High Performance Thin Layer Chromatography Method for Simultaneous Estimation of Cefepime Hydrochloride and Sulbactam Sodium

 

Laxman M. Prajapati*, Anjali Patel, Jimish R. Patel, Amit K. Joshi, Mohammadali Kharodiya

Department of Pharmaceutical Chemistry, Shri B M Shah College of Pharmaceutical Education and Research, College Campus, Modasa-383315, Gujarat, India.

*Corresponding Author E-mail: laxchem@rediffmail.com

 

ABSTRACT:

A Simple High Performance Thin Layer Chromatography method for the simultaneous estimation of cefepime hydrochloride and sulbactam sodium was developed. The determination was carried on Silica Gel 60 GF254 HPTLC Plates using the mobile phase of chloroform:ethyl alcohol: Diethyl amine: water (12:7:1:0.4V/V). The absorbances of the spots were measured by densitometry at 254nm. The Retention Factor (Rf) was found to be 0.17 for cefepime hydrochloride and 0.76 for sulbactam sodium respectively. Cefepime hydrochloride and sulbactam sodium showed linear response at concentration range 4-20 g/band and 2-10 g/band repepectively. The Correlation co-efficient (r2) for cefepime hydrochloride and sulbactam sodium was found to be 0.9997 and 0.9997 respectively. The percentage recoveries obtained for cefepime hydrochloride and sulbactam sodium in range form 99.87-100.12 and 98.91-100.50.

 

KEYWORDS: Combined dosage, HPTLC, cefepime hydrochloride, sulbactam sodium, validation.

 

 


INTRODUCTION:

Cefepime hydrochloride is chemically (6R,7R)-7-[[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-methoxyiminoacetyl]amino]-3-[(1-methylpyrrolidin-1-ium-1-yl)methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid; chloride; hydrate; hydrochloride. It is a semi-synthetic, broad spectrum, cephalosporin antibiotic for parenteral administration Figure 11-2. The drug is official in Indian Pharmacopoeia, British Pharmacopoeia and United State Pharmacopoeia2-4. Literature survey revealed that several analytical methods have been developed for cefepime hydrochloride alone and in combination with several other drugs 5-8.

 

Sulbactam sodium is a β-lactamase inhibitor. This drug is given in combination with β-lactam antibiotics to inhibit β-lactamase, an enzyme produced by bacteria that destroys the antibiotics. Chemically sulbactam sodium is Sodium (2S,5R)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo (3.2.0)heptane-2-carboxylate 4,4-dioxide1,10. The drug is official in The United States Pharmacopoeia, and British Pharmacopoeia10-11. Several analytical methods including UV, HPTLC, and RP-HPLC have found to be reported for sulbactam sodium 12-17.Detailed literature survey of analytical methods revealed a HPLC and couple of UV-spectrophotometric methods have been reported for combination of cefepime hydrochloride and sulbactam sodium 18-20. The present work describes the HPTLC method for Simultaneous estimation of cefepime hydrochloride and sulbactam sodium. The method further was validated as per ICH guidelines 21.

 


 


Figure 1: Chemical structure of (A) Cefepime hydrochloride (B) Sulbactam sodium

 

MATERIALS AND METHODS:

Instrumentation

A Camag HPTLC system (Camag, Muttenz, Switzerland) with Lanomate V automatic sample applicator and Camag -Scanner III.

Flat bottom and twin trough developing chamber

(1010 cm)

100 L Hamilton syringe (Hamilton, Switzerland).

UV cabinate with dual wavelength UV lamp

(254 nm and 366 nm)

Camag win-CATS software

Sonicator FS4

Analytical balance (Shimadzu ATX224)

Precoated silica gel 60 F254 TLC plates

(1010 cm, layer thickness 0.2 mm, E. Merck, Germany).

