Nanoparticles as carrier for improve therapeutic efficacy of pioglitazone in ocular inflammatory disorders: development and validation of a high throughput LC–MS/MS method for quantitation in ocular tissues.

Pioglitazone is an oral anti-hyperglycemic agent and it is used for the treatment of diabetes mellitus type 2. The anti-inflammatory activity has also been demonstrated in the literature. Pioglitazone belongs to Class II of Biopharmaceutical Classification System, i.e., low soluble and high permeable. Polymeric nanoparticles formulations play an important role in the improvement of the efficacy of ocular therapies. These systems are non-toxic and biodegradable, show appropriate physicochemical characteristics as well as prolonged release profile suitable for ocular delivery.

An accurate, sensitive, selective, reproducible and high throughput LC-MS/MS method was validated to quantitate pioglitazone in ocular swine tissues (cornea, sclera, lens, aqueous humour and vitreous humour). The chromatographic separation was achieved in 10 min on a Kinetex C18 column at 35°C, using a mobile phase composed of formic acid 0.1% in water and formic acid 0.1% in acetonitrile in gradient mode at a flow rate of 0.6 mL/min. The detection in the MRM mode for pioglitazone was 357.2/134.1 for quantitation (most sensitive), and 357.2/119.1 for confirmation. Linear response of pioglitazone was observed over the range of 5–100 ng/mL. Tissues were spiked with pioglitazone to obtain final extract levels in ng. The limit of quantitation was in 10 ng/ml in extract. The recovery of pioglitazone was in the range 70-120% % in all tissues and levels tested. The intra-day precision was < 3% and the inter-day precision was <7%. The obtained extracts demonstrated to be stable under various experimental conditions in all the studied matrices. The recovery of pioglitazone spiked at the levels of the validation study, in the form of a nanoparticle formulation of polylactic-co-glycolic acid-polyethyleneglycol (PLGA-PEG), was also found in the range 70-120 % with a precision <7%. This method can be applied to in vivo and ex vivo biodistribution studies related to the ocular administration of pioglitazone nanoparticles.