2.1. Chemicals and Reagents
All the Chemicals and reagents are of analytical grade. PAR & ORP were obtained as a gift from Egyptian International Pharmaceutical Industries Co. (EIPICO), Egypt. Acetonitrile (ACN) & methanol are HPLC grade and were obtained from Germany ((Riedel-de Haën Laboratory Chemicals, Selzer). Analytical grade ammonium formate and formic acid were bought from Germany (Merck company in Darmstadt, Germany). All stock, working and buffer solutions which are used for the mobile phase (aqueous solutions) are prepared by Milli-Q grade water (Millipore, Milford, MA, USA).
2.2. Chromatographic separation and MS/MS conditions
An Agilent 1260 series LC system (Agilent Technologies, Waldbronn, Germany) coupled with degasser (G4212B), Thermostat (G1330B), Quaternary pump (G1311B/C) along with auto-sampler (G1329B) was used to inject 10 µL aliquots of the used samples on an Agilent Eclipse Plus ODS (4.60 x 100 mm, 3.50 μm) column (Agilent Technologies, USA), which was kept at room temperature (25 ± 2◦C) . A gradient elution was carried out by altering the proportion of mixture A (6 mM ammonium with 0.1% formic acid buffer) and mixture B (ACN) as follow: 0–6.50 min (90% A - 10% B), at 6.5 min (60% A - 40% B), at 8 min (10% A - 90% B), at 10 min (0% A - 100% B), at 12 min (20% A - 80% B), and at 14 min (98% A - 2% B). The gradient mobile phase was delivered at a flow rate of 0.50 mL/min into the Electrospray ionisation chamber of mass spectrometer. PAR and ORP were identified using a triple–quadruple LC/tandem mass spectrometric detection system (Agilent 6460 QQQ LCMS) with a positive ionization electrospray ionization (ESI) interface. With a dwell time of 200 ns, the multiple reaction monitoring (MRM) transitions of m/z 152 → 110 & 65 for PAR and 270.3→ 181 for ORP were employed (Fig 2). At a flow rate of 600 Lh-1, nitrogen was used as a desolvating gas. The desolvation line temperature was 400 degrees Celsius, whereas the source temperature was 300 degrees Celsius, and the nebulizer pressure was 45 pounds per square inch. The collision gas flow, which was Argon, was 11 mL.min-1, with a capillary voltage of 4 kV. For PAR, the cone voltage and collision energy were set at 113 V and 39 eV, respectively, while for ORP, they were set at 181V and 17 eV. Quantitative Analysis B.07.00 (the Mass Hunter software) was utilized to manage the UPLC–MS/MS system, and the Target Mass Hunter program was used for data collecting and processing .
2.3. Preparation of stock solution
PAR and ORP stock solutions (100 g mL-1) were made by dissolving accurately weighed amounts of their respective standards in methanol and then using these solutions to make the calibration curve and quality control sample (QC).
2.4. Preparation of Calibration standards, QC samples
The PAR and ORP stock solutions were then diluted with ammonium formate-ACN (90:10, v/v) to make the calibration standards and QC sample working solutions. Plasma calibration standards (eight concentration levels) were prepared by dissolving in mobile phase and then spiking in rat blank plasma to reach final concentration levels of 1, 10, 50, 100, 500, 1000, 2500, 5000 ng mL-1 for PAR and 1, 5, 10, 20, 50, 100, 200, 500 ng mL-1 for ORP. In the same way, three QC samples were made at 10, 500, 2500 ng mL-1 for PAR and 10, 100, 500 ng mL-1 for ORP and treated as LQC, MQC, and HQC by dissolving in mobile phase or spiking in the blank plasma. Both plasma standards and QC samples were stored at -80 degrees Celsius until validation and/or optimization. The mobile phase samples could be stored at 4℃ for a month without changing.
2.5. Plasma sample preparation
Before preparation, plasma samples (calibration curve standards, QCs) were thawed at room temperature and vortexed for 30 seconds. One hundred microliters of plasma (QCs, blanks, and calibration standards) were added to a 1 mL Eppendorf tube, vortexed for 30 seconds, and deproteinized with 290 µL of ACN. At room temperature, the samples were centrifuged for 10 minutes at 10,000 g. The supernatant was separated and transferred to a new tube, which was maintained in the refrigerator until analysis, where 10 µL was injected into the LC-MS/MS.
2.6. Application to PK and DDI study in rats
The Suez Canal University Faculty of Pharmacy's Research Ethics Committee approved all the study's experimental protocols (Approved no. 201808RH1). All methods were carried out in accordance with the ARRIVE guidelines. Male Wistar Albino rats, 8 weeks old and weighing 200–250 g, were randomly placed into three groups, each with three rats. After an overnight fast, rats in Group I were given ORP (50 mg/Kg); rats in Group II were given PAR (450 mg/Kg); and rats in Group III were given ORP (50 mg/Kg) + PAR (450 mg/Kg) orally. After 0.5, 1, 2, 3, 4, 6, 8, 12, 24 hours, blood samples (each 300 µL) were obtained via the femoral artery into EDTA containers. Following blood collection, the rats were given an identical volume of saline in each case. The blood samples were centrifuged for 10 minutes at 10,000 g. The plasma layer was removed and transported to clean tubes, where it was kept until analysis at -80°C. To examine method development and validation, blank plasma was taken from a rat that had not been given any drugs. The non-compartmental pharmacokinetic analysis was carried out using the PK Solver tool, which is a free add-in for Microsoft Excel. The linear trapezoidal approach was used to compute the area under the plasma concentration-time curve (AUC0-t). The experimental data was used to compute Cmax (peak plasma concentration) and Tmax (time necessary to achieve peak plasma concentration). Regression analysis was used to get Kel (the elimination rate constant) from the slope of the best fit line, and T1/2 (the drug's half-life) was calculated using 0.693/ Kel. The PK Solver program was used to compute MRT (mean residence time), CL/F (total clearance of the drug from plasma), and Vd/F (volume of distribution) .
2.7. Method Validation
The following parameters were used to validate the method: selectivity and specificity were determined by comparing the chromatograms of blank rat plasma, plasma spiked with target analytes, and plasma samples collected from rats given PAR and ORP. LOD (limit of detection) and LOQ (limit of quantitation), which are concentrations with a signal-to-noise (S/N) ratio of at least 3.3 and 10, respectively, have been used to define sensitivity. A least squares regression line was used to assess linearity, which was reported as r (correlation coefficient). On three distinct days, the linearity of the analytes was resolved by at least 8 concentration levels other than the blank . The effect of plasma constituents on analyte ionization was assessed by comparing the responses of post-extracted plasma standard QC samples to the responses of analytes from neat samples at equivalent concentrations. Three QC samples were produced at low, middle, and high concentrations of 10, 500, 2500 ng mL-1 for PAR and 10, 100, 500 ng mL-1 for ORP, respectively, to assess intra- and inter-day precision and accuracy. The intraday precision was determined by calculating the percent RSD for the evaluation of QC samples in triplicates, whereas the interday precision was determined by examining QC samples on three different days. The relative error percentage ((measured conc - true conc / true conc) * 100) was obtained by comparing the averaged measurements to the nominal values. The peak area ratios of analytes in rat plasma at QC concentrations were compared to those in the mobile phase at corresponding concentrations and represented in percentages to demonstrate the recovery of PAR and ORP. The stability of the target analytes in rat plasma was examined for short periods of time (up to 24 hours) at room temperature (RT) and at 4°C, as well as for extended periods of time (up to two weeks) at -80 ℃ and after three freeze-thaw cycles .