Optimization and Establishment of Validated stability indicating RP-HPLC Method for Simultaneous Estimation of Mefenamic acid and Paracetamol from Tablet Dosage Form

 

Sudesh D. Shambharkar*, Purnima D. Hamrapurkar

Department of Pharmaceutical Analysis, Prin K.M. Kundnani College of Pharmacy, Jote Joy Building,

Rambhau Salgaokar Marg, Cuffe Parade, Colaba, Mumbai-400 005, India

*Corresponding Author E-mail: sudeshshambharkar@gmail.com

 

A simple, selective and precise gradient HPLC method has been developed for simultaneous estimation of Mefenamic acid and Paracetamol  combination from pharmaceutical dosage form. The separation was accomplished on an HiQSil reversed phase C18 column, 250 mm ×4.6mm I.D., 5µm column using two mobile phases i.e. A as methanol: 50mM potassium dihydrogen orthophosphate (pH = 7.5)(20:80 v/v) and B as methanol in a gradient elution mode. The flow rate was 1mL/min. The eluents were monitored with a UV detector set at 245 nm as detection wavelength. The investigated validation elements showed the method has acceptable specificity, accuracy, linearity, precision, robustness and high sensitivity. Detection and quantification limits were established at 0.02 µg/ml and 0.05 µg/ml respectively. A stability indicating HPLC method has been established for analysis of Mefenamic acid and Paracetamol combination. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis, and thermal decomposition.

 

 

1. INTRODUCTION:

Mefenamic acid (figure 1) is 2-[(2,3-dimethylphenyl)amino] benzoic acid. Mefenamic acid is a non-steroidal anti-inflammatory drug used to treat pain, including menstrual pain. It is typically prescribed for oral administration. Mefenamic acid decreases inflammation (swelling) and uterine contractions by a still unknown mechanism. However it is thought to be related to the inhibition of prostaglandin synthesis. There is also evidence that supports the use of Mefenamic acid for perimenstrual migraine headache prophylaxis.^[1,2,3]

 

Paracetamol (figure 2) is N-(4-hydroxyphenyl) acetamide. Paracetamol (acetaminophen) is generally considered to be a weak inhibitor of the synthesis of prostaglandins (PGs). However, the in-vivo effects of Paracetamol are similar to those of the selective cyclooxygenase-2 (COX-2) inhibitors. Paracetamol also decreases PG concentrations in -vivo, but, unlike the selective COX-2 inhibitors.^[4,5,6]

 

fig. 1: Structure of  Mefenamic acid

 

fig. 2: Structure of Paracetamol

 

Stability is considered one of the most important criteria in pharmaceutical QC. Only stable preparation promise accurate delivery of drugs to patients. Expiration dating on any drug product is based upon scientific studies at normal and stressed conditions.

 

A literature survey revealed that analytical methods are available for determination of Mefenamic acid and Paracetamol combination using spectroscopic method^[7], Pharmaceutical preparations and biological samples.^[8,9,10] No validated stability indicating analytical method for the determination of degraded substances in Mefenamic acid and Paracetamol combination available. Therefore the objective of the research was to study degradation of mefenamic acid and paracetamol combination under different International Conference on Harmonization (ICH) recommends stress condition^[11] and to established a validated stability indicating HPLC method for accurate quantification of degradation product. The stress conditions studied for forced degradation of Mefenamic acid and Paracetamol combination were acid hydrolysis, base hydrolysis, oxidation, heat and UV light. Fixed dose combination containing Mefenamic acid (500 mg) and Paracetamol (450 mg) is available in the tablet form in the market.

 

2. EXPERIMENTAL:

2.1 Chemicals and reagents

The working standard of Mefenamic acid and Paracetamol were procured from Alexo chemical Pvt. Ltd. India, and ANQIU LU AN, china Respectively. HPLC grade methanol was purchased from Merck (Darmstadt Germany). Deionised and ultrapure water was used in all experiments was obtained from Milli-Q system (Millipore). Potassium dihydrogen orthophosphate used as buffer AR grade (Qualigens). NaOH used for adjusting the PH of buffer solution AR grade (S. D. Fine chemicals).

 

2.2 Instrumentation and Equipment

pH of the mobile phase was checked on a pH/ion analyser (Lab India, PHAN, India). Refluxing of drug in hydrolysis condition was carried out in round bottom flask-condenser assembly. The HPLC system employed in method development, forced degradation studies, and assay method validation was Jasco PU -2089 Plus Quaternary gradient HPLC pump (Japan). Jasco UV-2070 Plus Intelligent UV-Vis Detector (Japan) and Chrompass software as data integrator.

