INTRODUCTION:

HPLC has been used in research (e.g., separating the components of a complex biological sample, or of similar synthetic chemicals from each other), manufacturing (e.g., during the production process of pharmaceuticals and biological products), legal (e.g., detecting performance enhancement of drugs in urine), and medical (e.g., detecting vitamin D levels in blood serum).¹ Analytical chemistry may extract the structural factors found in a science or artwork to discover the fabric structure. The same division of analytical chemistry was drug analysis. The study of drugs through request relies on their concepts in the areas of chemistry, mechanics, microbiology, atomic sciences, and telecommunications, respectively.²

Piroxicam belonging to a new class of compounds and the Food and Drug Administration recognized as NSAID (Non-steroidal anti-inflammatory drug) used as an analgesic and anti-inflammatory in rheumatoid arthritis, osteoarthritis, acute pain in musculoskeletal illnesses, and acute gout. It works by preventing two cyclooxygenase isozymes (COX-1 and COX-2) from catalyzing the creation of prostaglandins, which are involved in many physiological processes in the body, such as blood pressure regulation and the regulation of smooth muscles in the gastrointestinal and respiratory tracts. While COX-2 is engaged in pain and inflammation, COX-1 is essential in preserving the physiological function of the renal and gastrointestinal systems. So, anti-inflammatory actions of Piroxicam are due to the inhibition of COX-2 only whereas the inhibition of COX-1 leads to the unwanted side-effects like dyspepsia, heartburn, nausea and vomiting.³Chemically, 4-hydroxy-2-methyl-1,1-dioxo-N-pyridin-2-yl-1λ 6,2-benzothiazine-3-carboxamide. Three different forms of 4-hydroxy-2-methyl-N-(2-pyridyl)2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide, the anti-inflammatory drug piroxicam, crystallize two anhydrates and one monohydrate.⁴

The literature survey shows that no technique is utilized in the testing of piroxicam gel compositions for high-performance liquid chromatography (HPLC). Consequently, this research aimed to improve a simple and accurate HPLC Reverse Phase (RP) process for piroxicam evaluation activities.

Piroxicam:

Figure 1: Complex of Piroxicam⁵

Diluent preparation:

MeOH was the diluent used.

Formulation of Mobile Phase:

The mobile phase is 25 mM KH₂PO₄ buffer (pH 3.0) and ACN has been used in the interaction of (60:40) (v/v).

Typical standard Stock Solution Prep of Piroxicam:

A 100mL volumetric flask of Piroxicam 100mg was drawn. The diluent has been reduced by applying 50mL, and the diluent volume made up to mark (1000μg/ml) then diluted (100μg/ml) 1milliliter of the aforementioned solution in a 10ml volumetric flask to determine the volume. Pipette 2ml in 10ml volume flask with a dilution of 20μg/ml volume (20μg/ml). Pipette 4ml in 10ml volume flask with a dilution of 8μg/ml volume (8μg/ml).⁶

MATERIALS AND METHODS:

Table 1: Reagent and Chemicals

S. No.	Chemicals	Grade	Manufacturer
1	Potassium dihydrogen orthophosphate	LR	Chemspure
2	Methanol	HPLC	Standard Reagents Pvt Ltd
3	Acetonitrile	HPLC	Merck
4	Distilled Water	HPLC	Rajco
5	Piroxicam	API	Orchid Chemicals Pvt. ltd.
6	Pirox gel	SAMPLE	Cipla

Table 2: Instruments Listed

S. No.	Name of the Equipment	Manufacturer	Model
1	UV- Spectrophotometer	Shimadzu	UV- 1800 240V
2	HPLC	Jasco	2580
3	pH Meter	Toshniwal	UG-PA D-02
4	Sonicator	Vibracell	PGPA M-01
5	Analytical Balance	Scaltec	SPB-31

Table 3: Chromatographic Conditions

Optimized Chromatographic Conditions
HPLC system	:	Jasco, Japan
HPLC software	:	Borwin Version No. 1.5
Column	:	Kromacil C18 (250 x 4.6)mm 5µ
Flow rate range	:	0.000 to 10.000 mL /min
Maximum operating pressure	:	5000 psi (345 bar)
Injector	:	Manual Rheodyne injector
Injector loop volume	:	20 µL
Detection Wavelength	:	325 nm

Preparation of solutions for linearity:

Linearity solutions are produced by diluting with inventory product diluents. Piroxicam has been approved with levels of 4, 6, 8, 10, and 12µg/mL. In a 10ml volumetric flask pipette 2, 3, 4, 5, 6mL, and extract the quantity in the above grades.

