A Reliable and Effective UPLC-MS/MS Method for the Determination of Oprozomib in Rat Plasma

Oprozomib, as a second-generation orally bioavailable protease inhibitor (PI), is undergoing clinical evaluation for the treatment of haematological malignancies. In relapsed refractory multiple myeloma (RRMM) patients, oprozomib has shown good efficacy as a single agent or combination therapy. In this experiment, our purpose was to validate a sensitive and rapid method for the determination of oprozomib concentration in rat plasma by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The samples were treated with acetonitrile as the precipitant and separated by gradient elution using a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm). Using the selective reaction monitoring (SRM) method, the measurement was finished with the ion transitions of m/z 533.18 ⟶ 199.01 for oprozomib and m/z 493.03 ⟶ 112.03 for tepotinib (internal standard, IS), respectively. Meanwhile, acetonitrile and 0.1% formic acid aqueous solution were used as the mobile phase, and the flow rate was 0.3 mL/min. The lower limit of quantification (LLOQ) of the method was 1.0 ng/mL, and the linear relationship was good in the range of 1.0–100 ng/mL. In addition, the precision of four concentration levels was determined with the values of 3.1–7.3% and the accuracy was from −14.9% to 12.9%. Moreover, the recovery was determined to be from 85.1% to 96.1%, and the values of matrix effect were no more than 110.4%. The optimized UPLC-MS/MS method was also suitable for the pharmacokinetic study of rats after a single oral administration of 21 mg/kg oprozomib.


Introduction
Following lymphoma, multiple myeloma (MM) is the most common hematologic malignancy, which is a neoplasm of clonal plasma cells and belongs to postgerminal lymphoid Bcell lineage [1,2]. At the present medical level, MM is still not completely cured, but with the development of modern medicine, protease inhibitor (PI), immunomodulatory drugs, monoclonal antibody (MoAb), stem cell transplantation therapy, and other drugs and treatment methods appear for MM patients with the treatment and prolong the survival of a new hope [3].
PIs have been proved to be an efective and common treatment for MM [4]. Oprozomib (Figure 1(a)), as a second-generation orally bioavailable PI, is undergoing clinical evaluation for the treatment of haematological malignancies [5,6]. In relapsed refractory multiple myeloma (RRMM) patients, oprozomib has shown good efcacy as a single agent or combination therapy. Te pharmacokinetic and pharmacodynamic characteristics of oprozomib have previously been studied in phase I studies in solid tumor patients, which demonstrated oral bioavailability and showed dose-dependent proteasome inhibition [5,7,8].
In most cases, oprozomib is used in combination with other agents, including frst-generation agents, when treating MM/RRMM, and drug-drug interactions (DDIs) have to be considered [9]. Studies have shown that physiology-based pharmacokinetic (PBPK) models have been well used to promote the design and extrapolate DDIs [10]. Although good progress has been made in simulating the interaction between oprozomib and other drugs based on PBPK model, in vitro metabolism study of oprozomib has shown that it was unlikely to be afected by coadministered cytochrome P450 (CYP) [11].
In order to explore the pharmacokinetics and DDIs of oprozomib in vivo, it is necessary to build a sensitive quantitative detection method for oprozomib in biological fuids to provide a basis for subsequent clinical research. However, until now, the method of bioanalysis and determination of oprozomib in biological liquid has not been established. Tus, in this experiment, our purpose was to validate a sensitive and rapid method for the determination of oprozomib concentration in rat plasma by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Te newly established UPLC-MS/MS technique was also suitable for the pharmacokinetics of oprozomib in rats.

Chemical Materials and Reagents.
Te purity of both oprozomib and tepotinib (used as internal standard, IS, Figure 1(b)) was >98.0% and was provided by Shanghai Chuangsai Technology Co., Ltd. (Shanghai, China). In addition, sodium carboxymethyl cellulose (CMC-Na) was also purchased from Shanghai Chuangsai Technology Co., Ltd. (Shanghai, China). Methanol and acetonitrile supplied by Merck (Darmstadt, Germany) in this study were of liquid chromatography (LC) grade. Moreover, formic acid was of LC grade, which was supplied by Anaqua Chemicals Supply (ACS, American). Te ultrapure water used in the experiment was prepared by Milli-Q Water Purifcation System (Millipore, Bedford, USA).

