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Sofia Begum et al. J Sci Res Pharm, 2017;6(12):185-189

World Inventia Publishers

J

ournal of

S

cientific

R

esearch in

P

harmacy

http://www.jsrponline.com/

Vol. 6, Issue 12, 2017 ISSN: 2277-9469

USA CODEN: JSRPCJ

Research Article

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF

CEFUROXIME AND LINEZOLID BY RP-HPLC METHOD

Safia Begum 1_{*, T. Mahender}2

* 1 _{Asst.Professor St.Peter’s Institute of Pharmaceutical Sciences, Hanamkonda, Telangana, INDIA.}

2 _{Asst.Professor Jayamukhi Institute of Pharmaceutical Sciences, Narsampet, Warangal, Telangana, INDIA.}

Received on: 10-11-2017; Revised and Accepted on: 25-12-2017

ABSTRACT

A

new method was established for simultaneous estimation of Cefuroxime and Linezolid by RP-HPLC method. The chromatographic

conditions were successfully developed for the separation of Cefuroxime and Linezolid by using Agilent C18 5µm (4.6*250mm) column, flow rate was 1ml/min, mobile phase ratio was Phosphate buffer ph 4.0 : ACN (30:70%v/v), detection wave length was 254nm. The instrument used was WATERS HPLC Auto Sampler, Separation module 2695, PDA Detector 996, Empower-software version-2.The retention times were found to be 3.503 mins and 2.577 mins. The % purity of Cefuroxime and Linezolid was found to be 100.3% and 101.1% respectively. The system suitability parameters for Cefuroxime and Linezolid such as theoretical plates and tailing factor were found to be 1.3, 5824.4 and 1.2, 2936.0 the resolution was found to be 9.4. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study for Cefuroxime and Linezolid was found in concentration range of 20μg-100μg and 20μg-100μg and correlation coefficient (r2) was found to be 0.999 and 0.999, % mean recovery was found to be 102.5% and 101.0%, %RSD for repeatability was 0.6 and 0.5, % RSD for intermediate precision was 0.7 and 0.6 respectively. The precision study was precise, robust, and repeatable. LOD value was 3.1 and 3.02, and LOQ value was 10.1 and 10 respectively. Hence the suggested RP-HPLC method can be used for routine analysis of Cefuroxime and Linezolid in API and Pharmaceutical dosage form.

KEYWORDS: Agilent C18, Cefuroxime and Linezolid, RP-HPLC.

INTRODUCTION

B

road-spectrum cephalosporin antibiotic resistant to beta-lactamase. It has been proposed for infections with gram-negative and

gram-positive organisms, gonorrhea, and haemophilus Cefuroxime, like

the penicillins, is a beta-lactam antibiotic. By binding to specific penicillin-binding proteins (PBPs) located inside the bacterial cell wall, it inhibits the third and last stage of bacterial cell wall synthesis. Cell lysis is then mediated by bacterial cell wall autolytic enzymes such as autolysins; it is possible that cefuroxime interferes with an autolysin inhibitor [1-7]_.

Linezolid is a synthetic antibiotic, the first of the oxazolidinone class, used for the treatment of infections caused by multi-resistant bacteria including streptococcus and methicillin-resistant Staphylococcus aureus (MRSA). The drug works by inhibiting the initiation of bacterial protein synthesis. Linezolid is a synthetic antibacterial agent of the oxazolidinone class of antibiotics. It has in vitro activity against aerobic Gram positive bacteria, certain Gram negative bacteria and anaerobic microorganisms. It selectively inhibits bacterial protein synthesis through binding to sites on the bacterial ribosome and prevents the formation of a functional 70S-initiation complex. Specifically, linezolid binds to a site on the bacterial 23S

*Corresponding author:

Safia Begum Asst. Professor,

St. Peter’s Institute of Pharmaceutical Sciences, Hanamkonda, Telangana, INDIA.

