RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF LORNOXICAM AND EPERISONE HYDROCHLORIDE

RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF LORNOXICAM AND EPERISONE HYDROCHLORIDE

Zenab Bohra ^*, Vivek Jain and Navjot Singh

Department of Pharmaceutical Chemistry, NRI Institute of Pharmacy, Bhopal - 462021, Madhya Pradesh, India.

ABSTRACT: Several spectrophotometric and HPLC methods for the determination of lornoxicam and eperisone hydrochloride have been documented individually or in combination with other drugs in pharmaceutical dosage forms. Therefore, in the present study, a reasonable and practical reverse phase HPLC method was developed and validated for lornoxicam and eperisone hydro-chloride in bulk formulation. In RP-HPLC method, the analyte was resolved by using an isocratic system, Methanol: Water (55:45) with 0.1% v/v Ammonium Hydroxide, pH 7.4 was used as mobile phase, at a flow rate of 1.0 ml/min, on HPLC system containing C18 analytical column (25 × 0.46 cm, 5 µm). The detection was carried out at 274 nm. The retention time was found to be 7.4 min and 9.2 min for lornoxicam and eperisone hydrochloride, respectively. Good resolution and retention time were observed. The results of the analysis in the method were validated with respect to following parameters linearity, precision, specificity and system suitability, limit of detection and limit of quantification.

RP-HPLC, Lornoxicam, Eperisone hydrochloride, Methanol, Ammonium hydroxide, Validation

INTRODUCTION: Lornoxicam ¹ (chlortenoxicam) is a non-steroidal anti-inflammatory drug (NSAID) of the oxicam class with analgesic, anti-inflammatory and antipyretic properties. Lornoxicam differs from other oxicam compounds in its potent inhibition of prostaglandin biosynthesis, a property that explains the particularly pronounced efficacy of the drug. Lornoxicam is absorbed rapidly and almost completely from the GI tract (90-100%). It is 99% bound to plasma proteins. Lornoxicam is metabolized completely by cyp 2C92, with the principal metabolite being 5'-hydroxy-lornoxicam, and only negligible amounts of intact lornoxicam are excreted unchanged in the urine. Approximately 2/3 of the drug is eliminated via the liver and 1/3 via the kidneys in the active form.

FIG. 1: CHEMICAL STRUCTURE OF LORNOXICAM

Eperisone ³ is an antispasmodic drug that relaxes both skeletal muscles and vascular smooth muscles and demonstrates a variety of effects such as reduction of myotonia, improvement of circulation, and suppression of the pain reflex ⁴.

FIG. 2: CHEMICAL STRUCTURE OF EPERISONE HYDROCHLORIDE

Literature review reveals that Different methods like UV spectrophotometry, HPLC, HPTLC for the estimation of Lornoxicam as single component and combinations with other drugs excluding Eperisone have been reported. Very few methods have been reported for estimation of Eperisone hydrochloride as single component like HPLC, GCMS. Very few methods have been reported so far in literature for simultaneous estimation of Lornoxicam and Eperisone hydrochloride as a combination in any dosage forms or bulk formulation ^5-23.

MATERIALS AND METHODS:

Instruments and Apparatus: Separation was achieved using Phenomenex C₁₈ Analytical Column (25 × 0.46 cm, 5 µm) on HPLC Shimadzu LC-20AD. Digital weighing balance (A & D company, GR (200)), pH meter (Lab India digital pH meter), Enertech ultrasonicator and pipettes and volumetric flasks (Borosilicate) used during the study.

Reagents and Chemicals: Lornoxicam and Eperisone were procured from Innova captab and Macleod’s, respectively. Methanol HPLC grade, HPLC grade water, LR grade ammonium hydroxide was obtained from Merck Ltd. India.

Chromatographic Conditions: Stationary phase: Phenomenex C₁₈ Analytical Column (25×0.46 cm, 5 µm).

Mobile Phase: Methanol: Water (55:45) with 0.1% v/v Ammonium Hydroxide.

Flow Rate: 1.0ml/min

Injection Volume: 20µl

Wavelength used: 260nm

Run Time: 15 min.

