Abstract
The work presented here demonstrates two unique approaches in the application of LC/MS for the analysis of drugs and endogenous compounds in biofluids. First, the use of basic aqueous mobile phases in conjunction with both methanol and acetonitrile by LC/MS operating in electrospray positive ionization mode was investigated. Second, the design and development of a prototype ceramic micro-fluidic device (CMFD) and optimized MS source was carried out. The CMFD was packed with sub 2 μm bridged ethyl hybrid (BEH) chromatographic particles that could withstand operating pressures of up to 12,000 psi. The MS source was built to operate in both positive and negative electrospray ionization mode with the operating flow rates corresponding to the 300 um i.d. of the CMFD.The results generated from studies utilizing the addition of a base, such as ammonium hydroxide to modify the aqueous mobile phase, showed significant benefits for LC/MS/MS based bioanalytical assays when analyzed with electrospray positive ionization mode. Increases in the signal-to-noise values were observed for twenty-two out of twenty-four of the probe pharmaceuticals tested. Increase in the chromatographic retention of poorly retained compounds was also observed however, this increase in analyte retention did not occur for many highly polar compounds that eluted in the column void when chromatographed with the basic mobile phase conditions. The effect of the pH of the mobile phase further showed that the phospholipid fraction, present in protein precipitated plasma, was only slightly affected by the change in mobile phase pH. It provided complementary data to that obtained with traditional acidic based mobile phases and results in an increased number of ions detected overall for metabonomic studies. Further it was observed that by changing the pH to a basic system it was possible to resolve compounds that had previously co-eluted on traditional acidic reversed-phase LC/MS.
A 0.3 x 100 mm i.d. CMFD and compatible MS source was successfully designed for the analysis of biological samples. The device showed average chromatographic efficiencies of 9038 plates compared to 10219 plates for standard silica capillary columns. Gradient performance utilizing a diverse mix of compounds yielded a peak capacity of 55 as the average peak widths for all analytes was 0.11 minutes for a 6 minute separation. Resolution of the probe pharmaceutical alprazolam and associated hydroxylated metabolite was maintained between 1.2 and 1.5 for four different devices. Testing of the device with plasma samples prepared by protein precipitation resulted in over 1000 injections being carried out over approximately a one week period. The system was however unable to withstand the high pH (10.5) utilized in the previous study but could operate with a mobile phase pH of 10. The resulting MS source built to operate under these conditions and with the flow rates required to operate the 0.3 x 100 mm CMFD yielded signal to noise increase in the range of 8.3 to 38.6 for the molecules tested.
The CMFD/MS system was successful in the analysis of biofluid samples in both ESI + and ESI – ionization modes and was shown to allow for the analysis of small sample injection volumes from plasma prepared by protein precipitation, and dried blood spots. This device was successfully utilized for the profiling of metabolites from the beta blocker drug, propranolol. Further separation of complex biofluid samples derived from axenic rats and bile from dogs again illustrated the separation and sensitivity capabilities offered by the CMFD/MS system.
| Date of Award | Nov 2013 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Norman Smith (Supervisor) & David Cowan (Supervisor) |