Advanced UPLC-MS/MS Method for the Quantification of SIPI6398 in Rat Plasma and Its Pharmacokinetic Characterization

SIPI6398 is a novel anti-schizophrenia agent with a new mechanism of action and demonstrates better target selectivity and safety compared to its competitors. However, few in vivo studies on the pharmacokinetics and bioavailability of SIPI6398 have been performed. A rapid and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach was developed for accurate quantification of SIPI6398 in rat plasma. A simple protein precipitation of acetonitrile-methanol (9 : 1, v/v) was used to treat plasma. Chromatography was performed on a UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm) at a flow rate of 0.4 ml/min. The mobile phase consisted of acetonitrile-water (with 0.1% formic acid) and gradient elution was used, and the elution time was 4 minutes. Quantitative analysis was performed using electrospray ionization (ESI) in positive ion detection mode with multiple reaction monitoring (MRM) mode. To evaluate the pharmacokinetics and bioavailability, SIPI6398 was administered to rats in two different ways: oral (4 mg/kg) and intravenous (2 mg/kg) administration. The calibration curve for the UPLC-MS/MS approach shows excellent linearity in the range of 1–2000 ng/mL with an r value above 0.99. The precision, accuracy, recovery, matrix effect, and stability results all meet the criteria established for biological analytical methods. The UPLC-MS/MS method was successfully applied it to pharmacokinetics study of SIPI6398. The bioavailability of SIPI6398 was calculated to be 13.2%. These studies have the potential to contribute towards a more comprehensive comprehension of the pharmacokinetics and bioavailability of SIPI6398.


Introduction
Schizophrenia signifcantly afects human health and is an important cause of disability in patients with mental illness, with a disability rate of 2.8% [1,2].Te global incidence of schizophrenia is approximately 1%, which represents a signifcant burden for both patients and their families [3,4].Terefore, overcoming schizophrenia, the frst "cause of death" of mental illness, is not only a global mental health challenge but also a concern for the entire society.It is also a responsibility and challenge that medical professionals must face.Schizophrenia is a serious mental illness characterized mainly by positive symptoms, negative symptoms, and cognitive defcits [5,6].While anti-schizophrenia medications currently on the market can relieve positive and negative symptoms, they cannot efectively combat cognitive impairment, often leading to more serious side efects.
SIPI6398, chemically N-(trans-4- (2-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl) ethyl)cyclohexyl)furan-2-carboxamide [7], is a new anti-Schizophrenia drug.Te innovative drug received clinical trial approval from the National Medical Products Administration (NMPA) in November 2020 [8].Te approval underscores the drug's potential to confer substantial therapeutic benefts in the management of diverse ailments.Te novel active multitarget molecule SIPI6398 exhibits a new mechanism of action, efectively ameliorating both positive and negative symptoms of schizophrenia while signifcantly enhancing cognitive dysfunction.It has the value of further development as a new drug candidate against schizophrenia.
Pharmacokinetics involves the application of kinetic principles and mathematical models to quantitatively describe and summarize the absorption, distribution, metabolism, and excretion (ADME) of drugs administered via diferent routes (e.g., intravenous injection, intravenous infusion, and oral administration) [9,10].Pharmacokinetic testing is an essential process in drug research for new drugs, improved formulations, or of-patent generics.Terefore, to comprehensively understand the in vivo dynamic changes of SIPI6398, it is imperative to investigate its mechanism of action.
Te in vivo pharmacokinetics of SIPI6398 have been previously reported [7]; however, the LC-MS/MS method used was not fully validated.Te primary objective of this study is to develop a highly accurate UPLC-MS/MS detection method for quantifying the concentration of SIPI6398 in rat plasma.By comprehensively investigating drug concentrations in blood and understanding the pharmacokinetic principles, a better comprehension of the mechanism of action for novel drugs within the body can be achieved.Furthermore, by calculating absolute bioavailability, more precise reference data can be provided for the development and application of new pharmaceuticals.

Instruments and Conditions.
Te Waters XEVO TQ-S micro-triple quadrupole mass spectrometer and ACQUITY H-Class UPLC (Waters Corp., Milford, MA, USA) were used to detect SIPI6398.Te process involved the use of Mas-sLynx 4.1 software (Waters Corp.) for both data acquisition and device control.
UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm) was employed in this work, and the column temperature was set at 40 °C.Acetonitrile-water (with 0.1% formic acid) was chosen as the mobile phase.Te fow rate was set at 0.4 mL/ min.Te entire elution process took 4.5 minutes.Te detailed elution program is as follows: from 0 to 3.0 min, it increases from 5% to 60% acetonitrile; from 3.0 to 3.5 min, it remains at 60% acetonitrile; from 3.5 to 3.6 min, it decreases from 60% to 5% acetonitrile; and from 3.6 to 4.5 min, it remains at 5% acetonitrile.Te cone gas employed was nitrogen, fowing at a rate of 50 L/h, while the desolvation gas utilized was nitrogen, with a fow rate of 993 L/h.Te capillary voltage was set to 3.96 kV, the ion source temperature was maintained at 148 °C, and the desolvation temperature was optimized to 498 °C.Quantitative analysis was conducted using MRM mode in ESI with positive detection, specifcally, m/z 439.30⟶ 176.90 for SIPI6398.For this analyte, the cone voltage and collision voltage were set to 30 V and 20 V, respectively.Additionally, m/z 326.20 ⟶ 291.40 served as the internal standard with a cone voltage of 30 V and a collision voltage of 32 V (Figure 2).