Preparation of Mobile Phase (Methanol and Water (55:45) and 0.1% Ammonium Hydroxide solution): Mobile phase prepared by mixing Methanol (HPLC grade) and water (HPLC grade) in selected proportion. Prepared Mobile phase taken separately filtered through membrane nylon filters of size 4.5 μ, to the filtered solution 1.5 ml of Ammonium Hydroxide Solution added and the mixed solution then sonicated for 15 min and filtered through membrane nylon filters of size 4.5 μ.

Preparation of Working Standard Mixture:

Standard Stock Solution of Lornoxicam (1000 µg/ml): Weigh accurately 10 mg of Lornoxicam into a 10ml volumetric flask. Add a sufficient amount of methanol, sonicate to dissolve, cool, and dilute up to the mark with methanol.

Standard Stock Solution of Eperisone (1000 µg/ml): Weigh accurately 10 mg of Eperisone into a 10ml volumetric flask. Add a sufficient amount of methanol, sonicate to dissolve, cool, and dilute up to the mark with methanol.

Standard Working Solution of Lornoxicam and Eperisone: The above stock solutions were further diluted 1 ml to 10 ml with methanol.

Method Validation: ^24-33

Specificity and System Suitability: Specificity can be defined as the ability to measure the concentration of an analyte in the presence of all other sample materials accurately. Blank and standard samples were injected to perform the specificity. Specificity of the procedure was established by proving that the excipients did not interfere. System suitability was determined by calculating parameters like theoretical plates, resolution, tailing factor, and % RSD.

Linearity: The linearity of the analytical technique is defined as the ability to obtain variable data test results, which is directly proportional to the analyte concentration in the sample. A series of standard solutions 25-100µg/ml was prepared for both drugs. Mix the equal volumes of lornoxicam and eperisone of each sample respectively, then injected into HPLC and recorded it. The plot of average peak area versus concentration is plotted, and from this the coefficient of correlation and the equation of regression are developed.

Precision: The closeness of agreement (degree of scatter) between a series of measurements obtained under the prescribed conditions from multiple sampling of the same homogenous sample. Method precision was calculated by repeatability test where %RSD should not be more than 2.0%.

Limit of Detection: The lowest analyte concentration the analytical technique can reliably discern from the background noise called the limit of detection. The limit of detection (LOD) was calculated using the following formulae:

LOD = 3.3(SD)/S

Where, SD = the standard deviation of response and S = Slope of the calibration curve.

Limit of Quantification: LOQ is defined as the lowest analyte concentration in a sample, which can be calculated with reasonable accuracy and precision. The Limit of Quantification was calculated using the following formulae:

LOQ = 10 (SD)/S

Where, SD = the standard deviation of response and S = Slope of the calibration curve.

RESULTS AND DISCUSSION:

Method Development and Optimization: Method development work was started with the use of UV-spectra in the range 200-400 nm of Lornoxicam and Eperisone (10µg/ml) standard solution. Then samples were scanned on a UV-Visible spectro-photometer between wavelength ranges of 200 nm to 400 nm. Two isosbestic points found in overlain spectra of Lornoxicam and Eperisone are 243 nm and 274 nm. From which, 274 nm was selected as detection wavelength.

FIG. 3: OVERLAIN UV SPECTRA OF LORNOXICAM & EPERISONE

The retention time for Lornoxicam was found to be 7.4 min and for Eperisone 9.2 min as shown in Fig. 4.

FIG. 4: HPLC CHROMATOGRAM OF MIXTURE OF WORKING STANDARDS

Method Validation:

TABLE 1: DATA FOR SPECIFICITY TEST OF LORNOXICAM

TABLE 2: DATA FOR SPECIFICITY TEST OF EPERISONE

TABLE 3: DATA FOR SYSTEM SUITABILITY

Linearity: The linearity for Lornoxicam and Eperisone was found between 25-100µg/ml. The result is shown in Table 4.