 

Chemicals and Reagents:

Pure sample of cefepime hydrochloride and sulbactam sodium were kindly gifted from Montage Laboratories Pvt. Ltd. Himatnagar, Gujarat, India. Commercial injection formulation-Supime (Venus Remedies Limited) was purchased from local market. Chloroform, ethyl alcohol, diethyl amine and water used were of AR Grade (Finar Chemicals Pvt. Ltd., Ahmedabad, India).

 

Experimental:

Chromatographic Condition

The analysis was carried out by HPTLC using chloroform: ethyl alcohol : diethyl amine : water (12:7:1:0.4 v/v) as a mobile phase and silica gel 60 F254 HPTLC plates (1010cm) as a stationary phase. The samples were applied on HPTLC plates as 6 mm bands, by means of Linomat V automatic sample applicator fitted with 100 ml Hamilton syringe and the nitrogen flow. The plate was developed in Camag twin-trough glass chamber previously saturated for 20 min. The length of densitogram run was 7.5 cm. The plates were dried with air dryer and scanned at 254 nm by means of camag TLC scanner (Table2).

 

Preparation of stock solution:

Preparation of Standard stock solution of cefepime HCL (10,000 g/ml)

Standard stock solutions prepared by dissolving 100 mg of cefepime HCL in 10 ml water to get the concentration of 10000g/ml of cefepime HCL.

 

Preparation of standard stock solution of sulbactum sodium (10,000 g/ml)

Standard stock solutions prepared by dissolving 100 mg of sulbactam sodium in 10 ml water to get the concentration of 10000g/ml of sulbactam sodium.

 

Preparation of working standard:

2 ml from standard stock solution of cefepime HCL was taken in10 ml volumetric flask and diluted with distil water to get the concentration of 2000 g/ml of cefepime HCL. 1ml from standard stock solution of sulbactum sodium was taken in to 10 ml volumetric flask and diluted with distil water to get 1000g/ml of sulbactum sodium.

 

Method validation

Linearity

For linearity aliquots were taken and diluted to get different concentration range for the cefepime hydrochloride and sulbactam sodium. Solutions were scanned at 254 nm. Slope, intercept and correlation coefficient (R2) was calculated from the calibration curve.

 

Precision

The intra-day and inter-day precision studies were carried out by estimating the responses of three quality control (QC) standards in triplicates under same experimental conditions three times on the same day and on three different days. The results obtained, the precision was expressed as percentage relative standard deviations (% RSD) from mean intra and inter-day assays.

 

Accuracy:

Accuracy of the method was studied by recovery experiments. The recovery experiments were performed by adding known amounts of standard drug to formulation samples. The recovery was performed at three different concentrations levels (i.e. 80%, 100% and 120%). This procedure was repeated for three times for each concentration. The results of recovery studies were calculated for % RSD.

Specificity:

Specificity is the ability of the method to measure the analyte in the presence of other relevant components. The evaluation of specificity of the method was determined against placebo.

 

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The limit of detection (LOD) and the limit of quantitation (LOQ) of all selected combination of drugs were derived by calculating the signal to-noise ratio using the following equations as per the ICH guidelines.

LOD = 3.3 σ/S

LOQ = 10 σ/S

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

 

Sample solution stability:

Sample Solution stability was performed by analyzing the sample solution containing 4 g/spot and 2μg /spot on 0 hrs, 24 hrs. and 48 hrs. for cefepime hydrochloride and sulbactam sodium respectively.

 

Robustness:

In proposed method, determinations of cefepime hydrochloride and sulbactam sodium were carried out in sample solution (4μg/spot cefepine hydrochloride and 2μg/spot of sulbactam sodium) by using different conditions.