Standard
Curve.SIPI6398 and midazolam were each dissolved in methanol to prepare a stock solution with a concentration of 500 μg/mL.To obtain a series of SIPI6398 concentration standard working solutions of 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, and 20000 ng/mL, the reserve solution was diluted with methanol.Dilute the stock solution with acetonitrile to prepare a working solution of midazolam at a concentration of 50 ng/mL.All reserve and working solutions should be stored at 4 °C to ensure stability.
Te appropriate amount of SIPI6398 working solution was added to the blank plasma of rats to prepare plasma samples with concentrations of 2, 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng/mL.Te standard curve was created with a concentration range of 2-2000 ng/mL.At the same time, quality control (QC) samples were prepared using the same method with plasma concentrations of 4, 160, and 1600 ng/mL.

Sample Treatment Procedure. Te plasma sample (50 μL)
was added to a sterile 1.5 mL Eppendorf tube, followed by the addition of a solution (150 μL) containing the internal standard midazolam (50 ng/ml).Te solution consisted of acetonitrile and methanol in a ratio of 9 : 1.After thorough mixing using a vortex mixer for 1.0 min, the mixture was centrifuged at 11063 g for 10 minutes at 4 °C.Subsequently, the supernatant (100 μL) was transferred to the inner tube of a sample vial for UPLC-MS/MS analysis.Finally, an injection volume of 2 μL was used.

Pharmacokinetic Studies. Twelve healthy male Sprague
Dawley rats weighing between 220 and 250 g were obtained from the Animal Experimental Center of Wenzhou Medical University.Tese animals were housed in sterile cages with a controlled ambient temperature of 24-26 °C, following a rotating light-dark cycle.Tey had ad libitum access to rodent food and water.Prior to the experiment, the animals underwent an overnight fast but were allowed unrestricted access to water.All experimental procedures adhered to the animal experiment guidelines of Wenzhou Medical University, and the research protocol was approved by the university's animal ethics committee (xmsq2023-0656).Te experimental animals were randomly divided into two groups consisting of six rats each.One group received intravenous administration of SIPI6398 at a dose of 2 mg/kg, while the other group received oral administration at a dose of 4 mg/kg SIPI6398.At time points corresponding to 0.0833 h, 0.5 h, 1 h, Journal of Analytical Methods in Chemistry 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h post-administration, respectively, blood samples (0.3 ml) were collected from the tail vein using heparinized tubes followed by centrifugation at a speed of 11063 g for 10 minutes.Te resulting supernatant plasma (100 µL) was then transferred into new 1.5 mL Eppendorf tubes and stored at −20 °C until analysis.Te plasma was treated with a simple protein precipitation method using acetonitrile-methanol (9 :1, v/v).Subsequently, the treated plasma was analyzed by UPLC-MS/MS to determine the concentration of SIPI6398 using a standard curve.Te plasma concentration-time curves for each rat were obtained and pharmacokinetic parameters were calculated using DAS software (version 2.0, China Pharmaceutical University), including area under the concentration-time curve (AUC), mean residence time (MRT), half-life (t 1/2 ), volume of distribution (V), clearance (CL), and peak plasma concentrations (C max ).Tese parameters were calculated based on a non-compartment model.Absolute bioavailability (%) � 100 × AUCpo × Div/ (AUC iv × D po ), where AUC iv and AUC PO represent the AUC of the drug after intravenous and oral administration, respectively, while D iv and D po represent a single dosage of SIPI6398 for intravenous and oral administration, respectively.

3.1.
Selectivity.According to Figure 3, the retention time of SIPI6398 and the internal standard was determined as 2.81 min and 2.71 min, respectively.By employing an optimized gradient elution program, efective separation of these two substances was achieved without any interference from endogenous components within the observed retention time range of SIPI6398 and the internal standard.Hence, this method exhibits exceptional selectivity.

Standard Curve.
Te calibration curve of SIPI6398 in rat plasma showed remarkable linearity in the concentration range of 2-2000 ng/mL with an r value above 0.99.In rat plasma, the regression equation for SIPI6398 is typically expressed as y � 0.00148x − 0.02144 (r � 0.9990), where x represents the concentration of SIPI6398 in plasma and y indicates the ratio of the peak area of SIPI6398 to the internal standard.Te lower limit of quantifcation for SIPI6398 in rat plasma was 2 ng/mL, and the limit of detection was 0.5 ng/mL.

