HPTLC-Densitometric Analysis of Eperisone Hydrochloride and Paracetamol in Their Combined Tablet Dosage Form

A simple, precise, accurate, and reliable HPTLC method has been developed and validated for the analysis of EPE-Eperisone hydrochloride and PCM-Paracetamol in their combined dosage form. Identification and analysis were performed on 100mm × 100mm layer thickness 0.2mm, precoated silica gel G 60 -F 254 aluminum sheet, prewashed with methanol, and dried in an oven at 50C for 5 min. Toluene : methanol : ethyl acetate : glacial acetic acid (4 : 3.5 : 2.5 : 0.05) (v/v/v/v) was used as mobile phase. Calibration plots were established showing the dependence of response (peak area) on the amount chromatographed.The validated calibration ranges were 200–700 ng/spot and 1300–4550 ng/spot for EPE and PCMwith correlation coefficient (R) 0.994 and 0.996, respectively. Average % recovery was between 98.61–100.94% and 99.18–100.57% for EPE and PCM, respectively. The spots were scanned at 248 nm in a reflectance mode. The proposed method was validated as per ICH guidelines and successfully applied to the estimation of EPE and PCM in their combined tablet dosage form.


Introduction
EPE is chemically (2RS)-1-(4-ethylphenyl)-2-methyl-3-(1-piperidinyl)propan-1-one and hydrochloride (1 : 1) (Figure 1(a)) [1,2]. Eperisone HCl is a centrally acting muscle relaxant; it acts at the level of spinal cord by blocking sodium channels and calcium channels. Eperisone HCl exerts its spinal reflex inhibitory action predominantly via a presynaptic inhibition of the transmitter release from the primary afferent endings via a combined action on voltage-gated sodium and calcium channels. Eperisone HCl increases the blood supply to skeletal muscles; this action is noteworthy since a muscle contracture may compress the small blood vessels and induce an ischemia leading to release of pain stimulating compounds [3]. EPE is official in Japanese pharmacopoeia [1]. Chemically PCM is N-(4-hydroxyphenyl)acetamide (Figure 1(b)). Paracetamol is a weak inhibitor of PG-Prostaglandin synthesis of COX-1 and COX-2. Cyclooxygenase serves as a pain activator, is responsible for the biosynthesis of prostaglandins, is used for the relief of headaches, fever, pains, and is a major ingredient in numerous cold and flu remedies [4]. Paracetamol is official in Japanese Pharmacopoeia [1], British Pharmacopoeia [5], United States Pharmacopoeia [6], and Indian Pharmacopoeia [7].
To the best of our knowledge, there is no published HPTLC method for this combination. So, the present paper describes a simple, accurate, and precise method for simultaneous estimation of EPE and PCM in combined tablet dosage form by HPTLC method. The developed method was validated in accordance with ICH guidelines [20] and successfully employed for the assay of EPE and PCM in their combined dosage form.  and PCM 325 mg were weighed and transferred to a 100 mL volumetric flask and dissolved in methanol. The flask was sonicated, and volume was made up to the mark with methanol to give a solution containing 500 g/mL EPE and 3250 g/mL PCM. One mL of this aliquot was added to 10 mL volumetric flask, and volume was made up to the mark with

Analysis of Marketed Formulation.
Twenty tablets were weighed accurately; average weight was found and finely powered. A quantity equivalent to 50 mg EPE and 325 mg PCM was accurately weighed and transferred to volumetric flask of 100 mL capacity. 80 mL of methanol was transferred to this volumetric flask and sonicated for 20 min to dissolve the drug. Resulting solution was filtered through Whatman filter paper (0.45 ) into a 100 mL volumetric flask. The flask was shaken, and volume was made up to the mark with methanol to give a solution containing 500 g/mL of EPE and 3250 g/mL of PCM. One mL of this aliquot was added to 10 mL volumetric flask, and volume was made up to the mark with methanol to give a solution containing 50 g/mL of EPE and 325 g/mL of PCM. Now this prepared sample solution was applied on TLC plate, developed, dried in air, and photometrically analyzed as described above. From the peak area obtained in the chromatogram, the amounts of both of the drugs were calculated.      to prequantitated sample solutions of tablet dosage forms. The amounts of EPE and PCM were estimated by applying values of peak area to the regression equations of the calibration graph.

