SIMULTANEOUS ESTIMATION OF MEFENAMIC ACID AND DICYCLOMINE HYDROCHLORIDE BY SPECTROSCOPIC METHODS

SIMULTANEOUS ESTIMATION OF MEFENAMIC ACID AND DICYCLOMINE HYDROCHLORIDE BY SPECTROSCOPIC METHODS

Dipal Prajapati* and Hasumati Raj

Department of Quality Assurance, Shree Dhanvantary Pharmacy College, Kim-Kudsad Road, Kim, Surat-394110, Gujarat, India

ABSTRACT

A novel, simple, accurate, sensitive, reproducible, economical spectroscopic method was developed and validated for the determination of Mefenamic acid and Dicyclomine hydrochloride in combined dosage form. Three different analytical methods, Absorption correction method, Differential derivative method, Simultaneous equation method were developed for estimation of  Dicyclomine hydrochloride(10mg) and Mefenamic acid (250mg) in tablet dosage form. wavelength for estimation was 223nm for Dicyclomine hydrochloride and 308.60nm for Mefenamic acid in absorption correction method. 211.60nm was Zero crossing point of Mefenamic acid and 308.80nm was Zero crossing point of Dicyclomine hydrochloride which can estimate in differential derivative method. Simultaneous equation method was developed in NaOH which was linear in the range of  1-6µg/ml for Dicyclomine hydrochloride and 25-150µg/ml for Mefenamic acid, the correlation coefficient obtained was nearer to one. The method was validated for linearity, accuracy and precision as per ICH guidelines. The developed and validated method was successfully used for the quantitative analysis of commercially available dosage form.

Combined dosage form

INTRODUCTION: Mefenamic acid [N-(2, 3-xylyl) anthranilic acid] is an Aminobenzoate, a subclass of analgesic with Non steroidal anti-Inflammatory properties ¹. It acts by binds the prostaglandin synthetase receptors COX-1 and COX-2, inhibiting the action of prostaglandin synthetase ^{2, 3}. It is used for the treatment of rheumatoid arthritis, osteoarthritis, dysmenorrhea, and mild to moderate pain, inflammation, and fever ⁴.

Dicyclomine (bicyclohexyl]-1-carboxylic acid is an antispasmodic and anticholinergic (antimuscarinic) agent ⁵. It Action is achieved via a dual mechanism: a specific anticholinergic effect (antimuscarinic) at the acetylcholine-receptor sites, a direct effect upon smooth muscle (musculotropic) ⁵.

It is used to treat a certain type of intestinal problem called irritable bowel syndrome. It helps to reduce the symptoms of stomach and intestinal cramping. This medication works by slowing the natural movements of the gut and by relaxing the muscles in the stomach and intestines ^{6, 7}. Combination of Mefenamic Acid and Dicyclomine Hydrochloride has a Synergistic effect.

This Combination is a highly effective and used in the treatment of spasmodic dysmenorrhoea, intestinal colic, biliary colic, ureteric colic ⁸. Mefenamic Acid is a medication that helps in reducing the pain in the body.Antipyretic is a drug that helps in reducing the fever by reducing the temperature of the body to the normal temperature. Anti-inflammatory is a kind of medication that is mainly for reducing the inflammation by controlling the pain. Lastly Dicyclomine HCl helps in offering relief to the cramps of the stomach, bladder and intestine ⁸.

Both the drugs are official in Indian pharmacopoeia 2010 ¹¹, United State Pharmacopoeia ¹² and British Pharmacopoeia ¹³. Literature survey reveals that RP-HPLC ¹⁴, Liquid Chromatography ¹⁵, Voltametry ¹⁸, UV-Visible Spectrophotometry ^{20, 21}, methods were reported for the estimation of Mefenamic Acid alone or in combination with other drugs except Dicyclomine Hydrochloride and Capillary Gas-Chromatography ³⁰, Stability indicating gas-liquid chromatography ³⁰, HPTLC³¹, methods were reported for the estimation of Dicyclomine Hydrochloride alone or in combination with other drugs except Mefenamic Acid.

As per literature survey, no analytical method has been reported for simultaneous estimation of Mefenamic Acid and Dicyclomine Hydrochloride in pharmaceutical dosage forms. Therefore the present research work, our aim is to develop a novel, simple, accurate, sensitive, reproducible, economical analytical method to estimate Mefenamic Acid & Dicyclomine Hydrochloride in their combined dosage form in routine analysis.

