INTRODUCTION:

NSAIDS are the most frequently prescribed drugs worldwide for the treatment of short-term pain relief (headache, toothache, etc.) as well as chronic conditions like osteoarthritis.^1-2 Globally, non-steroidal anti-inflammatory medications are taken daily by more than 30 million people.³ The availability of over-the-counter NSAIDS also aids in the market's expansion. Throughout the forecast period of 2023–2030, it is anticipated that the global NSAIDS market would expand by around 6%.⁴ The growing interest in selective cyclooxygenase-2 (COX-2) inhibitors is one of the key market trends as it is used in the management of chronic pain.⁵ The development of novel cyclooxygenase-2 (COX-2) inhibitors is currently receiving major funding from numerous pharmaceutical firms. The rise in demand for NSAIDS increases the responsibility of pharmaceutical manufacturing industries to produce high quality NSAIDS at a low cost.

Pharmaceutical analysis is essential to ensure the quality of bulk medications and other pharma preparations. The quality of the drug depends on the analytical methods used.⁶

In the group of COX-2 inhibitors, Loxoprofen sodium was recently emerged derivative of propionic acid.^7-8 It is used as a NSAID to relieve pain and inflammation related to musculoskeletal, joint disorders and operative procedures.^9-11

Loxoprofen acts as a prodrug. It is absorbed from GIT and get converted to an active metabolite.¹² Loxoprofen inhibits prostaglandin biosynthesis by acting on cyclooxygenase.^13-15

The chemical name of Loxoprofen sodium is sodium-2-[4-(2-oxocyclopentyl1-methyl) phenyl] propionate dehydrate (Figure 1).

Figure 1: Chemical structure of Loxoprofen Sodium

It is recognized by the Japanese Pharmacopoeia as short acting NSAID.^16-17 Loxoprofen is marketed in India under the brand name ‘Loxomac’.¹⁸ A literature survey revealed that several HPLC methods are available for the analysis of Loxoprofen sodium. But we found some problems in those developed methods, which are as follows:

· The majority of techniques used acetonitrile (organic solvent) as mobile phase at concentration of more than 50%, which is not economical.

· More than 50% organic solvent in the mobile phase is hazardous to the HPLC column.

· In some methods, the peak shapes observed are not appropriate and have a higher retention time

To overcome these drawbacks, the study was undertaken with the objective of using a cost-effective mobile phase and developing a method with a short retention time.¹⁹ In the underlined study quick and reliable RP-HPLC method has been established by using methanol and buffer solution as mobile phase. Methanol is cost-effective, and the lack of interpretation behavior is an added advantage over existing mobile phases used to develop HPLC methods.²⁰

MATERIALS AND METHODS:

Materials:

Loxoprofen sodium, a pure drug, is obtained from Dr. Reddy Labs, Hyderabad, India, as a gift sample. Solvents (HPLC grade) required for chromatographic estimation were obtained from store house of KLE College of Pharmacy, Belagavi. Roxon in 60mg tablets (Mfg. Date: 03/06/2020, Exp. Date: 03/06/2024, Company: SAJA Pharmaceuticals) were purchased from a local pharmacy store.

Instruments:

A UV-Spectrophotometer of the make Shimadzu and model 1800 having UV probe software was used for the determination of λ max. The reverse phased HPLC method was developed and validated using an Agilent Technologies 1100 series model, equipped with a G-13148 (DAD) detector. A computer connected to the instrument was used to capture the chromatograms using Chemstation software. Loxoprofen sodium was analysed by chromatography using a PRIMESIL C18 column (id 4.6 x 250mm length).

Preparation of Stock solution and working standard solutions:

The procedure used to prepare stock solution and working standard solutions is summarized in Figure 2.

Figure 2: Flow chart of preparation of stock solution and working standard

Chromatographic Conditions:

The stationary phase used for the separation of Loxoprofen sodium is a PRIMESIL C18 column (id 4.6 x 250mm length). A mixture of methanol and 0.05% OPA buffer in the ratios of 75:25 v/v was selected as a mobile phase. The 20µl samples were injected at a flow rate of 1ml/min. The detector used is a G-13148 (DAD) detector, with a wavelength observed of 225nm at ambient column temperature.

RP-HPLC Method Development:

Methanol is selected as a mobile phase for the development of a novel RP-HPLC technique for analyzing Loxoprofen sodium. Four different trails were taken with different mobile phase combinations. The details of the trials are given in the Table No.1.

RP-HPLC Method Validation^21-31

Based on the results obtained, trail 4 was found to be giving acceptable results. The method parameters in Trial 4 were validated with reference to ICH guidelines. The parameters studied during the validation of an analytical method include linearity, precision, accuracy, repeatability, specificity, selectivity, limit of detection, limit of quantification, and robustness of the method.