* E-Mail: [email protected]

DOI:https://doi.org/10.5281/zenodo.1432830

ribosomal RNA of the 50S subunit and prevents the formation of a functional 70S initiation complex, which is an essential component of the bacterial translation process. The results of time-kill studies have shown linezolid to be bacteriostatic against enterococci and staphylococci. For streptococci, linezolid was found to be bactericidal for the majority of strains. Linezolid is also a reversible, nonselective inhibitor of monoamine oxidase. Therefore, linezolid has the potential for interaction with adrenergic and serotonergic agents [8-15]_.

Fig. 1: Chemical Structure of Cefuroxime

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MATERIALS AND METHODS

C

efuroxime and Linezolid API, Potassium dihydrogen,

Acetonitrile, Methanol, Water.

Instruments:

HPLC-auto sampler –UV detector Separation module2695, UV.detector2487 Empower-software version-2 Waters U.V double beam spectrometer Digital weighing balance(sensitivity 5mg) pH meter Sonicator.

Chromatographic conditions (optimised method):

Column : Agilent C18 5µm (4.6*250mm)

Mobile phase ratio : Phosphate buffer ph 4.0: ACN (70:30%v/v)

Detection wavelength: 254nm

Flow rate : 1ml/min

Injection volume : 10μl Column temperature : Ambient

Fig. 3: Chromatogram of Trail-Observation

The chromatogram is perfect with clear separation of components. The peak symmetry and system suitability parameters are within the limits. Hence this method is chosen as optimized one.

Method Development:

Method development f o r simultaneous e s t i m a t i o n o f Cefuroxime and Linezolid Pharmaceutical dosage forms includes the following steps:

1. Selection of detection wavelength (λmax) 2. Selection of column

3. Selection of mobile phase 4. Selection of flow rate 5. Preparations and procedure

Preparation of the individual Cefuroxime standard preparation: 10mg of Cefuroxime working standard was accurately weighed and transferred into a 10ml clean dry volumetric flask and about 2ml of DMF is added. Then it is sonicated to dissolve it completely and made volume up to the mark with the diluent. (Stock solution). Further 10.0 ml from the above stock solution is pipette into a 100 ml volumetric flask and was diluted up to the mark with diluent.

Preparation of the individual Linezolid s t a n d a r d preparation: 10mg of Linezolid working standard was accurately weighed and transferred into a 10ml clean dry volumetric flask and about 2ml of DMF is added. Then it is sonicated to dissolve it completely and made volume up to the mark with the diluent. (Stock solution). Further 10.0 ml from the above stock solution is pipette into a 100 ml volumetric flask and was diluted up to the mark with diluent.

Preparation of Sample Solution :( Tablet):

Accurately 10 tablets are weighed and crushed in mortar and pestle and weight equivalent to 10 mg of Linezolid and Cefuroxime (marketed formulation) sample into a 10mL clean dry volumetric flask and about 7mL of Diluents is added and sonicated to dissolve it completely and made volume up to the mark with

Method Validation:

Accuracy:

The accuracy study was performed for 50%, 100% and 150 % for Cefuroxime and Linezolid. Each level was injected in triplicate into chromatographic system. The area of each level was used for calculation of % recovery.

Precision:

The precision study was performed for five injections of Cefuroxime and Linezolid. Each standard injection was injected in to chromatographic system. The area of each Standard injection was used for calculation of % RSD.

Intermediate precision/Ruggedness:

The intermediate precision study was performed for five injections of Cefuroxime and Linezolid. Each standard injection was injected into chromatographic system. The area of each standard injection was used for calculation of % RSD.

Specificity:

The system suitability for specificity was carried out to determine whether there is any interference of any impurities in retention time of analytical peak. The study was performed by injecting blank.

Detection of limit:

LOD’s can be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve (S) at levels approximating the LOD according to the formula. The standard deviation of the response can be determined b a s e d on the standard deviation of y-intercepts of regression lines.

Quantification Limit:

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used for calculation of correlation coefficient.

