A RELIABLE RP-UPLC-TUV METHOD FOR SIMULTANEOUS ESTIMATION OF CLARITHROMYCIN, AMOXICILLIN, AND VONOPRAZAN IN CO-PACKED PHARMACEUTICAL DOSAGE FORMS: METHOD DEVELOPMENT AND VALIDATION WITH STABILITY INDICATING PROPERTIES

Objective: The study aims to develop a reliable RP-UPLC-TUV method for simultaneous estimation of Clarithromycin, Amoxicillin, and Vonoprazan in bulk and combined dosage. Methods: A simple, specific, and reliable method for determining Clarithromycin, Amoxicillin, and Vonoprazan has been developed using the RP-UPLC method. In order to successfully separate Clarithromycin, Amoxicillin, and Vonoprazan, 1.0 µl of a 100 % level solution was injected into a Hibar C18 (100 x 2.1 mm and 2 µm) column. The mobile phase consisted of Ammonium Acetate and Acetonitrile in equal volumes, and the flow rate was kept at 0.3 ml/min while the detection wavelength was set to 210 nm. Both the column and the injection port were kept at a temperature of 30 °C at all times. Results: The retention time (RT) of Clarithromycin, Amoxicillin, and Vonoprazan was observed at 1.24 min, 0.97 min and 1.66 min, correspondingly with accepted system suitability. The linear responses were observed for Clarithromycin, Amoxicillin, and Vonoprazan in the range of 25 to 150 µg/ml, 25 to 150 µg/ml and 1 to 6 µg/ml, respectively. The LOD and LOQ values were calculated to 0.07 µg/ml and 0.22 µg/ml for Clarithromycin, 0.81 µg/ml and 2.45 µg/ml for Amoxicillin and 0.03 µg/ml and 0.09 µg/ml for Vonoprazan. The % RSD values of both precision were assessed in the range of 0.8-1.4. The mean recovery of Clarithromycin, Amoxicillin, and Vonoprazan was in the range of 99.66 %-100.88 %. The statistical analysis of the validation parameters confirmed that the approach was reliable in terms of its accuracy, sensitivity, and precision while also exhibiting a high degree of sensitivity. The study of analytes in a variety of stressful situations guarantees the stability of the substances, ensuring that they represent the method's stability indication. Conclusion: The newly established technique is quite effective in separating Clarithromycin, Amoxicillin, and Vonoprazan from one another. Also separated with excellent resolution were the degradation products that were formed as a result of the stress conditions. The study concluded that the developed method has considerable adoption in the pharmaceutical sector.


INTRODUCTION
In order to treat a wide range of bacterial infections, a popular antibiotic that is used for this purpose is called Clarithromycin [1].It is an antibiotic that comes from erythromycin and is part of the macrolide family [1][2][3].It is via this inhibition of bacterial protein synthesis that Clarithromycin is able to exert its antibiotic actions [1,2].It does this by attaching itself to the 50S component of the bacterial ribosome, which prevents the production of protein synthesis complexes that are really useful.Because of this interference with protein synthesis, bacterial growth and reproduction are both stifled as a result [2,3].The antibacterial properties of Amoxicillin come from the drug's ability to block the production of cell walls in bacteria [1][2][3][4].It accomplishes this goal by causing disruptions in the cross-linking of peptidoglycan chains, which are necessary constituents of the cell walls of bacteria [4,5].The bacterial cell is rendered more vulnerable to osmotic lysis as a result of the effects of Amoxicillin, which works by preventing the production of a cell wall that is capable of maintaining its structural integrity.The germs will perish as a consequence of this action in the end.Clarithromycin and Amoxicillin are the two antibiotics that are indicated as part of a treatment plan for eliminating Helicobacter pylori [1][2][3][4][5].This bacterium is linked to peptic ulcers [5].In order for Vonoprazan to have its effect, it inhibits in a way that is both selective and irreversible an enzyme known as H+/K+-ATPase [6,7].This enzyme is what is ultimately responsible for the formation of acid in the stomach.Because it inhibits the activity of this enzyme, Vonoprazan is superior to other proton pump inhibitors (PPIs) in its ability to lower stomach acid production [6,7].It has an acidsuppressing effect that lasts for a longer period of time, which makes it effective for treating diseases where there is a worry for excessive acid generation [6].Ulcers, also known as sores in the lining of the stomach or intestine, may be caused by a specific kind of bacterium known as H. pylori [6,7].The combination of Clarithromycin, Amoxicillin and Vonoprazan is effectively used to treat ulcers and stop their recurrence [8,9].The three combined drugs were approved by FDA in May 2023 [9].The IUPAC names and chemical structures of Clarithromycin, Amoxicillin and Vonoprazan were publicized in fig. 1 [10][11][12].
A large number of analytical procedures were observed in the review of literature for analysis of Clarithromycin and Amoxicillin as solo entity and in a mixture with other agents [13][14][15].In the same way, numerous methods have been pronounced in the literature to estimate Clarithromycin and Amoxicillin combination with other anti-ulcer drugs such as omeprazole, lansoprazole, and rabeprazole [16][17][18][19].Clarithromycin, Amoxicillin and Vonoprazan are a novel combination in the pharmaceutical industry, effectively used to treat ulcers and stop their recurrence than former combinations and hence, a reliable liquid chromatographic approach is necessary in order to evaluate the quality and amount of Clarithromycin, Amoxicillin, and Vonoprazan in bulk as well as formulations.A review of the relevant literature found that, as of this moment, there has not been a single analytical method documented for the examination of Clarithromycin, Amoxicillin, or Vonoprazan.UPLC is a technology that requires shorter run periods and boosts sensitivity, in addition to providing selectivity, sensibility, and a wide range of analysis.As a result, the establishment and validation of a new UPLC technique was chosen and prioritized.