TABLE 4: LINEARITY OF STANDARDS FOR LORNOXICAM

FIG. 7: CALIBRATION CURVE FOR LORNOXICAM

TABLE 5: LINEARITY OF STANDARDS FOR EPERISONE

FIG. 8: CALIBRATION CURVE FOR EPERISONE

Precision: The % RSD for repeatability study for Lornoxicam and Eperisone was found to be 0.064 and 0.47, respectively. The results are shown in Table 5.

TABLE 6: DATA OF REPEATABILITY TEST FOR LORNOXICAM

TABLE 7: DATA OF REPEATABILITY TEST FOR EPERISONE

TABLE 8: SUMMARY OF REPEATABILITY

TABLE 9: LOD FOR LORNOXICAM AND EPERISONE

TABLE 10: LOQ FOR LORNOXICAM AND EPERISONE

CONCLUSION: The method developed is specific, linear, precise, and there was no interference in the chromatogram. Thus, it can be concluded that this newly developed approach can be used for the routine study of combined dosage forms due to easily accessible and cost-effective reagents.

ACKNOWLEDGEMENT: Special acknowledge to NRI Institute of pharmacy Bhopal and Bhabha Pharmacy Research Institute Bhopal, which provided us with the required equipment’s, chemicals needed to achieve this work.

CONFLICTS OF INTEREST: The authors declare there is no conflict of interest.

REFERENCES:

