**3. Materials and Methods 3. Materials and Methods**

**and** 

*3.1. Experimental*

**3. Materials**

Baricitinib (>99% purity) was purchased from Enzo Life Sciences, Inc. (Exeter, UK) and irbersartan (Figure **??**B) was purchased from Beijing Mesochem Technology Co., Ltd. (Beijing, China). HPLC-grade methanol and acetonitrile were obtained from Avonchem Ltd., (Macclesfield, UK) and Winlab Pty. Ltd. (Brendale, Australia), respectively. Analytical-grade formic acid, dichloromethane, n-hexane, and ammonium acetate were obtained from the BDH Laboratory (Lutterworth, UK). Blank rat plasma was collected and separated from the blood of healthy rats.

#### *3.2. Equipment*

Waters Acquity TQD UPLC/ Mass spectrometer (Waters Co., Milford, MA, USA) was used in the study. Other equipment used included vortex mixer Taboys ® model AP 56 (TROEMNER. Hingham, MA, USA), analytical balance Mettler Toledo ® model XS 205 (Greifensee, Switzerland), and a sample concentrator Thermo ® Savant SC210A speed Vac (Waltham, MA, USA). Deionized water was prepared by Milli-Q reverse osmosis (Millipore ®, Bedford, MA, USA).

#### *3.3. Chromatographic Conditions*

Sample analysis was performed on an Acquity TQD UPLC-MS/MS system (Waters Co., Milford, MA, USA). The triple quadrupole mass spectrometer was operated in the MRM mode, and detection was carried out using electrospray ionization (ESI) in the positive ion mode. In addition, the mass spectrometry parameters including parent to daughter ion transition, collision energy, cone voltage, and dwell time were optimized to determine the analyte and IS (Table **??**). The collision gas (argon) flow rate was kept at 0.1 mL/min and the desolvation gas (nitrogen) flow rate was optimized to 600 L/h. Notably, chromatographic separation was achieved using a Waters Acquity UPLC HILIC BEH 1.7 μm 2.1 × 50 mm column with the mobile phase consisting of 0.1% formic acid in acetonitrile and 20 mM ammonium acetate (pH 3). The flow rate was 0.2 mL/min.


**Table 5.** Mass optimization parameter for, baracitinib and irbersartan (IS).

#### *3.4. Preparation of Stock and Working Solutions*

The standard stock solutions of baricitinib and irbesartan were prepared individually by dissolving 5 mg of each drug in dimethyl sulfoxide (DMSO, Loba Chemi, Mumbai, India). Stock solutions were then diluted with methanol to obtain working solutions of 1000 μg/mL for both baricitinib and IS. Further dilution was carried out in acetonitrile to yield working solutions (20 μg/mL) for both the analyte and IS. The calibration range was 0.2 to 500 ng/mL (0.2, 1.0, 5.0, 1.0, 20.0, 100.0, 200.0, and 500.0 ng/mL). Three quality control samples at three concentration levels 0.6, 40.0, and 400.0 ng/mL and lower limit of quantitation (LLOQ; 0.2 ng/mL) were prepared.

#### *3.5. Sample Preparation*

Baricitinib and IS were extracted from rat plasma using liquid–liquid extraction. 20 μL of irbesartan working solution (20 μg/mL) was added to 100 μL plasma samples. After vortex-mixing for 20 s, 1 mL of *n*-hexane:dichlromethane mixture (1:1) was added. Samples were then mixed for 1 min and centrifuged for 5 min at 5600× *g*. The supernatant (0.8 mL) was transferred to a 5-mL tube and evaporated to dryness at 40 ◦C. Residues were reconstituted in 100 μL of the mobile phase and 5 μL was injected into the LC-MS/MS system.

#### *3.6. Method Validation*

The method was validated according to the FDA and EMA guidelines for bioanalytical method validation [**? ?** ].

The selectivity of the proposed method was estimated by analyzing rat blank plasma samples from 6 di fferent sources to detect the interfering peaks at the same retention times of the analyte and IS. The sensitivity of the method is expressed by the LLOQ, which is defined as the lowest concentration of an analyte that can be accurately measured.

The plasma calibration curves were constructed by plotting the peak area ratios (baricitinib/IS) against baricitinib concentrations covering the expected range (0.2–500 ng/mL), including the LLOQ. The correlation coe fficient (r2) of calibration curves should not less than 0.99. The limit of detection (LOD) is defined as the concentration of an analyte yielding a peak with a signal to noise ratio of 3, while this ratio is 5 times for the low limit of quantification (LOQ).

