Chemical and physical stability of selected drugs for aerosol therapy with Sirmione thermal water

Respiratory diseases represent an important public and private expense. However, the impact is not only economic, but other factors must be considered: in pediatric patients, diseases such as rhinosinusitis, rhinitis, asthma, and upper and lower respiratory tract infections have extremely relevant social and clinical implications. In Italy, a quarter of the medical consultations relating to primary care in pediatric patients concern respiratory problems [1]. The airway allows the medication to arrive directly at the nasal fossa without systemic distribution, with a high therapeutic effect and low undesirable effects [2-4]. The most-commonly prescribed nebulizers for the treatment of upper and middle respiratory tract diseases are corticosteroids, mucolytics agents, and antibiotics [5-7]. In order to improve the drug administration, the molecules were mingled with 0.9% or 3% sodium chloride solutions and subsequently administered via aerosol. The use of these sodium chloride solutions enhances not only the inhaled drug’s permeability and bioavailability [8], but also the therapeutic effect, leading to the improvement of the mucociliary clearance time [9]. Currently, in common clinical practice, Sirmione thermal water is also used as a diluent, especially for mucolytic agents and corticosteroids. Because of the high concentration of sodium chloride, carbonates and calcium salts, the saltybromine-iodine thermal water of Sirmione exhibits hypertonic properties that lead to a fl uidifying and cleansing action (Table 1). Abstract

The water of Sirmione has an adjuvant action in the treatment of both infectious and allergic rhinitis and sinusitis thanks to: a) its mucolytic and unblocking action, due to the sulfur content, especially present as hydrogen sulfi de, moreover, it has a vasodilating and stimulating action on the secretory IgA of the nasal mucus and it can improve the rheological characteristics of the mucus, b) its antiseptic action, due to iodine content, c) its moisturizing action, due to the mineral complex [10]. These properties make the thermal water of Sirmione suitable not only for aerosol therapy, but it can also be used as a diluent for active drugs for inhalation use. This study aims to evaluate the stability in aerosol containers of two mucolytic agents, N-acetylcysteine (300 mg/3 ml) and ambroxol (15 mg /2ml), and two corticosteroids beclomethasone dipropionate (800 μg /2ml) and budesonide (0.50 mg /2ml and 0.25 mg /2ml), after dilution with Sirmione thermal water stored in plastic ampoules, for aerosol therapy. A large number of analytical methods for the determination and quantifi cation of N-acetylcysteine [11,12], ambroxol [13,14], budesonide [15] and beclomethasone dipropionate [16,17] in pharmaceutical preparations have been developed. However, in spite of the structural diversity and different chemical-physical properties of these drugs (Figure 1), mainly due to the presence of excipients (sodium edetate, polysorbate 80, polysorbate 20, and sorbitan laurate) that can interfere with the separation process, no method was found for their simultaneous analysis. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) allowed the optimization of the method and the separation of the four drug in a short analysis time.

Sam ple preparation
Triplicate samples of tested drugs were analyzed immediately after dilution with thermal water of Sirmione (fi nal volume 5 ml) at 1 hour, 6 hours, 12 hours and 24 hours.
Samples were stored in aerosol ampoules at room temperature and exposed to normal fl uorescent light. The fi nal concentration of the solutions obtained were 30 mg/ml for ambroxol, 0.16 mg/ml for beclomethasone dipropionate, 0.05 mg/ml and 0.10 mg/ml for budesonide and 60 mg/ml for N-acetylcysteine, respectively. To avoid the solvent's evaporation, the aerosol ampoules were sealed with Parafi lm ® . The pH of the solutions was determined before each analysis using a pH meter (HBasic 20, Crison Instruments S.p.A., Modena, Italy). Analyzes were performed in triplicate (n = 45).The pH was measured immediately after dilution with thermal water of Sirmione at 1 hour, 6 hours, 12 hours and 24. The sterility of the samples was not assessed.

