Method Development and Validation of RP-HPLC Method for Determination of Meloxicam and Paracetamol in Bulk and Pharmaceutical Formulation

 

M Venkataramana1*, P. Sankeerthana1, Ganesh Akula1, Yerrolla Soundarya1, Salla Pujitha2

1Department of Pharmaceutical Analysis, Surabhi Dayakar Rao College of Pharmacy,

Rimmanaguda (V), Gajwel (M), Siddipet (D), Telangana – 502312, India.

2Department of Pharmacology, Surabhi Dayakar Rao College of Pharmacy,

Rimmanaguda (V), Gajwel (M), Siddipet (D), Telangana – 502312, India.

*Corresponding Author E-mail: m_ramana100@yahoo.com

 

ABSTRACT:

A simple, accurate, and reliable Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the simultaneous estimation of Meloxicam and Paracetamol in bulk and pharmaceutical dosage forms. The chromatographic analysis was carried out using a Waters HPLC system equipped with an auto sampler and PDA detector (Model 996). Separation was achieved on a Zodiac C18 column (4.6mm × 150mm, 5μm) maintained at a temperature of 38°C. The mobile phase consisted of Acetonitrile and Water in the ratio of 40:60v/v, delivered at a flow rate of 1.0mL/min. Detection was performed at a wavelength of 223nm, with a 10µL injection volume and a run time of 10minutes. The method provided effective resolution of Meloxicam and Paracetamol with sharp and well-defined peaks. Validation of the method was performed according to ICH guidelines, and the method was found to be linear, precise, accurate, robust, and specific. The results confirm that the developed RP-HPLC method is suitable for routine quality control analysis of Meloxicam and Paracetamol in combined pharmaceutical formulations.

 

KEYWORDS: RP-HPLC, Meloxicam, Paracetamol, Zodiac C18 column, PDA detector (Model 996), simultaneous estimation, validation.

 

 

INTRODUCTION:

Pharmaceutical Analysis plays a vital role in quality assurance and quality control of bulk drug and their formulations. When it comes to bulk medications and their formulations, pharmaceutical analysis is essential to quality assurance and control. A specific area of analytical chemistry called pharmaceutical analysis involves separating, identifying, and calculating the relative concentrations of various substances in a sample. It is focused on quantitative and qualitative chemical characterization of materials. Numerous analytical methods have been developed in the last few years. An analytical method is a specific application of a process to address a problem. The creation and assessment of new products, as well as the preservation of the environment and consumers, depend heavily on analytical equipment. The lower detection levels needed to guarantee safe food, medicine, water, and air are provided by this equipment1,8.

 

Meloxicam2,6 is a nonsteroidal anti-inflammatory drug (NSAID) used to relieve various types of pain, including pain caused by mucoskeletal conditions, rheumatoid arthritis, osteoarthritis. It is available in oral, intravenous, transdermal formulations. It is preferential COX-23,7 inhibitor and reducing the risk of adverse gastrointestinal tract effects. Molecular formula of meloxicam:C14H13N3O4S

 

Acetaminophen4,5,12 is a widely used nonprescription analgesic and antipyretic medication for mild-to-moderate pain and fever. Harmless at low doses, acetaminophen has direct hepatotoxic potential when taken as overdose and can cause acute liver injury and death from acute liver failure. Even in therapeutic doses, acetaminophen can cause transient serum aminotransferase elevations. Molecular formula of paracetamol: C8H9NO2

 

The availability of an HPLC9 method with high sensitivity and selectivity will be very useful for the determination of Aprepitant in pharmaceutical formulations. The objective of the work was to develop10,11 simple, accurate, precise and economic RP-HPLC method with lesser run time to estimate the meloxicam and paracetamol in bulk and pharmaceutical dosage forms.

 

MATERIALS AND METHODS:

The experimental work was carried out using various analytical instruments and high-quality glassware to ensure accuracy and precision. A WATERS Alliance 2695 HPLC system equipped with a 996 PDA detector and Empower 2 software was employed for chromatographic analysis. Additional insruments included a Lab India pH meter, Sartorious weighing machine a Labman Digital ultra sonicator. All Volumetric flasks, Pippets and Burrets, Beakers used were of Borosil make. The chemicals used in the study were of analytical grade including Meloxicam and Paracitamol Procured from Sun pharma, Provided by Sura Pharma Labs, Water and Methanol for HPLC from LICHROSOLV (MERCK), Acetonitrile for HPLC from Merck.

