Review for Analytical Methods for the Determination of Mefenamic Acid

Mefenamic acid (MFA) is a non-steroidal anti-inflammatory drug that belongs to the anthranilic acid derivative family. It is used to relieve mild to moderate pain. The present review article includes a compilation of articles on the various properties along with an extensive literature survey on the reported analytical methods of MFA. Using a comprehensive computer assisted literature review; this article discusses the analytical methodologies for quantifying MFA both in active pharmaceutical ingredient and pharmaceutical dosage forms. This is the first review article in this series with focus on the analytical profile of MFA. Although, several methods like High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), spectrophotometry, fluorimetry, turbidimetry, Atomic Absorption Spectroscopy (AAS), Mass Spectroscopy (MS) and electro analytical methods were reported in the literature, HPLC stands out first for the quantification of MFA. Review Article Ashira et al.; JPRI, 33(43B): 426-437, 2021; Article no.JPRI.73896 427


INTRODUCTION
MFA (Fig. 1) is a non-steroidal anti-inflammatory drug. It is an analgesic, antipyretic and weaker anti-inflammatory drug. It is used as pain reliever for tooth ache, menstrual pain etc [1]. MFA is official in IP [2], BP [3], USP [4] and EP [5]. MFA designated as anthranilic acid, which is a white to off-white, crystalline powder with a melting point ranging from 230-231°C. MFA is structurally 2-(2,3-dimethylanilino) benzoic acid [6] with molecular formula C15H15NO2 and molecular mass 241.28 g/mol respectively [6,7]. The MFA is an achiral compound and the optical activity is unspecified [8]. A review of the literature shows that many methodological approaches are in the process of being formulated and validated for MFA may be used alone or in conjunction with other medications [9]. The drug is a Biopharmaceutical Classification System (BCS) type II drug which means it has a low water solubility and high permeability [10]. This review article provides readers with an abundance of details on the different analytical methods for determining MFA. Among the published analytical methods for estimating MFA, HPLC was found to be the most efficient and validated, followed by spectrophotometric and other methods. Distribution of analytical methods described in the literature for the determination of MFA is shown in Fig. 2. This review shows the highlight of analytical method for quantitation and determination of MFA in both pharmaceutical products and biological samples described in the literature. For this purpose the search focused on the following database: PubMed, Scopus and Web of Science whose period ranged from 1990 to 2021.

Mechanism of Action
MFA binds to the COX-1 and COX-2 prostaglandin synthetase receptors inhibiting prostaglandin synthetase activity. Pain effects are temporarily reduced when these receptors play a role as a key mediator of inflammation and a role for prostanoid signaling in activitydependent plasticity [11].

ANALYTICAL STATUS ON MFA
Drug research is crucial in the pharmaceutical industry as it is used from the early stages of drug development to the post-marketing stages. Analytical methods lead to valuable knowledge about medicines such as bioavailability and bioequivalence tests, drug molecule physical and chemical stability and dosage form design, impurity quantification and detection as well as substance quality quantification of branded products and also used to test pharmacokinetic criteria for medicinal drug control. As per the WHO, drug quality management is a set of approaches used to guarantee the quality and purity of pharmaceuticals. These methods are widely carried out by using chromatography (e.g., High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC) and Thin-Layer Chromatography (TLC). In addition, many analytical techniques such as Ultra Violet Spectroscopy (UV), Mass Spectroscopy (MS) and fluorimetry are used to assess drug purity from the reports available on the literature, many instrumental analytical tools like atomic absorption spectrometry and turbidimetry are also used.