Quantitative analysis using UHPLC/MS/MS
The UHPLC system consisted of an Agilent Technologies 1200 series system equipped with a degasser, binary pump and a high-performance autosampler (HiP ALS SL+) with a thermostatic column compartment. Separation was carried out using a reversed-phase Zorbax Eclipse Plus, 2.1 x 100 mm, 1.8 μm (Agilent, Santa Clara, CA, USA), operating at 0.3 ml/min. The mobile phase consisted of 10mM ammonium formate/ formic acid 0.1% in acetonitrile 80:20 (v/v) as solvent A and 10mM ammonium formate/formic acid 0.1% in acetonitrile 20:80 (v/v) as solvent B. The initial conditions were 95% A and 5% B. The mobile phase B linear gradient was applied as follows: mobile phase B (5%) was held for 2 min, then it was increased to 95% in 6.0 min and held at this percentage for 0.5 min. This procedure was followed by the reconstitution of starting conditions within 0.1 min, and by the equilibration with 5% of B for 0.5 min, resulting in a total analysis time of 11 min. UHPLC fl ow rate was set at 400 μL/min and the column temperature at 40 °C.The i njection volume was 5 μl. The analysis was performed with the UHPLC tandem 6460 triple quadrupole mass spectrometer (Agilent Technologies, Inc.). An Agilent Mass Hunter workstation was used for the control of equipment, data acquisition, and analysis. The 6460 Agilent featuring an electro-spray ionization (ESI) interface served for MS/MS analysis. The source operated with the Jet Stream technology and a super-heated sheath gas helped to enhance sensitivity. The ion source parameters for negative mode were as follows: vaporizer temperature, 250 °C; sheath gas, 11 mL/ min; temperature, 250 °C; nozzle voltage, 400 V and capillary voltage, 3000 V. Finally, nitrogen was used as nebulizer gas and was set at 45 psi with a fl ow rate of 5 L/min.

Data a n alysis
The initial concentration was considered as 100% and the subsequent concentrations were expressed as a percentage of the initial value. Following the acceptability criteria indicated by the Offi cial Italian Pharmacopoeia, the degradation of a molecule greater than 10% of the initial concentration is considered unacceptable in terms of loss of activity [18]. The accuracy and precision of our assay met standards set for bioanalytical method validation [19].

Results and discussion
The dev elopment of the analytical method allowed the simultaneous analysis of the four different drugs with a total analysis time of 11 minutes. In this study, a triple quadrupole mass spectrometer was used as a detector that allowed the optimization of the mass transitions of the individual compounds. In particular, the following selected reaction monitoring (SRM) transitions were used: N-acetylcysteine 164→59, ambroxol 376→261, budesonide 521→503, and beclomethasone dipropionate 431→413. Figure 2 shows the separation of the analytes in the Total Ion Chromatogram (TIC).
The method was found to be linear in concentrations between 500-5000 ng/ml for ambroxol, budesonide and beclomethasone dipropionate (r 2 = 0.9975, 0.9964 and 0.9975, respectively) and between 50-500 ng/ml for N-acetylcysteine (r 2 = 0.9962). Peak areas were used for the determination of the different drug concentrations. The coeffi cients of regression (r 2 ) for all of the standard curves were found to be between 0.98 and 0.99, during the whole study. Intraday and interday coeffi cients of variation for acetylcysteine samples were ranked from <6% at 80 μg/mL to <2% at 220 μg/mL. Table  1 shows the stability data of the four molecules analyzed in aerosol ampoules at room temperature under light exposure, after dilution with Sirmione thermal water. As we can see in Table 2, all drugs exhibited optimal stability at +1 hours, +6 hours, +12 hours, and +24 hours.
The average percentage of degradation found for each molecule was: 0.3% for N-acetylcysteine, 0.6% for ambroxol, 0.7% for budesonide (0.25 mg/ml), 0.5% for budesonide (0.50 mg/ml) and 0.2% for beclomethasone dipropionate. All solutions had a pH of 6.82+0.4 for the entire study. UHPLC belongs to a new category of separative techniques based on established principles of liquid chromatography. The stationary phase is composed by particles smaller than 2 μm. Furthermore, the mobile phase can operate at high speeds and this positively infl uences the resolution, sensitivity, and speed of the analysis. Due to its speed and sensitivity, this technique has gained considerable attention in recent years in the fi eld of pharmaceutical and biomedical analysis [20]. A large number of factors can affect the stability of a pharmaceutical product, that includes manufacturing process, stability of the molecule, storage conditions (temperature, exposure to light and humidity), as well as chemical reactions such as oxidation, reduction, hydrolysis, and racemization, which could occur after dilution of the drug before administration. The analyses carried out allowed not only the determination of the stability of the four drugs studied, but also the exclusion of degradation products, and impurities already known in literature for all tested drugs [12,15,16,21] (Figure 3).

Conclusion
The proposed method was successfully developed and allowed to defi ne an effi cient, simple, rapid, sensitive, and economical protocol for the evaluation of the stability of ambroxol (15 mg/2ml), beclomethasone dipropionate (800 μg/2ml), budesonide (0.50 mg/2ml and 0.25 mg/2ml) and