 

Method Development:

Preparation of standard solution:

Accurately weigh and transfer 10mg of Meloxicam and Paracetamol working standard into a 10ml of clean dry volumetric flasks add about 7ml of Methanol and sonicate to dissolve and removal of air completely and make volume up to the mark with the same Methanol.Further pipette 1.0ml of the above Meloxicam and 0.5ml of the Paracetamol stock solutions into a 10ml volumetric flask and dilute up to the mark with Methanol.

 

Procedure:

Inject the samples by changing the chromatographic conditions and record the chromatograms, note the conditions of proper peak elution for performing validation parameters as per ICH guidelines.

 

Mobile Phase Optimization:

Initially the mobile phase tried was Methanol: Water and Water: Acetonitrile and Methanol: TEA Buffer: ACN with varying proportions. Finally, the mobile phase was optimized to Acetonitrile: Water in proportion 40:60v/v respectively.

 

Optimization of Column:

The method was performed with various columns like C18 column, Symmetry and Symmetry ODS. Zodiac column C18 (4.6mm×150mm, 5µ Particle Size) was found to be ideal as it gave good peak shape and resolution at 1ml/min flow.

 

Method Validation:

Preparation of Buffer and Mobile Phase:

Preparation of Mobile Phase:

Accurately measured 400ml (40%) of Acetonitrile and 600ml of Water (60%) were mixed and degassed in digital ultra sonicator for 15minutes and then filtered through 0.45µ filter under vacuum filtration.

 

Diluent Preparation:

The Mobile phase was used as the diluent.

 

Method Validation Parameters:

System Suitability:

Procedure:

The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits.

 

Specificity study of drug:

Procedure:

Inject the three replicate injections of standard and sample solutions and calculate the assay.

 

Preparation of Drug Solutions for Linearity:

Level – I -0.2ml Meloxicam and 0.3ml Paracetamol

Level – II -0.4ml Meloxicam and 0.4ml Paracetamol

Level – III -1.6ml Meloxicam and 0.5ml Paracetamol

Level – IV -0.8ml Meloxicam and 0.6ml Paracetamol

Level – V -1.0ml Meloxicam and 0.7ml Paracetamol

 

Procedure:

Inject each level into the chromatographic system and measure the peak area.

 

Precision:

Repeatability:

The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits.

 

Accuracy:

For preparation of 50-150% Standard stock Solution:

Accurately weigh and transfer 10mg of Meloxicam and 10mg of Paracetamol working standard into a 10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution) Further pipette 0.5ml (50%), 1.0ml (100%), 1.5ml (150%) Meloxicam and 0.25ml (50%), 0.5(100%), 0.75(150%) Paracetamol stock solution into 10ml volumetric flask and dilute up to the mark with Diluent. Inject the Three replicate injections of individual concentrations (50%, 100%, 150%) were made under the optimized conditions. Recorded the chromatograms and measured the peak responses. Calculate the Amount found and Amount added for Meloxicam and Paracetamol and calculate the individual recovery and mean recovery values.

 

Robustness:

The analysis was performed in different conditions to find the variability of test results. The following conditions are checked for variation of results.

 

Effect of Variation of flow conditions:

The sample was analyzed at 0.9ml/min and 1.1ml/min instead of 1ml/min, remaining conditions are same. 10µl of the above sample was injected and chromatograms were recorded.

 

Effect of Variation of mobile phase organic composition:

The sample was analyzed by variation of mobile phase i.e. Acetonitrile: Water was taken in the ratio and 47:53, 37:63 instead (40:60), remaining conditions are same. 10µl of the above sample was injected and chromatograms were recorded.

 

RESULTS AND DISCUSSIONS:

Figure-1: Optimized Chromatogram (Sample)

 

Table-1: Optimized Chromatogram (Sample)

S. No.

Peak Name

Rt

Area

Height

USP Resolution

USP Tailing

USP plate count

1

Meloxicam

2.122

67825

68476

-

1.07

6589

2

Paracetamol

3.520

832456

867345

4.56

1.19

8956

 

Acceptance Criteria:

·       Resolution between two drugs must be not less than 2.