Instruments
This chromatographic examination took place using the aid of an Acquity UPLC separation unit that was mounted with a TUV detector.In order to do tests and perform processing on the data that had been generated, the Empower 2 software was employed.The pH meter obtained from BVK enterprises, India.

Chemicals and reagents
Gift samples of Clarithromycin, Amoxicillin, and Vonoprazan were given by Spectrum Research Lab, Hyderabad.Ammonium acetate, acetic acid, and acetonitrile were purchased from Merck, India.

Preparation of solutions
Standard preparation 50 mg Clarithromycin, 50 mg Amoxicillin, and 2 mg Vonoprazan pure powders were accurately weighed and transferred into a 50 ml volumetric flask, where they were dissolved with diluent to obtain a solution with concentrations of 1000, 1000, and 40 µg/ml for Clarithromycin, Amoxicillin, and Vonoprazan, respectively.It is illustrated as stock solution.1 ml of the above-mentioned stock solution was diluted to 100 ml to get concentrations of 100, 100, and 4 µg/ml for Clarithromycin, Amoxicillin, and Vonoprazan, respectively (100 % solution).

Preparation of sample solution
The tablets and capsule powder equivalent to 50 mg of Clarithromycin, 2 mg of Vonoprazan and 50 mg of Amoxicillin was precisely weighed and taken in a 50 ml volumetric flask, where they were dissolved with diluent to obtain a solution with concentrations of 1000 µg/ml, 1000 µg/ml, and 40 µg/ml for Clarithromycin, Amoxicillin, and Vonoprazan, respectively.After filtration, 1 ml of the above-mentioned stock solution was diluted to 100 ml to get concentrations of 100, 100, and 4µg/ml for Clarithromycin, Amoxicillin, and Vonoprazan, respectively.Before the sample solution was introduced into the device, it was filtered via a 0.45 µ nylon filter so that any particulate matter could be removed.

Method development
This was accomplished by a variety of trials (table 1), which involved modifying the mobile system, columns, and buffers, among other things.Initially, method development was started with SB 100 C18 column and mobile phase of OPA: Acetonitrile (40:60 v/v), where peak shapes were not good along with the less plate count for Vonoprazan.Further, OPA was replaced with KH2PO4 in mobile system and column was changed to HSS C18 and Hibar C18.Successful separation of Clarithromycin, Amoxicillin, and Vonoprazan were not done with the altered conditions.Finally, with Hibar C18 (100 mm x 2.1 mm and 2 µm) column, Ammonium Acetate and Acetonitrile (60:40 v/v), all three drugs separated effectively.

Method validation
Validation of the technique was performed in accordance with the Q2 standards of the ICH guidelines [30].

