DEVELOPMENT OF SELECTIVE AND SPECIFIC SOLID-PHASE EXTRACTION METHODS FOR DETERMINATION OF SALBUTAMOL AND ITS MAJOR METABOLITE IN BIOLOGICAL SAMPLES

DEVELOPMENT OF SELECTIVE AND SPECIFIC SOLID-PHASE EXTRACTION METHODS FOR DETERMINATION OF SALBUTAMOL AND ITS MAJOR METABOLITE IN BIOLOGICAL SAMPLES

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dc.contributor.author	KOH, YEW MING
dc.date.accessioned	2016-01-22T07:31:34Z
dc.date.available	2016-01-22T07:31:34Z
dc.date.issued	2002-12
dc.description.abstract	Selective and specific sample preparation has been the focus of attention in recent rears, particularly in chromatographic analysis. Conventional sample preparation methods such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE) had been widely and Commonly used in the analysis of salbutamol in biological samples for the past 50 years. However, these methods lack selectivity to extract salbutamol from "dirty" samples. fherefore, one of the main objectives was to develop a selective sample preparation method. The method involved the fonnation of salbutamolphenylboronate complex using phenylboronic acid reagent under basic condition prior to the solid phase clean-up step. The complex was retained in the end-capped CIS solid phase and the endogenous interference was removed by washing the CIS cartridges with 50 mM sodium carbonate buffer pH 9.60. Salbutamol was then eluted from the solid phase after dissociation of the complex using acidic buffer depending on the sample matrix and the drug was analyzed by reversed phase high perfonnance liquid chromatography (HPLC) with fluorimetric detection. The extracti~n efficiency of the phenylboronic acid/CIS SPE was approximately 88 - 90 %. This selective clean-up method was applied to study the difference between oral and inhaled administration of salbutamol in human by means of chiral separation. The final eluent from SPE was dried and reconstituted with the mobile phase and analyzed by nonnal phase chiral HPLC with fluorimetric detection. The enantiomeric ratio of S( + )-salbutamollR( -)-salbutamol and S( + )-salbutamol SlllfatelR( -)-salbutamol sulfate of the urine and plasma samples after oral and inhaled administration of salbutamol were calculated and analyzed statistically. The Abstract result shows that there is a significant difference between the two administration routes of salbutamol in human. The development of immunoaffinity SPE method as the specific sample preparation for biological samples was the other one of the main objectives of this study. By ll:Sing the model compound salbutamol, production of polyclonal antibody was carried out. The salbutamol antiserum produced was coupled to solid matrix namely Cyanogen bromide (CNBr) activated Sepharose gel and this immunosorbent was eventually optimized for application to biological samples. The optimization of the immunoextractionprocedures placed emphasis on the loading of samples into the immunocolumns, waShing to remove the unbound materials and the elution of salbutamol from the columns in the minimwn volume possible. The antibody-drug interaction is specific, mainly due to hydrophobic and ionic interaction as indicated by the use of mild pH elution buffer in association with polarity reducing agents (organic modifiers). The extraction efficiency of the immunoaffinity SPE was more than 98 %. The immunoextraction procedure for salbutamol was optimized for elution in a single 1 mL elution fraction that was analyzed directly by HPLC. High column capacity was desirable in the immunoaffinity columns as this made the columns reusable. This immunoextraction procedure was applied to isolate the major metabolite of salbutamol from human urine samples. Identification of the metabolite was done using liquid chromatograph-mass spectrometer (LC-MS). From the results obtained, the major metabolite ofsalbutamol was.identified as salbutamol-4'-O-sulfate and thus further investigation should be carried out to quantify the metabolite in both urine and plasma samples in future.	en_US
dc.identifier.uri	http://hdl.handle.net/123456789/1685
dc.subject	SALBUTAMOL	en_US
dc.subject	METABOLITE	en_US
dc.title	DEVELOPMENT OF SELECTIVE AND SPECIFIC SOLID-PHASE EXTRACTION METHODS FOR DETERMINATION OF SALBUTAMOL AND ITS MAJOR METABOLITE IN BIOLOGICAL SAMPLES	en_US
dc.type	Thesis	en_US

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