Precision, Accuracy, Recovery, and Matrix Efects.
Te intra-and inter-day precisions of SIPI6398 were both within 11%, demonstrating excellent reproducibility.Te intra-and inter-day accuracies ranged from 90% to 106%, indicating a close agreement between the measured values and true values.Moreover, the recovery of SIPI6398 exceeded 80%, while the matrix efects were observed to be in the range of 89-93%.Detailed data can be found in Table 1.Te results demonstrated that the accuracy of SIPI6398 ranged from 86% to 113%, with a relative standard deviation (RSD) within 13% (Table 2).Tese fndings highlight the exceptional stability exhibited by SIPI6398.

Pharmacokinetics.
Te concentration-time curve of SIPI6398 in rat plasma is shown in Figure 4. Te fgure clearly shows the concentration of SIPI6398 in rat plasma at diferent time points.Table 3 lists the main pharmacokinetic parameters.AUC (0-t) was 2092.8 ± 264.0 ng/mL * h and 550.7 ± 39.5 ng/mL * h for intravenous (i.v., 2 mg/kg) and oral (p.o., 4 mg/kg) administration of SIPI6398 in rats, respectively.t 1/2z was 2.6 ± 0.8 h and 2.8 ± 1.2 h; CLz was 0.9 ± 0.1 L/h/kg and 6.9 ± 0.4 L/h/kg; C max was 1208.1 ± 218.8 ng/mL and 151.4 ± 26.5 ng/mL for intravenous and oral administration of SIPI6398 in rats, respectively.It should be noted that the oral bioavailability of this compound is low, only 13.2%.

Discussion
To accurately determine SIPI6398 and internal standard substances based on the chemical structure properties of these two analytes, we employed the positive ESI source mode in conjunction with the MRM mode.Te ESI positive ion mode, which is well suited for most organic compounds, was utilized for detection purposes.For quantitative analysis, an MRM mode was adopted to efectively minimize background interference while enhancing detection sensitivity and specifcity.Troughout this process, meticulous optimization of parameters such as cone voltage, collision voltage, ion source temperature, nebulization gas fow, and drying gas fow was conducted to enhance the mass spectrometry detection performance of each analyte.UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm) was employed due to its exceptional stability and separation efcacy, rendering it particularly suitable for precise compound separation and analysis.To improve the symmetry of the chromatographic peaks and the retention time of the analytes, the composition and ratio of the mobile phase were studied and optimized based on the selected chromatographic column.After comparing diferent organic solvents (such as methanol and acetonitrile) and continuously adjusting the gradient program, we found that using an acetonitrile-water system as a mobile phase can make the retention time of each analyte more stable.In addition, to further improve the symmetry and detection sensitivity of chromatographic peaks, we considered the addition of formic acid to improve the peak shape and response value based on the chemical properties of the analyte.After comparing diferent volume fractions (0.1% and 0.5%) of formic acid, we found that 0.1% formic acid not only effectively improved the peak shape but also signifcantly   improved the detection sensitivity.Terefore, we ultimately decided on 0.1% formic acid as the optimal modifer.When selecting an internal standard, due to the difculty of obtaining a homologue of SIPI6398, we tried to use midazolam, which has a similar molecular weight and structure to SIPI6398.After verifcation, we found that midazolam had similar retention behavior to SIPI6398, with moderate retention time, good peak shape, and high extraction yield.Terefore, we believe that midazolam is an appropriate internal standard.
Te pharmacokinetics of SIPI6398, including plasma and brain, were measured in rats [7].Te half-life of the brain samples was >15 hours regardless of intravenous or oral administration.Te half-life of the plasma samples was 0.8 hours and 1.7 hours for intravenous and oral administration.SIPI6398 demonstrated high brain penetration with a brain-plasma AUC ratio of 16.1 : 1 despite low oral bioavailability (13.0%) in rats.While the half-life of the plasma samples in this study was 2.6 ± 0.8 hours and 2.8 ± 1.2 hours when administered intravenously and orally, it was not the same as reported.Te oral bioavailability of SIPI6398 was 13.2% in this study and was consistent with the literature [7].

Conclusion
Trough the development of UPLC-MS/MS technology, this study successfully determined the accurate measurement of SIPI6398 in rat plasma.After rigorous validation, the method showed excellent performance in terms of accuracy, precision, specifcity, and linear response.Te pharmacokinetic properties of SIPI6398 in rats and relevant bioavailability data were determined.In the future, the pharmacokinetic properties of SIPI6398 will be studied in detail in other animal models and humans to provide more reliable evidence for optimal drug design and clinical application.

Figure 3 :
Figure 3: UPLC-MS/MS of SIPI6398 and IS in rat plasma: (a) blank rat plasma; (b) blank rat plasma spiked with LLOQ and IS; (c) rat plasma after intravenous administration of SIPI6398.

Table 1 :
Accuracy, precision, matrix efect, and recovery of SIPI6398 in rat plasma.

Table 2 :
Stability of SIPI6398 in rat plasma.