INTRODUCTION:

1Cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7 (MOX) is the chemical name for Moxifloxacin (MOX), which is the active ingredient of Zithromax (ZIOP) (4as,7as) -octahydro-6H-pyrrolo(3,4-b) pyridin-6-yl) 4 oxo-3 quinolinecarboxylic acid is a chemical compound. DNA gyrase and topoisomerase IV are bacterial enzymes inhibited by this fluoroquinolone family synthetic antibiotic.

One of the most common uses for it is gramme positive and gramme negative bacteria infections¹.

()-5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-carboxylic acid is chemically 2-amino-2-hydroxymethyl 1,3-propanediol, ketorolac tromethamine (KET) (Fig. 2). KET is considered to have analgesic and anti-inflammatory properties because it inhibits prostaglandin biosynthesis².

The combinational ophthalmic solution of Moxifloxacin and Ketorlac is recommended for the treatment of post-operative inflammatory eye diseases. These medications can be estimated either separately or in combination with other pharmaceuticals using spectrophotometric^3-8, HPLC^9-16analytical techniques. However, for the simultaneous estimate of these medications, only a small number of analytical methods^17-31are available. There are a few disadvantages to the disclosed techniques, such as higher organic phase concentrations, longer run durations, and lower sensitivity. Even the stated approaches for assessing stability are limited to single-drug evaluation. A regular pattern of stress concentrations has been described in the published methods for simultaneous estimation as the only one that works. This is why we have been working on developing simple spectrophotometric and RP-HPLC techniques for estimating both medications simultaneously. The techniques created were validated in accordance with ICH^32-33 standards. Forced degradation of pharmacological compounds was done under stress circumstances in order to establish the RP-HPLC method's stability indicating natures (peroxide, acid, base, thermal, ultraviolet and neutral hydrolysis). Furthermore, we experimented with various stress situations and stress concentrations to determine the degree of deterioration under various circumstances.

MATERIALS AND METHODS:

Instrumentation and analytical conditions:

LABINDIA 3092 UV-Visible Spectrophotometer with 2 nm spectral bandwidth was used for the UV technique. The absorbance of a solution was measured with 0.5 nm wavelength accuracy and a pair of matched 1cm quartz cells. For the current investigation, an RP-HPLC technique utilising a rheodyne injector and a 20-milliliter fixed-loop HPLC system (Shimadzu) with a binary gradient pump and UV detector (LC-20AD) was used for analysis. RP-HPLC was used to separate the two medicines using a 5 micron SHISHEDO C18 column with a mobile phase of acetonitrile and water (40:60 v/v) and two drops of OPA at flow rate of 0.9 ml/min and UV detection at 308 nm, which was achieved on a SHISHEDO C18 column.

Chemicals and reagents:

Standard MOX and KET were purchased from Yarrow Chemicals Ltd. in Mumbai (India). The neighbourhood drugstore sold commercially supplied M Quin KT eye drops. Merck Specialities Pvt. Ltd., Mumbai, supplied the HPLC grade acetonitrile used in the experiment. Fisher Scientifics Ltd. in Mumbai provided the HPLC grade water and methanol. In addition to orthophosphoric acid, Merck Specialties Pvt. Ltd. in Mumbai provided hydrochloric acid, sodium hydroxide pellets filtered, hydrogen peroxide 30 percent AR grade, sodium acetate, and acetic acid for the project.

METHODOLOGY:

UV Spectrophotometric method:

In a clean, dry, calibrated 10 ml volumetric flask were transferred 10 mg of MOX and 10 mg of KET and dissolved in acetonitrile: water in the ratio of 60:40 V/V to achieve a concentration of MOX of 100 g/ml and a concentration of KET of 100 g/ml, respectively. We produced the required concentrations of MOX and KET using appropriate dilutions, which were each 10 ng/ml. Dilutions of other working standards were made to meet the specifications.

Simultaneous equation method:

A scan in the UV region of 200-400 nm was performed on the reference solutions in order to determine the medicines' maximum absorbance values. When it comes to MOX and KET, the max wavelengths were determined to be 295 nm and 316 nm, respectively. The diluent was used to make five standard solutions ranging from 1-9 g/ml for both MOX and KET. The medicines' respective maximum absorbances were measured. By plotting concentration on the x-axis and their corresponding absorbance on the y-axis, linearity charts might be produced. It was discovered that the selected medicines' absorptivity was affected by the wavelength they were exposed to. Equations (1&2) for calculating the amount of medication contained in a particular combination or formulation may be designed using the same values.

