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 E. RUIZ; JUAN F. MORALES; ANDREA V. ENRIQUE ; MARÍA L. SBARAGLINI ; ALAN TALEVI; GERMÁN A. ISLAN ; MAXIMILIANO L. CAICEDO ; LUIS E. BRUNO-BLANCH ; GUILLERMO CASTRO

Córdoba

Otro; III Reunión Internacional de Ciencias Farmacéuticas ? RICIFA 2014; 2014

: Departamento de Farmacia, Universidad Nacional de Córdoba

A HPLC-UV method for determination of Levofloxacin (LFX) in mouse lung tissue was developed and validated. Conditions: C18 column, acetonitrile/KH2PO4 30 mM buffer pH 2-80 (22/78) mobile phase, 1.0 ml/min and 300 nm detection. Enrofloxacin (EFX) was used as internal standard (IS). Sample preparation comprised the homogenization of excised mouse lungs spiked with the 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 organic layer was separated and evaporated to dryness, and the residue resuspended in 200 μl of mobile phase prior to inyection. In an initial validation stage, samples were prepared in blank homogenized mouse lung tissue by the adition of known concentrations of LFX and EFX in methanolic solution, and linearity, precision, accuracy, limit of quantification (LQ), specificity and stability were assessed. The response was linear in the range 0,05 (LQ) ? 30.00 μg/ml. Precision RSD values were < 5.0% and accuracy was between 90?100%. The method was specific to the biological matrix, and the drugs were stable in methanolic solution, biological matrix and prepared samples. The recovery was constant and between 60-70% due to the drug loss in the lung tissue, since the extraction procedure had a 95-100% recovery. As the method is intended for the quantification of the drug after nasal administration of biopolymeric microparticles, precision, accuracy, recovery and stability were also determined using microparticles with a known concentration of LFX instead of the methanolic solution. Microparticles were prepared by co-precipitation of calcium carbonate in presence of the alginate biopolymer. The obtained hybrid microparticles showed a narrow size dispersion around the 5 micrometers, an ideal size for pulmonary delivery. Levofloxacin was incorporated by absorption, reaching a LFX loading of 40.0 µg per miligram of matrix.. Finally, the method was successfully applied to the quantification of LFX in a mouse lung after nasal administration of polymeric microparticles containing the drug.