Development and validation of a HPLC-UV method for determination of Levofloxacin in lung tissue. Application to the quantification of the drug after pulmonary administration into biopolymeric microparticles

MARÍA ESPERANZA RUIZ; JUAN FRANCISCO MORALES; ANDREA ENRIQUE; MARÍA LAURA SBARAGLINI; ALAN TALEVI; GERMAN ISLAN; MAXIMILIANO CACICEDO; LUIS BRUNO-BLANCH; GUILLERMO CASTRO

Córdoba

Congreso; 3a. Reunión Internacional de Ciencias Farmacéuticas - Ricifa 2014; 2014

Universidades Nacionales de Rosario y de Córdoba

A HPLC-UV method for determination of Levofloxacin (LFX) in mouse lung tissue was developed and validated, in order to quantify the drug after pulmonary administration into biopolymeric microparticles, as a targeted delivery strategy useful for the management of pulmonary infections in patients with cystic fibrosis. Conditions: C18 column, acetonitrile/KH2PO4 30 mM pH 2.80 (22/78) mobile phase, 1.0 ml/min and 300 nm detection. Enrofloxacin (EFX) was used as internal standard (IS). For sample preparation, excised mouse lungs were homogenized, spiked with IS and diluted in 2 ml of physiologic solution. After 24 hs at 4 °C, two 0.9 ml-aliquots were extracted with 2 ml of dichloromethane and centrifuged. The separated organic layer was evaporated to dryness, and the residue resuspended with 100 μl of mobile phase prior to injection. In an initial validation stage, samples were prepared in blank homogenized lung tissue by the addition of known concentrations of LFX and EFX in methanolic solution. The response was linear between 0,02 (LQ) ? 15.00 μg/ml, precision RSD values were < 4.0% and accuracy was 90?100%. The method was specific to the biological matrix, and the drugs were stable in the matrix, methanolic solution and prepared samples. Recovery was around 70% due to the drug loss in the tissue, since the extraction procedure had a 95-100% recovery. Microparticles were prepared by co-precipitation of CaCO3 in presence of the alginate biopolymer. The obtained hybrid microparticles showed narrow size dispersion around 5 μm, ideal for pulmonary delivery. LFX was incorporated by absorption, reaching a loading of 40.0 µg/mg of matrix. Therefore, in a second validation stage, validation parameters were re-assessed using these microparticles instead of the methanolic solution, with similar results. Finally, the method was successfully applied to the quantification of LFX in lungs of mice sacrificed after receiving pulmonary administration of the microparticles.