Stability Indicating RP-HPLC Method:

Ten milligrams MOX and ten milligram KET were measured correctly and placed into a calibrated, clean and dry, 10 millilitre volume flask, where they were dissolved in the solvent to produce a MOX/ket concentration of 1000 micrograms/milliliter. Dilutions were used to generate the required MOX and KET concentrations of 4 ng/ml and 4 ng/ml, respectively. Dilutions of other working standards were made to meet the specifications. To achieve a concentration of 100 g/ml of MOX and 100 g/ml of KET, one millilitre of an ophthalmic formulation combination (containing 5% w/v MOX and 5% w/v KET) was added to a 50-milliliter volumetric flask. This solution was then diluted 100 times. MOX and KET concentrations of 4 g/ml were achieved by repeated dilutions with diluent.

Table 1: Optimized conditions of RP-HPLC method

Chromatograph	Shimadzu HPLC system
Mobile phase	Water: ACN (40:60, V/V) + 2 DROPS OF OPA
Diluent	Mobile phase
Column	Shiseido ODS (250 x 4.6 mm, 5 µm)
Column temp.	Ambient
Wave length	308 nm
Injection volume	20 µl
Flow rate	0.9 ml/min
Run time	8 min
Retention times	2.080 min for MOX; 4.400 min for KET

Fig 1: Chromatogram showing well resolved peaks of Moxifloxacin & Ketorolac

RESULTS:

UV Method:

With the use of the overlaid spectrum, we were able to determine the max of the medicines. Both medications have a corresponding absorbance at these wavelengths. MOX's and KET's maxes both had less outliers, therefore they were used to build the simultaneous equations. It was discovered that for MOX at 295 nm and 392 nm, the absoptivity values were 1486 and 392 respectively; for KET, the values were 609 and 321 at 295 and 316 nm, respectively. A1=392C1+321C2---(1) and A2=1486C1+609C2---(2) were developed, with A1 representing the sample's absorbance at 295 nm and A2 its concentration of MOX and KET at 316 nm, respectively; C1 and C2 being concentrations of the analytes. For MOX and KET, good linearity responses were found in the concentration ranges of 1-9 g/ml. Standard deviation and slope values were used to determine the LOD and LOQ values.

Three concentrations at various levels were used to assess precision in terms of inter-day and intra-day analyses. In the same way, the toughness of the system was examined by changing the analysts. The accuracy of the technique is described by the lower percentage of RSD. Table summarises the findings.

To test the method's accuracy, different concentrations of commercial formulation samples were introduced to a standard sample of a set concentration at levels of 50%, 100%, and 150%. To determine accuracy, researchers looked at recovery time and absorbance. a percentage of The values for recovery and RSD were computed and shown in the table.

RSD values were generated for the test of a commercially available ophthalmic medication. During the formulation analysis used to describe the method's specificity, no interferences were discovered. Table summarises the results of the assay tests. Table contains an overview of the method's parameters. To estimate MOX and KET in bulk and pharmaceutical dose forms, the new approach may be utilised successfully for routine routine analysis.

Table 2: Summary of UV method and Validation parameters

Parameter	MOX	KET
Lambda maximum (nm)	295	316
Linearity range (µg/ml)	1.0-9.0	1.0-9.0
Molar extinction coefficient, (ɛ) (295 nm)	55812±3414.72	7849.39±1678.06
Sandell’s sensitivity (S.S) (295 nm)	0.00067±0.00003	0.0032±0.00056
Absorptivity (E^1%_1cm) (295 nm)	1486.76±90.96	321.35±68.70
Molar extinction coefficient, (ɛ) (316 nm)	14746±1871.36	14891.86±2646.45
Sandell’s sensitivity (S.S) (316 nm)	0.002575±0.00028	0.001675±0.00024
Absorptivity (E^1%_1cm) (316 nm)	392.81±49.85	609.67±108.35
LOD (µg/ml)	0.02	0.12
LOQ (µg/ml)	0.07	0.37

HPLC Method:

It was necessary to analyse the created technique in accordance with the guidelines of the International Conference on Harmonization (ICH) in order to validate it.