Range:

The linearity study was performed for concentration range of 20μg - 100μg and 20μg-100μg of Cefuroxime and Linezolid and the correlation coefficient was found to be 0.999 and 0.999. (NLT 0.999).

Robustness:

As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method.

RESULTS AND DISCUSSION Accuracy:

Table No. 1: Accuracy results of Cefuroxime

% Concentration (at

specification Level) Area added (m) Amount found (m) Amount Recovey % Mean Recovery

50% 1426646 5 4.9 101.8%

102.5%

100% 2551005 10 9.98 99.9%

150% 2139845 15 15.0 100.0%

Table No. 2: Accuracy results of Linezolid % Concentration (at

specification Level) Area added (m) Amount found (m) Amount Recovey % Mean Recovery

50% 975578 5 5.0 101.3%

101.0%

100% 1718370 10 9.96 99.6%

150% 1465857 15 14.9 99.3%

Precision:

Table No. 3: Repeatability results of Cefuroxime and Linezolid

S. No. Peak name RT Area Peak name RT Area

1 Cefuroxime 2.506 1553631 Linezolid 3.230 2790868

Mean 1542200 2711560

Std.dev 20490.0 47796.3

%RSD 1.33 1.76

Intermediate precision/Ruggedness:

Table No. 4: Ruggedness results

S. No. Peak name RT Area Peak name RT Area

Mean 1786782 2586764

Std.dev 10795.03 19163.75

%RSD 0.6041 0.7408

Linearity:

Table No. 5: Linearity Results Linezolid

S. No. Linearity Level Concentration Area

1 I 20 ppm 471543

2 II 40 ppm 656277

3 III 60 ppm 794999

4 IV 80 ppm 946124

5 V 100 ppm 1002139

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Table No. 6: Linearity Results Cefuroxime

S. No. Linearity Level Concentration Area

1 I 20 ppm 471543

2 II 40 ppm 656277

3 III 60 ppm 794999

4 IV 80 ppm 946124

5 V 100 ppm 1002139

Correlation Coefficient 0.999

Acceptance Criteria: Correlation coefficient should be not less than 0.999

Plotting of calibration graphs:

The resultant areas of linearity peaks are plotted against Concentration

Fig. 4: Calibration curve of Linezolid Fig. 5: Calibration curve of Cefuroxime

Robustness:

Table No. 7: System suitability results For Linezolid(Flow rate)

S. No. Flow Rate(ml/min) System suitability results USP Plate count USP Tailing

1 0.8 3483 1.26

2 1.0 2936 1.3

3 1.2 2832 1.1

Table No. 8: System suitability results for Cefuroxime (Flow rate)

S. No. Flow Rate(ml/min) System suitability results USP Plate count USP Tailing

1 0.8 6645 1.3

2 1.0 5824.4 1.3

3 1.2 6059.0 1.2

Mobile Phase:

Table No. 9: System suitability results for Linezolid (Mobile phase)

S. No. Change in Organic Composition in the

Mobile Phase

System suitability results USP Plate count USP Tailing

1 10% Less 3254.5 1.1

2 Actual 3516 1.2

3 10% More 3215 1.2

Table 10 System suitability results for Cefuroxime (Mobile phase)