Spectroscopy Techniques of MFA
Spectrophotometric instruments are one of the most reliable instruments in pharmaceutical science for qualitative and quantitative drug analysis because they are simple, inexpensive and precise.
Asad Raza studied the spectrophotometric determination of MFA in pharmaceutical preparations using a Hitachi U-1100 UV-Visible spectrophotometer with a 1.00 cm glass cell based on the charge transfer complexation of MFA as an n-electron donor and chloranil as a π -acceptor results in a violet chromogen with a wavelength of 540 nm. The absorbance and concentration of the analysed drug in the range of 10-60 µg/ml were found to have a linear relationship with a strong correlation coefficient (0.9996) under ideal conditions [12]. Istabraq Qahtan Rashad et al studied the spectrophotometric determination of MFA using metol reagent by oxidative coupling reaction which involves an oxidative coupling reaction of MFA with metol in an alkaline medium with potassium periodate as an oxidizing agent to produce a water-soluble and stable substance with a maximum absorption at 533 nm. In a concentration range of 2.4-24 µg/ml MFA with a molar absorptivity of 7.2x103 L/mol cm, Beer's law holds true which proves the suggested approach to be efficient. This procedure has been used to determine MFA in a variety of pharmaceutical formulations with great success [13].
Harinder singh et al established development of UV spectrophotometric method for estimation of MFA in bulk and pharmaceutical dosage forms. The slope, intercept, correlation coefficient, identification and quantization limits were determined using Beer's law. The suggested approach has been successfully used to analyze the drug in both its pure and tablet delivery forms [14]. Hendriwasito et al studied MFA determination in tablet formulations using a selective and accurate spectrophotometric method based on prussian blue formation. This method showed that after 15 minutes of incubation, visible spectrophotometric measurements at 715 nm were obtained under optimal conditions of 15 mmol/L potassium ferricyanide and 2.5 mmol/L ferric chlorides in 0.5 mol/L acetic acids [ Nerdy Nerdy studied the UV spectrophotometry method for the determination of MFA in suspension dosage form and the solvent is sodium hydroxide (NaOH) 0.1 N solution. The determination of the Mefenemic acid suspension preparation uPondex®, Omestan® and Novastan® were that contain not less than 90.0% and not more than 110.0% of the amount stated on the label. The results meet the requirements of the validation test of analysis method with the parameter percent recovery 100.08% for accuracy, relative standard deviation 0.04% for precision, the correlation coefficient 1.0000 for linearity, range 8 µg/ml to 12 µg/ml, limit of detection limit 0.0118 µg/ml and limit of quantitation 0.0356 µg/ml [18]. Mallinath M. Langade developed spectrophotometric determination of MFA in bulk and tablet formulation. The reaction of the drug with ferric chloride in the presence of potassium ferricyanide is the basis of the process. Beer's law was followed in the concentration range of 10 to 40 µg/ml [19]. N. A. Alarfaj et al. described spectrophotometric determination of MFA in pharmaceutical preparations. The first method (I) was based on the reaction of MFA as N-donor with p-chloranilic acid as a π-acceptor. The absorbance concentration ranges from 10-300 g/mL for a redcolored substance with a peak at 520 nm. The oxidation of MFA with N-bromosuccinamide is used in the second method (II). The third method (III) is based on the creation of an oxidative binding compound in the presence of ferric chloride solution by reacting MFA with 3methylbenzo-thiazolin-2-one hydrazone as a chromogenic reagent [20].
Nief R. Ahmed et al studied indirect spectrophotometric method for the determination of MFA in pharmaceutical formulations. The method involves the oxidation of MFA by iron(III) followed by the complexation of iron(II) with ophenanthroline resulting in a red-colored complex (ferroin) with a maximum absorbance at 510 nm. Beer's law is obeyed in the concentration range of 0.4-2.0 µg/ml [21]. Raju Chandra et al developed validation for the estimation of MFA from marketed tablet and the λmax was found to be 370 nm, the limit of detection and limit of quantification was 0.3 and 0.9 ppm respectively. The inter-day and intra-day mean recoveries by 98.56 % and 97.13 % for UV-spectrophotometer [22].
S O Idowu et al established novel colorimetric assay of MFA. This study was based on a di azo coupling reaction with diazotized 4-amino-3,5dinitrobenzoic acid (ADBA) as the chromogenic derivatizing reagent. According to optimization tests, the coupling reaction was extremely fast, taking less than one minute to complete [23] ( Table 1).

Thin layer chromatography (TLC)
For the simultaneous determination of MFA (MFA) and its two toxic impurities, Martha M. Morcoss et al established and validated a process. The proposed TLC-densitometric system using a mobile phase consisted of of chloroform: acetone: acetic acid: ammonia (70:30:2:2, v/v/v/v) and TLC aluminum plates 60 F254 was used as a stationary phase and the separated bands were UV-scanned at 225 nm [24]. Harrizul Rivai et al. studied and validated thin layer chromatography-densitometry method for analysis of MFA in tablet. The accuracy and reliability of the method was assessed by evaluation of linearity (50-300 µg/ml), precision intra-day and inter-day relative standard deviation values were always less than 2, accuracy (102.45 % ± 1.36% for Sample A and 100.28% ± 1.90% for Sample B) in accordance with ICH guidelines [25]. The detailed information is depicted in Table 2 [35]. Studies are demonstrated in Table 3.   The process includes oxidizing MFA using cerium (IV) to create cerium (III), The fluorescence of cerium (III) after stimulation at 255nm was measured at 354nm. [36] Spectrofluorim etric determination of anthranilic acid derivatives based on terbium sensitized fluorescence The process is based on the conversion of radiative energy from anthranilates to terbium ions in alkaline methanolic solutions.