·       Theoretical plates must be not less than 2000.

·       Tailing factor must be not less than 0.9 and not more than 2.

·       It was found from above data that all the system suitability parameters for developed method were within the limit.


 

Method Validation:

System Suitability:

Table-2: Results of system suitability for Meloxicam and Paracetamol

S.No.

Name

Rt

Area

Height

USP plate count

USP Tailing

 

1

Meloxicam

2.120

658658

67854

6895

1.06

Meloxicam

Mean-658836.6

 

Paracetamol

3.521

8658485

845250

8542

1.18

2

Meloxicam

2.123

657893

67582

6847

1.07

Std. Dev-707.2067

 

Paracetamol

3.523

8695847

847584

8574

1.19

% RSD-0.107342

3

Meloxicam

2.127

658985

67895

6875

1.06

 

 

Paracetamol

3.526

8657474

847612

8569

1.18

 

4

Meloxicam

2.122

659863

67852

6845

1.06

Paracetamol

Mean-8662669

 

Paracetamol

3.520

8625698

846985

8532

1.18

5

Meloxicam

2.125

658784

67456

6865

1.07

Std. Dev-25911.66

 

Paracetamol

3.525

8675842

847526

8541

1.19

% RSD-0.299119

 

Acceptance criteria:

·       %RSD of five different sample solutions should not more than 2.

·       The %RSD obtained is within the limit, hence the method is suitable.

 

Specificity:

Table-3: Peak Results for Assay Standard

S. No.

Name

Rt

Area

Height

USP Resolution

USP Tailing

USP plate count

Injection

1

Meloxicam

2.125

658985

67854

-

1.06

6859

1

2

Paracetamol

3.525

8659852

845798

4.68

1.18

8643

1

3

Meloxicam

2.122

657542

67259

-

1.07

6874

2

4

Paracetamol

3.520

8652874

846354

4.69

1.19

8596

2

5

Meloxicam

2.127

658935

67823

-

1.06

6982

3

6

Paracetamol

3.526

8659875

849653

4.68

1.18

8569

3

 

Table-4: Peak results for Assay sample

S.No.

Name

Rt

Area

Height

USP Resolution

USP Tailing

USP plate count

Injection

1

Meloxicam

2.125

665985

685983

-

1.06

6965

1

2

Paracetamol

3.525

8758985

8546985

4.68

1.19

8659

1

3

Meloxicam

2.127

662974

685986

-

1.07

6928

2

4

Paracetamol

3.526

8759864

8569854

4.69

1.18

8647

2

5

Meloxicam

2.122

669852

685482

-

1.06

6975

3

6

Paracetamol

3.520

8756598

8569853

4.68

1.18

8624

3

 

Linearity:

Chromatographic Data for Linearity Study:

Table-5 Meloxicam and Paracetamol

Meloxicam

Concentration mg/ml

Average Peak Area

0

0

20

215688

40

423569

60

609752

80

791290

100

997991

 

Paracetamol

Concentration mg/ml

Average Peak Area

0

0

30

5648990

40

7379860

50

9195830

60

10965990

70

12858660

 

Figure 2: Calibration graph for Meloxicam

 

Figure 3: Calibration graph for Paracetamol

 

Conclusion:

Correlation Coefficient (r) is 0.99, and the intercept is 55981. These   values meet the validation criteria.

 

Repeatability:

Table-6: Results of repeatability for Meloxicam and Paracetamol

S. No.

Name

Rt

Area

Height

USP plate count

USP Tailing

 

1

Meloxicam

2.125

654879

67598

6825

1.06

Meloxicam- Mean

656335.2

 

Paracetamol

3.525

8659854

845865

8569

1.19

2

Meloxicam

2.127

658498

67259

6849

1.06

Std. Dev-2686.993

 

Paracetamol

3.526

8645985

845798

8575

1.18

% RSD-0.409393

3

Meloxicam

2.122

653593

67254

6826

1.07

 

 

Paracetamol

3.520

8657494

847584

8597

1.19

 

4

Meloxicam

2.120

654854

67369

6879

1.06

Paracetamol -Mean

8656616

 

Paracetamol

3.521

8659873

847592

8549

1.18

5

Meloxicam

2.123

659852

67458

6845

1.07

Std. Dev-6031.092

 

Paracetamol

3.523

8659874

845685

8543

1.19

% RSD-0.06967

 

Acceptance Criteria:

·       %RSD for sample should be NMT 2.

