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.