INTRODUCTION:

Cefepime (CEF) is chemically 1-{[(6R,7R)-7-[2-(2-amino-4-thiazolyl)glyoxylamido]-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl]-1-methylpyrro-lidiniumchloride,72-(Z)-(O-methyloxime), monohydrochloride, monohydrate salt^[1].It is a fourth generation cephalosporin and is used as a broad spectrum antibiotic with improved activity against gram-negative bacteria over other commercially available cephalosporin drugs.

Figure 1: Chemical structure of cefepime

Tazobactam (TZB) is chemically sodium (2S,3S,5R)-3-methyl-7-oxo-3-(1H-1,2,3triazol-1-ylmethyl)-4-Thia-1-azabicyclo[3.2.0] heptanes-2-carboxylate 4,4dioxide^[2]. It is a beta-lactamase antibiotic and is used in combination with other beta-lactamase antibiotic as anti-bacterial.

Figure 2: Chemical structure of Tazobactam

CEF and TZB are available in combined pharmaceutical dosage form for the treatment of lower respiratory tract infections, skin infections and urinary tract infections and usually in pediatric infections^[3]. Literature survey reveals several spectroscopic^[4], HPLC^[5]and HPTLC methods for the estimation of CEF individually as well as in combination with other drugs^[6].Also survey reveals UV spectroscopic^[7]and HPLC ^[8]methods for the estimation of TZB individually as well as in combination with other drugs ^{[9, 10]}. Not even a single HPLC method is reported so far for the simultaneous analysis of CEF and TZB in their combined dosage form. So a need was felt to develop new methods to analyze the drugs simultaneously. A successful attempt has been made to estimate the two drugs simultaneously by HPLC analysis. This paper describes a simple, rapid, accurate, reproducible and economical method for the simultaneous determination of CEF and TZB in injection formulation.

EXPERIMENTAL:

Materials

Analytical pure samples of CEF (Smilax Pharmaceuticals Ltd., Hyderabad, India) and TZB (Smilax Pharmaceuticals Ltd., Hyderabad, India) were used in the study. The pharmaceutical dosage form used in this study was MAGNOVA (Lyka Labs Limited, Ankaleshwar, marketed by Lupin Ltd., Mumbai, India) labeled to contain 1000mg of CEF and 125mg of TZB. Acetonitrile, water, methanol used are of HPLC grade.

Instrumentation

Waters 2695 Series Alliance HPLC system consisting of an inbuilt auto sampler, a column oven, a quaternary pump and a photo-diode array detector (PDA) was employed throughout the analysis. The data was acquired through the Empower-2 software. The column used was ODS symmetry C18 (150 × 4.6mm, 5µm). Meltronics sonicator was used for enhancing the dissolution of the compounds. Wensar electronic analytical balance was used for weighing the sample. Digisun pH meter was used for adjusting the pH of buffer solution.

Method Development and Optimization

Preparation of solutions

Preparation of diluent:

The mixture of Acetonitrile (HPLC) and water were prepared in the ratio of 50:50.

Preparation of Mobile phase:

By transferring 3.4g of potassium dihydrogen orthophosphate into 1000ml. volumetric flask and about 900ml of water was added, sonicated for 15min and madeup the final volume with water. Finally pH was adjusted to 2.4 with dilute orthophosphoric acid solution. This prepared buffer and mixture of methanol and acetonitrile in the ratio of 90:5:5 was used as mobile phase.

Optimized Chromatographic Conditions:

The chromatographic elution was carried out in the isocratic mode using a mobile phase consisting of phosphate buffer (pH 2.4, adjusted with orthophosphoric acid), methanol and acetonitrile in the ratio of 90:5:5. The analysis was performed at ambient temperature using a flow rate 1.1 mL/min with a run time of 10 min. The eluent was monitored using PDA at a wavelength of 260nm. The mobile phase was filtered through whatmann filter paper No.41 prior to use.

Preparation of Standard Stock Solution:

Accurately weighed and transferred 250mg of Cefepime and 31.25mg of Tazobactam working Standards into separate each 100 ml clean dry volumetric flasks, 70ml of diluent was added , sonicated for 5 minutes and made up to the final volume with diluent.

Preparation of Sample Stock Solution:

10ml of single dose injection contained 1000mg of Cefepime and 125mg Tazobactam were transferred into a 100 mL volumetric flask, 70mL of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered using Nylon membrane filter. From the filtered solution 0.5ml was pipetted out into a 10 ml volumetric flask and made up to 10ml with diluent. From this stock solution different aliquots were prepared.

