent response ratios.

The origin of this deviation was identified using a post column standard infusion test highlighting a region of ionisation suppression co-eluting with the analyte. Full scan acquisition analysis revealed co-elution of endogenous glycerophospholipids. The relative expression of these compounds between species was investigated, employing a precursor ion scanning method of a common product ion indicative of phosphotidyl choline compounds; the human diversity was also investigated. Distribution was found to differ between animal species, which was further tested by construction of a model using partial least squares regression which correctly identified the species origin of all non-primate species tested. The mechanism of differentiation between compound and deuterated analogue by endogenous phosphotidyl choline was proposed as micellar phase equilibrium following elution from the HPLC column.

A new Turboflow LC-MS/MS assay was optimised with attention to the resolution of glycerophospholipid interferences and retested for applicability between species. It was simultaneously applied to the analysis of small volume whole blood samples via dried blood spots on filter paper. Precision and bias were acceptable for the analysis of rat and mouse samples using human control plasma. Analysis of incurred ex-vivo samples from rats was successful in demonstrating equivalence between calibration sources.

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