MATERIALS AND METHODS:

Reagents and Chemicals: Methanol (AR Grade) and NaOH (S.D. fine Chemicals) were used as solvent. Pure Standard gift sample of Mefenamic Acid (MEF) and Dicyclomine Hydrochloride (DICY) provided by Shree Dhanvantary Pharmaceutical Analysis & Research Centre, (Kim) and Mercury labs Ltd., Vadodara. Tablets of MEFTAL-SPAS (Mefenamic Acid- 250 mg, Dicyclomine Hydrochloride- 10 mg) were purchased from local market.

 Instruments: Shimadzu UV/Vis-2450 and UV/Vis-1800 double beam UV/Vis spectrophotometer with a fixed slit width of 2 nm, 1 cm quartz cells was used for recording derivative spectra of standard and test samples. Sartorius CD2250 balance was used for weighing the samples. Class ’A’ volumetric glassware were used.

Preparation of standard solution:

1. Absorption Correction and Differential Derivative Method:
   1. Preparation of stock solution of MEF: Weighed accurately about 10mg of MEF & transferred into 100 mL volumetric flask add 20mL of methanol and sonicated for about 15 min than diluted up to the mark with methanol to get a stock solution having strength 100 μg/mL.

• Preparation of working Standard solution of MEF: 100 μg/mL of MEF solution was prepared by diluting stock solution to 10mL with Methanol: NaOH (50:50) and NaOH. This solution was diluted further to get the concentration range of 10, 25, 30, 40, 50, 75 μg/mL of MEF.

1. Preparation of stock solution of DICY: Weighed accurately about 10mg of DICY & transferred into 100 mL volumetric flask add 20mL of methanol and sonicated for about 15 min than diluted up to the mark with methanol to get a stock solution having strength 100µg/mL.

• Preparation of Working Standard Solution of DICY: 100 μg/mL of DICY solution was prepared by diluting stock solution to 10 mL with Methanol: NaOH (50:50) and NaOH. This solution was diluted further to get the concentration range of 1, 2, 3, 4, 5, 6 μg/ml of DICY.

2. Simultaneous Equation method: An accurately weighed quantity of MEF (50 mg) and DICY (10 mg) were transferred to a separate 100 mL volumetric flask, respectively and dissolved and diluted to the mark with Methanol to obtain standard solution having concentration of MEF (500μg/mL) and DICY (100 μg/mL). Further dilution with NaOH as a solvent.

Procedure for Determination of Wavelength for Measurement:

1. Absorption Correction and Differential Derivative Method: 1 mL of working standard solution of Dicyclomine HCl(100µg/mL) and 2.5 mL of working standard solution of Mefenamic acid(2500μg/mL) was pipetted out in two different 10 mL volumetric flask to have a concentration of 10μg/mL and 250μg/mL for DICY and MEF respectively. One flask dilute up to make with 0.1N NaOH and labelled as sample cell & Second flask were dilute up to mark with mixture of Methanol: NaOH (50:50) and labelled as reference cell. Prepared solutions were scanned in the range of 200-400 nm on Shimadzu double beam UV visible spectrophotometer using Reference compartment solution in reference cell & sample compartment in sample cell. Wavelength estimation was selected from scan spectra. According to same procedure the derivative spectra were recorded by using digital differentiation (Convolution method) with a derivative wavelength difference (Δλ (N)) of 2 nm in   the range of 200-400 nm.
2. Simultaneous equation Method: By appropriate dilution of two standard drug stock solutions with methanol, solutions containing 500 μg/mL of MEF and 100 μg/mL of DICY were scanned separately in the range of 200-400 nm to determine the wavelength of maximum absorption for both the drugs. MEF showed absorbance maxima at 335 nm and DICY at 218.40 nm.

Validation: Sample preparation for validation was given in following manner but dilutions were made according to solvent required for differential & simultaneous equation method.

The methods were validated with respect to linearity, precision, accuracy, robustness, LOD & LOQ and assay.

Linearity: Standard stock solutions were prepared by dissolving 25 mg MEF and 10 mg of DICY in 100 ml volumetric flasks in 25 mL Methanol and the volume was made up with Methanol to get a concentration of 250 μg/mL of MEF and 100 μg/mL of DICY. From this, suitable dilutions were made in NaOH to get the working standard solutions of 10-75 μg/mL for MEF and 1-6 μg/mL for DICY. Absorbance of each solution measured at 308.60nm for MEF, 223nm for DICY in Absorption Correction Method. The absorbance of the derivatised spectra was measured at 308.80 nm and 211.60 nm for MEF and DICY, respectively in Differential Derivative Method. Several aliquots of standard stock solutions were taken in different 10mL volumetric flask and diluted up to mark with NaOH, such that the final linearity concentration of MEF and DICY were 25-150 µg/mL and 1-6 µg/mL in Simultaneous Method. Five replicate analyses were carried out. Plot the calibration curve of absorbance Vs respective concentration for MEF and DICY. Find out correlation coefficient and regression line equations for MEF and DICY.