Preparation of linearity solutions:

From the standard stock solution 1ml, 1.5ml, 2ml, 2.5ml and 3ml were pipetted out separately into five 10ml volumetric flasks, then made up to the mark with diluent. These prepared solutions were having concentrations of 250, 375, 500, 625 and 750 and 31.25, 46.87, 62.5, 78.125 and 93.75 for CEF and TZB in ppm respectively.

Method Validation:

The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines ^[11].

System Suitability:

Having optimized the efficiency of a chromatographic separation the quality of the chromatography was monitored by applying the following system suitability tests: capacity factor, tailing factor and theoretical plates. The system suitability method acceptance criteria set in each validation run were: capacity factor >2.0, tailing factor ≤2.0 and theoretical plates >2000. In all cases, the relative standard deviation (R.S.D) for the analytic peak area for two consecutive injections was < 2.0%. The system suitability test was performed using five replicate injections of standards before analysis of samples. System suitability parameters were shown in table 1. Standard chromatogram of cefepime and tazobactam was shown in figure 4.

Specificity:

Solutions of standard and Sample were prepared as per the test method and injected into the chromatographic system and the chromatograms were recorded and compared to know that there is no interference of excipients.

Linearity:

Standard curves were constructed daily, for three consecutive days (Twice a day), using six standard concentrations in a range of 250-750µg/ml (for Cefepime) and 31.25-93.75μg/ml (for Tazobactam). The linearity of peak area responses versus concentrations was demonstrated by linear least square regression analysis. The linear regression equations were y = 13720x + 134118(for Cefepime) and y = 20872x - 20186 (for Tazobactam) Linearity values were shown in table 2. Calibration curves of cefepime and tazobactam were shown in figures 5and 6.Analytical performance parameters were shown in table 3.

Precision:

System precision: (Repeatability)

To study precision, six replicate standard solutions of 500µg/ml of Cefepime and 62.5µg/ml of Tazobactam were prepared and analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responses was calculated and it was found to be which is well within the acceptance criteria of not more than 2.0%.system precision values were shown in table 4.

Method precision: (Reproducibility)

The intraday and inter-day precision of the proposed method was determined by analyzing the corresponding responses 3 times for concentration of sample solutions of 500µg/ml of Cefepime and 62.5µg/ml of Tazobactam. The result was reported in terms of relative standard deviation (% RSD). Method precision values were shown in table 5.

Limit of Detection and Limit of Quantification:

LOD and LOQ of drug were calculated using the following equations designated by International Conference on Harmonization (ICH) guidelines.

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

Where, σ is the standard deviation of response

S is slope of the calibration curve

Results of LOD and LOQ were shown in table 5.

Accuracy (Recovery study):

The accuracy of an analytical method is the closeness of test results obtained by method to the assay value. Accuracy must be established across the specified range of the analytical procedure. Accuracy was determined over the range of 50%, 100% and 150% of the sample concentration.

The accuracy was then calculated as the percentage of analyte recovered by the assay.

% Recovery = [(Ct – Cpa)/ Cs] × 100

Where Ct = Total concentration of analyte

Cpa = Concentration of pre-analysed sample

Cs = Concentration of standard added to pre-analysed sample.

Recovery studies were shown in table 7.

Robustness:

The robustness study was performed to evaluate the influence of small but deliberate variation in the chromatographic condition. The robustness was checked by changing parameters like flow rate of mobile phase and mobile phase combination.

 Change in the organic phase of mobile phase ± 0.5ml (methanol : Acetonitrile 4.5:5.5 and 5.5 : 4.5)

 Change in flow rate by ± 0.1 ml/minute (1.0 ml/min and 1.2 ml/minute).After each change, sample solution was injected and % RSD was checked. Robustness values were shown in tables 8-11.

Assay of pharmaceutical formulation:

The proposed validated method was successfully applied to determine in their dosage form. And the % Assay results were shown in table 12.

