Application of Box-Behnken design for optimization of A RP-HPLC method for determination of palonosetron and netupitant in their combined dosage form in presence of their impurities

Abstract The use of (Netupitant and Palonosetron) combination to treat nausea and vomiting in cancer chemotherapy patients has been authorized by the Food and Drug Administration. For the simultaneous determination of Netupitant (NET) and palonosetron (PAL) in the presence of two of their related substances and in their dosage form, a sensitive and selective RP-HPLC method has been developed and validated. The aforementioned medications were separated and quantified with the help of experimental design. The Box-Behnken design was used in the experiment to optimize the chromatographic method’s analytical parameters. It employed RP-HPLC with a UV detector. Waters ODS-C18 column (3.5 µm, 75 × 4.6 mm) with a mobile phase composed of acetonitrile: 25 mM phosphate buffer (pH = 3.5) in a gradient mode at 254 nm was employed to separate the cited drugs and their impurities. Palonosetron was linear over the concentration range (1–50 µg/mL) and Netupitant (10–100 µg/mL). According to ICH guidelines, the new method underwent thorough validation. Between the proposed method’s results and those from the reported method, there was no significant difference. It is easy to apply the technique to the analysis of the specified drugs in their combination dosage form for quality control considerations. Graphical Abstract


Introduction
Palonosetron (PAL), (3aS)-2-[(3S)-1-azabicyclo [2.2.2] octan-3-yl]-3a,4,5,6-tetrahydro-3H-benzo [de]isoquinolin-1-one, is an isoquinolone with an empirical formula of C 19 H 24 N 2 O and a molecular mass of 332.8 (Figure 1a). [1] PAL is a 5-HT3 receptor antagonist which has anti-emetic activity at both central and GI sites. [2] It is commonly recommended for the acute and delayed stages of chemotherapy-induced nausea and vomiting (CINV) after moderately emetogenic chemotherapy (MEC), as well as the acute phase of CINV after highly emetogenic chemotherapy (HEC). Although PAL is the sole allosteric antagonist, it has a higher affinity for the 5-HT3 receptor than the other 5-HT3 receptor antagonists and has a lengthy duration of action. [3] The use of AKYNZEO V R (NET and PAL) to treat nausea and vomiting in cancer chemotherapy patients has been approved by the US Food and Drug Administration in 2014. [4] Netupitant (NET), 2-[3,5-bis(trifluoromethyl)phenyl]-N,2dimethyl-N-[4-(2-methylphenyl)-6-(4-methylpiperazin-1-yl) pyridin-3-yl] propenamide (Figure 1b), has been shown to be beneficial for patients in circumstances including chemotherapy-induced nausea and vomiting, where blocking neurokinin 1 (NK1) receptors has shown clinical value. [5] When combined with palonosetron, it is used to relieve nausea and vomiting in cancer chemotherapy patients. [4] Pharmacological evidence shows that it only inhibits NK1-to-NK1 receptor binding, not NK2 or NK3 receptors. High brain penetration, oral activity, and a potent and focused NK1 receptor antagonist are all characteristics of NET. [5] 5,6,7,8 Tetrahydronaphthalene-1-carboxylic acid (IMP A) ( Figure  1c) [6] is one of the intermediate compounds in the synthesis of PAL while 2-chloro-5-nitropyridine (IMP B) (Figure 1d), is an intermediate during the synthesis of NET. [7] Many HPLC methods coupled with UV-detector have been reported for the simultaneous assay of PAL and NET with or without their degradation product in stability studies, either in bulk or in its pharmaceutical dosage form [8][9][10][11][12][13][14][15][16][17][18][19] or in spiked human plasma. [20] UPLC methods have been reported for their quantitative assay. [21][22][23] A chromatographic method coupled with mass detector (LC-MS/MS) has been reported for their simultaneous quantitative assay in human plasma. [24] Based on the previous finding, there are many chromatographic methods have been reported for the simultaneous assay of PAL and NET in presence of their forced degradation products, but there was no single method for their quantitative assay in the presence of their intermediate impurities which may be included in dosage form during the industrial process of preparation of the pharmaceutical products. PAL impurity 11, namely 5,6,7,8 tetrahydronaphthalene-1-carboxylic acid, is one of the intermediates involved in the synthesis of PAL. The first step in PAL synthesis includes reduction of 1-naphthoic acid using 10% Pd/C in the presence of an alcohol or an organic acid, to provide 5,6,7,8-tetrahydro-naphthalene-1-carboxylic acid as described by European Patent Office. [25] In addition, the synthesis of NET was initiated with a nucleophilic aromatic substitution reaction involving 2-chloro-5-nitropyridine. [26] Consequently, the aim of this study is to develop new, rapid, sensitive HPLC method for the quantitative assay of both drugs in presence of their intermediate impurities; 5,6,7,8 tetrahydronaphthalene-1-carboxylic acid (IMP A) and 2chloro 5-nitropyridine (IMP B).
DoE (Design of Experiments) is a broad paradigm that has the main objective to develop a detailed process through techniques for experiment design that are well-established. DoE is described as "a systematic approach to pharmaceutical development that begins with established objectives and emphasize product and process understanding and process control, based on strong science and quality risk management" by the International Conference of Harmonization (ICH). [27] Numerous statistical experimental designs have been acknowledged as helpful methods for comprehending the variables and their interactions. [28] A variety of experimental designs may be employed to decrease the number of investigations while producing more useful results. If the objective is to filter a large number of variables, first-order models, such as the Plackett-Burman design, can be used. If the goal is to correctly optimize a process or quantitatively estimate a reaction, second-order models, like the Box-Behnken design (BBD), are chosen. [29] BBD is an independent, rotatable quadratic design where the design combinations are at the midpoints of the process space's edges and at its center and which excludes embedded factorial or fractional factorial design. [29][30][31] Using a three-level BBD design with a center point, the effects of the independent experimental variables that were chosen on the dependent variables were evaluated. The developed method has been validated in compliance with ICH recommendations [32] and is suitable for the quality control assessment of the drugs specified.

