The Estimation of Ceftaroline Fosamil in Lyophillized Powder for Injection by RP-HPLC.

 

P. Govardhan Reddy¹, V. Kiran Kumar¹, VVSS. Appala Raju², J. Raghu Ram ³, N. Appala Raju³*

¹Department of Pharmaceutics, Unity College of Pharmacy, Raigir (V), Bhongir (M), Yadagiri(D), Telanagan State.

²Deparment of Chemistry, Faculty of Pharmacy, MAHSA University, Koula Lumpur, Malaysia

³Department of Pharmaceutical Chemistry, Sultan Ul-Uloom College of Pharmacy, Mount Pleasant,

8-2-249, Road No.3, Banjara Hills, Hyderabad, Telangana – 50034, India

 

ABSTRACT:

A simple, precise, rapid and accurate reverse phase HPLC method developed for the estimation of Ceftaroline Fosamil in sterile powder for injection. An RP C-18 Hypersil BDS C18, 150x4.6 mm i.d, 5 μm partical size, with mobile phase consisting of mixture of 0.03 M ammonium acetate in water (solvent A) and methanol (solvent B) was set in the gradient mode over 30 minutes, was found to be most suitable for gradient mode was used to separate ceftaroline fosamil. The flow rate was kept at 1 ml/min and the effluents were monitored at 205 nm. The retention time was 13.12 min. The detector response was linear in the concentration of 20-240 mcg/ml. The respective linear regression equation being Y=25740x+70940. The limit of detection and limit of quantification was 0.25 and 0.75 mcg/ml respectively. The percentage assay of Ceftaroline Fosamil was 99.75%. The method was validated by determining its accuracy, precision and system suitability. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of Ceftaroline Fosamil in bulk drug and in its pharmaceutical dosage form.

 

 

 

INTRODUCTION:

Ceftaroline fosamil is a sterile, semi-synthetic, prodrug antibacterial drug of the cephalosporin class of beta-lactams (βlactams) ^1-3. Chemically, the prodrug, ceftaroline fosamil monoacetate monohydrate is (6R,7R)-7-{(2Z)-2(ethoxyimino)-2-[5-(phosphonoamino)-1,2,4-thiadiazol-3-yl]acetamido}-3-{[4-(1-methylpyridin-1-ium-4yl)-1,3-thiazol-2-yl] sulfanyl}-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylate mono acetate monohydrate^4-6.  Its molecular weight is 762.75. The empirical formula is C₂₂H₂₁N₈O₈PS₄.C₂H₄O₂.H₂O. The bactericidal action of ceftaroline is mediated through binding to essential penicillin-binding proteins (PBPs) ⁷. Ceftaroline is bactericidal against S. aureus due to its affinity for PBP2a and against Streptococcus pneumoniae due to its affinity for PBP2x ⁸. Teflaro is indicated for the treatment of acute bacterial skin and skin structure infections (ABSSSI) caused by susceptible isolates of the following Gram-positive and Gram-negative microorganisms: Staphylococcus aureus (including methicillin-susceptible and -resistant isolates), Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca. Literature survey reveals many Chromatographic methods for the determination of Ceftaroline Fosamil in biological fluids. So far, very few assay procedures has been reported for the estimation of Ceftaroline Fosamil from pharmaceutical dosage forms. The availability of an HPLC method with high sensitivity and selectivity will be very useful for the determination of Ceftaroline Fosamil in pharmaceutical formulations. The aim of the study was to develop a simple, precise, and accurate reversed-phase HPLC method for the estimation of Ceftaroline Fosamil in bulk drug samples and in pharmaceutical dosage form.

 

Figure-1: Chemical structure of Ceftaroline Fosamil.

 

 

EXPERIMENTAL:

Materials and Methods:

Ceftaroline as active pharmaceutical ingredient was obtained from Gyma laboratories as reference standards for present investigation. Teflaro® (ceftaroline fosamil-sterile powder for Injection) vials contain 400 mg of anhydrous ceftaroline fosamil was purchased from local market. All the reagents were of analytical-reagent grade unless stated otherwise. Glass-distilled and de-ionized water (Nanopure, Barnstead, USA), HPLC-grade acetonitrile, ammonium acetate (S.D.Fine chem, Mumbai, India) were used. Acetonitrile (HLC grade) and ammonium acetate were obtained from Loba Chem, Mumbai, and SD fine-Chem ltd, Mumbai, respectively. All other chemicals used in the analysis were AR grade.