Linearity study:

Linearity is determined at 5 concentrations, viz., 5µg/ml to 25µg/ml of the analyte, under test conditions. The graph was plotted between the peak area and concentration in ppm. A further trend line was plotted, and the equation of the line and correlation coefficient (r²) were determined.

Detection Limit and Quantitation Limit:

LOD and LOQ of method was computed using following formula.

3.3x avgSD 10x avgSD

LOD= -------------- LOQ= -----------------

Slope Slope

Precision:

The method's precision was assessed on the basis of intraday (Thrice a day) and interday (three times on consecutive days) precision. The reproducibility of results was observed at different time intervals on same day and also on different days. The percent RSD was calculated from the obtained results, and precision was determined.

Accuracy:

The recovery study was used to investigate the method’s accuracy. In brief, to the known quantity (5mg) of standard drug, powdered samples of marketed tablets equivalent to 80% (4mg), 100% (5mg), and 120% (6 mg) were added. The mixture is diluted sufficiently, and the percent recovery of added quality was determined by a developed method.

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, variations in method parameters and provides an indication of its reliability during normal usage.¹⁶ Several parameters viz., flow rate (±0.1ml), composition (74:26v/v, 76:24v/v), and wavelength (±1nm) of the developed method were deliberately changed, and analysis was done to study the effect of these variations on results.

Assay of marketed formulation:

The assay test was carried out to access the quantity of Loxoprofen sodium in the marketed formulation. 20 tablets of Roxonin were taken and triturated. A powdered sample equivalent to 5mg of drug was weighed and transferred to a 10ml volumetric flask. The sample was diluted with the mobile phase. The obtained solution was further diluted to get a test solution of 25µg/ml. The 0.5ml of test solution was injected. The procedure was repeated twice. Based on the results obtained, the percentage assay was calculated.

RESULTS AND DISCUSSION:

RP-HPLC method development:

In RP-HPLC, methanol and acetonitrile are the two most commonly utilised organic modifiers. A literature survey revealed that acetonitrile is the common mobile phase, which is widely used in combination with buffer solutions for the analysis of Loxoprofen sodium. Methanol is cost effective in comparison to acetonitrile. In this context, methanol is selected as a mobile phase to develop a novel RP-HPLC technique for analyzing Loxoprofen sodium. The observations of the trial runs are given in the Table No.2

The peak observed for the mobile phase in trial 4 was well defined with good resolution. Therefore, this chromatographic condition kept constant throughout the Loxoprofen sodium drug estimation. The optimized chromatographic condition are given in Table No.3 and chromatogram is shown in Figure 3.

Table No: 2: Observations of the Trial runs

Trial No.	Retention time	Area	Theoretical plates	Resolution	Height of peak	Width of the peak	Selectivity	Observations
1	5.090	1271.00781	10918	8.74	165.19722	0.1147	1.40	Peak height is more.
2	7.046	1166.55847	10101	12.96	109.04530	0.1650	2.03	Retention time is more.
3	9.450	904.70343	9629	21.99	61.72464	0.2267	3.52	Peak width &retention time is more.
4	4.060	819.61066	9551	1.31	126.79233	0.0978	1.09	Well defined peak with good resolution& low retention time.

Table No: 3 Optimized Chromatographic condition

Mobile Phase	Methanol : 0.05% OPA 75 : 25	Area	819.61066
Stationary Phase	Primesil C18 column	Theoretical plates	9551
Wavelength used	225nm	Resolution	1.31
Flow Rate	1 ml/min	Height of peak	126.79233
Temperature	Ambient	Width of peak	0.0978
Sample size	20µl	Selectivity	1.09
Retention time	4.060

Figure 3: Chromatogram of Trial 4

RP-HPLC method validation:

Linearity:

The method's linearity was investigated in a concentration range of 5 to 25µg/ml, wherein the results were found to be linear with correlation coefficient (r²) values of 0.999. The results of the linearity study are shown in Figure 4.

Figure 4: Standard calibration curve of Loxoprofen sodium

LOD and LOQ:

Based on the results obtained during the development of the calibration curve, limits of detection and quantitation for developed method were observed to be 0.09175 and 0.278032µg/ml respectively.

Precision:

To determine intraday and interday precision, three distinct concentrations, viz. 10, 15 and 20µg/ml, were analysed at three different times on same day (for intraday) and three different days (for interday). The percent RSD value for intraday study was ranged between 0.09% to 0.16%, while for interday study it was ranged between 0.16% to 0.62%, which is below the acceptance limit of 2%. Thus, the developed method was found to be precise. The results of the precision study are as shown in Table No. 4

Table No. 4: Observation Table for Intraday and Interday Precision

Intraday Precision
Conc (µg/ml)	Area I	Area II	Area III	Mean	% RSD
10	373.4038	372.2188	372.8834	372.84	0.16
15	559.7612	561.3866	560.6543	560.60	0.15
20	768.2559	766.879	767.6785	767.60	0.09
Interday Precision
10	381.0605	376.3238	378.6745	378.68	0.62
15	580.777	583.9671	582.632	582.45	0.27
20	769.2219	771.7639	770.987	770.65	0.16

