Masters Degrees (Pharmacology)
Permanent URI for this collection
Browse
Browsing Masters Degrees (Pharmacology) by Author "Hundt, H. K. L."
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Open Access Development and validation of assay methods for the quantitative determination of drugs and their metabolites in biological specimens (piroxicam and nabumetone)(University of the Free State, 2001-05) De Jager, Andrew David; Hundt, H. K. L.; Swart, K. J.English: The development and validation of bioanalytical assay methods suitable for the quantitation of drugs in biological matrices is discussed, as well as general principles applicable to this particular aspect of drug development. Relevant literature is consulted, with a view to exemplifying what constitutes good assay method development strategy, as well as to reflect current international policy in this field, with particular reference to bioequivalence studies. Comparisons are made between international practices and those in place at FARMOVSPAREXEL Bioanalytical Services Division®. Attention is given inter alia to detector selection, chromatographic optimisation, extraction procedures and method validation, with reference to new assay methods for two drugs in particular that have been developed and validated according internationally acceptable standards. In the first instance, a highperformance liquid chromatographic method with ultraviolet detection was developed for the determination of 6-methoxy-2-naphthylacetic acid (6-MNA, the active metabolite of nabumetone ). The sample preparation involved a simple but effective protein precipitation procedure. Reversed-phase liquid chromatography was optimised, and full resolution between the analyte and endogenous matrix peaks achieved in a chromatographic runtime of five minutes. The assay method was validated over a range of plasma concentrations between 0.070 and 145 µg/ml. 1242 Plasma samples generated during a comparative bioequivalence study were then assayed and the performance of the assay method shown to be well within accepted international norms. The coefficient of variation for quality control standards over the range of 0.18 to 39 µg/ml processed during the assaying of the study samples, varied between 3.6 and 8.1 %. In the second instance, a novel method for the determination of piroxicam in four biological matrices (sub-cutaneous tissue (SCT), synovial fluid (SF), synovial capsule (SC) and plasma) was developed. A double back-extraction procedure was followed by reversed-phase liquid chromatography and electrochemical detection (ECD). Extracts from all four biological matrices were injected onto a single HPLC system. Ratios between plasma and the three remaining matrices were used to characterise transdermal absorption of two topical preparations of piroxicam when applied to the knee. Low systemic levels associated with topical formulations necessitated the development and validation of a highly sensitive assay method. Plasma was used as a surrogate matrix for all the processed tissue samples and the assay method was validated over a range of plasma concentrations between 1.24 and 600 ng/ml. 168 Samples generated during a multi-centre study involving knee replacement surgery, were assayed and the performance of the assay method shown to be well within accepted international norms. The coefficient of variation for quality control standards over the range of 1.74 to 300 ng/ml processed during the assaying of the study samples, varied between 7.7 and 13.5 % which can be considered excellent in the light of the complexity of the sample preparation process. Analytical data generated during the above-mentioned two research projects are discussed, with novelties and improvements to existing assay methods being elucidated. Both assay methods were presented and accepted for publication in peer reviewed scientific journals. Both full-length publications are included in an appendix in this dissertation, together with the correspondence entered into with journal editors and referees. Furthermore, a section containing copies of the slides used to present the latter HPLC assay method as an oral presentation at the 1998 Annual Congress of the South African Pharmacological Society, is included.Item Open Access The development and validation of quantitative methods for the determination of stavudine and alfuzosin in plasma and monic acid in urine(University of the Free State, 2003-05) Wiesner, Joachim Lubbe; Swart, K. J.; Hundt, H. K. L.English: The development and validation of bio-analytica) assay methods suitable for the quantification of stavudine in plasma, alfuzosin in plasma and monic acid in urine is discussed. A short summary of these methods are given: • A sensitive method for the determination of stavudine in plasma was developed, using highperformance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from plasma with Waters, Sep-Pak®Vac, 100 mg, tC18®solid phase extraction (SPE) columns. Chromatography was performed on a Supelco Discovery" C18, 5 urn, 150 x 2 mm column with a mobile phase consisting of ammonium acetate (0.01 M): acetonitrile: methanol (800:100:100, v/v/v) at a flow rate of 0.3 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS/MS) set at unit resolution in the multiple reaction monitoring mode (MRM). Atmospheric pressure chemical ionization (APCl) was used to obtain deprotonated ions (molecular ion m/z 223.1 to the product ion m/z 42.01). The mean recovery for stavudine was 94 % with a lower limit of quantification set at 4 ng/ml. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS/MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of stavudine in human plasma than has previously been published. The assay metod was used to quantitatively determine stavudine concentrations in plasma samples to follow the concentration vs. time profile for at least five half lives of the drug after a single 40 mg oral dose of stavudine was given to healthy adult male human subjects. • A selective, sensitive and rapid liquid chromatography-tandem mass speetrometry method for the determination of alfuzosin in plasma was developed. An Applied Biosystems API 2000 triple quadrupole mass speetrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray (TIS) with positive ionisation was used (molecular ion of alfuzosin m/z 390.2 to the product ion m/z 71.2; molecular ion of prazosin m/z 384.2 to the product ion m/z 95.0). Using prazosin as an internal standard, liquid-liquid extraction was followed by CI8 reversed phase liquid chromatography and tandem mass spectrometry. The mean recovery for alfuzosin was 82.9 % with a lower limit of quantification set at 0.298 ng/ml, the calibration range being between 0.298 and 38.1 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric (MS/MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of alfuzosin in human plasma than has previously been published. The assay method was used to quantify alfuzosin in human plasma samples generated in a multiple-dose (5 mg bd.) study at steady state. • Two selective methods for the determination of monic acid (a metabolite of mupirocin) in urine were developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. An Applied Biosystems API 2000 triple quadrupole mass spetrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray (TIS) with positive ionisation, was used (molecular ion of monic acid m/z 345.2 to the product ion m/z 327.0) The minimal sample preparation and short chromatography time (retention time ~ 3.8 min.) makes these methods suitable for the assay of large numbers of samples per day. Linearity (weighted 1/concentratiorr²) was established from 50.1 to 1001 ng/ml for the direct injection method, and linearity (weighted 1/concentration) was established from 15.8 to 1013 ng/ml for the SPE method. The assay method was used for the determination of monic acid concentrations in human urine in order to detect and quantify the absorption of mupirocin after multiple topical applications ofO.5 g of a 2 % ointment. Analytical data that were generated during these three research projects are discussed in this dissertation, improvements and novelties to existing methods are elucidated. The methods for the determination of stavudine and alfuzosin have been published. Both full-length publications are included in this dissertation, together with correspondence between myself and the journal editors and referees. The assay methods for monic acid will soon be submitted for publication in the Journal of Chromatography B.