Instrumentation
An HPLC instrument Waters (Alliance e 2695) with Waters detector (PDA 2998) and Auto-injector (Alliance e 2695) have been utilized. Waters ODS-C 18 column (3.5 mm,75 Â 4.6 mm) and an Elmasonic EASY 180 H (220-240 V) sonicator were employed. The pH measurements were carried out using pH meter (AQUASEARCHER TM AB33PH Bench Meter). The mobile phase was filtered using nylon membrane filters with 0.45 mm pore sizes from Sigma-Aldrich Company in Germany. Digital balance (OHAUS, PR series, PR224) were used throughout the current experiment.

Preparation of stock and working solutions
A precisely weighted quantity of 56.15 mg of standard PAL HCl (equal to 50 mg PAL) was transferred into a 50 mL volumetric flask, and 25 mL of methanol was then added to create a standard stock solution of PAL (1 mg/mL) in methanol. After performing sonication for 10 min, the volume was finished using the same solvent.
By properly weighing out 10 mg of standard NET into a 10 mL volumetric flask and adding 5 mL of methanol, a standard stock solution of NET (1 mg/mL) was prepared. After performing sonication for 10 min, the volume was finished using the same solvent.
A stock solution of 2-chloro-5-nitropyridine (IMP B) and 5,6,7,8 tetrahydronaphthalene-1-carboxylic acid (IMP A), each of concentration of 1 mg/mL solution was prepared by transferring the accurately weighted amount of 10 mg of each into a 10 mL volumetric flask and adding 5 mL methanol in each one. The volume was ultimately finished with the same solvent after 10 min of sonication.
All the prepared stock solutions are stored at a refrigerated temperature (2-8 C).

DoE for chromatographic conditions improvement
Using a three-level Box-Behnken design with a center point, the effects of the independent experimental variables that were chosen on the dependent variables were examined. The 15 experimental runs that were carried out by injecting a mixture of the listed medications are listed down in Table 1. The theoretical plates of the first peak (N1) and retention time of the fourth peak (RT4) were the chosen responses, they were recorded and used to build the model. The data were examined using Minitab V R 17 software. Using the polynomial equations and the built models, it was possible to examine both the direct effects and the interactions of the independent variables.

Chromatographic conditions
Waters ODS-C 18 column (3.5 mm,75 Â 4.6 mm) was used for the simultaneous determination of PAL and NET in the presence of their intermediate impurities in their capsule, with a constant flow rate at 1 mL/min throughout the whole run. The method was conducted at gradient elution as follow; from 0 to 3 min, the mobile phase was composed of acetonitrile: 25 mmol phosphate buffer (pH ¼ 3.5) with a ratio of (40:60, v/v), then it was changed from 3 to 10 min to be composed of acetonitrile: 25 mM phosphate buffer with a ratio of (100:0, % v/v). After 10 min, reconditioning to the column occurred with the first composition of the mobile phase. The investigation was performed using a UVdetector at wave length (254 nm) and at room temperature. Twenty microliters was the injection volume.

Preparation of laboratory prepared mixtures
To obtain various ratios of the concentrations of the four components, accurate aliquots of the PAL working solutions, NET working solutions, and their intermediate impurities (Imp A and Imp B) were transferred into a series of 10 mL volumetric flasks, completed to volume with methanol, and thoroughly mixed (PAL, NET, IMP A, and IMP B).

