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Mefenamic acid has the capacity to convert Cu²⁺ ions into Cu¹⁺ ions, so the development of an analytical approach based on Neocuproine reagent by flow injection method for mefenamic acid measurement in aqueous solution and tablets is extremely promising [8]. Furthermore, the suggested technique was applied to the quantitative evaluation of mefenamic acid in the actual samples (aqueous and pharmaceutical). The suggested method’s quick reaction time, straightforward chemical and equipment requirements. The drugs industry has a significant challenge today in the form of the development of quicker, cheaper, and more precise methods. novel analytical techniques to ensure drug content and detect substances in medications that pose health hazards. and eliminate (or swap out) any materials dangerous to the environment and public health. Flow systems are great instruments for handling solutions in wet chemical analysis for these reasons. since one of the main requirements for analytical chemistry is automation.

In this proposed flow injection method, we used uncommon reagents and were not tuned to achieve the lowest possible waste production, Limit of Detection (LOD), Limit of Quantification (LOQ), standard deviation (SD), maximum sensitivity and wide ranges of concentration 1.00-80.00 µg/mL.

Fundamental benefits of the suggested flow injection method for measuring mefenamic acid in pharmaceutical and aqueous solutions include speed, ease of use, precision in findings, and low cost of equipment determination samples in very low concentrations 1.00-80.00 µg/mL. Accordingly, they are easier to use as well as quicker than the majority of the documented ways. Since common excipients included in commercial preparations do not interfere with the measurement of mefenamic acid, the proposed procedures can be effectively used for routine pharmaceutical quality control and research purposes.

Introduction

Mefenamic acid consider as a class of a nonsteroidal anti-inflammatory medicine (NSAID), and owing to the powerful analgesic and anti-inflammatory qualities it has, it has garnered a large amount of interest in recent years. As a consequence of this, it is used extensively in the therapy of a wide variety of unpleasant diseases, including menstrual cramps, rheumatoid arthritis, and osteoarthritis, amongst others⁽¹⁾. It contains numbing, anti-pyretic, and anti-inflammatory characteristics, at higher concentrations, this molecule reduces inflammation⁽²⁾. The chemical formula of mefenamic acid (MEF) is 2-,[(2,3,-Dimethylphenyl)amino] benzoic acid (C15H15NO2). It is a weak organic acid that also includes the related (3-hydroxyanthranilic acids), a tryptophan (a standard amino acid) natural metabolite⁽³⁾.

Neocuproine is a chelating substance and heterocyclic chemical molecule C₁₄H₁₂N₂. The derivatives substituted at the location 2 and 9 positions are among the most researched of the substituted phenanthrolines, having been initially published in the late 19th century⁽²²⁾. Similarly, In chemistry of copper(I), Neocuproine as a (NN ligands) with somewhat large substituents crucial. Because of its selectivity for copper(I) and potent visual absorbance of the Cu(DMP)²⁺ adduct, 2,9-dimethyl-1,10-phenanthroline (NC) is typically the reagent chosen for the colorimetric measurement of copper(I)⁽¹⁴⁾

Water-insoluble mefenamic acid is somewhat soluble in (chloroform) and (ethers), faintly soluble in ethanol (4.6 mg/mL) and water (1:9), and more soluble in dimethylformamidee (38.5 mg/mL)⁽⁴⁾ Mefenamic acid reduces inflammation and pain by inhibiting COX and prostaglandin synthesis since it provides cerebral and peripheral analgesia. Its nonselective COX inhibitors inhibit both COX-1 and COX-2 enzymes⁽⁵⁾

