Development and Validation of RP-HPLC Method for Simultaneous Estimation of Eperisone Hydrochloride and Lornoxicam in Synthetic Mixture

 

Sejal K. Patel, Harshil R. Patel*

Department of Quality Assurance, S.K. Patel College of Pharmaceutical Education & Research,

Ganpat University, Ganpat Vidyanagar – 384012, Mehsana, Gujarat, India.

*Corresponding Author E-mail: harshil285@gmail.com

 

A simple, accurate, and rapid reversed phase high performance liquid chromatographic method has been developed and validated for the simultaneous estimation of Eperisone Hydrochloride (EPE) and Lornoxicam (LOR) in synthetic mixture. The separation was carried out using mobile phase consisting of methanol: ACN: water (60: 30: 10, v/v/v) (pH-3, adjusted with OPA). The column used was Phenomenex C18, (250 mm x 4.6 mm i.d., 5 µm) with flow rate 1 ml/min using PDA detection at 255 nm. The method was linear over a concentration range 10 – 100 μg/ml for EPE and 2 – 20 μg/ml for LOR. The retention time of Eperisone hydrochloride and Lornoxicam were found to be 2.2 min and 3.15 min respectively. Results of analysis were validated statistically and by recovery studies. The mean recovery was 99.07 ± 0.42 and 100.79 ± 1.05 for EPE and LOR respectively. The limit of detection (LOD) and the limit of quantification (LOQ) for EPE and LOR were found to be 0.3504 and 1.0618 µg/ml and 0.1903 and 0.5769 µg/ml, respectively. The results of the study showed that the proposed RP-HPLC method was found to be simple, sensitive, precise and accurate and also useful for the routine determination of EPE and LOR in mixture.

 

 

INTRODUCTION:

Eperisone hydrochloride (EPE) is a well known antispasmodic drug¹. Chemically it is 4-ethyl-2-methyl-3- piperidinopropiophenone hydrochloride (Figure 1). It is official in Japanese Pharmacopoeia (JP)². JP describes potentiometric titration method for its estimation. Literature survey reveals liquid chromatography-ESI- tandem mass spectrometry for determination of eperisone in human plasma³. Literature survey also reveals Liquid Chromatography – Electrospray Ionization - Mass Spectrometry⁴, GC-MS⁵ method for the determination of Eperisone in human plasma. Lornoxicam (LOR) (3E)-6-chloro-3-[hydroxy (pyridin-2- ylamino)  methylene]-2-methyl-2,  3-dihydro-4H-thieno  [2,3-e]  [1,  2]  thiazin-4-one  1,  1-dioxide  is  a  novel  non- steroidal  anti-inflammatory  drug  (NSAID)  with  marked analgesic  activity⁶ (Figure 2). Various analytical methods, such as RPHPLC⁷, Extractionless HPLC⁸, UV spectroscopy⁹, HPTLC¹⁰, LC-MS-MS¹¹  determination of Lornoxicam in dosage forms and human plasma.

 

Lornoxicam in combination with other drug like Diacerin¹² and Thiocolchicoside¹³ have been also detected. The combination of these two drugs is not official in any pharmacopoeia; hence no official method is available for the simultaneous estimation of EPE and LOR in their combined synthetic mixture. The present communication describes simple, sensitive, rapid, accurate, precise and cost effective RP-HPLC method for simultaneous estimation of both drugs in synthetic mixture.

 

Figure 1: Chemical structure of Eperisone hydrochloride (EPE)

 

Figure 2: Chemical structure of Lornoxicam (LOR)

MATERIALS AND METHODS:

Apparatus

RP-HPLC instrument (Shimadzu, LC-2010CHT, Japan) equipped with a UV-Visible detector and a photodiode array detector, auto sampler, phenomenex C18 column (250 x 4.6 mm, 5 µ particle size) was used. Chromatograms were automatically obtained by LC-Solution system software. A Sartorius CP224S analytical balance (Gottingen, Germany), an ultrasonic bath (Frontline FS 4, Mumbai, India), Whatman filter paper no. 41 (Millipore, USA) were used in the study.

 

Reagent and materials

EPE bulk powder was kindly gifted by Sun Pharmaceuticals Ltd., Vadodara, Gujarat, India and LOR bulk powder was kindly gifted by Acme Pharmaceuticals Ltd., Mehsana, Gujarat, India. Methanol (AR Grade), ACN (AR Grade) was procured from Finar Chemicals Ltd., Ahmedabad, India.

