DEVELOPMENT OF SELECTIVE AND SPECIFIC SOLID-PHASE EXTRACTION METHODS FOR DETERMINATION OF SALBUTAMOL AND ITS MAJOR METABOLITE IN BIOLOGICAL SAMPLES
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Date
2002-12
Authors
KOH, YEW MING
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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.
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Keywords
SALBUTAMOL , METABOLITE