Mefenamic Acid (MFA) operates in the same way as other nonsteroidal anti-inflammatory medicines by which blocking the enzymatic prostaglandin synthase⁽⁶⁾ There are several known and reported analytical methods for measuring MEF acid in biological and pharmacological products⁽⁷⁾⁾ Spectrophotometry⁽⁸⁾ Gas Chromatography⁽⁹⁾ Extraction method of Micro-electrodriven Membranes in 2 Phases⁽¹⁰⁾ capillary electrophoresis with two channels⁽¹¹⁾ RP-HPLC⁽¹²⁾ Fluorometric method⁽¹³⁾ Hyphenated Techniques⁽¹⁴⁾ Electrospray Ionization Mass Spectrometry⁽¹⁵⁾ Turbidity Measurement⁽¹⁶⁾ flow injection method⁽¹⁷⁾

MEF is determined by different flow injection technique, it has been estimated in pharmaceuticals by flow-injection⁽¹⁸⁾⁾ and estimation of Mefenamic Acid in Aqueous Solutions Using Reverse - Continuous Flow Injection Analysis⁽⁶⁾ determination of MEF in pharmaceuticals formulation by flow-injection Fluorometry^(21)). However, in recent years there has been a growing interest in the use of Neocuproine (2,9-dimethyl-1,10-phenanthroline) as a complexing agent in analytical chemistry. Neocuproine exhibits strong and selective chelation with various metal ions, including copper (II)⁽¹⁸⁾ This property has been effectively utilized in the development of novel analytical methods, particularly in flow injection analysis (FIA)⁽¹⁹⁾⁾ Similarly, in terms of mefenamic acid analysis, the creation of a sensitive flow injection technique that uses neocuproine as a complexing agent is a breakthrough⁽²⁰⁾

Materials and methods

All materials were of the analytical reagent grade; and the solutions were prepared with deionized water.

Instrumental Condition

Flow Injection System

We used in this flow injection system double beam Shimadzuu UVa-1700 Japan for absorption measurements result, x-y Recorder type Siemens C 1032, Germany, and we used Ismatic, Germany peristaltic pump. To provide the solution fluid, the flow injection analysis system (FIA) employed in this project consists of peristaltic pump and injection valve with 6-way port that are connected to one another by a single low-load connection, as illustrated in Figure 1.

Figure 1. The model of flow injection system that we designed in our laboratory. 

Results and discussion

The current work employs as a novel method for determination of MEF. However, when the Cu(II) reacted with Neocuproine reagent, the colorless complex will form Cu(IІ)-Neocuproine, this formed complex will react with mefenamic acid (reduced form) to produce yellow-orange products Cu (I)-Neocuproine⁽²²⁾ As showed in Figure 2.

Figure 2. Schematic diagram for proposed method. 

Determination of maximum wavelength (λmax)

Absorbance of coloured products was measured in the 190-1100 nm range against a reagent blank. However, we conclude that Mefenamic acid absorption has a maximum wavelength of 454 nm. Under the testing circumstances, each reagent blank had a negligible absorbance at the relevant λ_max.

The influence of pH changes

To investigate the effect of pH (1.0-9.0) on peak Hight (cm) and coloured complexes, various amounts 0-2 mL of 0.004 M standardized hydrochloric acid solution were added to an aliquot of solution containing 50 µg/mL of MFA as shown in Figure 3.

Figure 3. The Effect of pH changes, λmax= 454nm, NC.Conc. = 50 µg/mL, Cu(ІІ)Conc.=350 µg/mL ,Flow rate =2.3 mL/min , MEF Conc.=50 µg/mL. 

As shown above the optimum pH with higher absorption intensity at peack Hight 5.6 cm at pH 4.0 due to good intensity.

Cu(II) Concentration effect on absorbance

Effects of copper (II) concentration (50-500) µg/mL on complex’s absorbance was studied. It was observed that 350 µg/mL of Cu(II) gave the highest absorption with peack Hight 6 cm, which is strongly suggested for experimental procedures as we showed in Figure 4.

Figure 4. The Effect of Cu(II) concentration , λmax= 454nm,NC.Conc. = 50 µg/mL , pH=4.0, Flow rate =2.3 mL/min, MEF Conc.=50 µg/mL. 

