IJUP08 - Universidade do Porto

Implementation of automatic methodologies for quality control of
pharmaceutical formulations containing allopurinol based on
biocatalysts
C. I. C. Silvestre, M. A. Segundo, M. L. M. F. S. Saraiva and J. L. F. C. Lima
REQUIMTE, Serviço de Química-Física, Faculdade de Farmácia, Universidade do Porto,
Rua Aníbal Cunha, 164, 4099-030, Porto, Portugal
Email: cristina.silvestre@gmail.com
Allopurinol is widely used in the management of hyperuricaemia, due to its capacity to
inhibit xanthine oxidase [1]. Xanthine oxidase, a metal flavoprotein, is the last enzyme in
the pathway for the degradation of purine derivatives and therefore the rate limiting step in
purine metabolism. Its main role is to oxidise hypoxanthine and xanthine to uric acid.
In the present work, two automatic sequential injection analysis methodologies were
developed for the enzymatic determination of allopurinol. Both methodologies were based
on the inhibition by allopurinol of the reaction catalysed by xanthine oxidase in vivo,
assessed by spectrophotometric detection of the reaction product (uric acid) at 295 nm.
Furthermore, one methodology was based on a single readout at a fixed-time after reaction
while the other was based on the decrease of the rate of formation of uric acid, evaluated
by monitoring the absorbance change during a pre-set time interval.
Both methodologies allowed fast quantification of allopurinol in pharmaceutical samples
(n = 8), providing results that were not significantly different from those obtained by the
British Pharmacopoeia procedure. The determination frequency was 15 and 34
determinations per hour for the fixed-time and kinetic method, respectively.
The developed methodologies allowed the automation of the determination of allopurinol
with the purpose of its quality control, implementing Green Chemistry concepts [2] in the
Analytical Chemistry area. In this particular application, low quantities of reagents (µL
range) were used and a low volume of effluent was produced (< 5 mL) per assay.
Furthermore, the utilization of a biocatalyst for this determination, instead of rather toxic
reagents, also makes it more environmentally friendly.
References:
[1] Borges, F., Fernandes, E., Roleira, F. (2002), Progress towards the discovery of xanthine
oxidase inhibitors, Current Medicinal Chemistry, 9 (2), 195-217.
[2] Prado, A. G. S. (2003), Química Verde, os desafios da química do novo milénio, Química Nova,
26 (5), 738-744.
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