2.1. Chemicals and reagents
Tiropramide hydrochloride was procured from TCI chemicals (Hyderabad, India). All solvents used for the studies are of HPLC grade. Chemicals, solvents and reagent were obtained from following suppliers: Sodium hydroxide (NaOH) and ammonium formate (SRL, Pvt. Ltd., Maharashtra, India); Hydrochloric acid (HCl) (Merck, Mumbai, India); HPLC grade methanol (Duksan Pure chemicals, Hyderabad, India); Hydrogen peroxide (H2O2) (s.d. Fine-Chem Ltd., Mumbai, India); Type 1 ultrapure water was generated from ultra-clear TWF system (Evoqua Water Technologies, Chennai, India).
2.2. Apparatus and equipment
Hotplate (IKA RCT Basic, Bengaluru, India) is used to carry out forced degradation studies. The weighing balance was from Sartorius (Mumbai, India). An ultrasonicator from PCI Analytics PVT Ltd., (Mumbai, India), pH of the mobile phase and buffer solutions were adjusted using pH meter from Eutech Instruments (Hyderabad, India). Thermal degradation was carried out in the Osworld laboratory oven (Osworld Scientific Pvt. Ltd). For photostability studies, samples were kept in photostability chamber (Newtronic Lifecare Equipment PVT Ltd., Mumbai, Maharashtra, India).
The study samples were analysed by using the HPLC-PDA system (Waters, 2695 series, Milford, MA, USA) equipped with Agilent Eclipse (250 × 4.6mm, 5 µm) which is controlled by Empower 3 software to separate all the degradation products formed. The high-resolution mass spectra of tiropramide and its degradation products were acquired by using Agilent 1200 infinity series UHPLC connected with G6540B Q-TOF LC-MS/MS (Agilent Technologies, USA). Mass Hunter Workstation software was used for the acquisition and processing of the data, respectively. The mass spectrometric data was acquired with electrospray ionization (ESI) in positive mode. Parameters optimised for mass spectrometric studies include, fragmentation voltage (175 V), capillary voltage (3500 V), skimmer voltage (65 V), octopole RF (750 V), nitrogen as drying (12 L/min) and nebulizing gas (55 psig). Mass range of 50–1700 m/z for full scan mode and 50–1200 m/z for MS/MS studies and nitrogen as collision gas (10–30 eV) were used to obtain data with source temperature of 300°C.
Preparative HPLC (Waters, MA, US), which was equipped with a UV/VIS detector (Waters 2489) was employed for isolation of degradation products for NMR studies. Data acquisition was performed under the control of empower software. Waters XBridge Prep C18 (19 x 250 mm, 5 µm column was used for the preparative isolation of major degradation products. The isolated samples were dried using rotary evaporator (Buchi Pvt. Ltd., Mumbai, India) and submitted for further characterization studies.
Nuclear Magnetic Resonance (NMR) experiments (1D and 2D NMR) were carried out using AVANCE III HD 500 MHz NMR, Bruker (Switzerland). DMSO-d6 was used as a solvent. 1H and 13C chemical shift values were reported on the δ scale in ppm relative to that of TMS (δ = 0.00 ppm) as an internal standard. The data acquisition and processing of NMR spectra were performed using Topspin software (3.2 version).
2.3. Forced degradation studies
Tiropramide was subjected to different stress conditions like hydrolysis (acidic, basic and neutral pH levels), oxidative, thermal and photolytic conditions as per ICH Q1A (R2) and ICH Q1B. All the stress studies were carried out at a final drug concentration of 1 mg/ml which was prepared in methanol: water (50:50, v/v) as diluent. Acidic and alkaline hydrolytic studies were initiated by adding equal volumes of drug stock solution and degradation medium i.e., 1N HCl for acidic pH and 1N NaOH for basic pH. For neutral pH level, same proportions of drug stock solution and diluent were added. All the samples prepared were subjected to heating at 70 ºC on an oil bath heated on hotplate. The samples were withdrawn at their respective time points, and were neutralized with either acid or base (500µL stress sample + 300µL diluent + 200µL acid/base). Oxidative degradation was carried out by adding 3% H2O2 to the drug stock solution and the same was kept at room temperature for 1 hour in dark place. The reaction was stopped by adding 50 µL of sodium thiosulphate (10 mM) a reducing agent for quenching the oxidative reaction. Thermal decomposition studies were performed on solid sample of the drug conducted in amber coloured vial and exposed to dry heat at 60 ºC for 30 days by keeping it in hot air oven. Photolytic studies were carried in solution (acidic – 0.1 N HCl, basic – 0.1 N NaOH and neutral conditions) as well as in solid form of drug, by exposing them to ultraviolet (NLT 200W h/m2) and fluorescent lamp (NLT 1.2 million lux hours) for 7 days. All the samples after withdrawal were carefully preserved in refrigerator for further analysis.
2.4. HPLC and HRMS studies
The method development was started with literature review before conducting the stress studies. Information was gained about the maximum wavelength, solubility of drug in different solvents, type of buffers to be used, previous HPLC methods if present. Various chromatographic conditions like pH of buffers, gradient conditions, flow rate and column temperature were varied systematically to achieve final optimised HPLC method. The final optimised method for the separation of DPs generated in stress study comprises of Agilent Eclipse plus C18 (4.6 x 250mm; 5µm) column as stationary phase along with methanol and ammonium formate (10 mM, 3.60 pH) as organic and aqueous mobile phases in gradient elution (% B composition was maintained at 10% up to 3.00 min, then it was linearly increased to 90% in 32 min, % B composition was kept constant at 90% up to 35 min, then % B was adjusted back to 10% in 40 min and was equilibrated up to 45 min. Flow rate and injection volume were optimised as 1.0 mL/min and 10 µL, respectively. The photo diode array (PDA) chromatogram of the drug was extracted from 210–400 nm range which gave the maximum UV absorption at 230 nm in the spectrum.
Mass spectrometric studies of the drug substance and DPs were carried out by using LC/ESI/QTOF/MS/MS for acquiring high resolution mass spectrometry (HRMS) data. Same HPLC method was transferred for mass studies by adjusting flow rate to 0.4 mL using T-union to mass spectrometer and 0.6 mL was routed to waste. Other parameters were optimized in order to get good spectra showing the important information regarding fragments which was further used to draw fragmentation pattern of drug aiding for structural characterization of DPs.
2.5. Isolation of DPs and NMR studies
Isolation of DPs were carried out for DP2, DP3 and DP5 for NMR studies. DP1 and DP4 could not be isolated as they were formed in very less percentages as, DP1 and DP4 were formed in very less percentages. Stock solution of drug with the concentration of 3 mg/mL was prepared and exposed with degradation media i.e., HCl and H2O2 for acidic hydrolytic and oxidative conditions, respectively for the isolation of DPs. The generated DPs were separated by preparative HPLC. All the collected fractions were dried on a rotary evaporator. The dried residues of all DPs were dissolved in DMSO-d6 as solvent in each individual 5 mm NMR tubes. Subsequently, for drug and DP5, 1 D (1H, 13C and Distortionless Enhancement by Polarization Transfer-135 (DEPT-135)) and 2D (Correlation spectroscopy (COSY), Heteronuclear Single Quantum Coherence (HSQC) and Heteronuclear Multiple Bond Correlation (HMBC) were acquired and 1H NMR spectrum for DP2 and DP3 were acquired. NMR spectra were obtained at the temperature of 298 K by using 5 mm broad band observe (BBO) probe and the frequency of 500 MHz for 1H nucleus and 125 MHz for 13C nucleus was used.