 

2.3 Preparation of mobile phase

Since gradient elution was applied Two mobile phases were used. Mobile phase -A consisted of 5.44 g of potassium dihydrogen orthophosphate was dissolved in 800 mL water. The pH was adjusted to 7.5 using NaOH solution. And further mixed with 200 mL methanol. Mobile phase-B consisted of methanol. The mobile phases were sonicated for 15 min.

 

2.4 Preparation of standard solution

A stock solution of Mefenamic acid (1 mg/mL) and Paracetamol (0.9 mg/ mL) was prepared in methanol. Standard solutions were prepared by dilution of stock solution with mobile phase to concentration range 100 µg/mL to 0.05 µg/mL.

 

Table 1.  Gradient  program from Analysis of Mefenamic acid and Paracetamol combination.

Time	Mobile phase A(%v/v)	Mobile phase B (%v/v)	Gradient  Program
0	60	40	Isocratic
0- 10	60-10	40-90	Linear gradient
10-12	10-60	90-40	Linear gradient
12-15	60	40	Isocratic

 

Table 2.  Linearity and range of  methods.

Linearity and range	Mefenamic acid	Paracetamol
Range (μg mL-1)	2.50- 7.50	2.25-6.75
r2	0.9995	0.9996
Slope	68.732	115.62
Intercept	6.8341	0.7393

 

2.5 Optimized chromatographic conditions

The chromatographic separation was achieved on an HIQ SIL RP-C₁₈ column (250×4.6 mm, 5 μm particle size) using mobile phase i.e. mobile phase A consisted of 5.44 g of potassium dihydrogen orthophosphate was dissolved in 800 mL water. The pH was adjusted to 7.5 using NaOH solution. And further mixed with 200 mL methanol. Mobile phase B consisted of methanol. All reagents were filtered through 0.45 µm filter paper and sonicated before use. The injection volume was 20 µL. The detection wave length was set at 245 nm. The assay was performed at room temperature, and the flow was 1.0 ml/min. The gradient program of the HPLC method shows the change in mobile phase concentration with respect to time (Table 1).

 

2.6 Validation of the method

A stock solution of drug was prepared at concentration 1 mg/mL for Mefenamic acid and 0.9 mg/mL for Paracetamol. It was diluted to prepare solutions containing 100- 0.05µg/mL of drug. The solutions were injected into triplicate into the HPLC column, keeping the injection volume constant (20µL). (Table 2)

 

Twelve injections of three different concentration (4.00, 5.00, 6.00 µg/mL for Mefenamic acid and 3.60, 4.50, 5.40 µg/mL for Paracetamol) were made one the same day, and the RSD values were calculated to determine intraday precision. These study were also repeated on different days to determine interday precision. (Table 3,4)

 

Accuracy was evaluated by recovery studies with known concentration of added drug. The specificity of the method was ascertained by analyzing the standard drug, and sample retention time (RT) of Mefenamic acid and Paracetamol was confirmed by comparing the RT with that of standard.

 

 

Table 3. Precision and Recovery data of  Mefenamic acid

Precision			% Recovery
Actual Concentration(μg/mL)	Measured Concentration  (μg/mL) ± S.D.; % R.S.D.
	Intra-day	Inter-day	Intra-day	Inter-day
4.00	4.000±0.047; 1.178	4.011±0.039; 0.988	100.01	100.28
5.00	5.00 ±0.053 ;  1.060	5.006±0.031; 0.624	100.00	100.13
6.00	6.025±0.032; 0.542	6.014±0.022; 0.367	100.41	100.24

 

Table 4. Precision and Recovery data of  Paracetamol

Precision			% Recovery
Actual Concentration (μg/mL)	Measured Concentration (μg/mL) ± S.D.; % R.S.D.
	Intra-day	Inter-day	Intra-day	Inter-day
3.60	3.601±0.031; 0.875	3.605±0.021; 0.593	100.04	100.14
4.50	4.500 ±0.045 ;  1.012	4.508±0.027; 0.610	100.02	100.17
5.40	5.410±0.011 ; 0.208	5.411±0.012; 0.227	100.20	100.21

 

The LOD and LOQ were determine at an S/N of 3:1 and 10:1 respectively, by injecting a series of dilute solutions with known concentration.

