Simultaneous Determination and Validation of Flupirtine Maleate and Paracetamol in Combined Dosage Form by Chromatographic Technique

Objective: The focus of this research was to establish a validated high-performance thin layer chromatographic (HPTLC) method for analysing Flupirtine maleate and Paracetamol in a combined dosage form. Method: Paracetamol and Flupirtine maleate were measured using a mobile phase of Ethyl acetate: Chloroform (7:5 v/v) at 286 nm. This technique was validated in accordance with the International Conference on Harmonization (ICH) guidelines. Results: The Rf value for paracetamol was 0.31 and 0.52 for Flupirtine maleate in this existing technique. Paracetamol's linearity was found to be in the range of 3250-6500 ng/band, while Flupirtine maleate's linearity was found to be in the range of 1000-2000 ng/band. The method's accuracy was determined by recovery experiments, which revealed a percent recovery of 98 to 102 percent. The % RSD was determined to be less than 2 in the Precision investigation, and the assay result for both compounds was within the limit. Original Research Article Dash et al.; JPRI, 33(36A): 91-98, 2021; Article no.JPRI.70410 92


INTRODUCTION
Flupiritine maleate is a non opoid, centrally acting analgesic used to treat acute and chronic pain.
Various analytical methodologies using spectrophotometry, HPLC, and stability studies have been mentioned for Flupirtine maleate alone and in combination with other compounds, according to a review of the literature [7-10]. For paracetamol with various combinations, analytical methods such as spectrophotometry, HPLC, UPLC, LC-MS, and stability studies have been published [11][12][13][14][15][16][17][18][19]. For the determination of Flupirtine maleate and Paracetamol in mixed dosage form, numerous spectrophotometric, chromatographic and stability indicating approaches have been identified in the literature [20][21][22][23][24][25]. A HPTLC method for estimating Flupirtine maleate and Paracetamol in mixed dosage form is provided in the current work. The proposed approach has been validated in accordance with ICH criteria [26].

Materials
Reference standard of Flupirtine maleate was obtained from Datt Chemicals, Gujarat and Paracetamol was obtained from Yarrow Chem Laboratories, Maharashtra. The marketed formulation of Flupirtine maleate and Paracetamol in combination (Lupirtin-P) was procured from local pharmacy. Methanol, Ethyl acetate, Chloroform and distilled water were used throughout the study are AR grade.

Chromatographic Conditions
The HPTLC system consist of TLC Aluminum Sheet precoated with Silica Gel 60 F 254 as stationary Phase, Win-CATS software, 100 µl Hamilton syringe with CAMAG Linomat 5 applicator and 286 nm was selected as the wavelength for measurement of Paracetamol and Flupirtine maleate. On the basis of optimum resolution and R f value, the mobile phase was selected as a mixture containing Ethyl acetate: Chloroform (7:5 v/v).

Preparation of Standard Stock Solution of Flupirtine Maleate and Paracetamol
Accurately weighed quantity of Flupirtine maleate and Paracetamol (50 mg) was taken in a 50 ml volumetric flask separately, dissolved and volume made up to mark with methanol (1000 µg/ml

Selection of Wavelength
Standard stock solutions of Paracetamol (3250 ng/band) and Flupirtine maleate (1000 ng/band) were prepared and applied on the precoated TLC plate and scanned using CAMAG HPTLC scanner III. It was observed that Paracetamol and Flupirtine maleate showed considerable absorbance at 286 nm. So, 286 nm was selected as the wavelength for measurement of Paracetamol and Flupirtine maleate throughout the method.

Preparation of Calibration Curve
Different concentrations of standard Flupirtine maleate solution ranging from 1000-2000 ng/band and Paracetamol solution ranging from 3250-6500 ng/band were prepared by taken 10, 12, 14, 16, 18 and 20 µl from mixed standard stock solution, applied to the plate for the calibration curve of these drugs. Peak area of the spots was measured at 286 nm in the absorbance mode with CAMAG TLC scanner.

Linearity and range
The linearity of response was determined in concentration range of 1000-2000 ng/band for Flupirtine maleate and 3250-6500 for ng/band Paracetamol. The calibration curve was plotted using peak areas vs. concentrations to get correlation-coefficient and regression line equations for Flupirtine maleate and Paracetamol. Linearity is expressed in terms of correlation co-efficient of linear regression line.

Repeatability
Mixed standard solution containing Flupirtine maleate (1600 ng/band) and Paracetamol (5200 ng/band) was applied six times, scanned, peak area was measured and % RSD was calculated.

Intra-day precision
Variation of results within same day is called Intraday precision. The Intra-day precision was determined for standard solution of Flupirtine maleate and Paracetamol for the three different concentrations three times on the same day. Peak areas was measured and % RSD was calculated.

Inter-day precision
The Inter day precision was determined for standard solution of Flupirtine maleate and Paracetamol for the three different concentrations were analyzed 3 times on the three different days. The % RSD was calculated.

Accuracy
1600 ng/band and 5200 ng/band drug solution of Flupirtine maleate and Paracetamol was taken in three different flask labelled as A, B and C respectively. Spiked 80%, 100%, 120% of standards solution in it and diluted up to 10 ml. The peak area of each solution was measured at 286 nm. The amount of Flupirtine maleate and Paracetamol was calculated at each level and % recoveries were computed.

Limit of detection and limit of quantification
In order to determine the detection and quantification limits, following equations designated by International Conference of Harmonization (ICH) guidelines.

Mobile Phase Optimisation
Ethyl acetate: Chloroform (7:5 v/v) was selected as mobile phase for the separation of Paracetamol and Flupirtine maleate after tried different solvent system. The Rf values are found to be 0.31 for Paracetamol and 0.52 for Flupirtine maleate.

Linearity and range
Flupirtine maleate linearity was observed in the concentration range of 1000-2000 ng/band, while Paracetamol linearity was found in the range of 3250-6500 ng/band (Table 1). Flupirtine maleate and Paracetamol obtained correlation coefficients of 0.996 and 0.994, respectively, for the calibration curve. Fig. 3 depicts the calibration curve for Flupirtine maleate and Paracetamol. The regression line equation was Y=0.864x+2623 for Flupirtine maleate and Y=1.049x+8071 for Paracetamol. Fig. 4 shows the linearity chromatograms of Flupirtine maleate and Paracetamol.

Precision
The repeatability, interday precision and intraday precision of the proposed approach were all evaluated. In Repeatability study same concentration analyzed six times. In intraday precision, the method was analyzed three times on the same day and in interday precision, the method was analyzed three times on the different days. The results shown that the percentage RSD is less than 2% at each level, clearly indicates that the proposed method is precise enough for the analysis of drug (Table 2).

Limit of detection (LOD) and limit of quantitation (LOQ)
In this proposed method the LOD and LOQ were determined to be 300.38 and 910.24 for Flupirtine maleate, 1042.90 and 3160.32 for Paracetamol respectively ( Table 2).

Accuracy
Accuracy of the analytical procedure was established by percentage recovery study from marketed formulation at three level of standard addition. Values of recovery in the range of 98 -102 % indicate that proposed method is accurate for the analysis of drug (Table 2).

Estimation of Drug Content in Formulation
The proposed method was used to estimate the drug content in commercially available formulation. The results are shown Table 3. The chromatograms were shown in Fig. 5.   Peak Area