  

Instrument:

Waters separation 2996 liquid Chromatograph with PDA detector module equipped with automatic injector with injection volume 20 µl, and 2693 pump with Empower-2 solutions software was used for the analysis. The chromatographic and the integrated data were recorded using HP-Vectra (Hewlett Packard, Waldbronn, Germany) computer system.  Chromatographic separation of ceftaroline fosamil was achieved on RP C-18 Hypersil BDS (150x4.6 mm; particle size 5 µm). The pH measurements were carried out with Elico; model LI 120, pH meter equipped with a combined glass-calomel electrode calibrated using standard buffer solutions of pH 4.0, 7.0 and 9.2.

 

HPLC Conditions:

The contents of the mobile phase were mixture of 0.03 M ammonium acetate in water (solvent A) and methanol (solvent B) was set in the proportion of 10:90 over 12 minutes, was found to be most suitable for gradient mode. They were filtered before use through a 0.45 μm membrane filter, and pumped from the respective solvent reservoirs to the column at a flow rate of 1.0 ml/min. The run time was set at 30.0 min and the column temperature was ambient. Prior to the injection of the drug solution, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The eluents were monitored at 265 nm. The gradient program is show in Table:

 

Table-1: Time programming of the gradient elution to determine Ceftaroline fosamil

Time(min)	Flow rate (ml/min)	% Solvent A	% Solvent B
0.01-1.0 1.0-5.0 5.0-12.0 12.0-18.0 18.0-20.0 20.0	1.0 1.0 1.0 1.0 1.0 1.0	95.0 95.0 10.0 10.0 95.0 95.0	5.0 5.0 90.0 90.0 5.0 5.0

 

Preparation of Standard Stock solution:

A standard stock solution of the drug was prepared by dissolving 100 mg of Ceftaroline fosamil in 100 ml volumetric flask containing 30 ml of diluent (50:50 v/v acetonitrile: water), sonicated for about 15 min and then made up to 100 ml with diluent to get a  1mg/ml standard stock solution.

 

Working Standard solution:

10 ml of the above stock solution was taken in 50 ml volumetric flask and made up to 50 ml with diluent to get a concentration 200 µg/ml.

 

Preparation of Sample solution:

Transfer 119.8 mg of sample of the powder for injection, which is equivalent to 100 mg of the active ingredient, was mixed with 50 ml of diluent. The mixture was allowed to stand for 15 min with intermittent sonication to ensure complete solubility of the drug, and then filtered through a 0.45 µm membrane filter, followed by adding diluent to obtain a stock solution of 1.0 mg/ml. An aliquot of this solution was transferred to a 10 ml volumetric flask and made up to sufficient volume with mobile phase to give the concentration of 200mcg/ml.

Linearity:  

Aliquots of standard Ceftaroline Fosamil stock solution were taken in different 10 ml volumetric flasks and diluted up to the mark with the diluent such that the final concentrations of Ceftaroline Fosamil are in the range of 20-240 mcg/ml. Each of these drug solutions (20 μL) was injected three times into the column, and the peak area and retention time were recorded. Evaluation was performed with PDA detector at 265 nm and a calibration graph was obtained by plotting peak area versus concentration of Ceftaroline Fosamil (Fig-2). The plot of peak area of each sample against respective concentration of Ceftaroline Fosamil was found to be linear in the range of 20–240 µg/ml with correlation coefficient of 0.9999. Linear regression least square fit data obtained from the measurements are given in table I.  The respective linear regression equation being Y=25740x+70940. The regression characteristics, such as slope, intercept, and %RSD were calculated for this method and given in Table I.

 

Table I: Linear Regression Data for Calibration curves.

