CONICET | Buscador de Institutos y Recursos Humanos

Introduction Pipemidic acid (PA) is a synthetic quinolone that belongs to the first generation of this kind of compounds, which is used as antibacterial agent. The antibacterial property of the quinolones is associated with their potential to inhibit the bacterial topoisomerase II (1). PA is widely used for the treatment of urinary tract infections, showing high activity against gram-positive and negative bacteria. Several methods for the determination of PA have been developed (2-4) but the use of microfluidic enzymatic sensors with electrochemical detection represents an interesting option to be considered for the PA determination, because these devices offer many potential advantages (5). For these reasons, we have developed a very sensitive device based in the presence of tyrosinase immobilized on APCPG particles contained into of the central channel (CC) of the microfluidic system, where occurs the enzymatic reaction for the indirect-PA determination. Materials and methods All reagents used were of analytical reagent grade. The main body of the sensor was made of Plexiglas. The gold electrode is at the end of the CC. The CC containing 0.3 mg of controlled-pore glass, and the end of the CC was blocked with glass fibers. The diameter of the CC was 150 µm and the diameter of the accessory channels were 100 µm. The potential applied to the gold electrode was 0 V vs the Ag/AgCl wire pseudo-reference electrode and a Pt wire was the auxiliary electrode. Amperometric detection was performed using a BAS LC-4C potentiostat and BAS 100 B/W (USA), which was used to voltammetric determinations. Syringe Pumps Systems were used for pumping, sample introduction, and stopping flow. Results and discussion The measuring principle of this biosensor for the determination of PA in pharmaceutical formulations is as follows. First, the tyrosinase immobilized on APCPG particles catalyzes the oxidation of catechol (Q) to o-benzoquinone (P), whose electrochemical reduction back to Q was obtained at potential of 0 V. Second, the detection of the PA was accomplished for the suppression of the substrate recycling process between tyrosinase and the electrode (denoted by the dotted arrow), decreasing the peak current obtained proportionally to the increase of the PA concentration. A linear relation, ΔI (nA) = 311.37 4.443 [CPA] was observed between the ΔI and the PA concentration in the range of 0.02 to 70 µM. The linear regression coefficient and the detection limit (DL) were r = 0.998 and 18 nM, respectively. Reproducibility assays were made using repetitive standards solutions (n=5) containing 1.0 mM Q and 10 µM PA, and the coefficient of variation (CV) for this study was below 3%. The developed microfluidic-biosensor for the PA determination was applied to two commercial preparations. There is no need for any extraction procedure before of the analysis. No change of the peak height in the presence of the excipients was observed. Conclusions In this article we have showed the usefulness of the microfluidic-enzimatic sensor with electrochemical detection, applied to the determination of very low concentrations of pipemidic acid in pharmaceutical samples. This sensor provides a cost effective solution to obtain good quantitative information and wide applicability in the pharmaceutical industry as quality control method. Acknowledgements The authors wish to thank the Universidad Nacional de San Luis, the Agencia Nacional de Promoción Científica y Tecnológica, and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) for their financial support. References (1) Barrett J.F., Bernstein J.I., Krause H.M., Hilliard J.J., Ohemeng K.A., Testing potential gyrase inhibitors of bacterial DNA gyrase: A comparison of the super coiling inhibition assay and ´cleavable complex´ assay, Anal. Biochem. 214 (1993) 313-317. (2) Meras I.D., De La Pena A.M., Lopez F.S., Caceres Ma.I.R. Complexation of antibacterial quinolonic acid and cinolonic derivatives with Zn(II) and Al(III): Application to their determination in human urine, Analyst 125 (2000) 1471-1476. (3) Cañada-Cañada F., Espinosa-Mansilla A., Muñoz de la Peña A., Separation of fifteen quinolones by high performance liquid chromatography: Application to pharmaceuticals and ofloxacin determination in urine, Journal of Separation Science 30 (2007) 1242-1249. (4) Llorent-Martínez E.J., Ortega-Barrales P., Molina-Díaz A., Multicommuted optosensor for the determination of pipemidic acid in biological fluids, Anal. Biochem. 347 (2005) 330-332. (5) Park, K.H., Park, H.G., Kim, J.H., Seong, K.H., Poly(dimethyl siloxane)-based protein chip for simultaneous detection of multiple samples: Use of glycidyl methacrylate photopolymer for site-specific protein immobilization, Biosens. Bioelectron. 22 (2006) 613-620.