Table 1: System suitability parameters

PARAMETERS	CEFEPIME	TAZOBACTAM	ACCEPTANCE CRITERIA
Peak asymmetric factor	1.39	0.95	NMT 2.0
Number of theoretical plates	8268	4402	NLT 2000
Retention time (min)	2.236	4.498

Table 2: Linearity of Cefepime and Tazobactam

Concentration of Cefepime (μg/ml)	Cefepime Peak Area (mV.sec)	Concentration of Tazobactam (μg/ml)	Tazobactam Peak Area (mV.sec)
250	3645928	31.25	621769
375	5294743	46.87	932041
500	7077915	62.50	1289303
625	8672776	78.125	1598881
750	10313787	93.75	1929428

Table 3: Analytical performance parameters

PARAMETERS	CEFEPIME	TAZOBACTAM
Linearity range	250-750(μg/ml)	31.25-93.75(μg/ml)
Correlation coefficient (r²)	0.999	0.999
slope (m)	13371	21005
Intercept(C)	315529	38518
Regression Equation	y = 13371x + 315529	y = 21005x + 38518

Table 4: System Precision

S. No	CEFEPIME		TAZOBACTAM
S. No	Rt (min)	AREA (mV.sec)	Rt(min)	AREA (mV.sec)
1.	2.240	7076893	4.811	1289156
2.	2.252	7077357	4.985	1289427
3.	2.253	7077584	4.887	1289274
4.	2.253	7077693	4.892	1288947
5.	2.255	7077474	4.923	1289068
6.	2.261	7077347	4.873	1289192
Mean	2.2523	7077391	4.895167	1289177
S.D	0.0068	278.0062	0.05744	165.744
%RSD	0.3019	0.0039	1.173	0.0128

Table 5: Method Precision

S.NO	CEFEPIME Standard area = 7077915(mV.sec)		TAZOBACTAM Standard area = 1289303(mV.sec)
S.NO	PEAK AREA	% LABELLED CLAIM	PEAK AREA	% LABELLED CLAIM
1	7074323	99.95	1286243	99.76
2	7075692	99.97	1285493	99.70
3	7073259	99.93	1288932	99.97
4	7075932	99.97	1287393	99.85
5	7076324	99.98	1287643	99.87
6.	7076293	99.98	1285496	99.70
Mean	7075304	99.963	1286867	99.808
S.D	1241.599	0.0197	1363.816	0.1072
%RSD	0.0175	0.0197	0.105	0.1074

Table 6: LOD and LOQ

SAMPLE	LOD (µg/ml)	LOQ (µg/ml)
Cefepime	(3.3 × 8.4478) / 13371 = 0.0020	(10 × 8.4478) / 13371 = 0.0063
Tazobactam	(3.3 × 6.1644) / 21005 = 0.0009	(3.3 × 6.1644) / 21005 = 0.0029

Figure 3: Recovery studies for Cefepime and Thazobactam

Table 7: Recovery Studies

Name

Pre Analysed Sample Concentration(µg/ml)

Spiked amount (µg/ml)

Area

(mV.sec)

Amount Recovered

(µg/ml)

% Recovery

Average % Recovery ± SD

CEFEPIME

250

3790117

3790671

3790773

259.887

259.925

259.932

98.87

99.25

99.32

99.147 ± 0.242

500

7436686

7436920

509.905

509.931

509.947

99.05

99.31

99.47

99.277 ± 0.211

750

11082440

11082557

11082250

759.919

759.927

759.906

99.19

99.27

99.06

99.173 ± 0.106

TAZOBACTAM

31.25

720178

720311

720530

36.196

36.202

36.213

98.93

99.04

99.26

99.076 ± 0.168

62.50

1341913

1341893

1342092

67.443

67.442

67.452

98.87

98.84

99.04

98.917 ± 0.108

93.75

1964252

1964112

1963953

98.721

98.714

98.706

99.42

99.29

99.12

99.277 ± 0.150

Figure 4: Standard Chromatogram for Cefepime and Thazobactam

Figure 5: Calibration curve of Cefepime

Figure 6: Calibration curve of Tazobactam

Table 8: Effect of change in flow rate for Cefepime

S. No	1.0ml/min	1.2ml/min
S. No	AREA (mV.sec)	AREA (mV.sec)
1	6824256	5615370
2	6827645	5616470
3	6836892	5617342
Mean	6829597	5616394
S.D	6540.398	988.1943
%RSD	0.0957	0.017

Table 9: Effect of change in flow rate for Tazobactam

S. No	1.0ml/min	1.2ml/min
S. No	AREA (mV.sec)	AREA (mV.sec)
1	1155660	973552
2	1176076	1003180
3	1164723	996723
Mean	1165507	991151.7
S.D	10227.13	15579.94
%RSD	0.8774	1.571