System suitability
Standard solution was thereafter injected in six separate duplicates in order to verify that the present procedure is suitability for the system.System suitability parameters such as USP plates, resolution, tailing and % RSD were assessed computed from the resultant chromatograms.

Linearity
The linear response of the current approach was assessed by analyzing a series of concentrations of 25, 50, 75, 100, 125, 150 µg/ml for both Clarithromycin, Amoxicillin and 1, 2, 3, 4, 5, and 6 µg/ml for Vonoprazan.At last, a calibration curve was plotted for each Clarithromycin, Amoxicillin, and Vonoprazan in between given concentrations and resultant peak responses to compute the regression coefficient (R 2 ).The identical process was repeated for two more time to calculate the standard deviation (SD) of yintercept and mean of slope.

Sensitivity
Sensitivity in terms of limit of detection (LOD) and limit of quantification (LOQ) were computed by following equations.
Where σ-SD of the y-intercepts of linear plot (n=3)

Specificity
The approach is considered as specific when the drug under investigation can be identified by the method to be present in the presence of other matter efficiently and without any interference.Injecting a volume of 1.0 µl of each of the separate solutions of blank, standard preparation, and standard solution spiked with excipients was needed.It was determined if there were any interferences between the retention times (RT) of the blank and the placebo and the RTs of the Clarithromycin, Amoxicillin, and Vonoprazan in the chromatograms that were recorded.The approach was able to achieve a higher level of specificity by comparing the chromatograms of the standard and forced degradation solutions.This was done in order to check for interferences between the RTs of the degradation products and active ingredients Clarithromycin, Amoxicillin, and Vonoprazan.

Precision
The findings of the homogeneous solution on multiple analyses under similar environments are shown to have an excellent correlation with one another; one may assert that the methodology in concern possesses high levels of precision.Analyzing the 100 % level six times brought the system precision of the anticipated method up to the expected level while injecting the sample solution six times brought the method precision up to the expected level.Calculations were done to determine the % RSD values of observed peak regions (system precision) as well as % assay (method precision).

Accuracy
To validate the accuracy parameter, the percentage recovery procedure was chosen as the approach to use.During the course of this procedure, a predetermined quantity of sample solution was mixed in with three distinct percentages of standard solution (50, 100, and 150 %).Each solution that had been spiked was examined three times.At each of the three levels that were indicated, the typical % recovery of Clarithromycin, Amoxicillin, and Vonoprazan was calculated.

Robustness
The ability of the technique to generate similar outcomes after changing the method conditions deliberately to some amount is termed as "robustness."A number of minor modifications were made to the flow rate (±0.1 ml/min), Temperature (±5 °C), and mobile phase ratio (±1 part) in order to verify the reliability of the approach that is now being used.

Forced degradation (FD) studies
In order to assess the stability indicating quality of the procedure, it was decided to purposefully subject the drug solution to circumstances that would cause its degradation.Research may readily make accurate predictions about the pathways of degradation and the necessary storage conditions for drug material and drug product by using these investigations [20][21][22].FD studies were done in accordance with the requirements of ICH's Q1A, QIB, and Q2B [28][29][30].

Acid degradation
In the process of acid degradation, stock solution of blended Clarithromycin, Amoxicillin, and Vonoprazan and 2N HCl solution were equally mixed together and allowed to reflux for thirty minutes at a temperature of 60 °C.Further, the mixed solution was neutralized with 2N NaOH and again diluted to obtain a solution of 100, 100 and 4 µg/ml for Clarithromycin, Amoxicillin, and Vonoprazan correspondingly.After injecting a volume of 10 µl of the prepared solution above, chromatograms were obtained and then assessed to determine the amounts of Clarithromycin, Amoxicillin, and Vonoprazan that had been degraded by comparing them to the control solution [23].

Alkali degradation
In the process of Alkali degradation, stock solution of blended Clarithromycin, Amoxicillin, and Vonoprazan and 2N NaOH solution were equally mixed together and allowed to reflux for thirty minutes at a temperature of 60 °C.Further, the mixed solution was neutralized with 2N HCl and again diluted to obtain a solution of 100, 100 and 4 µg/ml for Clarithromycin, Amoxicillin, and Vonoprazan correspondingly.After injecting a volume of 10 µl of the prepared solution above, chromatograms were obtained and then assessed to determine the amounts of Clarithromycin, Amoxicillin, and Vonoprazan that had been degraded by comparing them to the control solution [24].

