Assay of Bio Analytical Method for the Estimation of Avelumab and Axitinib Using UPLC and Its Application to Pharmacokinetic Studies

Aims: Bioanalytical methods for the estimation of Avelumab and Axitinib using UPLC have been validated and may be used in Pharmacokinetic investigations. Place and Duration of Study: Department of Engineering Chemistry, AUCE (A), Visakhapatnam, Andhra Pradesh, between September 2021 to February 2022. Methodology: Using a C18 column (50x2.1 mm, 1.7 ) and an organic mobile phase containing 0.1 percent formic acid and Acetonitrile in a 70:30 ratio, this article provides an overview of recent advances in bioanalytical UPLC procedures. Results: Avelumab and axitnib had linear calibration curves in the 5-100 ng/ml and 5-100 ng/ml ranges, respectively. Avelumab's precision and accuracy trial results were from 100.18 to 103.94, whereas Axitinib's were 99.39 to 100.81, according to the findings. The LQC and HQC of Avelumab and axitinib were 99.9 and 99.1 percent, respectively, in the matrix effect. There was a 0.67 and a 0.59 percent CV for the two medicines at the LQC and HQC levels respectively. In pharmacokinetic investigations, a simple and effective approach was created and used to observe the analyte of interest in bodily fluids. Conclusion: It shows that the system's appropriateness, precision, linearity, and accuracy are all in accordance with USFDA criteria and may be used successfully for pharmacokinetic research in rabbits. Original Research Article Prasanthi and Himabindu; JPRI, 34(27A): 1-10, 2022; Article no.JPRI.84631 2

Pfizer developed axitinib, marketed as Inlyta, as a small molecule tyrosine kinase inhibitor [20,21]. It has been proven in animal (xenograft) models [22] to dramatically limit the development of breast cancer [23], as well as in human studies with renal cell carcinoma (RCC) and numerous other tumour types [24]. The most frequent adverse effects include diarrhoea, hypertension [25], tiredness, reduced appetite, nausea, dysphonia, hand-foot syndrome [26], weight loss, vomiting, asthenia, and constipation, which occur in more than 20% of individuals. Coadministration with powerful CYP3A4/CYP3A5 [27] inhibitors should be avoided as it may impair axitinib plasma clearance. The structural formula of Axitinib was C 22 H 18 N 4 OS.
To date, there have been no bio analytical UPLC methods for Avelumab and Axitinib estimation. Thus, the goal of the study is to predict Avelumab and Axitinib, which is a pharmaceutical component, using RP-UPLC.

Chemicals and Reagents
Acetonitrile, HPLC-grade formic acid, water were purchased from Merck India Ltd, Mumbai, India. APIs of Avelumab and Axitinib standards were procured from Glenmark, Mumbai.

Equipment
Agilent1290 Infinity II LC System with quaternary pump, PDA detector with empower 2.0 software was used.

Chromatographic Conditions
When utilising a chromatography column of size C18 (50mmx2.1mm,1.7), the separation was performed in an isocratic mode at temperature using a C18 (50mmx2.1mm,1.7) column in a chromatographic condition of isocratic operation. In this experiment, formic acid (0.1 percent) and acetonitrile (70:30 v/v) were utilised as the mobile phase. As the greatest concentrations of Avelumab and Axitinib were observed at 220 nm, the injection volume was 5 µl, and the eluent was measured at that wavelength. So, the wavelength of 220 nm was chosen.

Preparation of standard stock solution
In a 100ml volumetric flask, add 5 mg of Avelumab working standards and 70 mg of diluents; sonicate for 10 minutes to dissolve the contents fully and bring the diluent volume to the mark. Add 4 ml to a 100 ml volumetric flask for further dilution. 1 ml of the aforementioned solution is placed into a 10 ml volumetric flask, and the diluent is added to bring the volume to the mark.

Preparation of internal standard stock solution
Daunorubicin, 5 mg, is dissolved in 10 minutes by ultrasonically dissolving it in 100 millilitres of diluent, which is then transferred to a volumetric flask for analysis. To get 100 mL of this solution, use a volumetric flask with a neck diameter of 100 mm. One millilitre of the aforementioned solution is transferred to a 10 millilitre volumetric flask and diluted with the diluent.

Preparation of standard solution
Preparation for a typical meal A 2 ml centrifuge tube was filled with 200 µl of plasma, 300 µl of acetonitrile, and 500 µl of standard stock solutions, IS, and diluents, then vortexed for 10 minutes. Centrifuged for 30 minutes at 2000 rpm on these samples. The solution was collected, filtered via a 0.45 nylon syringe filter, and then put into a vial before being injected into the machine.