Parameter	Results of proposed HPLC method
Detection wavelength (nm)	265
Linearity range (mcg/ml)	20-240
Regression Equation (y=mx + c)	Y=25740x+70940
Slope (m)	25740
Standard deviation on slope (Sm)	3.87x102
Intercept ©	70940
Standard deviation on intercept (Sc)	7.20x104
Correlation coefficient	0.9999

Assay:

20 µl of sample solution (Teflaro® sterile powder for Injection) was introduced into the injector of liquid chromatograph. The retention time was found to be 6.14 mins. The amount of drug present per vial of lyophilized powder for injection was calculated by comparing the peak area of the sample solution with that of the standard solution. The data are presented in Table II.

 

Recovery Studies:

Accuracy was determined by recovery studies of Ceftaroline Fosamil, known amount of standard was added to the pre-analyzed sample and subjected to the proposed HPLC analysis ⁹. Results of recovery study are shown in Table II. The study was done at three different concentration levels.

 

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

The detection limit of the method was investigated by injecting standard solutions Ceftaroline into the HPLC column. By using the signal-to-noise method the peak-to-peak noise around the analyte retention time is measured, and subsequently, the concentration of the analyte that would yield a signal equal to certain value of noise to signal ratio is estimated.

 

 

Table II: Results of HPLC assay and Recovery studies

S.No	Label  claim (mg)	Proposed RP-HPLC Method			% Found by the reference method	% Recovery by the proposed method**
		% found ± S.D*	t - test	F-test	± S.D	± S.D
1	400	99.43±0.8	2.14	1.38	100.21±0.68	103.4±0.23
2	400	99.96±0.27	1.64	1.77	100.23±0.36	99.81±0.38
3	400	99.86±0.43	1.94	1.69	100.3±0.33	102.75±0.61

Average ± standard deviation of six determinations, the t- and F-test values refer to comparison of the proposed method with the reference method.  Theoretical values at 95% confidence limit, t=2.57, F= 5.05. ** Average of three different concentration levels.

 

 

Fig 2:  Typical Chromatogram of Ceftaroline Fosamil by RP-HPLC.

RESULTS AND DISCUSSION:

The system suitability tests were carried out on freshly prepared standard stock solution of Ceftaroline Fosamil. Parameters that were studied to evaluate the suitability of the system are given in Table II.  From the typical chromatogram of Ceftaroline Fosamil as shown in fig 1, it was found that the retention time was 13.310 min. A mixture of mixture of 0.03 M ammonium acetate in water (solvent A) and methanol (solvent B) was set in the proportion of 10:90 over 30 minutes in gradient mode of separation was found to be most suitable to obtain a peak well defined and free from tailing. In the present developed HPLC method, the standard and sample preparation required less time and no tedious extraction were involved. A good linear relationship (r=0.9999) was observed between the concentration range of 20-240 mcg/ml. Low values of standard deviation are indicative of the high precision of the method. The assay of Ceftaroline Fosamil lyophilized powder for injection was found to be 99.75%. From the recovery studies it was found that about 101.98 % of Ceftaroline Fosamil was recovered which indicates high accuracy of the method. The absence of additional peaks in the chromatogram indicates non-interference of the common excipients used in the sterile powder for injection. The limit of detection (LOD) and limit of quantification (LOQ) for Ceftaroline Fosamil were found to be 0.2 and 0.6 µg/ml respectively. The signal to noise ratio is 3 for LOD and 10 for LOQ. This demonstrates that the developed HPLC method is simple, linear, accurate, sensitive and reproducible. Thus, the developed method can be easily used for the routine quality control of bulk and Tablets dosage form of Ceftaroline Fosamil within a short analysis time.

 

 

Fig 3: Calibration curve of Ceftaroline Fosamil by RP-HPLC.

 

ACKNOWLEDGEMENTS:

The authors are grateful to Gyma Laboratories for the supply of Ceftaroline Fosamil as a gift sample and to the Management, Sultan-Ul-Uloom College of Pharmacy, Hyderabad for providing the necessary facilities to carry out the research work.

 

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Accepted on 04.10.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(2): 455-458.