Oxidative degradation
In the process of Oxidative degradation, stock solution of blended Clarithromycin, Amoxicillin, and Vonoprazan and 20 % H2O2 solution were equally mixed together and allowed to reflux for thirty minutes at a temperature of 60˚C.The solution again diluted to obtain a solution of 100, 100 and 4 µg/ml for Clarithromycin, Amoxicillin, and Vonoprazan correspondingly.After injecting a volume of 10 µl of the prepared solution above, chromatograms were obtained and then assessed to determine the amounts of Clarithromycin, Amoxicillin, and Vonoprazan that had been degraded by comparing them to the control solution [25].

Photodegradation
During the course of the Photodegradation process, the stock solution of blended Clarithromycin, Amoxicillin, and Vonoprazan was placed in an UV cabinet at shorter wavelength (254 nm) over a period of seven days while a dark control was maintained.The solution again diluted to obtain a solution of 100, 100 and 4 µg/ml for Clarithromycin, Amoxicillin, and Vonoprazan correspondingly.After injecting a volume of 10 µl of the prepared solution above, chromatograms were obtained and then assessed to determine the amounts of Clarithromycin, Amoxicillin, and Vonoprazan that had been degraded by comparing them to the control solution [26].

Dry heat degradation or thermal degradation
During the course of the dry heat degradation process, a standard stock solution volume about 10 ml was kept in oven at 105 °C for 6 h.The solution again diluted to obtain a solution of 100, 100 and 4 µg/ml for Clarithromycin, Amoxicillin, and Vonoprazan correspondingly.After injecting a volume of 10 µl of the prepared solution above, chromatograms were obtained and then assessed to determine the amounts of Clarithromycin, Amoxicillin, and Vonoprazan that had been degraded by comparing them to the control solution [27].

Neutral degradation by water
In the process of oxidative degradation, stock solution of blended Clarithromycin, Amoxicillin, and Vonoprazan and 2N NaOH solution were equally mixed together and allowed to reflux for thirty minutes at a temperature of 60˚C.The solution again diluted to obtain a solution of 100, 100 and 4 µg/ml for Clarithromycin, Amoxicillin, and Vonoprazan correspondingly.After injecting a volume of 10 µl of the prepared solution above, chromatograms were obtained and then assessed to determine the amounts of Clarithromycin, Amoxicillin, and Vonoprazan that had been degraded by comparing them to the control solution.

Application of current method for assay of commercial dosage form
The stated approach was utilized to determine the % purity of Clarithromycin, Amoxicillin, and Vonoprazan in commercially available formulation.This was accomplished by performing the analysis of same concentrations of standard and sample solution.The % purities of Clarithromycin, Amoxicillin, and Vonoprazan were determined by comparing the peak responses of Clarithromycin, Amoxicillin, and Vonoprazan in both the standard solution and the sample solution.

Optimized method conditions
In the aim of successful separation of Clarithromycin, Amoxicillin, and Vonoprazan, 1.0 µl of a 100 % level solution was injected into a Hibar C18 column with dimensions of (100 mm x 2.1 mm and 2 µm).The mobile system consisted of ammonium acetate and acetonitrile in equal volumes, and the flow rate was kept at 0.3 ml/min while the detection wavelength was set to 210 nm.As compared to the reported methods [16][17][18][19], this method has more organic phase, which was good for the column life.Both the column and the injection port were kept at a temperature of 30 °C at all times.The mobile phase and all prepared solutions were filtered through the 0.45 microns filters before being introduced to the instrument.This was done to eliminate any particulate debris.The RT of Clarithromycin, Amoxicillin, and Vonoprazan observed at 1.24 min, 0.97 min and 1.66 min, correspondingly with accepted system suitability (fig.2).The total run time was 3 min.Which was very short when compared to the reported methods [16,19].