Animal Experiments. Six Sprague Dawley (SD) rats
(weight 200 ± 20 g) were provided by the Laboratory Animal Center of Te First Afliated Hospital of Wenzhou Medical University (Wenzhou, China) and were fed under standard condition, which were temperature 25-28°C, humidity 50-60%, and 12 h light/12 h dark. All the rats were fed on a standard rodent diet, which supplied water and food without limit. Under the rules for the Care and Use of Laboratory Animals, all behaviors and operations in this experiment were reviewed and approved by the Institutional Ethics Committee of Te First Afliated Hospital of Wenzhou Medical University (Zhejiang, China).
Oprozomib was prepared into suspension with 0.5% CMC-Na solution and administered orally to rats at a dose of 21 mg/kg. Before the experiment, six rats were fasted for 12 h, while all of them were allowed to access water unrestrainedly. At diferent time points of 0.3, 0.7, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12, 24, and 48 h, about 0.3 mL of blood samples was taken from the caudal vein and placed in a 1.5 mL Eppendorf (EP) tube containing heparin. Ten, the blood samples were centrifuged rapidly at 4000 × g at 4°C for 10 min to obtain plasma samples. Subsequently, the treated plasma was stored at −80°C before the further analysis. After sample preparation, the plasma concentration of oprozomib in rats was evaluated and detected by UPLC-MS/MS method. Ten, pharmacokinetic parameters of oprozomib in noncompartmental analysis were calculated by DAS software (Drug and Statistics, version 3.0, Shanghai University of Traditional Chinese Medicine, China).

Instrumentations and Analytical
Conditions. Te UPLC-MS/MS system, coupled with an electrospray ionization (ESI) source (Milford, MA, USA), was composed of the Waters Xevo TQ-S triple quadrupole tandem mass spectrometer and the Waters Acquity UPLC I-Class system (Milford, MA, USA).
Mass spectrometry monitoring was performed using the Xevo TQ-S triple quadrupole tandem mass spectrometer, in conjunction with the Acquity UPLC system for analysis, in positive ion mode for the detection of oprozomib and IS. Te

Sample Preparation.
In preparing all samples, we performed protein precipitation method and acetonitrile was employed as the precipitant. First, 50 μL of plasma sample, in a 1.5 mL EP centrifuge tube, was spiked with 10 μL of IS working solution (100 ng/mL) and mixed for 0.5 min in a vortex. Ten, a total of 450 μL acetonitrile was added to precipitate the plasma protein, and the mixture was vigorously vortexed for 1.0 min, followed by a 10 min centrifugation at 13,000 × rpm under 4°C. Te next step was to transfer 100 μL of the supernatant into a clean automatic injection fask. Finally, the injection volume was set at 2.0 μL, and a chromatographic system for quantitative analysis was used.

Method
Validation. Tis analysis method followed FDA guidelines for the validation of bioanalytical tests, and the method validation procedures including the calibration curve, stability, matrix efect, LLOQ, precision, and accuracy were fully conducted [12].
In this experiment, in order to detect the selectivity of the method, blank plasma samples from six diferent rats were analyzed by chromatography to evaluate whether there was endogenous interference at the retention times of both the analyte and IS in the method.
We constructed the standard curve according to the principle of least square method and used the weighting factor of the reciprocal of concentration (1/x) to measure its linearity. LLOQ is the lowest concentration of the analyte on the standard curve that can be quantifed reliably, with acceptable accuracy and precision. At the LLOQ, the accuracy (relative error, RE, %) is ±20%, and the precision (relative standard deviation, RSD, %) is less than 20%.
Te precision and accuracy of QC samples were evaluated by fve repeated tests at three diferent concentration levels for three consecutive days. Under three concentration levels (fve samples were made in parallel for each concentration), the recovery rate was calculated by comparing the concentration ratio of the analyte before and after protein precipitation. As well, the response rate of extracted plasma matrix and analyte in pure solution was compared to calculate the matrix efect, and the matrix efect was also measured in fve repetitions.
LQC (2.0 ng/mL), MQC (40 ng/mL), and HQC (80 ng/ mL) were used for stability evaluation. Te specifc evaluation contents were divided into the following four parts [13,14]: one was the stability at room temperature for 3 h and −80°C for 3 weeks as another, the third was the stability of automatic sampler at 10°C for 4 h, and the last was three times of complete freezing and thawing from −80°C to room temperature.