Preparation of Calibration Curve:⁷

In a 10mL series of volumetric flasks, stock solution (100μg/ml) Piroxicam aliquots had been pipetted. The amount was processed to the limit and purified using an HPLC grade diluent with a 0.45μ pore membrane filter to obtain a concentration between 4 and 12μg/mL. The calibration curve is calculated by injecting 20μL maximal regions and reporting the results. The beer rule is tested between 4 and 12μg/mL.

RESULTS AND DISCUSSIONS:

Different parameters were undertaken for the development of this method. The solubility of the Piroxicam drug was initially developed. The RP-HPLC system for maintaining a high peak-controlled chromatographing. The drug was initially evaluated at different formulations in flowing processes. The mobile phases and level of flow are chosen based on high altitude, efficiency, plate theory, asymmetry of the tailing variable, runtime, and resolution. The 25mM KH₂PO₄ buffer (pH 3.0) in mobile phase A and the ACN in mobile phase B, both with a 60:40 (v/v) ratio and a 1.0ml/min flow rate, were both robust. The optimum wavelength for measurement was 325nm, to which the medication responded more strongly. Piroxicam retention time was observed in 5.525 mins, blank chromatogram, and Piroxicam, respectively, as shown in Figures 2 and 3.

Figure 2: Chromatogram of Blank (Diluent)

Figure 3: Chromatogram for Piroxicam (Standard)

System suitability:

The suitability checking of the device is used to control the chromatographic approach to reproductive efficiency. Efficiency has been tested for newly produced solutions to drug applications.

Table 4: Results for System Suitability of Piroxicam

Criteria	Acceptance criteria	Results
Tailing factor	Not More Than 2.0	0.8666
Theoretical plates	Not Less Than 2000	6359.016

Data interpretation:

Table 4 shows the system fitness parameters to be within the acceptability requirements of the information, as mentioned above.

Linearity:⁸

For standard solutions produced in the range of 4.00–12.00µg/mL at concentrations of 4.00, 6.00, 8.00, 10.00, and 12.00 at 50, 75, 100, 125, and 150% of working concentration, linearity experiments were carried out in triplicate. Piroxicam's correlation coefficient was found to be 0.9998, falling within the ICH requirements with an NLT of 0.998. A remarkable linear relationship was found in the piroxicam linearity regression data over the concentration range of 4.00–12.00µg/mL.

Table 5: Results for Linearity of Piroxicam by HPLC Method

Sample % Level	Conc. (µg/mL)	Average Peak area
50	4.00	123697.5987
75	6.00	165924.8687
100	8.00	214487.1467
125	10.00	256914.6433
150	12.00	297049.2403
Regression Equation		Y = 21855x + 36537
Correlation Coeff (r²)		0.9998
Slope		21855
Intercept		36537

Figure 4: Calibration curve of Piroxicam

Criteria of approval:

Not Less Than 0.99 should be the regression factor.

Data evaluation:

Table 5 of Piroxicam reveals that the reaction of Piroxicam is constant between 50% and 150% of the function rate through numerical analysis of its Linearness tests.

Precision:

An analytical method's accuracy is determined by how often it is used to different samples of a homogeneous material.⁹

System Reliability (Intraday Precision):

System reliability means ensuring the proper functioning of the analytics unit.

Table 6: Results for System precision studies of Piroxicam by HPLC Method

Conc (µg/mL)	Peak area
8	206484.500
8	207408.000
8	205022.641
8	199938.250
8	199321.661
8	206484.500
Mean	204109.9253
Std dev	3558.592601
%RSD	1.74

Criteria of approval:

RSD level should be less than 2.0 percent of the Piroxicam maximum region reaction collected from 6 normal preparation injections. Data evaluation: Table 6 above shows a coherent area response, shown by a relative standard deviation.

Method precision:

Accuracy of technique shows whether or not a single-material technique produces coherent outcomes.

Criteria of approval:

In Table 7 assessments, the amount RSD determined should be greater than 2.0%.

Table 7: Method precision studies for Piroxicam by HPLC Method

Different assay sample	% Assay	Acceptance Criteria
Assay	100.75	95-105%
Intraday	99.88
Interday	101.22
Average	100.613
St. dev	0.6798
% RSD	0.68	NMT 2%

Data evaluation:

It can be inferred from Table 7 above that the process is exact.

Accuracy:

The usual addition method was used to assess the accuracy of the procedure. To a given volume of previously examined standard solution, a known quantity of standard medication was added. 50%, 100%, and 150% of the conventional addition method were used. In accordance with the suggested methodology, the solutions were examined three times at each level. By using the suggested strategy, satisfactory recoveries were achieved. This suggests the accuracy of the suggested approach.

