https://doi.org/10.1016/j.jcoa.2022.100073
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2.2. UHPLC-MS/MS analytical condition
The chromatographic separation was achieved using NexeraX2 UHPLC system (Shimadzu Corporation, Kyoto, Japan). Processed samples were kept in a SIL-30AC MP autosampler and maintained at 5 °C. The eluent system containing 0.01 M ammonium acetate (aqueous) and acetonitrile (organic) was pumped by LC-30AD binary pump, in gradient mode of elution as follows: Initially, 20% of acetonitrile was maintained from 0 to 0.5 min, and then it was increased to 80% in 1 min, further it was maintained till 1.8 min, once again the 20% of acetonitrile was back at 1.81 min followed by re-equilibration up to 2.5 min, with a flow rate of 500 µL min−1 into an Acquity UPLC® BEH C18 (2.1 × 50 mm, 1.7 µm) column (Waters Corporation, Milford, MA, USA) maintained at 50°C in CTO-30A oven. The retention time of SUVN-911 was 0.86 min which is away from calculated void time of 0.225 min.
QTRAP-6500 mass spectrometer (SCIEX, Singapore) operated in positive ionization, selective reaction monitoring (SRM) mode, Analyst® software version 1.6.3 was used to carry out the data acquisition and quantification of SUVN-911. The parent ion (Q1) and daughter ion (Q3) transitions (m/z) for both SUVN-911 and IS, with other compound and source-dependent parameters, were represented in Table 1.
Table 1. Mass spectrometry parameters of SUVN-911 and internal standard.
| Parameters | SUVN-911 | Empty Cell | Internal standard |
|---|---|---|---|
| Empty Cell | (SUVN-911-N15 D3) | ||
| Q1/Q3 (m/z) | 225.1→96 | 229.1→ 96.2 | |
| Resolution | Unit | Unit | |
| Declustering potential (V) | 40 | 71 | |
| Collision energy (V) | 21 | 21 | |
| Cell exit potential (V) | 10 | 8 | |
| Entrance potential (V) | 10 | 10 | |
| Ionspray temperature (°C) | 450 | ||
| Ionspray voltage (V) | 5500 | ||
| Gas 1 (psi) | 40 | ||
| Gas 2 (psi) | 20 | ||
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C: Degree Celsius; V: Volts; psi:pounds per square inch.
2.3. Preparation of CS and QC stock solutions
Two independent standard stock solutions (2000 µg mL−1) of SUVN-911 in methanol (100%) were used to prepare the calibration, and quality controls. The working solutions were prepared from respective stocks by serial dilutions in water/methanol (50:50, v/v). The internal standard stock solution at a concentration of 1000 µg mL−1 was prepared in methanol (100%). The working solution of IS was also prepared in water/methanol (50:50, v/v). All the above solutions were kept in the refrigerator at 2–8°C until use.
2.4. Preparation of CC and QC samples
The working solutions of SUVN-911 were diluted with respective blank matrix (plasma/urine) to produce the calibration curve standard and quality control samples. The calibration curve (0.02 to 50 ng mL−1), quality control (0.02, 0.06, 20, and 35 ng mL−1), and Dilution integrity (DI) 350 ng mL−1 (10 x high QC) samples were prepared in human plasma. In a similar way, the calibration curve (5 to 5000 ng mL−1), quality control (5, 15, 2500, and 3500 ng mL−1) and DI samples 35,000 ng mL−1 (10 x high QC) were prepared in human urine. The bulk spiked quality control samples of SUVN-911 in human plasma and urine were kept at -70°C and -20°C for various stability experiments.
2.5. Sample preparation
The samples were prepared using a liquid-liquid extraction procedure. Briefly, 250 µL of plasma / 50 µL of urine (25 µL of 0.1 N NaOH was added to urine samples) study sample was transferred into 2.0 mL plastic tubes and 5 µL working solution of IS (1 µg mL−1) was spiked, and vortexed for approximately 30 s. The samples were extracted with 1.5 mL of diethyl ether: n-hexane (80:20, v/v) and vortexed for 3 min followed by centrifugation at 1300 x g for 2 min. The upper organic layer (1.2 mL) was carefully transferred into a 5 mL borosil glass tube. The organic layer was dried under nitrogen gas at 40°C, and 100 µL of 0.01M ammonium acetate: acetonitrile (80:20, v/v) was used to reconstitute the dried residue. The reconstituted samples were centrifuged at 1300 x g for 2 min. The clear supernatant solution was carefully transferred into 96 well plates and an injection volume of 2 µL (plasma) and 1 µL (urine) was used for analysis.
2.6. Pharmacokinetic study in healthy subjects
Phase I study of SUVN-911 (ClinicalTrials.gov Identifier NCT03155503 and NCT03551288) was conducted in the United States (US) as per Good Clinical Practice (GCP) guideline recommended by International Conference on Harmonisation (ICH), and the declaration of Helsinki. This study was conducted to evaluate the safety, tolerability, and pharmacokinetics of SUVN-911 [6]. The obtained human plasma (blood samples were centrifuged at 1300 x g for 10 min at 4°C) and urine samples from single and multiple ascending dose studies were stored at -70°C until analysis.
2.7. Method validation and acceptance criteria
The developed method was validated as per US FDA and EMA guidelines [7,8]. The sample types (CC, QCs, post spiked, extracted, etc.), and number of samples (n=) were prepared [9] in human plasma and urine to perform all the validation parameters. The validation parameters include selectivity, sensitivity, carryover, linearity, precision & accuracy, dilution integrity, recovery, matrix effect, and stability of SUVN-911 in solution, whole blood, plasma, and urine.
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