S. No. Change in Organic

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SUMMARY AND CONCLUSION

A

new method was established for simultaneous estimation

of Cefuroxime and Linezolid by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Cefuroxime and Linezolid by using Agilent C18 5µm (4.6*250mm) column, flow rate was 1ml/min, mobile phase ratio was Phosphate buffer ph 4.0 : ACN (30:70%v/v), detection wave length was 254nm. The instrument used was WATERS HPLC Auto Sampler, Separation module 2695, PDA Detector 996, Empower-software version-2.The retention times were found to be 3.503 mins and 2.577 mins. The % purity of Cefuroxime and Linezolid was found to be 100.3% and 101.1% respectively. The system suitability parameters for Cefuroxime and Linezolid such as theoretical plates and tailing factor were found to be 1.3, 5824.4 and 1.2, 2936.0 the resolution was found to be 9.4.The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study for Cefuroxime and Linezolid was found in concentration range of 20μg-100μg and 20μg-100μg and correlation coefficient (r2) was found to be 0.999 and 0.999, % mean recovery was found to be 102.5% and 101.0%, %RSD for repeatability was 0.6 and 0.5, % RSD for intermediate precision was 0.7 and 0.6 respectively. The precision study was precise, robust, and repeatable. LOD value was 3.1 and 3.02, and LOQ value was 10.1 and 10 respectively. Hence the suggested RP-HPLC method can be used for routine analysis of Cefuroxime and Linezolid in API and Pharmaceutical dosage form.

REFERENCES:

1. Shaikh Ka, Patil SD., et al. Simultaneous determination of

linezolid and chlordiazepoxide in pharmaceutical

preparations by spectrophotometric, RP-HPLC, and HPTLC

methods. JAOAC Int 2010;93(3):PP 904-10.

2. Surya Prakash Gupta. Development and validation of

spectrophotometric, HPTLC and HPLC methods for the determination of cefuroxime and Chlordiazepoxide in

pharmaceutical dosage forms. Der Pharmacia Sinica 2012;3(2):

185-192.

3. Fraga J, Abizanda A, Moreno F, Leon J. Simultaneous

determination of imipramine and amitrypline by derivative

spectrophotometry. J Pharm Biomed Anal 1991

;9(2):109-115.

4. Prajapati, Rajesh R. Parmar, Dushyant A. Shah. Development

and validation of uv spectrophotometric area under curve for simultaneous estimation of diazepam and cefuroxime in pharmaceutical dosage form inventi rapid: pharm analysis

and quality assurance. 2013, article id-

inventi:ppaqa/1046/13.

5. Nuthalapati mamatha, B. Thangabalan, S. Manoher Babu

Nuthalapati Mamatha. et al. Int J Res in Pharm Nano Sci

2014;3(3):200-214.

6. M. Senthil Raja, Shan S. Ha, P. Perumala. RP-HPLC method development and validation for the simultaneous estimation of linezolid. Int J PharmTech Res 2010;2(1)36-39.

7. Fathalla F. Belal, Mohie K. Sharaf, El-Din, Nahed M. El-Enany and

Samar Saad Belal et al. Chem Centr J 2013;7:162

8. Joana Sousa, Gilberto Alves. Validation of analytical methods, definitions and terminology, ICH Harmonized tripartite guideline, 1999.

9. Krishna Veni Nagappan. HPLC Principle and Practice, 1st _edition,

Academic press, 1997;24-37.

10. KA. Shaikha et al. HPLC for Pharmaceutical Scientists, 1st

edition, Wiley Interscience A JohnWiley & Sons, Inc., Publication,

2007;15-23.

11. Dr. Kealey and PJ Haines. Analytical Chemistry, 1st_{edition, Bios}

Publisher, 2002;1-7.

12. A. Braith Wait and FJ. Smith. Chromatographic Methods, 5th

edition, Kluwer Academic Publisher, 1996;1-2.

13. Andrea Weston and Phyllisr. Brown, HPLC Principle and

Practice, 1st _{edition, Academic press,}₁₉₉₇_;24-37.

14. Yuri Kazakevich and Rosario Lobrutto, HPLC for Pharmaceutical

Scientists, 1st_{edition, Wiley Interscience A John Wiley & Sons,}

Inc., Publication, 2007;15-23.

15. Chromatography, Online:

URL:http://en.wikipedia.org/wiki/Chromatography

How to cite this article:

Safia Begum, T. Mahender. ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF CEFUROXIME AND LINEZOLID BY RP-HPLC METHOD. J Sci Res Pharm 2017;6(12):185-189.

DOI: https://doi.org/10.5281/zenodo.1432830

Conflict of interest: The authors have declared that no conflict of interest exists.