Studies of UHPLC-MS performance and application to rapid sensitive and robust drug analysis

Student thesis: Doctoral ThesisDoctor of Philosophy


This thesis comprises studies of ultra high performance liquid chromatography (UHPLC) performance with a particular focus on the application of liquid chromatography-mass spectrometry (LC-MS) for the analysis of basic drugs. UHPLC-MS technologies are investigated to facilitate fast, sensitive and robust analysis of drugs in biological matrices, particularly those related to doping in human sport. Mobile phase solvent, pH and temperature significantly affect the chromatographic performance of hydrophilic, basic analytes. Manipulation of mobile phase pH suppresses the protonation of basic analytes for improved retention, peak shape and resolution. This is exemplified by the ephedrines, which are inherently difficult to separate by reversed-phase liquid chromatography (RPLC). A high pH RPLC separation is coupled with MS detection and validated for the identification and quantification of ephedrines in doping control analysis. The effects of mobile phase composition and pH on efficient and stable ionisation are studied for robust and sensitive analysis of basic analytes. Different mobile phase conditions are evaluated with electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI), with high pH eluents generating greater signal intensities for the basic analytes studied with ESI. Hydrophilic interaction liquid chromatography (HILIC) is evaluated as an alternative approach for accurate and robust quantification. A HILIC approach to separate the ephedrines is evaluated for robustness, and the validated method is compared with the high pH RPLC method in terms of linearity, accuracy, precision, matrix effects and sensitivity. Finally, a switching system comprising two different, complementary stationary phase materials is designed and evaluated to widen the elution window, allowing for the simultaneous analysis of both polar and non-polar analytes.
A combination of Hypercarb and Cig stationary phases are used with UHPLC column switching, and the suitability of the approach is illustrated by the analysis of selected doping agents covering a wide polarity range in a single injection.
Date of Award7 Mar 2013
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorNorman Smith (Supervisor) & David Cowan (Supervisor)

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