 

Robustness of method was investigated by varying the chromatographic conditions such as change of flow rate (± 10%), organic content in mobile in phase (±2%), wavelength of detection (±5%), and the pH of buffer in the mobile phase (±0.2%). Robustness of the developed method was indicated by the overall %RSD between the data at each variable condition.

 

The solution stability was carried out by leaving both test solution of sample and reference standard in tightly capped volumetric flask at -20°C for 7 days. The sample solution was assayed after 7 days with fresh sample.

 

2.7 stress degradation study

Mefenamic acid and Paracetamol combination dosage form used for the degradation studies. Mefenamic acid 50µg/mL and Paracetamol 45 µg/mL concentration of solution was used for degradation studies. For acid and base hydrolysis, 5 mL 0.1M HCl and 0.1M NaOH were added to 10 mL of stock solution separately. These mixtures were heated in a water bath for 80⁰c for 8Hrs. For the thermal degradation study 10 mL methanol was added to 10 mL stock solution. And the mixture was heated for 80⁰c for 8 Hrs. For oxidative degradation, 10 mL stock solution was treated with 5 mL 3%H₂O₂(v/v) solution, and this mixture was heated for 60 min for 60⁰c.The photolytic degradation was carried out by exposing solid drug substance to 245 nm UV light for 24 Hrs and the weighing and diluting to prepare a concentration of Mefenamic acid 50µg/mL and Paracetamol 45 µg/mL. 20 µL of resulting solution was injected into the HPLC system, and chromatographs were recorded. The stability samples were analysed using PDA detector to determine the peak purity.

 

3. RESULT AND DISCUSSION:

3.1 Degradation Behaviour

HPLC studies on Mefenamic acid and Paracetamol combination under different stress conditions suggested the following degradation behaviour (figure 3a, Table 5, 6).

 

Acid degradation

The drug gradually decrease with time upon heating at 80⁰c in 0.1M HCl. The Mefenamic degrade 99.54% while Paracetamol degrade 37.99% . (figure 3b)

 

Base degradation

The drug after treating with 0.1 M NaOH , Heated 80⁰c for 8Hrs. The Mefenamic acid degrade 99.00 % while Paracetamol produce less degradation as compared to acid degradation that is 17.05% (figure 3c).

 

Oxidative degradation

The drugs degrade more in oxidative condition than base degradation. The Mefenamic acid degrade 99.23 % while Paracetamol produce less degradation as compared to acid degradation that is 55.95% (figure 3d).

 

Thermal degradation

In thermal degradation Mefenamic acid produce less degradation 5.27% with peak purity of 99.619 %.but Paracetamol produce 55.77% degradation . (figure 3e).

 

Photolytic degradation

The Mefenamic degrade 28.64% while Paracetamol degrade 76.05% (figure 3f).

 

Table 5. Degradation of   Mefenamic acid and retention time

No.	condition	Retention time of drug, min	Peak area mv/s	Mass conc. µg/mL	Degradation of Drug  %
1	Untreated Stock solution (50 μg/mL)	7.57	3238.2	50
2	Acid hydrolysis	7.76	14.6	0.22	99.54
3	Base hydrolysis	7.66	32.1	0.49	99.00
4	Oxidation	7.40	24.8	0.38	99.23
5	Thermal	7.69	3067.3	47.36	5.27
6	Photolytic	7.65	2309.4	35.65	28.64

 

Table 6. Degradation of   Paracetamol  and retention time

No.	condition	Retention time of drug, min	Peakarea mv/s	Mass conc. µg/mL	Degradation of Drug%
1	Untreated Stock solution (45 μg/mL)	3.42	8932.0	45	……
2	Acid hydrolysis	3.42	5538.2	27.90	37.99
3	Base hydrolysis	3.42	7408.5	37.32	17.05
4	Oxidation	3.39	3934.3	19.82	58.83
5	Thermal	3.44	3950.5	19.90	55.77
6	Photolytic	3.44	2139.2	10.77	76.05

 

(a)

 

(b)

 

(c)

 

(d)

 

(e)

 

(f)

Figure. 3: Chromatogram of  degradation of Mefenamic acid and Paracetamol combination a) standard stock solution, b) acid degradation,  c) Base degradation, d) Oxidative degradation, e) thermal degradation, f) Photolytic degradation