Precision. Precisions of the proposed HPTLC methods
were determined by analyzing mixed standard solution of EPE and PCM at 3 different concentrations (300, 400, and 500 ng/spot for EPE and 1950, 2600, and 3250 ng/spot for PCM) 3 times in the same day and in 3 different days. The results are reported in terms of coefficient of variance (CV).

Repeatability.
Repeatability of method was assessed by applying the same sample solution 6 times on a plate with the automatic spotter using the same syringe and by taking 6 scans of the sample spot for both EPE and PCM (400 ng/spot of EPE and 2600 ng/spot of PCM) without changing the positions of the plate.

Specificity.
The specificity of the method was ascertained by analyzing standard drug and sample. The band of EPE and PCM in sample was confirmed by comparing the and spectra of the band with that of standard. The peak purity of both drugs was assessed by comparing the spectra at 3 different levels, that is, peak start (S), peak apex (M), and peak end (E) position of the band.

Limit of Detection and Limit of Quantification.
The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived by using the following equations as per International Conference on Harmonization (ICH) guidelines which is based on the calibration curve: where is the standard deviation of -intercepts of regression lines and is slope of calibration curve.

Method Optimization.
Several mobile phases were tried to accomplish good separation of EPE and PCM. Using the mobile phase Toluene : methanol : ethyl acetate : glacial acetic acid (4 : 3.5 : 2.5 : 0.05) (v/v/v/v) and 10 × 10 cm HPTLC silica gel 60 F254 aluminum-backed plates, good separation was attained with retention factor ( ) values of 0.26 for EPE and 0.79 for PCM. A wavelength of 248 nm was used for the quantification of the drugs. Figure 2 shows the detection of both of the drugs in their combined dosage form at 248 nm by HPTLC method. Resolution of the peaks with clear baseline separation was found. Figure 3 shows the densitogram of mixture which has a clear baseline. Figure 4 showed a good linearity when overlapped and scanned between 200 nm to 400 nm. Figure 5 shows a 3D overlapped spectrum of both drugs which has good linearity. The system suitability test parameters for the developed method are shown in Table 1. Figure 6 shows that linear correlation was obtained between peak areas and concentrations of EPE and PCM in the range of 200-700 ng/spot for EPE with 2 = 0.994 and 1300-4550 ng/spot for PCM with 2 = 0.996, respectively, and data are shown in Table 2.

Accuracy.
The recovery experiments were performed by the standard addition method. The HPTLC method was found to be accurate with % recovery of 98.61-100.94% for EPE and 99.18-100.57% for PCM, respectively, ( Table 3). The high values indicate that the method is accurate.

Precision.
The CV values were found to be <2%, which indicates that the proposed method is precise.

Conclusion
Thus, the objective of project work was development and comparison of analytical method of EPE and PCM in their combined dosage form. The developed and validated HPTLC method for EPE and PCM was found to be simple, specific, and cost effective and can be routinely applied for analysis of EPE and PCM in their combined dosage form. We can say that HPTLC method is more sensitive giving precise results (interday, intraday) for both drugs, and also HPTLC method is more sensitive in terms of LOD and LOQ. It also requires least solvents for analysis. The proposed method has the advantages of simplicity and convenience for the separation and quantitation of EPE and PCM in combination and can be used for the assay of their dosage form. Also, the low solvent consumption and short analytical run time lead to environmentally friendly chromatographic procedures. The additives usually present in the pharmaceutical formulations of the assayed analytes did not interfere with determination of EPE and PCM. The method can be used for the routine simultaneous analysis of EPE and PCM in pharmaceutical preparations.
pleasure. The authors are also thankful to Principal and Staff of Indukaka Ipcowala College of Pharmacy (New Vallabh Vidyanagar, India) and Director of SICART (New Vallabh Vidyanagar, India) for providing the necessary facilities for research work.