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Instrumentation and reagents
Two GC-MS instruments were used for purposes of this study. The first was a Hewlett Packard (HP, Inc.) GC 6890 gas chromatographer coupled to a MS 5973 series mass spectrometer. The initial phase of the method development and validation was conducted on this instrument. However, due to lengthy problems with the vacuum pump down time, pressure and equilibration irregularities, it was decided to complete the final phase of the metabolite quantification on a newer GC-MS instrument. Quality control and established validation parameters were ensured and carried over to the method on the newer instrument. This instrument was a Thermo Scientific (TS, Inc.) Trace 1300 Series GC coupled to a single quadropole ISQ mass spectrometer. Thermo Xcalibur (TS, Inc.) software was used for the method setup, instrument parameters, analyser tuning and sample data acquisition. A DB-5-MS capillary GC column and an electron impact (EI) MS ionisation source were used for the final sample analyses. Reagents with analytical grade greater than 98% purity were rchased pre-packaged as dry weighed powder. Structural chemistry and integrity of the reagents were crucial for the mass spectral analysis of the standards and unknown samples.
Sample preparation and derivatisation
The use of gas chromatography (GC) requires samples to be derivatised. This entails chemical modification of the analyte to increase volatility of otherwise non-detectable compounds. In addition, the polarity of water soluble functional groups such as COOH, OH, NH and SH should be changed to prevent adsorption to the stationery phase of the GC capillary column. The chromatographic behaviour of the compound of interest is thus improved upon. A number of derivatising reagents were tested during the method development stages. These included silylation, alkylation and acylation reagents. Owing to variation in the functional groups of the analytes measured in this study, it was decided to use a combination of alkylating reagents which were well suited to target all of the analytes. For the purpose of this study the fluorinated anhydride pentafluoropropionic anhydride (PFPA) was used. This derivatising reagent reacts with alcohols, amines and phenols.
Pentafluoropropanol (PFPOH) was used in conjunction with PFPA because it was shown, when tested, to be ideal for derivatisation of the remaining functional groups not targeted by PFPA. This combination enabled the simultaneous analysis of all the required metabolites from a single sample.
Mass spectral identification of the metabolites
Each of the analytes was at first analysed separately so as to identify the individual fragmentation pattern of the mass spectrum under electron impact ionisation conditions. The analyte samples were run parallel to matrix blank and solvent blank samples to differentiate between derivative responses on the chromatogram. Under normal GC-MS operating conditions the analyser was set at -70 eV in scan mode to obtain ions at a range of 50 to 600 mass to charge (m/z) units. The largest most prominent ion for each analyte was chosen for selective ion monitoring (SIM) mode to be used as a qualifier for identification. The most abundant mass ions 286, 266, 254 and 255 for tryptophan, kynurenine, quinolinic acid and nicotinamide (or nicotinic acid) respectively were selected for quantification.
Chapter 1 Introduction
1.1 Epidemiology and phases of HIV infection Tryptophan metabolism
1.3 Enzyme activity for the conversion of tryptophan to kynurenine
1.4 Tryptophan depletion and HIV infection
1.5 Niacin and HIV
1.6 References
Chapter 2 Materials and methods
2.1 Abstract
2.2 Patients
2.2.1 Patient inclusion and exclusion criteria
2.3 Controls
2.4 Sample collection and processing
2.5 Patient clinical evaluation and blood chemistry
2.6 Neopterin assay
2.7 Procalcitonin assay
2.8 Cytokine assays and flow cytometry
Chapter 3 Development and validation of a novel gas chromatography mass spectrometry method for the determination of tryptophan and metabolites of the kynurenine pathway
3.1 Abstract
3.2 Introduction
3.3 Method
3.4 Method validation
3.5 Validation results.
3.6 Summary
3.7 References
Chapter 4 Levels of procalcitonin, C-reactive protein and neopterin in patients with advanced HIV-1 infection
4.1 Abstract
4.2 Introduction
4.3 Methods
4.4 Results
4.5 Discussion
4.6 References
Chapter 5 Neopterin as pro-inflammatory indicator and as non-specific biomarker in HIV/AIDS
5.1 Abstract
5.2 Introduction
5.3 Methods
5.4 Results
5.5 Discussion
5.6 Summary/Conclusions
5.7 References
Chapter 6 Tryptophan depletion in a sub-Saharan HIV/AIDS population
6.1 Abstract
6.2 Introduction
6.3 Methods
6.4 Results
6.5 Discussion
6.6 Conclusions
6.7 References
Chapter 7 The kynurenine pathway in a sub-Saharan HIV/AIDS population
7.1 Abstract
7.2 Introduction
7.3 Methods
7.4 Results
7.5 Discussion
7.6 Summary
7.7 References
Chapter 8 Final summation
8.1 Contribution of the study
8.2 Shortcomings of the study
8.3 Suggestion for further study
8.4 References