3.2 Establishment of Stability indicating assay

Separation of Mefenamic acid and Paracetamol combination has been performed on an HIQ SIL RP-C₁₈ column. Different ratios of Potassium dihydrogen orthophosphate buffer and methanol solution (pH 7.5) of mobile phase A were tested. Increasing the methanol ratio was accompanied by a decrease in RT of drugs. However separation was still achieved. In order to ensure complete separation , the chosen ratio of mobile phase A is 50 mM Potassium dihydrogen orthophosphate buffer and methanol (80: 20 v/v) while mobile phase B is methanol. The detection was performed at 245 nm. where the maximum sensitivity was observed of both drugs. The specificity of the method is illustrated in figure 3. The calibration curve was constructed by transferring aliquots of Mefenamic acid Paracetamol stock and working standard solutions into a series of 10 mL volumetric flask a diluting to volume with the mobile phase to obtain solution in the concentration range of 0.02 – 100 µg/mL. A 20µL volume from each solution was injected in triplicate; chromatographic separation was obtained under the previously mentioned conditions.

 

All determinations were performed at ambient temperature; the average peak area obtained for each concentration was plotted versus concentration.

 

3.3 Validation of Method

The method was validated with respect to linearity, precision, Accuracy, specificity, and robustness. The response for the drugs was linear in the studied concentration range (r²= 0.9995 for Mefenamic acid and 0.9996 for Paracetamol.).

 

Table 3, table 4 provide data obtained from the precision experiments. The RSD values for intraday and interday precision were < 2. Thereby indicating that the method was sufficiently precise.

 

The method was found to be specific for drugs. The drugs peak was separated from any coeluting peak. Good separation were always achieved, which suggested that the method was selective for all component tested. The LOD and LOQ concentrations were found to be 0.02 and 0.05 µg/mL respectively.

 

The RSD values of the assay of Mefenamic acid and Paracetamol combination during solution stability experiments were within 2%. No significant changes were observed during the stability study. The solution stability data confirmed that the sample solution of Paracetamol was stable for at least 7 days but Mefenamic acid was stable atleast 24 Hrs.

 

4. CONCLUSION:

The study shows that Mefenamic acid is labile molecule when stressed with acid, alkali, oxidation, heat and UV light. While Paracetamol is more stable as compared to Mefenamic acid. A stability indicating method was developed that to provide degradation formed under variety of condition. The method prove to be simple, precise , specific and selective. Hence, it is recommended for analysis of drugs and product stability samples in the Pharmaceutical industry.

 

5. ACKNOWLEDGMENT:

The Authors are thankful to the Principal and Head of Pharmaceutical analysis Department, Prin. K.M. Kundnani College of Pharmacy, Cuffe Parade, Mumbai. We also thanks Alexo chemical Pvt Ltd India, and ANQIU LU AN, china for supplying authentic working standard of Mefenamic acid and Paracetamol Respectively.

 

6. REFERENCES:

1.        Maryadele J.O; Patricia E.H, Cherie B.K., Kristin J.R., Catherine M.K., Maryann R.D. The Merck index.14^th edition .United states Pharmaceutical Company Merck & co.2006, 5798 page no.1001.

2.        Sean Sweetman, Martindale: The complete Drug Reference, 36^th Edition, Page no.80.

3.        Pringsheim T, Davenport WJ, Dodick D. Acute treatment and prevention of menstrually related migraine headache: evidence-based review. Neurology. 2008;70(17):1555-1563.

4.        Sean Sweetman, Martindale: The complete Drug Reference, 36^th Edition, Page no.108.

5.        Hinz, B.; Cheremina, O.; Brune, K. (2008). The FASEB J. Federation of American Societies for Experimental Biology 22 (2): 383–390. 

6.        K.D. Tripathi, “Essentials of Medical pharmacology, 6^th-edition 2008.Page No.198-199

7.        Erdal Dinc¸ Cem Yu¨cesoy, Feyyaz Onur,. J. of Pharm. Biomed. Anal, 28(2002)1091-1100.

8.        Ali Babaei, Balal Khalilzadeh • Mohammad Afrasiabi, J Appl Electrochem (2010) 40:1537–1543.

9.        P.D. Sethi. “Quantitative Analysis of Drug in Pharmaceutical Formulation” 2001 Page No. 390-391.

10.     Harish L. Rau, A.R. Arora and P. Gundu Rao. Indian Drugs, 28(12) 563-565,1991.

11.     ICH Guidelines: Validation of Analytical Procedures: Test and Methodology, Q2(R1), (2005).

 

 

 

Received on 16.08.2013          Modified on 10.09.2013

Accepted on 15.09.2013         © AJRC All right reserved