Evaluation of paper spray mass spectrometry for direct analysis of small molecule drugs and hemoglobin constant spring peptide markers
Date of Issue2016
School of Biological Sciences
A significant limitation in performing preclinical studies using small animals is the need for macrovolume blood sampling, a similar situation encountered with newborn or neonates screening. Dried blood spot (DBS) method which requires small sample volume has overcome this limitation. However, the required extensive sample pre-treatment prior to chromatographic separation and detection is a major bottleneck. High throughput analysis is a priority in preclinical drug discovery and clinical settings. Paper spray mass spectrometry which is ideally suited to direct DBS analysis without sample pre-treatment and chromatographic separation prior to mass analysis at ambient environment could provide the required throughput. At present there is no report on the application of paper spray mass spectrometry for preclinical studies or biomarker analysis. The studies described in this thesis were undertaken to evaluate the application of paper spray mass spectrometry for direct analysis of small molecule drugs and biomarker in dried matrix spot samples. In the first part of the thesis, paper spray mass spectrometry (PS-MS) method was developed and validated. Potential analytical liabilities, such as carryover and ion suppression were systematically evaluated using structurally diverse test compounds. It was observed that ionization suppression was prevalent for the test compounds, but it could be effectively managed by using a stable isotope labeled internal standard (SIL-IS). Carryover was found to be minimal for some compounds and variable for others and could generally be overcome by cleaning the metal clip and inserting matrix blanks in-between samples. The results from the validation study indicated that the PS-MS assay was precise and accurate, obtaining a LLOQ of 1 and 5 ng/mL for sunitinib and benzethonium respectively in dried plasma spots (DPS). The test compound, sunitinib, was stable in all conditions studied and analyte extraction and detection sensitivity could be improved with sufficient off-line or on-line pre-moistening of the dried spot prior to PS-MS analysis after longer storage period. For quantitative bioanalysis, SIL-IS coated PCR tube sampler prepared by evaporation and concentration technique was used to sample microvolume (5 µL) of plasma, serum and whole blood and the results compared to that of conventional pre-mix method. The tube sampler results were found to correlate well with those from a conventional laboratory pre-mixed procedure and comparable accuracy and precision were achieved. Hence, < 10 µL blood could be sampled directly from small rodents during preclinical studies. Evaluation of the PS-MS assay to ascertain that it will provide comparable data to LC-MS/MS assay when used for in vivo bioanalytical analysis showed that PS-MS assay results correlate well with those from conventional LC-MS system, and comparable accuracy, precision, linearity and sensitivity (LLOQ of 1 ng/mL of sunitinib in DBS) were achieved with analysis speed of about 48-fold higher with sample volume of 5 µL. In addition, the presence of stable metabolite in the samples showed no impact on the parent quantitation for the test compound in PS-MS assay. Further application of the PS-MS assay to preclinical pharmacokinetic (PK) study found that, the assay was simple and rapid with overall analysis time of 1 min and a full plasma concentration vs. time profile of sunitinib could be obtained from a single mouse. The assay required small sample volume, thereby reducing the overall cost and the use of animals in the preclinical study. Therefore, PS-MS assay could be a potential valuable tool for high throughput in vivo screening of drug candidates. In the second part of the thesis, DBS method was developed as an alternative sampling method for the analysis of biomarkers in DBS samples and further investigated the direct detection of biomarkers from DBS samples using paper spray method. Hemoglobin Constant Spring (Hb CS) was the selected disease model used for this study, which was among a group of inherited blood diseases that were previously studied in our lab and the potential peptide markers identified using peptidomic approach. A DBS prepared by spotting 10 µL packed red cell on Schleicher & Schuell (S&S) 903 filter paper, dried for 4 h and punched-out spots of eight taken with subsequent solvent extraction and precipitation with 0.1 % formic acid in acetonitrile was sufficient to detect all Hb CS specific peptide markers using MALDI-MS and nanoLC-MS/MS analysis with comparable results to liquid packed red cell samples of 100 µL. Stability results also showed that peptide markers were stable in the DBS at 4 ℃ and room temperature for a period up to 12 months and at 37 ℃ and 40 ℃ for 1 month period. Direct analysis of the peptide markers from DBS using PS-MS method proved unsuccessful probably due to the nature of the paper substrate used or low percentage of Hb CS specific peptide markers present in the peripheral blood. However, this sampling method can potentially be used to screen Hb CS disease in newborns, critically ill patients or individuals from remote/resource-limited areas or be coupled to the discovery and validation study.
DRNTU::Science::Chemistry::Analytical chemistry::Qualitative analysis