Accuracy:

The developed method’s accuracy was assessed by the percent recovery method at three addition levels: 80%, 100%, and 120%. The present recovery for all the samples ranged between 97.71% to 101.09%. The results witnessed the accuracy of the method in predicting results. The results of the accuracy study are as shown in Table No. 5

Table No. 5: Results of Accuracy Study

Addition Level	Amount added (mg)	Area	Amount recovered	Mean Recovery (%)	% RSD
80%	4	333.838	3.91	97.91	0.21
	4	334.465	3.92
	4	334.152	3.92
100%	5	374.842	4.97	99.79	0.26
	5	375.851	5.00
	5	375.346	4.98
120%	6	416.542	6.06	101.07	0.02
	6	416.650	6.06
	6	416.596	6.06

Robustness:

In the robustness study, intentional minor changes in the optimised analytical parameters were made, viz., flow rate (±0.1ml). Mobile phase combination (±1%) and wavelength (±1nm). The findings of the robustness study revealed that the minor changes in the analytical parameters do not significantly affect the results (% RSD<2). Thus, the method was found to be robust. The results of the robustness study are as shown in Table No. 6.

Assay of marketed formulation:

The percent assay of the marketed formulation was determined based on the equation of the calibration curve. The % assay was found to be 101.85±0.090%, which witnesses that the tablet passes the compendial requirement. Thus, it can be ascertained that the method can be used to efficiently assess the quality of marketed formulations on a regular basis. The results of assay are given in Table No. 7.

Table No. 7 Observation table for Assay results

Concentration	Area	Amt Found	% Label Claim
25.00	968.6907	25.44532	101.78
25.00	969.9154	25.47722	101.91
25.00	969.3031	25.46127	101.85
Mean	969.30	25.46	101.85
SD	0.866	0.023	0.090
%RSD	0.089	0.089	0.089

CONCLUSION:

For the analysis of Loxoprofen sodium, a new RP-HPLC method was developed and validated. The developed method was accurate, sensitive, and precise. Methanol was used in the mobile phase, which is cost-effective and lacks any detrimental effects on the column. The developed method was validated according to ICH Q2(R1) guidelines. The outcomes of the validation study show that the method was sensitive for Loxoprofen sodium detection and quantification at low concentrations. The validation data further supports the method's linearity (over a range of 5µg/ml to 25µg/ml), and the fact that the RSD is less than 2% shows that the method is accurate and reliable. The shorter retention time of 4.06 minutes completes the analysis within a shorter duration of time with less consumption of chemicals. The outcomes of the study clearly demonstrate that method can be effectively utilized for rapid and economic estimation of Loxoprofen sodium in bulk and dosage form.

ABBREVIATIONS:

RP-HPLC: Reverse Phase High Performance Liquid Chromatography, UV: Ultra violet, LOD: Limit of detection, LOQ: Limit of quantification, ICH: International conference of harmonization, RSD: Relative Standard Deviation, GIT: Gastrointestinal Tract.

CONFLICT OF INTEREST:

The authors declare that there is no any conflict of interest.

ACKNOWLEDEMENTS:

The authors would like to thank to Dr. Reddy Laboratories for providing the drug sample. The authors also extend their sincere thanks to Principal KLE College of Pharmacy Belagavi and Nipani for availing the research facility during research work.

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Received on 27.06.2023 Modified on 08.09.2023

Research J. Pharm. and Tech 2024; 17(6):2889-2894.

DOI: 10.52711/0974-360X.2024.00453

Trial No.	Mobile phase composition	Ratio	Flow rate (ml/min)	Injection volume	Wavelength
1	Methanol and 0.05% OPA buffer	80: 20 %v/v	0.7ml/min	20µl	225nm
2	Methanol and 0.05% OPA buffer	70: 30 %v/v	0.7ml/min	20µl	225nm
3	Methanol and 0.05% OPA buffer	60: 40 %v/v	0.9ml/min	20µl	225nm
4	Methanol and 0.05% OPA buffer	75: 25 %v/v	1 ml/min	20µl	225nm

FLOW RATE (0.9 ml/min)				FLOW RATE (1.1 ml/min)
Conc. (µg/ml)	Area	Mean	%RSD	Area	Mean	% RSD
10	419.9682	422.01	0.68	355.9712	352.82	1.26
10	424.0482			349.6631
MOBILE PHASE Methanol : 0.05% OPA buffer (74:26 v/v)				MOBILE PHASE Methanol : 0.05% OPA buffer (76:24 v/v)
10	374.6719	372.5	0.84	371.1599	368.94	0.85
10	370.2668			366.7215
Change in Wavelength(224nm)				Change in Wavelength(226nm)
10	381.9029	385.8	1.42	360.5017	362.55	0.80
10	389.6729			364.6039