The determination of small hydrophilic pharmaceuticals in blood serum

This master thesis describes the development of two methods of analysis for very polar pharmaceutical compounds in blood serum based on high performance liquid chromatography with multiple stage mass detection (LC-MS/MS). These methods are very selective and sensitive, but the response can be highly affected by coeluting serum constituents, which necessitates clean sample extracts and a good chromatographic separation. Very polar compounds can, in theory, be sufficiently separated from serum constituents if little or no organic modifier is added to the mobile phase. In practise, this works counterproductive, as it causes the stationary phase to be desolvated (dewetting) which leads to a strong reduction of retention. There are specially designed reversed phase sorbents which are insensitive to these conditions, however, and in this thesis it is shown that one of these phases offers a good separation for all tested compounds. The first method of analysis was developed for ribavirin, which is a drug that is used for treating the hepatitis C virus. In this method, human serum samples were deproteinized using a 10% trichloroacetic acid solution. Since polar compounds usually have low protein binding, ultrafiltration can also be used as a means of sample preparation. This was applied successfully for the second method of analysis for the anti-tuberculosis drugs isoniazid, ethambutol and pyrazinamide. The described methods were validated and meet the requirements of the Food and Drug Administration. In addition, these methods were extensively tested for matrix effects.