The accuracy and precision of the method were determined by analyzing three di fferent QC concentrations (low, medium and high QC samples) representing the entire range of the calibration curve and LLOQ samples. Inter-day accuracy and precision were measured consecutively for three days, whereas intra-day accuracy and precision were measured in one day. Accuracy of QC samples should be within ±15% of the nominal concentrations of QC samples and 20% for LLOQ. Precision should not exceed 15% CV% for QC samples and 20% for LLOQ.

Stability of the analyte was assessed using five measurements of QC samples at low and high concentrations after exposure to di fferent conditions of storage and processing temperature. Baracitinib stability was evaluated after three freeze-thaw cycles, storage at room temperature (23–25 ◦C) for 6 h (short term stability), and subsequent storage for eight weeks at −80 ◦C (long term stability). Moreover, the stability of stock and working solutions of baricitinib and the IS at room temperature for 6 h (23–25 ◦C) and at refrigerator (2–8 ◦C) for 12 days was tested.

The absolute recovery of the method was determined by comparing the average of peak area measurements obtained from the blank plasma spiked before extraction to those obtained from the blank plasma spiked after extraction. The e ffect of the matrix was evaluated by comparing the peak area ratios of post-extracted plasma spiked with baricitinib and IS to peak area ratio of real solutions contain the same concentration.

#### *3.7. Animal*

Sprague Dawely rats weighing 200–230 g were obtained from the animal house at the National Organization for Drug Control and Research, Giza, Egypt. Rats were fed standard su fficient food and water and experiments were performed under the Guide for Care and Use of laboratory animals. experimental protocols were approved by the ethics committee of the Faculty of Pharmacy at Al-Azhar University, Cairo, Egypt (no. 206).

#### *3.8. Application to a Pharmacokinetic Study*

To demonstrate the utility of the present method, a pharmacokinetic study of baricitinib was performed on six male Sprague Dawely rats (200–230 g). Baricitinib was used to create a suspension using 1% carboxymethyl cellulose. Following overnight fasting, the drug was administered in a dose of 2 mg/10 mL/kg. Blood samples (approximately 0.25 mL) were then collected from the retro-orbital plexus into heparinized tubes at 0.0, 0.25, 0.5, 0.75, 1.0, 1.25, 2.0, 3.0, 5.0, 7.0, 9.0, and 11.0 h post-dosing. Plasma samples were obtained by centrifuging the blood at 4250× *g* for 5 min and stored frozen at −80 ◦C until analysis. The pharmacokinetic parameters Cmax, tmax, AUC, t 1 2 , and Kel were calculated using WinNonlin software.

#### **4. Conclusions**

A new rapid, sensitive, selective, reproducible, precise, and accurate UPLC-MS/MS method was developed and validated for the baricitinib plasma quantitation of. The method met the acceptance criteria for bioanalytical method validation defined by the recent FDA and EMA guidelines. The method was successfully applied in pharmacokinetic study following oral administration of a single dose of baricitinib in male rats under fasting conditions. This method demonstrates its applicability in relevant preclinical, therapeutic drug monitoring and potentially for pharmacokinetic studies of the baricitinib-drug interaction.

**Author Contributions:** Conceptualization, E.E. and T.E.-N.; Data curation, E.E., M.I., P.A. and T.E.-N.; Formal analysis, E.E. and M.I.; Funding acquisition, Y.A.A.; Investigation, E.E. and T.E.-N.; Methodology, E.E., M.I. and T.E.-N.; Project administration, E.E. and Y.A.A.; Software, T.E.-N.; Supervision, Y.A.A.; Validation, E.E. and M.I.; Writing—review and editing, E.E., T.E.-N., P.A. and A.A.A. All authors have read and agree to the published version of the manuscript.

**Funding:** This research was funded by the Research Supporting Project at King Saud University via gran<sup>t</sup> number RSP/2019/45 and article processing charge (APC) was also supported by the Research Supporting Project (RSP/2019/45).

**Acknowledgments:** The authors extend their sincere appreciation to the Research Supporting Project at King Saud University for funding this work through the gran<sup>t</sup> number RSP/2019/45.

**Conflicts of Interest:** The authors declare no conflict of interest.