 

Preparation of standard stock solutions of EPE and LOR (100 μg/ml)

An accurately weighed standard EPE and LOR powder (10 mg) were weighed and transferred to 100 ml separate volumetric flasks and dissolved in mobile phase with sonicator. The flasks were shaken and volumes were made up to mark with mobile phase to give a solution containing 100 μg/ml of each EPE and LOR.

 

Methodology

To optimize the RP-HPLC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for EPE and LOR was obtained with a mobile phase methanol: ACN: water (60: 30: 10, v/v/v) (pH-3, adjusted with OPA) at a flow rate 1 ml/min to get better reproducibility and repeatability. Quantification was carried out at 255 nm based on peak area. Complete resolution of the peaks with clear baseline was obtained (Figure 3). System suitability test parameters for EPE and LOR for the proposed method are reported in Table 1. Overlain UV spectrum showed that both drugs showed good absorbance at 255 nm, hence the wavelength of 255 nm was selected for quantification of EPE and LOR (Figure 4). 

 

Figure 3: Chromatogram of standard solution of EPE and LOR at 255 nm

 

Figure 4: U.V. Spectrum of EPE and LOR

Table 1: System suitability parameters of chromatogram

System suitability parameter	EPE ± RSD (n = 5)	LOR ± RSD (n = 5)
Retention time(min)	2.2 ± 0.79	3.15 ± 0.19
Theoretical plate (N)	2780 ± 0.47	3033 ± 1.12
Tailing factor (AS)	1.2 ± 0.16	1.6 ± 0.34
Resolution (RS)	3.16 ± 0.2

 

Validation of proposed method

The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines ¹⁴.

 

Calibration Curve (linearity)

Calibration curves were constructed by plotting peak area vs. concentration of EPE (Figure 5) and LOR (Figure 6), and regression equation were calculated. The calibration curves were plotted over the concentration range of 10 - 100 μg/ml for EPE and 2 – 20 μg/ml for LOR. From standard stock solution of EPE (1, 2, 3, 5, 7and 10 ml) and of LOR (0.2, 0.5, 0.8, 1, 1.5 and 2 ml) were transferred to a series of 10 ml volumetric flask and diluted to the mark with mobile phase. Aliquots (20 µl) of each solution were injected under the operating chromatographic condition as described above.

 

Method precision (Repeatability)

The precision of the instrument was checked by repeated injection (n = 6) of standard solutions of EPE (30 µg/ml) and LOR (10 µg/ml) under the same chromatographic condition and measurement of peak area, retention time and tailing factor. The low %RSD values(less than 2%) indicates that proposed method is repeatable.

 

Intermediate precision (Reproducibility)

The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of standard solutions of EPE (20, 30, 50 µg/ml) and LOR (5, 10, 15 µg/ml). The result was reported in terms of relative standard deviation (% RSD).

 

 

Figure 5: Calibration curve of EPE at 255 nm

 

Figure 6: Calibration curve of LOR at 255 nm

 

Limit of detection and Limit of quantification

LOD and LOQ of drugs can be calculated using the following equations designated by International Conference on Harmonization (ICH) guidelines¹⁴.

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

Where, σ = the standard deviation of the response and S = slope of the calibration curve.

 

Accuracy (Recovery study)

The accuracy of the method was determined by calculating recovery of EPE and LOR by the standard addition method. Known amounts of standard solutions of EPE and LOR (80, 100, 120 %) were added to pre quantified sample solutions of EPE and LOR. The amounts of EPE and LOR were estimated by applying obtained values to the respective regression equation of the calibration curve. The obtained value of recovery indicates that the proposed method is accurate. Results of recovery studies are shown in Table 2.

 

Estimation of EPE and LOR from synthetic mixture

EPE (50 mg) and LOR (4 mg) standard drug powder were accurately weighed and then mixed with commonly used formulation excipients like starch, lactose, magnesium stearate and talc in appropriate proportion. The mixture was then transferred to 100 ml volumetric flask containing 80 ml mobile phase and sonicated for 30 min. The solution was filtered through Whatman filter paper No. 41 and the volume was adjusted up to the mark with mobile phase. The above solution (1.5 ml) was transferred to 10 ml volumetric flask and diluted up to mark with mobile phase to obtain 75 µg/ml EPE and 6 µg/ml LOR. Aliquot (20 µl) of sample solution was injected under the operating chromatographic condition as described above and peak area was determined for both drugs (Figure 7). From these area values, the concentrations of EPE and LOR were determined using respective calibration graph. The analysis procedure was repeated six times with synthetic mixture (Table- 3).