Linearity:

In duplicate, six standard working solutions of MOX and KET were produced with concentrations ranging from 1.0 to 6.0 g/ml for MOX and 1.0 to 6.0 g/ml for KET. Under optimal circumstances, the material was given into the chromatograph. On the x-axis, concentration was plotted against mean peak area on the y-axis to create calibration plots.

Precision:

Method precision:

For the prepared concentrations, intra-day and inter-day analyses were used to check for repeatability. For this, MOX and KET were produced in duplicate and injected on the same day and on different days at concentrations that fell within the linearity range. The chromatograms' responses were analysed to determine the standard deviations.

System precision:

This was done to ensure that the system had a high degree of repeatability. A total of six injections of 4.0 g/ml of MOX and 4.0 g/ml of KET were used to test this hypothesis. In order to compare the percent RSD values with the tolerances, the repeated injections were computed.

Accuracy:

The method's accuracy was checked by adding a predetermined/fixed concentration of MOX and KET commercial formulation to the standard sample solution. In terms of recovery, the findings from constant concentrations were compared to those from spiked samples. Sample solution was spiked with the marketed sample at levels of 50%, 100%, and 150% to test this parameter.

Ruggedness:

The method's repeatability was tested by swapping out the analysts who worked on it. A total of six injections of 4.0 g/ml of MOX and 4.0 g/ml of KET were used to test this hypothesis. Researchers analysed and compared percent RSD results for multiple injections performed by several analyzers.

Specificity:

It was discovered that the excepients that were expected to interfere/interact with analytes really did not. Interfering peaks were checked to see if they responded at the analytes' retention periods using the interpretation.

Robustness:

The conditional parameters of the technique, such as pH, flow rate, and organic phase concentration, were changed to assess this validation parameter. The organic phase concentration was adjusted by 10%, i.e. 6010%, the flow rate was altered by 0.1 ml/min, and the wavelength was adjusted by 2 nm. These alterations had an impact on the output, which was analysed to determine the robustness of the approach.

System suitability:

The technique was found to be system-compatible. This was put to the test to see if the newly created approach produces findings that can be trusted. The asymmetric/tailing factor, retention duration, theoretical plate number, and resolution are a few of the factors that have been examined.

Assay of marketed formulation:

This formulation's MOX and KET concentration was assessed by comparing the acquired findings to the standard results. Six injections with the same target concentration were used to ascertain the concentration of the marketable sample.

Forced degradation studies:

Forced degradation tests were carried out under various stress conditions, such as acidic, basic, thermal, peroxide, UV and neutral environment, to verify the method's resilience. The degree of degradation and the interaction of the generated degradant peaks with the analyte were studied in this research. During the acidic forced degradation tests, 1 ml of stock analyte sample was placed into a calibrated volumetric flask of 10 ml capacity to measure deterioration. 1 ml of 0.1N HCl was added, and the flask was refluxed at 60 C for 30 minutes before the experiment was terminated. 0.1N NaOH was added to the solution to neutralise it, and the diluents were then used to bring it up to the required concentration. HPLC grade 0.45 micron syringe filters were used to filter the final solution, which was then injected into the system. Remaining diseases were treated in a similar manner. Basic experiments utilised 1 ml of 0.1N NaOH, and peroxide studies 3 percent H2O2. When it came to thermal studies, the sample was maintained at 105°C for 45 minutes under reflux. When it came to UV studies, the sample was kept in a UV chamber for seven days. After that, it was brought to the mark and injected into the system after filtering. 4.0 g/ml of MOX and 4.0 g/ml of KET were the target concentrations for the final concentrations.

DISCUSION:

Mobile phase optimization:

The chromatographic conditions for the estimation of MOX and KET in mixed forms were thoroughly optimised through many experiments. Various mobile phases were tested, including methanol: buffer, methanol: water, and acetonitrile: buffer. Finally, a 40:60 V/V water/acetonitrile mobile phase with two drops of OPA was set at a flow rate of 0.9 ml/min with a final concentration of acetonitrile. Perfect symmetry was found in the peaks. There are no interfering or additional peaks at MOX and KET retention periods in the chromatograms of blanks or placebos, showing the specificity of the technique. The analyte detection wavelength was set at 308 nm, and the detector responded well to this setting. MOX had a retention time of 2.080 min while KET had a retention duration of 4.400 min.