As shown above the optimum Cu(II) Conc µg/mL with higher absorption intensity at peack Hight 6.0 cm due to good intensity.

Standard Calibration Curve for Mefenamic Acid in Aqueous Solution

This is done by preparation a series of solutions containing the concentrations in the range from (1.0 to 80.0) µg/mL and by using the optimum conditions which is used in this project. The results we are getting shown in Figure 5.

Figure 5. standard Calibration curve of Mefenamic acid, λmax= 454nm, NC.Conc. = 50 µg/ ml pH=4.0 , Cu(ІІ)Conc. 350 µg/ ml, Flow rate =2.3 ml /min, MEF Conc.=50 µg/ ml. 

The statistical properties for our proposed method and Optimum conditions illustrated in Table 1.

Table 1. The Optimum conditions and statistical features for the proposed method. 

parameter	value
λ max	454 nm
flow rate	2.3 mL/min
pH of carrier solution	4.00
Neocuproine Conc.	50 µg/ ml
Cu(II) Conc.	350 µg/ ml
Linearity range	1.00-80.00 µg/ ml
Regression equation	y = 0.0711x + 0.0275
Regression coefficient (R2)	R² = 0.9998
Standard Deviation (SD)	0.0078
Standard Error	0.0074
Standard Error for Intercept	0.0103
LOD	0.360 µg/ ml
LOQ	1.08µg/ ml

LOD and LOQ

The sensitivity of the technique utilized in this method to identify mefenamic acid by the presentation limit of detection (L.O.D) and limit of quantification (L.O.Q) was highlighted. The fact that this FIA method could estimate the lowest concentration of mefenamic acid to be (1 µg/mL).

The limits of detection (LOD) and limit of quantitation (LOQ) were assessed as equation 3.3 and 3.4 ⁽²²⁾

………….3.3

………….3.4

Where:

b: Slope

So: Standard deviation of the regression line.

Discussion

This novel injection technique, characterized by its simplicity, accuracy, inexpensive and speed and time reducing was developed to determine mefenamic acid in aqueous solutions and pharmaceutical dosage form by using flow injection technique, the proposed method involved a direct redox reaction between Cu(II) and NC reagent, followed by an instantaneous reaction of Cu(II)-NC with mefenamic acid. However, this method has the potential to detect drugs at extremely at low concentrations (1µg/ml) in aqueous solutions and pharmaceutical dosage form. On the other hand, based on the findings, statistical analysis revealed a standard deviation was equal to (SD) 0.0078 and This FI unit’s data repeatability was accurate at low RSD levels. Additionally, the (LOD) was 0.360 µg/ mL, while the limit of quantification (LOQ) was 1.08 µg/ mL. The standard error was calculated to be 0.0074 µg/ml. Furthermore, the relationship between drug concentration in aqueous solutions and peak height (cm) was represented by the straight-line equation y = 0.0711x + 0.0275. The correlation equation for the standard specification yielded a value of 0.9998, indicating a strong linear relationship. Lastly, the linearity of this method was confirmed within the 1.00-80.00 µg/mL. The results of the calibration curve demonstrated a strong correlation coefficient, indicating a high degree of linearity. This suggests a robust relationship between the peak height (measured in centimeters) and the concentration (measured in micrograms per milliliter.

Conclusions

Based on the redox reaction, an effective, selective and precise flow injection technique for the measurement of mefenamic acid in aqueous solutions and pharmaceutical formulations was developed. The suggested approach, which has an excellent analytical performance, sensitive enough to enable estimation of lower amounts of (mefenamic acid), highly degree of repeatability, so can be used as a substitute method for the regular analysis of mefenamic acid in aqueous solutions and pharmaceutical raw and forms of medication. In conclusion, these flow injection techniques offer the benefits of increased selectivity, quick, straightforward performance, and low cost.