Method validation
The system suitability indicators, such as % RSD, R, N, and T, followed the previously established acceptable limits established by various regulatory bodies.Table 1 displays the acquired results together with the allowed ranges for the system suitability parameters.The R 2 values assessed from regression analysis for the Clarithromycin (25 to 150 µg/ml), Amoxicillin (25 to 150 µg/ml), and Vonoprazan (1 to 6 µg/ml) concentrations have been calculated to be 0.999, depicting the linearity of the procedure with a significant impact (fig.3).Clarithromycin, Amoxicillin, and Vonoprazan had mean slopes of 26188, 20093, and 38082, respectively.The SD values of the y-intercepts of Clarithromycin, Amoxicillin, and Vonoprazan were determined to be 9193, 1079.7, and 621.9, respectively.From the reported methods, it was concluded that, present method was very sensitive to estimate the drugs at low concentrations.Whereas the reported methods were having the linearity range at higher concentrations [19].The LOD and LOQ were assessed as 0.07 µg/ml and 0.22 µg/ml for Clarithromycin, 0.81µg/ml and 2.45 µg/ml for Amoxicillin and 0.03 µg/ml and 0.09 µg/ml for Vonoprazan.At the RT of Clarithromycin, Amoxicillin, and Vonoprazan, could not observe any interference from other solutions.It demonstrates the high degree of specificity of stated method determining the Clarithromycin, Amoxicillin, and Vonoprazan present in given bulk and formulation samples (fig.4  and 5).

Fig. 4: Chromatograms representing the specificity of the method
The % RSD of the peak area responses of Clarithromycin, Amoxicillin, and Vonoprazan in successive consecutive injections of 100 % level or standard solution was less than two (table 1).This is a major representation of the methods' precision.All the values were expressed in (n=3) mean±SD According to the opinions of experts, it is often acceptable for stabilityindicating approaches to tolerate up to 10 % degradation of the drugs material in most instances [23].Comparing the peak regions in the resulted chromatograms of control with those of the standard solution under stressful circumstances allowed for the calculation of an estimate of the proportion of Clarithromycin, Amoxicillin, and Vonoprazan that had degraded.The findings that were observed and calculated are shown in table 4. All the forced samples chromatograms were shown in fig. 5.The purity threshold values were greater than the purity angles of the produced degradants and intended drug substances.According to the findings of the observation, the procedure has a feature that indicates stability.Because degradation of any of the three analytes was not seen under settings of neutral pH, this indicates that the analytes were all stable under these conditions.

Application of current method for assay of marketed formulation
It was discovered that the % purity of the Clarithromycin, Vonoprazan, and Amoxicillin, marketed combination tablet and capsule, was 99.85-100.14% (table 5).This indicates that the assay values of the Clarithromycin, Amoxicillin, and Vonoprazan were in accordance with the guidelines set by the ICH.
There was no RP-UPLC approach that could be maintained with a simple solvent system and a low RT.Therefore, we attempted to find a technique that indicates stability using a simple solvent system (equal parts of ammonium acetate and acetonitrile) and a shorter amount of time at room temperature (less than two minutes).The RTs of Clarithromycin, Amoxicillin, and Vonoprazan were very low, which is considered to be cost-effective owing to the reduction in elution time and volume of solvent consumption.As a result, the amount of time spent analyzing samples is reduced, and the total number of samples that are tested may be raised.The FD investigations were helpful in determining the quantity of the medication that had been degraded, which was necessary in order to stipulate the stability-indicating characteristic of the approach.The parameters of the established method validation were in accordance with the requirements of ICH's Q2 standard.

CONCLUSION
To investigate Clarithromycin, Amoxicillin, and Vonoprazan simultaneously in mixed bulk form and in dosage form, an economical, low-cost, precise, and specific RP-UPLC technique with high sensitivity has been established.This approach has excellent sensitivity.Investigating analytes under different types of stress makes sure that the stability indication of the method is reflected in the stability of the analytes.The technique that was developed was successful in separating Clarithromycin, Amoxicillin, and Vonoprazan in addition to any potential degradation products of all three drugs.When it came to the newly developed approach, Clarithromycin, Amoxicillin, and Vonoprazan all had quicker separation times.This Method can be experimented for the bioanalysis of these drugs and indicates that the approach anticipated can often be used in commercial markets.

Fig. 5 :
Fig. 5: Chromatograms representing forced degradation studies of the method