Method Design and Optimization.
At the beginning of the study, we optimized the chromatographic conditions and improved the detection sensitivity to establish a simple UPLC-MS/MS method for the determination of oprozomib in rat plasma. As presented in Figure 1, mass spectra of oprozomib and IS were shown. In the process of quality transformation, we used m/z 199.01 as the quantitative method of product ions and with m/z 112.03 as the IS ion quantitative method. In addition, the experiment used 0.1% formic acid aqueous solution and acetonitrile solution as mobile phase and resulted in excellent separation efect and peak shape.
In the experiment, acetonitrile was used as the protein precipitator when we treated plasma. Te method was simple and the experimental time was short. In addition, we also comprehensively evaluated and analyzed the feasibility of this method in the detection of oprozomib from the aspects of accuracy, precision, and stability. Comprehensively, this was a simple and efective detection method. Figure 2, the retention times of oprozomib and IS were about 1.52 and 1.39 min, respectively. After comparing the representative SRM chromatograms of blank rat mixed plasma from 6 diferent sources, blank plasma supplemented with oprozomib and IS at LLOQ concentration level, and experimental plasma samples, no potential interfering substances were found. Based on the results, the method enabled to be used to determine the concentration of oprozomib since it had good selectivity for oprozomib and IS.

Linearity and Sensitivity.
Te concentration range of the standard curve was 1.0-100 ng/mL, and the linear regression equation between peak ratio (Y) and compatible concentration (X) was Y � 0.00683 × X + 0.00259 (R 2 � 0.9993), where good linear relationship met the requirements of methodological detection. Te LLOQ of the method was 1.0 ng/mL, where the precision was less than 7.3% and the accuracy was with ±5.3%.

Precision, Accuracy, Recovery, and Matrix Efect.
As part of the UPLC-MS/MS method, the precision and accuracy assessments were detected by calculating the QC samples with diferent concentration levels (n � 5) at 1.0, 2.0, 40, and 80 ng/mL for 3 consecutive days. Table 1 shows that the UPLC-MS/MS method established for quantitative analysis of oprozomib in rat plasma was highly precise and accurate.
In this study, HQC, MQC, and LQC were used to detect the recovery and matrix efect of fve repetitions. Te means of calculating extraction recovery were used to compare the plasma samples added to QC before protein precipitation with the plasma concentrations added after extraction. Using this method, the extraction recovery of three concentration levels ranged from 85.1% to 96.1% (  and no matrix efect was observed in the experiment to afect the determination of the analyte in plasma (  Table 3.

Animal Study.
Te UPLC-MS/MS method established in this study was suitable for the determination of plasma drug concentration in rats after a single oral dose of 21 mg/ kg oprozomib. Te average plasma concentration time curve of oprozomib in rats is shown in Figure 3. Te main pharmacokinetic parameters of oprozomib were analyzed by noncompartmental model and are summarized, as shown in Table 4. Te peak time (T max ) of the drug in rat plasma was about 1.1 ± 0.3 h and the half-life (t 1/2 ) was 13.8 ± 8.3 h. Considering that only 6 rats were used in our animal

Conclusion
Above all, we developed a simple and reliable UPLC-MS/MS approach for measuring the concentrations of oprozomib in plasma for the frst time. Tis method was demonstrated to have good reproducibility, high accuracy, precision, and good linearity and had the advantages of short analysis time, simple sample preparation, and low cost. It showed the value in the pharmacokinetic study of oprozomib in rats.

Data Availability
Te data used to support the study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.