Sample preparation
Each AKYNZEO V R Hard Capsule consists of one soft capsule of PAL (0.5 mg PAL) and three tablets of NET (each tablet contains 100 mg NET). So, the sample was prepared for each separately. For PAL, 10 soft capsules were sonicated with 5 mL methanol for 30 min before being extracted into a 10 mL volumetric flask. With methanol, the volume was finished to a total of 10 mL. To create a sample stock solution of (500 mg/mL), the mixture was then thoroughly mixed and filtered using a dry funnel and dry filter paper while discarding the first few milliliters.
For NET, 10 tablets were sonicated with 50 mL of methanol for 30 min in a 100 mL volumetric flask. Methanol was used to get the level to 100 mL. To obtain a sample stock solution of (10 mg/mL), the mixture was well mixed, filtered on dry funnel and dry filter paper, and the first few milliliters were discarded. This sample stock solution was then further diluted to produce several working solutions.

Validation of the proposed method
In compliance with ICH Q2R1 guideline, [31] the developed RP-HPLC technique for the simultaneous measurement of PAL and NET in dosage form was validated.

Results and discussion
The purpose of the current work was to create a time-efficient, sensitive, and selective analytical approach to determine PAL and NET in the presence of their synthetic intermediate impurities in their combined dosage form. In which method development is built upon the experimental design.

Method development
The main purpose of this chromatographic method was the separation of PAL, NET, and their impurities and the quantitation of PAL and NET in their combined dosage form.

Application of Box-Behnken design for optimization of the chromatographic conditions
With a center point, a three-level Box-Behnken design was used. The levels of each factor are shown in Table 1, and while all other experimental runs were carried out at random without replication, the response measurement at each factor's center point (level zero) was repeated three times to determine the experimental error. Following computation, the models' second order polynomial equations were determined to be: where RT4: is the retention time of peak number (4) (NET peak); N1: is the number of theoretical plates of peak number (1) (PAL peak); G1 is the % of acetonitrile in the first stage of gradient elution and G2 is the % of acetonitrile in the second stage of gradient elution, respectively. By simple observation of the equations, without conducting the trials, the reaction may be anticipated for each potential level of the components. This model considers interactions between the components under study as well as linear, quadratic, and other impacts. To cut down on the quantity of meaningless terms, a progressive backward elimination process was chosen.
For RT4 and N1; the minimum retention time and maximum number of theoretical plates are obtained using a pH 3.43, acetonitrile ratio in the first stage of the gradient elution (G1) is 40%, and acetonitrile ratio in the second stage of the gradient elution (G2) is 100%. Upon applying the proposed conditions for the optimization of the separation of the cited drugs, adequate separation was achieved. Therefore, the operated chromatographic conditions throughout the current study were gradient elution from 0 to 3 min with a mobile phase composed of acetonitrile: 25 mmol phosphate buffer (pH ¼ 3.5) with a ratio of (40:60, % v/v), then it was changed from 3 to 10 min to be composed of acetonitrile: 25 mmol phosphate buffer with a ratio of (100:0, % v/v). Contour plots and response surface plots were created for each response as shown in Figures 2 and 3, respectively.

Effect of factors
Equation (1) reveals that RT4 is inversely proportional to both of acetonitrile ratio in the first stage and in the second stage of the gradient elution and directly proportional to pH. While, Equation (2) reveals that N1 and pH are directly proportional, while pH's quadratic term is negatively skewed. As observed in Equation (2), the pH coefficient is a large number which concludes the great effect of pH on N1 and the quadratic term of pH is negative to indicate that as pH increases, N1 increases to a limit where further increase in pH leads to a decrease in N1 (Figure 4).

Statistical analysis of the model
Utilizing the statistical programme Minitab V R 17, experiment findings were statistically examined. The ANOVA test was used to validate the models. Which of the factors significantly affects the response has been assessed using the regression coefficients and their associated p values. Table 2 shows ANOVA results which reveal that the significant factors are G1 (% of acetonitrile in the first stage of gradient elution), G2 (% of acetonitrile in the second stage of gradient elution), and pH since their p values are <0.05. R 2 , adjusted R 2 , and predicted R 2 are used to gauge a model's ability to fit data and anticipate outcomes. Table 3 demonstrates that the R 2 , adjusted R 2 , and predicted R 2 are so near to one another, and they are all larger than 0.9, indicating that the models fit the data well and have good prediction capacities for further observations and optimization studies. Figure 5 displays the analysis response residual charts. The residuals typically follow a straight line in normal probability plots, indicating that the errors are normally distributed, supporting the idea that the models match the data. The histogram plots clearly show a normal distribution pattern, proving that the residuals are distributed normally. To confirm the notion that the residuals are independent from one   another, the residuals vs. order plot is employed. The fact that residuals are randomly distributed around the zero in vs. fit and vs. order plots indicates that the error terms are not connected to one another.