% Recovery =	Amount of drug recovered	X 100
	Amount of drug added

Criteria of approval:

The percentage recovery between individual and average should be between 98.0% and 102.0%.

Data evaluation:

Table 8 and 9 shows that the recovery is well within the limit. The technique is therefore correct.

Table 9: % Global Recovery in Piroxicam gel formulation by HPLC Method

% Levels	Piroxicam	Acceptance Criteria
50	101.20
100	100.75
150	100.67
Average	100.872	90-110%
Stdev	0.2902
%RSD	0.29	NMT 2%

Robustness:

The robustness of an analytical process measures its ability to remain uninfluenced by small but intentional process parameter differences and shows its reliability while using the technique normally.¹⁰

Table 10: Robustness effect of variation in mobile phase flow rate

Flow Rate	Rt (min)	Peak area	Asymmetry factor	Theoretical Plates
0.8 mL/min	5.717	343562.102	1.0	4306.9
1.0 mL/min	5.633	306974.768	0.8	5608.2
1.2 mL/min	4.575	175132.000	1.1	6021.3
Acceptance			1±0.2	NLT 2000

Table 11: Robustness effect of variation in Wavelength

Wavelength	Rt (min)	Peak area	Asymmetry factor	Theoretical Plates
320nm	4.667	156782.000	1.1	5546.4
325nm	5.633	306974.768	0.8	5608.2
330nm	5.025	238995.000	1.0	3951.2
Acceptance			1±0.2	NLT 2000

Data interpretation:

No important improvements could be found from Table 10 and 11 because of modifications in the circumstances mentioned above, and therefore the technique is robust.

Ruggedness:

By analyzing piroxicam on several analysts, the robustness of the approach was determined. Table 12 provides an overview of the outcomes. The results showed no discernible changes, indicating the robustness of the developed technique.¹¹

CONCLUSION:

A simple, quick, efficient RP-HPLC technique was effectively established to test piroxicam in its purest form. For various test conditions, the proposed procedure was tailored and tested. The results of pH, mobile stage proportion, and flow rate have been tested by piroxicam research. All analyses were well resolved and split in less than ten minutes. The design approach can be easily used in daily and stability tests utilizing quality control methods for evaluating the Piroxicam material. This technique could be used in pharmaceutical preparations for drug assessment and routine laboratory tests. Overall, the proposed method provides excellent reliability, accuracy, selectivity, and reproducibility for deciding piroxicam.

ACKNOWLEDGEMENTS:

The author greatly appreciates the Management of Sankaralingam Bhuvaneswari College of Pharmacy, Sivakasi, and the Department of Pharmaceutical Analysis for their assistance and provision of the tools required to complete the task effectively. We also thank the management of Dhanalakshmi Srinivasan University, Trichy, for encouraging the compilation and publication of the manuscript.

REFERENCE:

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Received on 17.04.2024 Revised on 12.08.2024

Accepted on 28.10.2024 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2090-2094.

DOI: 10.52711/0974-360X.2025.00299

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Parameters	Standard area	Sample area at different % levels			Acceptance criteria
Parameters	Standard area	50	100	150	Acceptance criteria
1	206484.500	105316.528	203520.361	310303.696
2	207408.000	102160.250	209855.504	307336.989
3	205022.641	102374.478	203520.361	306974.768
4	199938.250
5	199321.661
6	206484.500
Mean	204109.9253	103283.7520	205632.0753	308205.1510
Std.Dev.	3558.592601	1763.6913	3657.5965	1826.3952
%RSD	1.74	1.71	1.78	0.59	NMT 2 %
%Mean Recovery		101.40	101.20	100.75	98-102 %

Inj. No.	Analyst-A (Inter day)		Analyst-B		Acceptance Criteria
Inj. No.	Standard	Sample	Standard	Sample	Acceptance Criteria
1.	192148.881	198797.875	192489.635	193161.600
2.	192556.569	198064.750	190803.000	192415.850
3.	193097.500	193184.500	193911.313	192134.500
4.	191077.364		193567.986
5.	198833.723		189613.971
6.	198192.379		196463.000
Mean	194317.7360	196682.3750	192808.1508	192570.6500
STDEV	3322.6984	3051.3465	2428.0488	530.7597
%RSD	1.71	1.55	1.26	0.28	NMT 2%
% Assay		101.22		99.88	95-105%
Amount in mg		5.0608		4.9938	4.7500-5.2500
Difference		1.34			NMT 2%