Razvoj nove analitičke metode ultra brze tečne hromatografije sa tandem masenim detektorom za određivanje ambroksol hidrohlorida i njena primena
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Ambroxol hydrochloride is an expectorant agent, successfully applied in mucolytic therapy for acute and chronic bronchopulmonary diseases. The drug regulates not only mucus secretion but also showed antioxidant, anti-inflammatory and local anesthetic properties. Ambroxol is applied in mucolytic therapy of acute and chronic diseases associated with increased production and disruption of the formation and transportation of mucus. The purpose of this study was to develop an ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC–MS/MS) method for the simple determination of the ambroxol in serum with in vivo and in vitro application. The validation of the method was performed according the ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines Q2A/Q2B for the validation of bioanalytical methods. Linearity, precision, extraction recovery, carryover, and stability test of the spiked serums stored under diff...erent conditions satisfy the acceptance criteria. The presented method was found to be appropriate for all validation parameters. The chromatographic separation was achieved in a submicron Kinetex RP - C18 column (2.1 mm x 50 mm, 1.3μm) using the mobile phase methanol – 0.01% formic acid aqueous solution (90:10 v/v) at a flow rate of 0.3 mL/min. The ESI mass spectrometry in the MRM mode was used with a typical transitions m/z 378.9→263.8 for ambroxol and m/z 455.2→165.0 for IS at collision energies -20 V and -25 V, respectively. The admitted voltages for interface and detector were 4.5 kV and -1.92 kV. Linearity was determined with an average coefficient of determination >0.999 over the dynamic range from 0.5 - 200 ng/mL with LOD and LOQ of 0.25 ng/mL and 0.5 ng/mL, respectively. The results of the intra- and inter-day precision and accuracy determined in different days were all found to be within the acceptable limits ±15%. The reported UHPLC–MS/MS method was proved to be reliable and highly sensitive for the determinations of ambroxol in short analysis run time for an only minute and a half. Consequently, the method is enabled a hundred of injections per day which makes the validation process faster and more efficient. Developed a new UHPLC–MS/MS method of ultra high - performance liquid chromatography - tandem mass spectrometry for the determination of ambroxol, was proven useful in the in vivo and in vitro assays. The present method was applied to the in vivo pharmacokinetic study after a single oral dose administration of 30 mg ambroxol tablet to rabbits, and to the in vitro dissolution tests according the relevant pharmacopeia. The new method could be applied in the pharmaceutical industry in the study of new pharmaceutical forms of ambroxol. This also applies to the selection of the optimal pharmaceutical formulation ambroxol checking the pharmacokinetics of the drug on rabbits before the bioequivalence study in humans.