·       The %RSD for the standard solution is below 1, which is within the limits hence method is precise.

 

Precision:

Table-7: Results of Intermediate precision for Meloxicam and Paracetamol

S.No.

Name

Rt

Area

Height

USP plate count

USP Tailing

1

Meloxicam

2.127

648598

66598

6785

1.05

2

Meloxicam

2.122

648579

66985

6854

1.06

3

Meloxicam

2.120

659852

66879

6729

1.05

4

Meloxicam

2.125

648968

67985

6854

1.05

5

Meloxicam

2.123

658745

67498

6745

1.06

6

Meloxicam

2.120

649845

66934

6784

1.05

Mean

 

 

652431.2

 

 

 

Std. Dev

 

 

5350.652

 

 

 

% RSD

 

 

0.82011

 

 

 

 

Acceptance Criteria:

·       %RSD of Six different sample solutions should not more than 2.

 

Table-8: The accuracy results for Meloxicam and Paracetamol

 

%Concentration

(at specification Level)

Area

Amount Added

(ppm)

Amount Found (ppm)

% Recovery

Mean Recovery

Meloxicam

50%

334903

50

50.346

100.692%

Meloxicam-100.35%

Paracetamol

50%

515824.67

25

25.148

100.592%

Meloxicam

100%

665145

100

100.255

100.255%

Paracetamol

100%

975214.7

50

50.272

100.544%

Paracetamol-100.42%

Meloxicam

150%

995669.7

150

150.205

100.136%

Paracetamol

150%

1429742

75

75.130

100.173%

 

Acceptance Criteria:

·       The percentage recovery was found to be within the limit (98-102%).

·       The results obtained for recovery at 50%, 100%, 150% are within the limits. Hence method is accurate.

 

Limit of Detection:

Result - Meloxicam: 0.82µg/ml, Paracetamol:2.4µg/ml

Limit of Quantitation: Result - Meloxicam:0.86µg/ml, Paracetamol:2.8µg/ml

 

Robustness:

Table-9: Results for Robustness

Meloxicam:

Parameter used for sample analysis

Peak Area

Retention Time

Theoretical plates

Tailing factor

Actual Flow rate of 1.0mL/min

658748

2.122

6852

1.06

Less Flow rate of 0.9mL/min

725416

2.123

6985

1.05

More Flow rate of 1.1mL/min

648514

2.127

6548

1.02

Less organic phase

635254

2.125

6354

1.03

More organic phase

625098

2.127

6487

1.04

 

Paracetamol:

Parameter used for sample analysis

Peak Area

Retention Time

Theoretical plates

Tailing factor

Actual Flow rate of 1.0 mL/min

8695825

3.530

8548

1.18

Less Flow rate of 0.9 mL/min

9145487

3.523

8785

1.17

More Flow rate of 1.1 mL/min

8524583

3.526

8256

1.16

Less organic phase

8245147

3.525

8461

1.14

More organic phase

8365876

3.526

8199

1.15

 

Acceptance Criteria:

·       The tailing factor should be less than 2.0 and the number of theoretical plates (N) should be more than 2000.

 

CONCLUSION:

A precise and robust Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was successfully developed and validated for the simultaneous estimation of Meloxicam and Paracetamol in both bulk drug and pharmaceutical formulations. The analysis was performed using a Waters HPLC system equipped with an autosampler and PDA Detector (Model 996). Separation was achieved on a Zodiac C18 column (4.6mm × 150mm, 5μm particle size) maintained at 38°C.

 

The mobile phase consisted of Acetonitrile and Water in a 40:60 v/v ratio, delivered at a flow rate of 1.0mL/min. Detection was carried out at a wavelength of 223nm, with an injection volume of 10µL and a total run time of 10minutes.

 

Both Meloxicam and Paracetamol were well separated with sharp, symmetrical peaks and acceptable retention times. The method was validated as per ICH guidelines, demonstrating satisfactory results for linearity, accuracy, precision, specificity, robustness, and system suitability. These results confirm that the method is reliable and suitable for regular analysis in quality control laboratories.