Fluorimetric Method
A simple spectro fluorimetric procedure for assessment of MFA in pharmaceutical preparation and urine was reported by Ahad Bavili Tabriz. The process includes oxidizing MFA using cerium (IV) to create cerium (III) and the fluorescence of cerium (III) after stimulation at 255 nm was measured at 354 nm [36]. A sensitive and simple spectro fluorimetric approach was developed using terbium sensitized fluorescence. The process is based on the conversion of radiative energy from anthranilates to terbium ions in alkaline methanolic solutions, with detection limits 1.4x 10 -8 reported by Pinelopi C. Ioannou at el. [37] Studies are summarized in Table 4.

Atomic Absorption Spectrometry (AAS)
Atomic absorption spectrometry was used to measure MFA in tablet dosage type. Sunil Jawla et al explained the formation of metal complexes of Diclofenac sodium and MFA with cupric chloride and cobaltous chloride in these processes. The first approach involves reacting all drugs with cupric chloride to produce light blue metal complexes which are then separated with dichloromethane and digested with 0.1 M nitric acid. Both drugs are estimated indirectly using AAS to determine copper content in shaped complexes. The second approach is focused on the creation in pink-colored cobaltous chloride complexes of both drugs. In cupric chloride method, MFA can be determined in the concentration range 2.5-23.0 µg/ml with mean percentage recovery 100.31 ± 0.79%. In cobaltous chloride method MFA can be measured in the concentration range 3.0-24.5 µg/ml with mean percentage recovery was 100.26 ± 0.76% [38].

Hyphenated Techniques
The hyphenated approaches are sophisticated, repeatable and adaptable method for estimating analytes in a variety of biological and medicinal samples [39]. Using Thermo Hypurity C18 (50 x 4.6 mm, 5 µ) column and a flow-rate of 0.75 ml/min. 2 mM ammonium acetate buffer and methanol (pH 4.5 adjusted with glacial acetic acid; 15:85, v/v) was used as a mobile phase. Atmospheric pressure ionization source was used to introduce the sample and Mahadeo Mahadik et al reported that the drug was detected at m/z 240 to 196.3 [40].

Mass Spectrometry
Hani Nasser Abdelhamid et al investigated MFA and its metallo drug using electrospray ionization mass spectrometry (ESI-MS). ESI-MS can be used for the molecular characterization of these adducts as MFA has a nominal mass of 241.2Da [41].

Turbidimetric Method
Nagam S Turkie Al-Awadie et al published the determination of MFA in pure and pharmaceutical preparations using turbidimetric measurement (0-180 0 ) by Ayah 6SX1-ST-2D solar cell CFI analyzer which is a new advanced analytic tool characterized by its speed and sensitivity. The approach used was aqueous medium in which Ce(IV) Sulfate react with MFA to generate a bluish green color precipitate for the ion-pair complex. Turbidity was determined by measuring the reflection of incoming light colliding with surface precipitated particles at a range of 0-180 degree [42].
MFA using a new mode of irradiation (Array of Six Identical LEDs) and detection (Twin Solar Cells) through turbidity measurement by CFIA was developed and validated by Nagam S. Turkie Al-Awadie et al. The approach relied on phosphomolybdic acid reacting with MFA in aqueous medium to produce a blue color precipitate as an ion-pair complex. The reflection of incoming light colliding with the surface precipitated particles at 0-180 0 was used to determine turbidity [43]. Studies are summarized in Table 5.

CONCLUSION
MFA is an NSAID, which is a common and effective medication that is used as potent analgesic and anti-inflammatory agent in the treatment of osteoarthritis, rheumatoid arthritis and other painful musculoskeletal illnesses. Various analytical techniques for determining MFA in pharmaceutical formulations and biological fluids are described in the drug's analytical profile. The most developed and validated approach for determining MFA was found to be HPLC, which was accompanied by spectrophotometric and fluorimetric methods, hyphenated technique, turbidimetry, mass spectroscopy and electroanalytical methods.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.