Analysis of cefepime HCL and sulbactum sodium in marketed injection formulation:

Accurately weighed powder 1.5 gm (1000 mg cefepime HCL and 500 mg sulbactum sodium) was transferred to volumetric flask and dissolved in small quantity of water. Sonicate the solution for 10 minute and diluted up to 100 ml using water. The resulting solution was filtered using whattman filter paper. Take 1ml from the sample stock solution into 10 ml volumetric flask and diluted using water up to the mark to get the concentration of 1000 g/ml of cefepime HCL and 500 g/ml of sulbactum sodium. 4 μL of this solution applied on TLC plate followed by development and scanning at 254 nm.

 

RESULT AND DISCUSSION:

Linearity:

Linear regression data for the calibration plots revealed good linear relationships between area and concentration over the ranges 4-20 μg/spot for cefepime hydrochloride and 2-10 μg/spot for sulbactam sodium. The linear equations for the calibration plots were y = 552.15x - 908.1and y = 152.73x +824.67 with Regression (r2) being 0.999 and 0.999 for cefepime hydrochloride and sulbactum sodium, respectively (Figure 2, 3 and 4) (Table 1 and 2).


Figure 2 : HPTLC Chromatogram of Standard and sample of cefepime hydrochloride and sulbactum sodium.

 


 

Figure 3: 3D chromatogram of Cefepime HCL (4-20μg/spot) and Sulbactum Sodium (2-10μg/spot)

 

Figure 4: Calibration curve of cefepime hydrochloride and sulbactam sodium in distilled water at 254 nm.

 

Table 1 Calibration curves Detail

Standard Id

Volume (μL) used for spotting

Concentration of Cefepime HCL(μg/spot)

Concentration of Sulbactum Sodium (μg/spot)

S1

2

4

2

S2

4

8

4

S3

6

12

6

S4

8

16

8

S5

10

20

10

 

Table 2: Result of Calibration readings for cefepime hydrochloride and Sulbactam sodium

Cefepime hydrochloride

Sulbactam sodium.

Concentration

(ng/spot)

Rf

Area Mean (n=3)

SD

% RSD

Concentration (ng/spot)

Rf

Area Mean (n=3) SD

%RSD

4

0.17

1278.4

0.1315

2

0.76

1126.5

0.3539

8

0.16

3464.5

0.0539

4

0.76

1443.9

0.1355

12

0.17

5807.2

0.0528

6

0.76

1730.6

0.1898

16

0.17

7569.1

0.0285

8

0.76

2056.8

0.1645

20

0.16

10069

0.0487

10

0.76

2347.3

0.0680

 

 

Table 3: Results of accuracy study for cefepime hydrochloride and sulbactum sodium

Drug

Amount taken

(μg/spot)

Amount added

(μg/spot)

Total Amount

(μg/spot)

Amount found

(μg/spot)

% Recovery

%RSD

Cefepime HCL

4

3.2

7.2

7.19

99.92

0.05817

4

4

8.00

8.00

100.12

0.03047

4

4.8

8.8

8.78

99.87

0.03957

Sulbactum Sodium

2

1.6

3.6

3.61

100.50

0.05457

2

2

4

3.98

99.72

0.06611

2

2.4

4.4

4.35

98.91

0.596

 

 

Table 4: Results of Precision study for cefepime hydrochloride and sulbactum sodium

 

Cefepime hydrochloride

Sulbactum sodium

Repeatability(%RSD, n=5)

0.09449

0.0390

Inter-day Precision(%RSD, n = 3)

0.03535

0.1313

Intra-day precision(%RSD, n = 3)

0.0794

0.2264

 

 


Accuracy:

When the found the accuracy and spiked with 80, 100, and 120% of additional standard drug, the recovery was found to be 99.87- 100.12% for cefepime hydrochloride and 98.91- 100.50% for sulbactam sodium (Table 3).

 

Precision:

The precision of the method was expressed as relative standard deviation (RSD %). The % RSD values for intra-day precision study and inter-day study (Table 4) were <2.0%, confirming that the method was sufficiently precise.