Peak area was plotted on the x-axis versus the corresponding concentrations on a linearity curve. For MOX and KET, linearity was observed across the concentration range of 1.0-6.0 g/ml, with the corresponding findings shown in Table. The method's accuracy was assessed, and relative deviations were determined as a result. a lower percent RSD number indicates that the technique is more precise. Table shows the precision and toughness test results. As for MOX and KET, satisfactory recovery values were found in the range of 98.01% to 100.15%, with the findings given in the following table. When the concentration of the organic phase was altered by 10 percent, the optimised technique was shown to be resilient. The flow rate was modified from 0.9 ml/min to 0.1 ml fluctuation, and the wavelength was changed to 2 nm. Peak regions showed very minor shifts in temperature. Even a little adjustment in flow rate can have a small impact on analyte retention time. Figure 1 shows the robustness study findings. Precision samples' standard deviation values and slope from the linearity curve were used to compute the least quantity of drug detectable (LOD) and the limit of quantification (LOQ). These values were obtained from the formula: three times the LOD values (or) 3.3%; they were determined to be respectively 0.180 and 0.550 micrograms per millilitre (g/ml). Table shows the results of the system suitability performance for various parameters.

Assay of marketed formulation:

A percent RSD value was generated based on the test results for the commercial sample. Table shows the findings. When the RSD value is on the low side, the technique can be used for routine quality control. The specificity of the technique was demonstrated by the absence of interfering peaks in the test sample for degradants/excepients.

Forced degradation studies:

The medication sample was exposed to acidic, alkaline, peroxide, ultraviolet, and thermal environments during forced degradation experiments on the analyte. Seeing distinct peaks at various Rts corroborated the degradation peaks, as did seeing a decrease in the analyte's peak area. Figure 1 displayed the deterioration trend as a series of graphs. Data on MOX and KET's forced deterioration are shown in Table. As a result, the newly developed approach was shown to be cost-effective while also being time and space efficient.

The technique was able to successfully separate degradation products from analytes, demonstrating the method's specificity and stability. The method's appropriateness was demonstrated by the fact that the percent RSD values for all parameters were well within the recommendations' limitations. This method's test findings match the indicated quantities exactly. Consequently, the new technique may be used to regularly test MOX and KET in quality control labs.

Table 3: Summary of HPLC method and validation parameters

Parameter	Results
Parameter	MOX	KET and PIP5
Retention time (min)	2.080	4.400
Linearity range (μg/ml)	1.0-6.0	1.0-6.0
Correlation coefficient	0.9992	0.9994
Theoretical plates (N)	6645	10599
Resolution	-	11.045
Tailing factor	1.181	1.064
LOD (μg/mL)	0.240	0.180
LOQ (μg/mL)	0.720	0.550

Fig 2: Graphical representation of degradation of MOX

Fig 3: Graphical representation of degradation of KET

CONCLUSION:

The approach devised was straightforward, precise, and exact. To analyse Moxifloxacin HCl and Ketorolac Tromethamine in pharmaceutical dosage form, UV and RP-HPLC techniques were established. MOX has a max of 295 nm and KET has a max of 316 nm for the UV technique. Using a 40:60 diluent ratio of water and acetonitrile, a series of five standard solutions of MOX and KET were produced. On the other hand, the linearity responses of MOX and KET curves were good. The approach was simple, quick, cheap, sensitive, precise, and accurate as determined by statistical analysis and drug recovery data. The results indicated that the proposed approach may be used to determine drug concentration in pharmaceutical formulations with little influence from additives. For this reason, the RP-HPLC technique may be used to determine MOX and KET stability.

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Received on 30.09.2021 Modified on 28.11.2021

Asian J. Research Chem. 2022; 15(1):42-48.

DOI: 10.52711/0974-4150.2022.00006

Condition	MOX			KET
Condition	Std. area	*Sample area± S.D.**	% Degradation	Std. area	*Sample area± S.D.**	% Degradation
Acidic	1417747	1314677±3812.68	7.27	450257	374922±356.67	16.73
Basic	1417747	1333577±5797.50	5.94	450257	398967±42.62	11.39
Peroxide	1417747	1350589±3119.54	4.74	450257	370164±2065.26	17.79
UV	1417747	1196918±7896.25	15.58	450257	407604±426.45	9.47
Thermal	1417747	908282±553.73	35.93	450257	396575±154.46	11.92
Neutral	1417747	1416922±21345.0	0.97	450257	440983±146.19	1.94