Pharmacokinetic Study
All animals are fed and watered ad libitum overnight prior to research. The technique that was employed was a topical anaesthetic. These formulations were subjected to in-vitro testing for pharmacokinetics. Each of the rabbits was given the samples when they were fasting from food and water. 0.5, 2, 4, 8, 12, 16, 20, 24, 28, 32, and 36 hours after oral administration of avelumab and axitinib, blood samples were taken from the rabbit marginal ear vein using a 25gauge,5/8 inch needle by clipping it with a paper clip. The blood was drawn in an Eppendorf tube having an EDTA solution concentration of 10%. 5000 rpm for 30 minutes at 2-8°C was used to centrifuge the blood. The crystal-clear plasma supernatant was collected and kept at -30°C until it could be analysed. A new analytical approach was used to assess the plasma samples for drug content after liquid-liquid phase extraction. The animals were returned to the animal home for rehabilitation when the research was completed.
These characteristics were derived from plasma concentration data for avelumabs and axitinis that were administered orally. An AUC, C max , T max , the time at which the peak of the pharmacokinetic parameters K el , t 1/2 , were determined using this information. The concentration-time curve was plotted against time, and data was collected using the trapezoidal rule approach. From the graph, we were able to determine C max and T max .

RESULTS AND DISCUSSION
A recovery study, re-injection repeatability and stability were tested using the method's selectivity, sensitiveness, linearity, accuracy and preciseness, matrix condition, and recovery.

Specificity
Avelumab and Axitinib studies using this approach was shown to be very specific. Figs. 1 and 2 show the chromatograms of the blank and the standard. Analysis of the two samples showed no interference in their chromatograms.

Matrix Effect
The percent RSD (Relative standard deviation) for ion suppression/enhancement within the signal was reported to be 1.0 percent for Avelumab and Axitinib in UPLC, indicating that the matrix influence on analyte ionisation is within an acceptable range of ionisation under these conditions. Avelumab had 99.9 percent LQC (Low Quality Control) and 99.1 percent HQC (High Quality Control) in the matrix effect, whereas axitinib had 99.3, 100.2 percent LQC and HQC, respectively. The percent CV of the two medicines was 0.67 and 0.59 at the LQC level, and 0.73 and 1.24 at the HQC level, respectively. It demonstrates that the matrix influence on the ionisation of the analyte is within the acceptable range of values.

Linearity
The peak area ratio of calibration standards was proportional to the concentration of the calibration standard solution. Avelumab has a concentration range of 5 -100 ng/ml, whereas Axitinib has a concentration range of 5-100 ng/ml. In the accompanying table 1, the linearity findings of Avelumab and Axitinib are provided, as well as their calibration plots, which are displayed in picture 3. Calibration curves for Avelumab and Axitinib looked to be linear, and the coefficient of correlation was determined to be 0.999 for both drugs.

Precision and Accuracy
The accuracy and precision of internal control samples were determined by combining the findings of all individual assays. Based on the information presented, it was clear that the technique used was exact and successful. Table  2, 3 shows the precision findings for avelumab and axitinib.

Recovery
The bio-analytical method's extraction effectiveness was proven by the recoveries of Avelumab and

Stability
Diluents were added to Avelumab and Axitinib solutions for stability analysis and stored at 2-8 o C in a refrigerator. There was a correlation between recently created stock solutions and previously prepared stock solutions prepared 24 hours earlier. For a total of tswenty-four hours, the plasma on both the bench top and the auto sampler remained steady at 20 o C. Axitinib and Avelumab were found to be stable at -30 o C for up to 24 hours, based on the stability of the products in the future. Table 4 shows the overall stability data for avelumab and Axitinib.

In vivo Pharmacokinetic Evaluation
In both examples of experimental formulation, the graph showed a bell-shaped curve. The presence of Avelumab and Axitinib in the blood for 24 and 4 hours after oral treatment reflects the efficiency of the formulation's drug release. Table 6 displays the results of the calculations of the pharmacokinetic parameters C max , T max , T 1/2 , K el , AUC 0-t , and AUC 0-Both Avelumab and Axitinib had a maximum plasma concentration (C max ) of 45.203 ng/ml and 46.232 ng/ml. For Avelumab and Axitinib, the T max was determined to be 24 hours and four hours, respectively. ' Avelumab and Axitinib had t 1/2 values of 44 and 20 hours, respectively, in Table 6 and recovery plots were shown in Fig. 4.

CONCLUSION
For the first time, a more sensitive UPLC technique for the detection of Avelumab and Axitinib in rabbit plasma was developed and validated. Methods given here are robust, rapid and repeatable in the bio-analytical field. This approach has been shown to be safe and effective by the US Food and Drug Administration (FDA). For pharmacokinetic research and to observe the analyte in bodily fluids, an effective and simple approach has been devised.

CONSENT
It is not applicable.

ETHICAL APPROVAL
The protocol of animal study was approved by institute of animal ethics committee (reg.no:1074/po/re/s/05/cpcsea).