 

LOD AND LOQ:

LOD and LOQ were calculated by equation. The LOD and LOQ values were found to be 0.007214 and 0.02186 (g/spot) for cefepime hydrochloride and 0.009492 and 0.02876 g/spot for sulbactam sodium. (Table 5)

Table 5: Summary of Validation Parameter for Proposed method

Parameters

Cefepime hydrochloride

Sulbactam sodium

Linearity (μg/spot)

4-20

2-10

Slope

552.15

152.73

Intercept

908.1

824.67

Correlation coefficient

0.9997

0.9997

LOD (μg/ml)

0.007214

0.009492

LOQ (μg/ml)

0.02186

0.02876

Accuracy

80%

99.92

100.50

100%

100.12

99.72

120%

99.87

98.91

 

Robustness:

The results obtained in the new conditions were in accordance with the original results. The % RSD values for peak area was less than 1.0 indicating the highly robust nature of the developed method (Table 6).

 

 

 

 

 


 

Table 6: Robustness study for cefepime hydrochloride and sulbactum sodium

Name of drugs

Condition 1

Chloroform: Ethyl alcohol: Diethylamine :Water (12:7:1:0.4v/v)

Condition 2

Chloroform: Ethyl alcohol: Diethylamine :

Water (11:6.5:1:0.2v/v)

Mean of Peak area

%RSD

Mean of Peak area

%RSD

Cefepime HCL(4μg/spot)

1276.93

0.1315

1277.23

0.08659%

Sulbactum Sodium(2μg/ml)

1124.66

0.3539

1124.7

0.2185%

 

Table 7: Solution stability study for Cefepime hydrochloride and Sulbactam sodium

Time for stability

Std. solution of cefepime HCL(4μg/spot) measured peak area

%

RSD

Std. solution of Sulbactum Sodium (2μg/spot)

measured peak area

%

RSD

0 hrs.

1276.2

0.151%

1126.4

0.16%

24hrs.

1245

0.310%

1118.3

0.21%

48hrs.

1230.9

0.092%

1107.16

0.45%

 

 

Table 8: Assay results of marketed formulation (n =3)

Formulation

Label Claim (mg/tab)

Assay (% of label claim)

Cefepime HCL

Sulbactum Sodium

Cefepime HCL

Sulbactum Sodium

Supime injection

1000 mg

500 mg

99%

98.5%

 

 

 


Sample solution stability:

Sample solution stability was evaluated at room temperature for 0, 24 and 48 hrs. There was no significant deviation in peak area (RSD< 1.5%) observed on analysis up to 48 h (Table 7). There was no degradation of the drug observed during chromatogram development. These observations suggest that the drug is stable under the typical processing and storage conditions of the analytical procedure.

 

Analysis of cefepime hydrochloride and sulbactum sodium in marketed formulation:

When the marketed formulation was analyzed, cefepime hydrochloride and sulbactum sodium gave sharp and well defined peaks at Rf 0.17 and 0.72, respectively, scanned at 254 nm (Figure 5). There was no interference from the excipients commonly present in the injection formulation. The % purity was found to be 99% for cefepime hydrochloride and 98.5% for sulbactam sodium respectively (Table 8).

 

Figure 5: HPTLC Chromatogram of Sample cefepime HCl and sulbactam sodium mixture

 

 

CONCLUSION:

A new, simple, and sensitive HPTLC method has been successfully developed and validated for determination of cefepime hydrochloride and sulbactum sodium in injection dosage form. The method was found to be accurate, precise, and reproducible with good stability under various processing and storage conditions. Therefore this developed and validated method will help the industries as well as researchers for their simultaneous determination of cefepime hydrochloride and sulbactum sodium at low cost.

 

ACKNOWLEDGEMENTS:

The authors are thanks full to Montage Laboratories Pvt. Ltd. Himatnagar, Gujarat, India. For providing the gift sample of drug.

 

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Received on 19.07.2016 Accepted on 05.09.2016

Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2016; 6(4): 207-212.

DOI: 10.5958/2231-5675.2016.00031.4