Precision: The precision of the developed method was assessed in terms of repeatability (intra-day) and intermediate precision (inter-day) by analyzing three replicate of standard stock solutions at three levels that cover the calibration ranges for MEF and DICY. The % RSD value of the results corresponding to the absorbance was expressed for intra-day precision and on 3 days for intermediate (inter-day) precision.

Accuracy: Accuracy of the method was calculated by recovery studies at three levels (80%, 100% and 120%) by standard addition method. Twenty tablets were weighed and average weight was calculated. Twenty tablets were weighed and average weight was calculated. The tablets were crushed to obtain fine powder. Tablet powder equivalent to 125 mg MEF was transferred to 50 mL volumetric flask. 25 mL Methanol was added to dissolve the drugs and then volume was made up to the mark and sonicated for 15 minutes. The solution was then filtered through a Whatman filter paper (No. 41). From the filtrate 0.1 mL was transferred to three 10.0 mL volumetric flasks and add 0.08 mL (Flask 1), 0.1 mL (Flask 2), and 0.12 mL(Flask 3) of stock solution of API and then made up to the mark with suitable solvent to made them 80%, 100% and 120% spiking.

Robustness: The robustness of an analytical procedure is the measure of its capacity to remain unaffected by small, but deliberate, variations in method parameters and provides an indication of its reliability during normal usage. The parameters were change of volumetric flasks (10 mL, 50 mL and 100 mL) and Change in instrument (UV-Vis Spectrophotometer model no. 1800 and 2450). Three replicates were made for the same conc. (25μg/mL of MEF and 1μg/mL of DICY) in 10 mL, 50 mL and 100 mL volumetric flasks and the recording of absorbances were done on both the UV-Vis spectrophotometer. The result is expressed in Percentage RSD.

Limit of detection (LOD) and limit of quantization (LOQ): LOD and LOQ were calculated from the data obtained from the linearity studies. The slope of the linearity plot was determined. For each of the ten replicate determinations of same conc. (25 μg/mL of MEF and 1 μg/mL of DICY), standard deviation (SD) of the responses was calculated. From these values, the parameters Limit of Detection (LOD) and Limit of Quantitation were determined on the basis of standard deviation and slope of the regression equation.

LOD = (3.3 x SD) / Slope

LOQ = (10 x SD) / Slope

Assay: Twenty tablets were weighed and finely powdered. The average weight of tablets is determined with the help of weight of 20 tablets. A portion of powder equivalent to the weight of 125 mg of MEF was accurately weighed into 50 mL A-grade volumetric flask and 25 mL Methanol was added. The volumetric flask was sonicated for 15 min to effect complete dissolution of the DICY and MEF, the solution was then made up to volume with Methanol: NaOH & NaOH. The solution was filtered through Whatman filter paper. The aliquot portion of the filtrate was further diluted to get final concentration of 1μg/mL of DICY and 25 μg/mL of MEF. The % assay of the drugs was calculated.

RESULTS AND DISCUSSION:

1. Selection of Wavelength for Simultaneous Estimation of Mefenamic Acid and Dicyclomine HCl: (Absorption Correction and Differential Derivative Method): From the overlain spectrum of MEF and DICY in Methanol-NaOH and NaOH (Figure 1), it was observed that Dicyclomine has zero absorbance at 308.60nm; where as Mefenamic Acid has substantial absorbance. Thus, MEF was estimated directly at 308.40 nm with no interference of DICY. For the estimation of DICY, the absorbance of MEF was deducted from the total absorbance of sample mixture at 223 nm. The calculated absorbance was called as Corrected absorbance for DICY (eq. 2). The Concentration of DICY was determined from calibration curve at 223 nm using the corrected absorbance (Figure 1).

…………….(1)              ….....(2)

Where, A₁ and A₂ are the absorbances of mixture at 308.60 nm and 223 nm respectively, a_x1 and a_x2 are absorptivities of MEF and DICY at 308.60 nm and 223 nm respectively; a_y2 is absorptivity of DICY at 223 nm; C_MEF is concentration of Mefenamic Acid (MEF); C_DICY is concentration of Dicyclomine HCl (DICY).