Computation of the optimum separation conditions
Using the response optimizer tool and the final models obtained, the best conditions that simultaneously provide the best values of RT4 and N1 are calculated. The response optimizer determines the best way to achieve the intended aim of minimizing RT4 and maximizing N1, and then plots the optimization of that solution ( Figure 6). After using the previous design, symmetric peaks, and a reasonable separation were seen upon applying a gradient elution from 0 to 3 min with mobile phase composed of acetonitrile: 25 mmol phosphate buffer (pH ¼ 3.5) with a ratio of (40:60, % v/v), then it was changed from 3 to 10 min to be composed of  acetonitrile: 25 mmol phosphate buffer with a ratio of (100:0, % v/v) at flow rate of 1 mL/min. The detection wavelength was chosen to be the absorption wavelength (254 nm) after trying different wavelengths (220 and 254 nm) as it produced higher sensitivity. The retention times of PAL, IMP A, IMP B, and NET was found to be 2.128, 4.707, 7.500, and 9.160 min, respectively (Figure 7).

RP-HPLC method validation
According to ICH guideline Q2 (R1), the optimized chromatographic method's validity was assessed using the following metrics: linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), specificity, and system suitability parameters. [32] Linearity Seven concentration levels of the standard solutions of PAL and NET, respectively, were examined to determine the linearity; each concentration was examined three times. By using linear regression analysis, the linearity was assessed. For the developed approach, the correlation coefficients were higher than 0.999. Table 4 summarizes the analytical information for the calibration curves, including the standard deviations for the slope (S b ) and intercept (S a ), as well as the confidence limits for slope and intercept.

Precision
Three different concentrations of PAL and NET were examined three times on the same day (intra-day precision) and on three successive days to assess the method's precision (inter-day precision) ( Table 4).

Accuracy
By examining six concentrations of standard PAL and NET solutions, the proposed method's accuracy was examined. Table 5 confirms the accuracy of the method where recoveries >98% and lower than 102% were achieved.

Specificity
The analytical method's specificity refers to its capacity to judge the analyte response in the presence of any potential contaminants or excipients present in the dosage form. By testing PAL and NET in laboratory-made combinations with various ratios of intact pharmaceuticals and their intermediate impurities (IMP A and IMP B), specificity was proven (Table 6). Additionally, PAL and NET analyses in hard capsules revealed no excipient interference (Figure 8). The high specificity of the suggested approach was demonstrated by the good recovery% and low standard deviations (SD) ( Table 7).

Limit of detection and limit of quantitation
In accordance with ICH standards, the slope of the regression equation and the SD of the response were used to estimate the parameters LOD and LOQ (Table 4).

System suitability
The metrics for determining if a system is suitable for theoretical plate number, resolution factor, tailing factor, capacity factor, and selectivity factor were established (Table 8).

Statistics
Statistical analysis was used to compare the proposed analytical method with the reported method. [17] There was no discernible difference between the experimental values obtained in the pure sample analysis using the newly developed method according to the results of the student's t-test and F test, which were used. The suggested approach and the reported method are statistically compared in Table 9, where the computed t values and F values were both lower than the tabulated t values and F values. On the other hand, the proposed method has the advantage of separating the cited drugs from their possible impurities. A simple comparison between the results of one of the reported methods (17) and the proposed method for the analysis of NET and PAL is summarized in Table 10. As shown in Table 10, retention time was shorter in the reported method (17) than the proposed method but the proposed method could separate and quantify the cited drugs in presence of two synthetic impurities. Also, the proposed method was more sensitive than the reported method for the determination of NET, while, the reported method was more sensitive for the determination of PAL.

Conclusion
The simultaneous quantification of PAL and NET in the presence of two of their synthetic intermediates' impurities required the development, optimization, and validation of a stability-indicating, selective chromatographic technique using DOE. A three-level BBD design with a center point was used to optimize the proposed method. The suggested method was applied to determine PAL and NET in their combined dosage form. It is helpful in regular examination of the specified medications individually or in their combination dosage form in quality control laboratories due to good results and an appropriate run time.

Authors contributions
Ehab F. ElKady and Eman A. Mostafa contributed to the study conception and design and interpretation of the data. Material preparation, data collection, and analysis were performed by Mohamed A. ElHamid and Eman A. Mostafa. The first draft of the manuscript was written by Eman A. Mostafa and Mohamed A. ElHamid and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.