Chemicals and reagents
The following standard materials were used in this study: theobromine, theophylline, dropropizine, noscapine, and dexamethasone were purchased from Toronto Research Chemicals (TRC, Toronto, ON, Canada); guaifenesin, azelastine, and fexofenadine were purchased from United States Pharmacopeia (USP, Rockville, MD, USA); tramazoline was purchased from European Pharmacopoeia (Strasbourg, France); olopatadine was purchased from Sigma–Aldrich (St. Louis, MO, USA); and oxymetazoline was purchased from Clearsynth (Mumbai, India). Sodium 1-hexanesulfonate, phosphoric acid (reagent grade), and formic acid (LC-MS grade) were purchased from Sigma–Aldrich (St. Louis, MO, USA), deionized water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA), and acetonitrile and methanol (HPLC grade) were purchased from Merck (Darmstadt, Germany). All the solvents used in this study were filtered through a polytetrafluoroethylene (PTFE) filter (0.2 µm, Millipore) before analysis. Except for theobromine, standard stock solutions were prepared at a concentration of 1000 µg mL− 1 through dissolution in methanol. Theobromine was prepared at the same concentration through dissolution in an ACN:DW (60:40) solution. The stock solutions were stored in a refrigerator at approximately 4 ℃. For calibration curve and method validation, the stock solutions were diluted to the appropriate concentration using HPLC-grade methanol on the same day.
Instrumental conditions
The analysis method development and validation were conducted using the Waters Acquity UPLC System (Waters, Milford, CT, USA) equipped with a binary pump and photodiode array detector. An Acquity UPLC BEH C18 Column (2.1 × 150 mm, 1.7 µm) was used for separating the analytes. The column oven temperature was set as 35 ℃. Ultraviolet (UV) absorption was measured at a wavelength of 205 nm, and the temperature of the autosampler was 8 ℃. The flow rate of the mobile phase and injection volume of analytes were 0.2 mL/min and 1.0 µL, respectively. The following mobile phases were selected: (A) 0.5 mM sodium 1-hexanesulfonate buffer in deionized water containing 0.1% phosphoric acid and (B) acetonitrile:water (90:10). The gradient elution was set as follows: 0.0–1.0 min (A: 95%, B: 5%), 8.0–9.5 min (A: 55%, B: 45%), 11.0–13.0 min (A: 40%, B: 60%), 15.5–18.5 min (A: 0%, B: 100%), 19.0–22.0 min (A: 95%, B: 5%).
Comparison of clean-up methods
To optimize the sample preparation method, various clean-up methods were compared to minimize the impact of sample matrix components. The removal of polar matrix components, fatty acids, fats, and sterols was considered. [12, 13] A 0.2-µm PTFE filter and two types of dispersive SPE (d-SPE, commonly referred to as QuEChERS) kits were selected for comparison. For the comparison of the clean-up methods, testing samples were prepared at the same concentration: (a) filter only, (b) QuEChERS kit 1 (Thermo Fisher, Waltham, MA, USA, hereinafter referred as QuE 1), and (c) QuEChERS kit 2 (Waters, Rockwood, TN, USA, hereinafter referred as QuE 2) methods were compared to optimize the clean-up method (Fig. 3). The detailed comparison procedures are as follows.
Sample extraction
Sample types were categorized into solid and liquid, and the solid samples included hard capsules, tablets, and powder. For the solid samples, hard capsules and tablets were ground using a grinder (IKA Tube mill, Germany), including the outer shell. 1 g of each homogenized sample was weighed into a 50-mL volumetric flask, and 30 mL of 70% aqueous methanol was added. The mixture was extracted in an ultrasonic bath for 30 min and cooled at room temperature, and then 70% methanol was added to obtain a total volume of 50 mL. The extracts were centrifuged for 5 min at 4000 rpm at 8 ℃, and the supernatant was used as a sample extract.
(a) Filter only
The sample extract (2 mL, prepared as described in “Sample extraction” section) was filtered with a 0.2-µm PTFE syringe filter and then injected into the UPLC-PDA.
(b, c) QuEChERS Kits 1 and 2
The sample extract (5 mL) was added to QuE 1 (900 mg MgSO4, 150 mg PSA) and QuE 2 (900 mg MgSO4, 150 mg PSA, 150 mg C18) clean-up kits. It was strongly shaken for 1 min and centrifuged for 10 min at 4000 rpm at 8 ℃. The supernatant was filtered with a 0.2-µm PTFE syringe filter and then injected into the UPLC-PDA.
Method validation
To validate the developed UPLC-PDA method for analyzing 11 respiratory drug substances, the specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity, accuracy, precision, recovery, matrix effect, and stability were tested. The validation protocol was implemented with reference to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use [14].
The specificity was determined by comparing blank matrix and spiked sample chromatograms of solid and liquid samples, respectively. The LOD and LOQ were evaluated at signal-to-noise (S/N) ratios of ≥ 3 and ≥ 10. The linearity was evaluated by plotting the calibration curve using six concentrations, and the correlation coefficient (R2) was ≥ 0.999. The recovery was evaluated by calculating the ratio of the peak areas of the standard solution and the spiked sample at the same concentration. The precision and accuracy were measured by calculating the relative standard deviation (RSD) and recovery of each compound. Matrix effects were calculated by comparing the slope of the calibration curve for the standard solution with that of the spiked sample. The stability of the standard solution was confirmed through analysis after it was stored at room temperature for 6 h, and we checked whether the stability was maintained after 24 and 48 h. The intra-day and inter-day precision, accuracy, recovery, matrix effect, and stability were validated at three concentrations (low: LOQ, medium: 20 LOQ, high: 40 LOQ), and the entire analysis was performed three times.