Development and validation of a Reverse-phase Liquid Chromatographic method for simultaneous determination of timolol maleate and Human serum albumin in albumin nanoparticles

C. BOIERO; M. LONGHI; D. ALLEMANDI; J. LLABOT

Pamplona

Congreso; 19° Simposio Internacional de Microencapsulacion; 2013

International Microencapsulation Society

Topical application onto the eye´s surface is a common route of drugs administration; however physiology of the eye itself makes it difficult to achieve effective concentrations of drug for extended periods of time. Nanoparticles (Nps) have emerged as a suitable vehicle for the administration of drugs due to in many cases a significant increase of drug bioavailability was observed. Macromolecular substances (synthetic or natural polymers) can be used in the preparation of Nps, wherein human serum albumin (HSA) emerges as a suitable material (nontoxic, non-immunogenic, biocompatible and biodegradable). Our work focuses on the production of HSA-NP for the topical administration of Timolol Maleate (TM) for the treatment of glaucoma. One of the most important parameters to characterize these Nps is to evaluate the encapsulation yield and process performance besides the phisico-chemical characteristics like particle size, polydispersity index, Z potential, yield, and morphology (SEM). For this reason we have developed a one step HPLC method, which is simple, rapid and reproducible for the simultaneous determination of HSA and TM. HSA-Np were prepared by a desolvation method with addition of ethanol to a 2% solution of HSA (1:2) under continuous stirring. Then, coacervates were hardened by crosslinking with glutaraldehyde (1,56 μg/mg protein) for 5 h with continuous stirring. Then, the ethanol was eliminated by evaporation under reduced pressure, and the Nps were purified by centrifugation to eliminate the free HAS, TM nonencapsulated and excess cross-linking agent. Timolol maleate (TM, 55 μg /mg protein) was incorporated into the HSA solution before adding ethanol. Free HSA and TM were determined by HPLC. 0.05% (v/v) trifluoroacetic acid in acetonitrile [40:60 (v/v)] as mobile phase was pumped at a flow rate of 1 ml/min, a reverse phase Waters symmetry C18 (250 mm x 4.6 mm, 5 µm particle) column was used at 25°C and the peak response was monitored at a wavelength of 276 nm. 20 μL of sample were injected into the HPLC system and the data were acquired using the Empower Software. For the validation of the analytical method the following parameters were determined: linearity, accuracy, precision, selectivity, limits of detection and quantification, and stability for HSA and TM. The range of linearity was from 20 ? 0,01 mg/ml for HSA and 100 ? 0,3 µg/ml for TM. The peak area ratio and concentration of each drug was subjected to regression analysis to calculate the calibration equations and correlation coefficients. The results showed that within the concentration range mentioned above, there was an excellent correlation between peak area ratio and concentration. Also, this HPLC method showed good sensitivity and selectivity and is suitable for cuantification of HSA and TM simultaneus.