FIG. 1: OVERLAIN SPECTRA OF MEF AND DICY

In Differential Derivative Method, First order derivative spectrum for DICY showed zero crossing points: 211.60 nm. The wavelength selected for estimation of MEF was 210.59 nm because it showed adequate absorbance at this wavelength in mixture. Similarly, first order derivative spectrum for MEF was taken and it showed zero crossing point: 308.80 nm. The wavelength selected for estimation of DICY was 308.80 nm because it showed adequate absorbance at this wavelength in mixture (Figure 2).

FIG. 2: 1^STORDER DERIVATIVE OVERLAIN SPECTRA OF MEF AND DICY

FIG. 3: ZERO CROSSING POINT OF DICYF

FIG. 4: ZERO CROSSING POINT OF MEF

2. The proposed Simultaneous method provides a rapid, convenient and accurate way for routine quantitative determination of MEF and DICY. The stock solutions, working standards were prepared in methanol. The λ_max the both the drugs for analysis were determine by separately scan both the drugs in the range of 200-400 nm, wavelength selected for quantitation were 335.0 nm (λ_max of MEF) and 218.40 nm (λ_max of DICY) (Figure. 5). Two simultaneous equations (in two variables C_x and C_y) were formed using these absorptivity coefficient values.

A₁ = 1.275 C_x +0.0510 C_{y …………..…} (1)

A₂ = 0.490 C_x +0.0197 C_Y ………... (2)

Where, A₁ and A₂ are the absorbance of mixture at 335.0 (l₁) nm and 218.40 (l₂) nm wavelength respectively, C_x and C_y are the concentration of MEF and DICY measured in μg/mL, in sample solutions respectively, ax₁ and ax₂ are absorptivities of MEF at l₁ and l₂ respectively, ay1 and ay₂ are absorptivities of DICY at l₁ and l₂ respectively. By applying the Cramer’s rule to equation 1 and 2, the concentration of C_MEF and C_DICY, can be obtained as follows,

C_{DIC =} A₂ (0.0510) - A₁ (0.0197)/-0.00013

C_MEF= A₁ (0.490) - A₂ (1.275)/-0.00013

FIG. 5: ZERO ORDER OVERLAIN SPECTRA OF  DICY AND MEF

RESULTS:

 Absorption Correction and Differential Derivative Method:

FIG. 6: CALIBRATION CURVE OF MEF AND DICY. (ABSORPTION CORRECTION METHOD)

FIG. 7: CALIBRATION CURVE OF DICY AND MEF (DIFFERENTIAL DERIVATIVE METHOD)

TABLE 1: LINEARITY (n=5)

TABLE 2:  PRECISION (n=3)

TABLE 3: ASSAY (n=3)

TABLE 4: ACCURACY (RECOVERY STUDY)

Absorption correction method:

 Differential derivative method:

 TABLE 5: LOD and LOQ

TABLE 6: RUGGEDNESS & ROBUSTNESS

TABLE 7: SUMMARY OF VALIDATION PARAMETERS FOR PROPOSED METHOD

 Simultaneous equation method:

FIG. 8: CALIBRATION CURVE OF MEF AND DICY

TABLE 8 : LINEARITY DATA FOR MEF AND DICY* (n=6)

TABLE 9:  PRECISION (REPEATABILITY) *(n=3)

 TABLE 10: ACCURACY        

TABLE 11: ASSAY

 TABLE 11:  LOD & LOQ

 TABLE 12: RUGGEDNESS & ROBUSTNES

TABLE 13: SUMMARY OF VALIDATION PARAMETERS FOR PROPOSED METHOD

CONCLUSION: The developed method was novel, simple, accurate, precise reproducible, economical, which would be used to estimate MEF & DICY in their combined dosage form in routine analysis.

ACKNOWLEDGEMENTS: Authors are highly thankful to Shree Dhanvantary Pharmaceutical Analysis & Research Centre (kim, India) & Mercury labs Ltd. (Vadodara, India) for providing gift sample of MEF and DICY.

We acknowledge the Shree Dhanvantary Pharmacy College, Kim, for providing the facilities to complete the work.

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    How to cite this article:

    Prajapati D and Hasumati Raj: Simultaneous Estimation of Mefenamic Acid and Dicyclomine Hydrochloride by Spectroscopic Methods. Int J Pharm Sci Res 2012; Vol. 3(10): 3766-3775.

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