 

The developed RP-HPLC method is simple, sensitive, specific, and accurate for the simultaneous determination of Meloxicam and Paracetamol in bulk and pharmaceutical dosage forms. The use of a Zodiac C18 column and a mobile phase of Acetonitrile: Water (40:60 v/v) provides effective separation within 10minutes, making it ideal for routine quality control analysis. Validation results confirmed the method’s compliance with ICH guidelines, ensuring its reproducibility and reliability in pharmaceutical applications.

 

REFERENCES:

1.      Kapil Rana, Pushpendra Sharma. Analytical Method Development and Validation for the Simultaneous Estimation of Metformin Hydrochloride and Alogliptin by RP-HPLC in Bulk and Tablet Dosage Forms. Research Journal of Science and Technology. 2021; 13(2):111-8. doi: 10.52711/2349-2988.2021.00017

2.      Mahmoud M. Sebaiy, Abdullah A. El-Shanawany, Sobhy M. El-Adl, Lobna M. Abdel-Aziz, Hisham A. Hashem. Rapid RP-HPLC Method for Simultaneous Estimation of Norfloxacin and Tinidazole in Tablet Dosage Form. Asian J. Pharm. Ana. 2011; 1(4): 79-84.

3.      Nishant Sarode, G. S. Chhabra, Shailesh Luhar, Anil Jadhav. Development and Validation of RP-HPLC Method for the      Estimation of Montelukast Sodium in Bulk and In Tablet Dosage Form. Research J. Science and Tech. 2011; 3(5): 257-260.

4.      A. Suganthi, T.K. Ravi. Development and Validation of a Novel Stability Indicating High – Performance Liquid Chromatography Method for Simultaneous Determination of Paracetamol and Lornoxicam. Research J. Pharm. and Tech. 2011; 4(7): 1046-1051.

5.      MP Yeole, A J Asnani. Simultaneous Determination of Telmisartan and Amlodipine in Tablets by Reverse Phase High Performance Liquid Chromatography. Research J. Pharm. and Tech. 2011; 4 (1): 75-77.

6.      Poonam P. Patil, Mahesh M. Deshpande, Veena S. Kasture, Seema A. Gosavi. Simultaneous Estimation of Aceclofenac and Paracetamol in Bulk and Combined Tablet Dosage Form and From Biological Fluid by Planar Chromatography. Research J. Pharm. and Tech. 2010; 3(4): 1194-1199.

7.      M.R. Santhosh Kumar, K.P. Channa Basavaraj, C. Jose Gnana Babu, T. Tamizh Mani. Validated RP- HPLC Method for the Quantitation of Nebivolol in Bulk and Pharmaceutical Dosage Forms. Research J. Pharm. and Tech. 2010; 3(4): 1167-1169.

8.      The Estimation of Aprepitant in Capsules Dosage forms by RP-HPLC. V Kiran Kumar, N Appala Raju, Shabana Begum, JVLN Seshagiri Rao, T Satyanarayana. Research J. Pharm. and Tech. 2009; 2(2): 412-414.

9.      Syeda Kulsum, G. Vidya Sagar, M. Padmalatha. Validated RP-HPLC Method for the Quantitation of Candesartan in Bulk and Pharmaceutical Dosage Forms. Research J.  Pharm. and Tech. 2011; 4(6): 997-998.

10.   M. Padmalatha, Syeda Kulsum, K. Vanitha Prakash. High Performance Liquid Chromatographic Estimation of Prulifloxacin in Pharmaceutical Dosage Form. Research J. Pharm. and Tech. 2011; 4(5): 759-761.

11.   Kothapalli LP, Yadav AH, Pawar HI, Dewoolkar VC, Banerjee AG, Thomas AB, Deshpande AD. RP-HPLC Method for Determination of Aceclofenac, Chlorzoxazone and Paracetamol in Bulk and Pharmaceutical Formulation. Research J. Pharm. and Tech. 2009; 2(1): 161-163.

 

 

Received on 13.03.2026      Revised on 15.04.2026

Accepted on 12.05.2026      Published on 10.07.2026

Available online from July 25, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(3):191-196.

DOI: 10.52711/2231-5675.2026.00029

©Asian Pharma Press All Right Reserved

 

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