Options
Method development based on High Performance Liquid Chromatography (HPLC) separation using serial-coupling of Monolithic C18 and Hydrophilic interaction (HILIC) columns for simultaneous analysis of tuberculosis combination drug theraphy
Author
Tham, Lai Yee
Supervisor
Nowak, Steven A.
Abstract
Method development for combination drug products is particularly challenging when the analytes have significantly different polarities. This may often require multiple chromatographic methods for each active component. The aim of this research work is to resolve a wide range of compound polarities (non-polar and polar compounds) in a mixture, especially in combination drug therapies, with one High performance liquid chromatography (HPLC) method and equipment. Thus, the idea of serial-coupling of Hydrophilic Interaction Liquid Chromatography (HILIC) to a reversedphase column stationary phase was conceived. Tuberculosis (TB) first-line drug therapy consisting of rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA), streptomycin (STP) and ethambutol (EMB), was chosen for this purpose as these drugs are of a wide range of polarities.
The first part of this project focused on column selection and optimizing HPLC conditions. It was found that the correct choice of HILIC and RP i.e. SB-Phenyl stationary phases and dimensions of the columns to be serial-coupled for the separation was paramount for the success of the project. Sufficient length was needed to retain either very polar or very non-polar analytes. The gradient step was also critical to allow effective elution from the HILIC column and retention in the RP column and vice-versa. The use of a phenylethylisoyanate (PEIC) derivatization approach for INH, PZA, RIF and EMB was employed for UV detection.
The second part of the research examined on transferring the method to LC hyphenated with Mass Spectrometry (MS). LC-MS technique provides universal detection of analytes such as STP and un-derivatized EMB. This approach yielded better sensitivity and less sample preparation steps prior to analysis. Simultaneous determination of INH, PZA, RIF, EMB and STP was achieved with a single HPLC method within 16 minutes. With the column temperature and auto-sampler at ambient conditions and at a flow-rate of 0.6mL/min, the column backpressure achieved was approximately 135 bars. This method is therefore suitable for use with a standard HPLC system with UV and/or MS detection schemes.
The first part of this project focused on column selection and optimizing HPLC conditions. It was found that the correct choice of HILIC and RP i.e. SB-Phenyl stationary phases and dimensions of the columns to be serial-coupled for the separation was paramount for the success of the project. Sufficient length was needed to retain either very polar or very non-polar analytes. The gradient step was also critical to allow effective elution from the HILIC column and retention in the RP column and vice-versa. The use of a phenylethylisoyanate (PEIC) derivatization approach for INH, PZA, RIF and EMB was employed for UV detection.
The second part of the research examined on transferring the method to LC hyphenated with Mass Spectrometry (MS). LC-MS technique provides universal detection of analytes such as STP and un-derivatized EMB. This approach yielded better sensitivity and less sample preparation steps prior to analysis. Simultaneous determination of INH, PZA, RIF, EMB and STP was achieved with a single HPLC method within 16 minutes. With the column temperature and auto-sampler at ambient conditions and at a flow-rate of 0.6mL/min, the column backpressure achieved was approximately 135 bars. This method is therefore suitable for use with a standard HPLC system with UV and/or MS detection schemes.
Date Issued
2011
Call Number
RC311.3.C45 Tha
Date Submitted
2011