1. Sweetman SC: Martindale the complete Drug Reference, Pharmaceutical Press, London, Thirty 6^th Ed, 2007; 77-78.
2. Byrav PDS, Medhi B, Prakash A, Patyar S and Wadhwa S: Lornoxicam: A Newer NSAID. IJPMR 2009; 20(1): 27-31.
3. Sweetman SC: Martindale the complete Drug Reference, Pharmaceutical Press, London, Thirty Sixth Edition 2007; 1897.
4. Wikipedia, the Free Encyclopedia.
5. Kumbhar M, Bagde S, Karpe M and Kadam V: HPLC method development and validation for estimation of eperisone hydrochloride from bulk and marketed formulation. International Journal of Pharmacy Research & Technology 2019; 9: 6-14.
6. Taha EI, Al-Suwayeh SA and Mahrous GM: Simple, fast and reliable reversed phase hplc method for lornoxicam analysis in pharmaceutical formulations. World Journal of Pharmaceutical Research 2019; 8(2).
7. Muhammad Z: Development and validation of RP-HPLC method for determination of Lornoxicam in rabbit’s plasma. Pakistan Journal of Pharmaceutical Sciences 2019; 32: 333-38.
8. Vasavi A and Visagaperumal D: Development and validation of RP-HPLC method for simultaneous estimation of eperisone hydrochloride and diclofenac sodium in pharmaceutical dosage form. Journal of Advanced Pharmacy Education & Research 2019: 3(4): 550-55.
9. Kumar GV: A Stability indicating RP-HPLC method development and validation for simultaneous estimation of lornoxicam, paracetmol and serratiopeptidase in combined formulation. Indo Am J P Sci 2018; 05(12).
10. Divya A, Vishwanadham Y and Mounika: Development and validation of RP-HPLC method for simultaneous determination of diclofenac sodium and eperisone hydrochloride in pharmaceutical dosage form. Pharm Anal Acta 2017; 8: 552.
11. Sakhare RS, Pekamwar SS and Dannak KA: Stability indicating HPTLC method for simultaneous estimation of eperisone hydrochloride and diclofenac sodium in bulk and solid dosage form. Eura J Anal Chem 2017; 12(3): 245-56.
12. Gaur A, Yashwant and Singh R: Method development and its validation for simultaneous estimation of lornoxicam and paracetamol as API and in tablet dosage form by UV spectrophotometry using hydrotropic agents. International Journal of Pharmaceutical Quality Assurance 2016; 7: 66-74.
13. Akhtar J, Prajapati J, Ahmad S, Mujahid M and Elhassan G: Development and validation of derivative spectro-photometric method for simultaneous estimation of lornoxicam and eperisone in their synthetic mixture. Asian Journal of Research in Chemistry 2015; 8: 465.
14. Rajani B and Mukkanti K: RP-HPLC method development and validation for simultaneous estimation of thiocolchicoside and lornoxicam in tablet dosage form. Int J Chem Sci 2014; 12(2): 583-92.
15. Alagar RM, Godavari S, Banji D, Selva, Kumar D and Vanitha C: Anlytical method development & validation of Eperisone Hydrochloride and Diclofenac Sodium. Journal of Advanced Pharmacy Education & Research Apr-Jun 2013 Vol 3 Issue 2
16. Patel SK and Patel HR: “Development and validation of rp-hplc method for simultaneous estimation of eperisone hydrochloride and lornoxicam in synthetic mixture. Asian Journal of Research in Chemistry 2013; 6(4): 372-76.
17. Uchadadiya N, Mehta F and Sanchaniya P: Development and validation of derivative spectrophotometric method for simultaneous estimation of Eperisone hydrochloride and Paracetamol in combined tablet dosage form. Novus International Journal of Chemistry 2012; Vol. 1, No. 1.
18. Kumar P: RP-HPLC method and validation of lornoxicam in bulk and solid dosage form International Journal of Pharmaceutical and Chemical Sciences 2012; 1(2): 541-45.
19. Singh B: Estimation of lornoxicam in tablet dosage form by UV spectrophotometric method, International journal of Pharmaceutical Science and Research 2011; 2(1): 102-06.
20. Kuchekar BS: Development and validation of a RP-HPLC method for simultaneous determination of lornoxicam and thiocolchicoside in pharmaceutical dosage form and its application for dissolution study. International Journal of Research in Pharmaceutical Science 2011; 2(1): 1-7.
21. Vyas AJ: Simultaneous estimation of lornoxicam and paracetamol by vierodt’s method in Api and in synthetic mixture, international Journal of Chemtech Research 2011; 3(3): 1269-73.
22. Kumar DA, Tanuja S and Rao JVLNS: A validated RP-HPLC method for the determination of lornoxicam. Indian Journal of Pharmaceutical Research and Education 2011; 45(2): 153-56.
23. Patel DJ: Simultaneous determination of paracetamol and lornoxicam in tablets by thin layer chromatography combined with densitometry. International Journal of Chemtech Research 2010; 2(4): 1929-32.
24. Bhavsar SM, Patel DM, Khandhar AP and Patel CN: Validated RP-HPLC method for simultaneous estimation of Lornoxicam and Thiocolchicoside in solid dosage form. J Chem Pharm Res 2010; 2(2): 563-72.
25. Nash RA and Watcher AH: Pharmaceutical Process Validation, Marcel Dekker Inc.; New York 2003: 507-22.
26. Thomas S: Validating Analytical Methods Validating Based on Current ICH Guidance and USP/Industry Standards USP/Standards. Par Pharmaceuticals Spring Valley, New York 2001.
27. Épshtein NA: Validation of HPLC techniques for pharmaceutical analysis. Pharm Chem J 2004; 38: 4.
28. Text on Validation of Analytical Procedure, Q2A in ICH Harmonized Triplicate Guidelines, Oct 1994.
29. Asian Guidelines for Validation of Analytical Procedures. 1-12.
30. ICH Harmonised Tripartite Guideline Validation of analytical procedures: text and methodology Q2 (R1) (International Conference on Harmonization, Geneva, Switzerland Nov. 2005), available at https://www. academia.edu/34386868/ICH_HARMONISED_TRIPARTITE_GUIDELINE_VALIDATION_OF_ANALYTICAL_PROCEDURES_TEXT_AND_METHODOLOGY_Q2_R1,accessed Sept. 2017.
31. Snyder, Lloyd R and Joseph J: Kirkland, and Joseph L. Glajch. Practical HPLC method development. John Wiley & Sons, 2012; 4-10: 92-102.
32. Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), May 2001.
33. Center for Drug Evaluation, and Research (CDER). Reviewer Guidance; Validation of Chromatographic Methods November 1994.
    

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    How to cite this article:

    Bohra Z, Jain V and Singh N: RP-HPLC method development and validation of lornoxicam and eperisone hydrochloride. Int J Pharm Sci & Res 2021; 12(7): 3897-01. doi: 10.13040/IJPSR.0975-8232.12(7).3897-01.

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