 

Table 2: Determination of Recovery

Drug	Level	Amount of sample taken (µg/ml)	Amount of standard   spiked (%)	Mean % Recovery ± RSD
EPE	I	10	80 %	98.79 ± 0.73
	II	10	100 %	98.99 ± 0.31
	III	10	120 %	99.42 ± 0.22
LOR	I	4	80 %	99.79 ± 1.46
	II	4	100 %	101.57 ± 1.02
	III	4	120 %	101.01 ± 0.65

 

Figure 7: Chromatogram of sample solution of EPE (75 µg/ml) and LOR (6 µg/ml) at 255 nm

 

Table 3: Analysis of synthetic mixture of EPE and LOR by proposed method (n = 6)

Sample No.	Label Claim		Amount Found		% Label Claim
	EPE (mg)	LOR (mg)	EPE  (mg)	LOR (mg)	EPE  (%)	LOR (%)
1	50	4	49.19	4.01	98.39	100.18
2	50	4	49.79	3.97	99.58	99.39
3	50	4	49.58	4.03	99.17	100.89
4	50	4	50.14	4.06	100.29	101.56
5	50	4	49.49	3.99	98.99	99.88
6	50	4	50.78	4.07	101.56	101.81
Mean					99.67	100.62
S.D.					1.12	0.96

 

RESULTS AND DISCUSSION:

A RP-HPLC method was developed and validated for the determination of EPE and LOR in synthetic mixture on a Phenomenex C18, (250 mm x 4.6 mm i.d., 5 µm) with variable wavelength detection at 255 nm. The retention time of EPE and LOR was 2.2 min and 3.15 min, respectively. Linear correlation was obtained between area and concentration of EPE and LOR in the concentration range of 10 – 100 µg/ml and 2 – 20 µg/ml respectively. The low RSD value of interday (0.158 - 0.4677 % for EPE and 0.6118 – 1.1762 % for LOR) and intraday (0.1074 – 0.4929 % for EPE and 0.5526 - 1.0211 % for LOR) at 255 nm, reveal that proposed method is precise. The limit of detection (LOD) and limit of quantification (LOQ) for EPE and LOR were found to be 0.3506 and 1.0618 µg/ml and 0.1903 and 0.5769 µg/ml, respectively.

Table 4: Regression analysis data and summary of validation parameters for RP-HPLC method

Parameters		RP-HPLC method
		EPE	LOR
Detection wavelength (nm)		255	255
Beer’s law limit (µg/ml)		10 – 100	2 – 20
Regression equation                 y = mx + c		y = 66969x – 15461	y = 35056x + 7922
Slope		66969	35056
Intercept		15461	7922
Correlation coefficient		0.991	0.998
Repeatability (% RSD, n = 6)		0.6837	0.5946
Precision (%RSD)	Intraday (% RSD)	0.10 – 0.49	0.55 - 1.02
	Interday (% RSD)	0.15 – 0.46	0.61 – 1.17
LOD (µg/ml)		0.3504	0.1903
LOQ (µg/ml)		1.0618	0.5769
% Recovery  ±  SD  ( n = 6)		99.07 ± 0.42	100.79 ± 1.05
% Assay ± SD (n = 6)		99.67 ± 1.12	100.62 ± 0.96

 

These data show that method is sensitive for the determination of EPE and LOR. The recovery experiment was performed by the standard addition method. The mean recoveries were 99.07 ± 0.42 and 100.79 ± 1.05 for EPE and LOR, respectively (Table 4). The results of recovery studies indicate that the proposed method is highly accurate. The proposed validated method was successfully applied to determine EPE and LOR in synthetic mixture. No interference of the excipients with the retention time of drugs appeared; hence the proposed method is applicable for the routine simultaneous estimation of EPE and LOR.

 

CONCLUSION:

In this proposed RP-HPLC method, the linearity is observed in the concentration range of 10 - 100 µg/ml for EPE and 2 – 20 µg/ml for LOR with co-efficient of correlation, (r²) = 0.991 and (r²) = 0.998 for EPE and LOR, respectively at 255 nm. The results of the analysis of synthetic mixture by the proposed method are highly reproducible and reliable. The method can be used for the routine analysis of the EPE and LOR in mixture without any interference of excipients.

 

ACKNOWLEDGMENTS:

The authors are grateful to Sun Pharmaceuticals Ltd. Vadodara, Gujarat, India and Acme Pharmaceuticals Ltd. Ahmedabad, Gujarat, India for providing gift samples of Eperisone Hydrochloride and Lornoxicam and also to Department of Quality Assurance, S.K. Patel College of Pharmaceutical Education and Research, Ganpat University, Mehsana, Gujarat, India for providing the facilities to carry the research work.

 

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Received on 21.03.2013       Modified on 28.03.2013

Accepted on 31.03.2013      © AJRC All right reserved