Application of Solid Phase Microextraction for Quantitative Bioanalysis and Toxicokinetics: an Integrated Microsampling and Microanalysis Technique
Over the past decade, the growing field of microsampling has changed the way bioanalysis and preclinical studies are conducted. A variety of microsampling techniques have been adopted by the pharmaceutical industry and embedded into preclinical workflows. A technique known as solid phase microextraction (SPME) offers a distinctive advantage of measuring free drug concentrations within living organisms without the need for blood withdrawal. Despite its promise and potential advantages, SPME has not been extensively explored for preclinical use within the pharmaceutical industry. In this research, the application of SPME for quantitative bioanalysis and toxicokinetics was investigated for the first time within a pharmaceutical setting. This was performed through parallel in vitro and in vivo experiments. Initially three test compounds were selected (metoprolol, propranolol and diclofenac) and LC MS/MS methods were validated for all three. These were employed throughout the project to support quantitative analysis during the SPME in vitro and in vivo evaluation. SPME fibre blood exposure profiles and desorption profiles were constructed for the three tool compounds and parameters such as the impact of hematocrit levels, the effect of blood flow rate and on-fibre stability were investigated in vitro. SPME was then implemented in vivo. Practicalities of inserting the SPME fibre into the veins of animals was assessed using anesthetised rats and fibre blood exposure times were also determined during this first in vivo experiment. Since SPME measures free drug concentrations, its potential benefits as a tool to determine protein binding values of drugs were examined and compared to a gold standard approach for protein binding experiments known as rapid equilibrium dialysis (RED). The three tool analytes were studied as they cover a range of plasma protein binding levels (~ 30 - 99%) at three different physiologically relevant concentrations (10, 100 and 500 ng/mL). This was followed by an in vivo experiment to identify whether SPME measures free drug concentrations in conscious rats. In vivo SPME samples were compared with whole blood samples withdrawn from the same rats and analysed using the RED device. A full toxicology study was subsequently conducted in conscious rats for seven days to mimic a typical preclinical rodent study. SPME was compared with conventional caudal venipuncture whole blood sampling for generating toxicokinetic data. The impact and biocompatibility of SPME was studied through pathological endpoints and using an Irwin behavioural study. It was demonstrated that it may take up to 3 h for an analyte to reach equilibrium between the sample matrix and the SPME coating. This is not viable for in vivo applications due to ethical reasons and therefore pre-equilibrium conditions are more suited. Analyte desorption time of the SPME fibre was achieved between 15- 30 min. Levels of blood hematocrit had no impact on analyte response while blood flow rates may have an effect on analyte response and concentration. On-fibre stability was established for all three tool analytes for up to six weeks. It was found that consistent results were obtained by SPME when measuring protein binding values of all three analytes across three concentrations. The percentage difference between protein binding values determined by SPME and RED was within recommended limits for bioanalysis (<15 %) across all analytes and concentrations. The time required to obtain plasma protein values using SPME was considerably quicker than by using the RED device (1 h compared to 8 h). It was demonstrated that SPME provides a compelling alternative platform for the efficient generation of high quality plasma protein binding values. Pre-equilibrium conditions illustrated that using 2 min fibre exposure to systemic circulation was sufficient to produce reliable quantitative analysis. However, it was noted that current C18 fibre coatings did not detect metoprolol metabolite which exhibits a polar moiety. Mixed phase fibre coatings are required for metabolic analysis. The potential capacity of SPME to generate meaningful toxicokinetic data of free drug concentrations was shown. Biocompatibility of SPME was established by comparing pathological endpoints observed between SPME sampled and control rat groups. Finally, a novel approach was described for quantitative bioanalysis by direct SPME-MS. SPME was coupled to a mass spectrometer to enable direct elution of analytes from the SPME fibre onto the MS. This was characterised with two test analytes, metoprolol and propranolol, spiked into control rat blood. The data indicated the significance of this approach to enable rapid, selective and highly sensitive (10 ng/mL lower limit of quantification) qualitative and quantitative chemical analysis. Overall this research demonstrated that SPME could potentially provide a compelling alternative microsampling platform for preclinical studies.