Table 10: Effect of change in organic phase for Cefepime

S. No	Acetonitrile : Methanol (4.5 : 5.5)	Acetonitrile : Methanol (5.5 : 4.5)
S. No	AREA (mV.sec)	AREA (mV.sec)
1	6026111	6226413
2	6137839	6262323
3	6137496	6261763
Mean	6100482	6250166
S.D	64407.4	20572.9
%RSD	1.0557	0.329

Table 11: Effect of change in organic phase for Tazobactam

S. No	Acetonitrile: Methanol (4.5 : 5.5)	Acetonitrile : Methanol (5.5 : 4.5)
S. No	AREA (mV.sec)	AREA (mV.sec)
1	1058735	1084215
2	1081091	1074510
3	1065341	1079649
Mean	1068389	1079458
S.D	11485.44	4855.318
%RSD	1.075	0.4497

Table 12: Assay of Etravirine

Name

Dosage

Sample concentration

(mg)

Sample estimated

(µg/ml)

%Assay

(µg/ml)

CEF

1000

998.40

99.840

TAZ

125

124.75

99.81

Table 13: Summary of Validation Results

PARAMETERS	Cefepime hydrochloride	Tazobactam sodium
Linearity	250-750µg/ml	31.25-93.75 (μg/mL)
Correlation coefficient	0.999	0.999
System precision (% RSD)	0.0339	0.0128
Method precision (% RSD)	0.0175	0.105
% Recovery	99.20	99.09
LOD	0.0020	0.0009
LOQ	0.0063	0.0029

CONCLUSION:

In the present investigation simple, sensitive and economical new analytical method was developed for the Cefepime hydrochloride and Tazobactam sodium by RP-HPLC technique. The developed and validated RP-HPLC method was found to be more economical. The result of analysis of formulation and recovery studies obtained by HPLC method were statistically validated and high percentage of recovery studies suggest that the developed methods were free from interference of excipients used in formulation. The HPLC method was statistically validated in terms of accuracy, precision, linearity and reproducibility. Hence above methods can be employed in quality control laboratories to estimate the amount of Cefepime hydrochloride and Tazobactam sodium in bulk and in commercial formulations. summary of validation results were shown in table 13.

REFERENCES:

1. http://www.rxlist.com/maxipime-drugs.htm

2. http://www.rxlist.com/zosyn-drugs.htm

3. Sanders JrW, Tanney J, Kessler R. Efficacy of Cefepime in the treatment of infections due to multiply resistant Enterobacter species. Clin. Infect. Dis., 1996; 23:454-461.

4. Rodenas V, Parra A, Garcia-Villanova J, Gomez MD. Simultaneous determination of Cefepime and L-arginine in injections by second-derivative spectrophotometry. J. Pharm. Biomed. Anal., 1995; 13:1095-1099.

5. Elkhaili H, Linger L, Monteil H, Jehl F. High-performance liquid chromatographic assay for Cefepime in serum. J. Chromatogr. B Biomed. Sci. Appl., 1997; 690:181-188.

6. Patel SA, Patel NM, Patel MM. Spectrophotometric estimation of cefotaxime and cefetriaxone in pharmaceutical dosage forms. Indian J. Pharm. Sci., 2006; 68:101-103.

7. Haginaka J, Wakai J, Yasuda H, Uno T, Nakagawa T. Spectrophotometric determination of sulbactam by reaction with 1, 2, 4-triazole. Analystical, 1984; 109: 1057-1059.

8. Guillaume Y, Peyrin E, Guinchard C. Rapid determination of sulbactam and tazobactam in human serum by high-performance liquid chromatography. J. Chromatogr. B Biomed. Applied, 1995; 665:363-371.

9. Mahgoub H, Aly FA. UV determination of ampicillin sodium and tazobactam in two component mixture. J. Pharm. Biomed. Anal., 1998; 8:12731278.

10. Wang P, Yang J. A liquid chromatographic method for simultaneous determination of amoxicillin sodium and tazobactam in combination formulation. J. Pharm. Biomed. Anal., 2004; 36:565-569.

11. ICH, Q2 (R1), Harmonised tripartite guideline, Validation of analytical procedures: text and methodology International Conference Harmonization ICH, Geneva, Nov 2005.

Received on 09.11.2013 Accepted on 02.12.2013

Asian J. Pharm. Ana. 3(4): Oct. - Dec. 2013; Page 131-137