INTRODUCTION:

Faropenem is chemically 6-(1-hydroxyethyl)-2-[(2R)-tetrahydrofuran-2-yl]-2,3-didehydropenam -3-carboxylic acid. Faropenem is a novel β-lactam antimicrobial agent sharing structural similarities with both the penicillins and cephalosporins. It exhibits a broad spectrum of activity that includes Gram-negative, Gram-positive and some anaerobic bacteria. The primary mode of action of Faropenem is consistent with that of other β-lactam antibiotics, namely binding to penicillin-binding proteins^1-3. Literature survey reveals that the drug was determined by HPLC, and only few visible spectrophotometric methods are reported^4-6. Therefore the need for fast, low cost and selective method is obvious especially for routine quality control analysis of pharmaceutical formulation. Hence the proposed colorimetric estimation of Faropenem using FC reagent has been developed. The structure of Faropenem is shown in fig 1

Figure 1 Structure of Faropenem

EXPERIMENTAL:

APPARATUS

All spectral characteristics and absorbance measurements were made on ELICO (SL 244) double beam spectrophotometer with 10 mm matched quartz cell

CHEMICALS

 All chemicals used were of analytical grade and double distilled water was used throughout.

 F-C reagent 2N supplied by S.D. Fine chem. India, Ltd., was reagent used by diluting FC with double distilled water in the ratio of 1:2.

 Sodium carbonate solution (20%) was prepared by dissolving 20 g of sodium carbonate in 100 mL double distilled water.

 1mg/mL stock reference solution was freshly prepared from pure sample of Faropenem by dissolving 100 mg of Faropenem in 100 mL of double distilled water.

PROCEDURE FOR CALIBRATION CURVE:

Aliquots of working standard solution (0.5 to 3.0 mL) were transferred to a series of 10 ml volumetric flasks so as to provide final concentration range of 50 to 300 mcg/mL. To each flask, 1 mL of FC reagent and 2 mL of sodium carbonate were successively added and kept aside for 15 minutes for colour development. Later the solutions were made up to volume with distilled water. The absorbance of each solution was measured at 745 nm against the reagent blank. The calibration graph was then prepared by plotting the absorbance versus the concentration of the drug. The concentration of the unknown was read from the calibration graph shown in figure 2 or computed from the regression equation.

Figure 2 Calibration curve of faropenem with F.C reagent

PROCEDURE FOR TABLETS:

Twenty tablets of Faropenem (Faronem 200mg, marketed by Ranbaxy laboratories limited) were weighed accurately and ground into a fine powder. An amount of powder equivalent to 100 mg of Faropenem was weighed into a 100 mL volumetric flask, 50 mL of distilled water was added and shaken thoroughly for about 10 minutes, then the volume was made up to the mark with the distilled water, mixed well and filtered. The concentration of the resultant solution is 1mg/ml. The assay of the tablets was completed according to general procedure. None of the excipients usually employed in the formulation of tablets interfered in the analysis of Faropenem, by the proposed method.

METHOD DEVELOPMENT

The F-C reagent was used in the determination of many phenolic compounds and a large number of substances of pharmaceutical interest. The proposed method is based on the formation of a blue colored chromogen, following the reduction of phospho-molybdo tungstic mixed acids of the F-C reagent by faropenem, in the presence of sodium carbonate, which could be measured at 745 nm. The mixed acids in the F-C reagent were the final chromogen and involve the following chemical species:

3H₂O,P₂O₅,13WO₃,5MoO₃,10H₂O and

3H₂O,P₂O₅, 14WO₃,4MoO₃,10H₂O

Faropenem probably effects reduction of 1, 2 or 3 oxygen atoms from tungstate and / or molybdate in the F-C reagent, there by producing one or more possible reduced species which have characteristic intense blue color. The effect of different variables such as nature and strength of alkali i.e sodium carbonate and F-C reagent, reaction time and order of addition of reactants were studied and optimized for attainment of maximum color and stability of colored species.

Optimization of conditions and absorption spectrum of the reaction product:

Condition under which reaction of Faropenem with F-C reagent fulfills the essential requirements was investigated. All conditions studied were optimized at room temperature (32± 2 ⁰C).

Selection of reaction medium

To find a suitable medium for the reaction, different aqueous bases were used, such as borax, sodium hydroxide, sodium carbonate or bicarbonate, sodium acetate and sodium hydrogen phosphate. The best results were obtained when sodium carbonate was used. In order to determine the optimum concentration of sodium carbonate, different volumes of 20% sodium carbonate solution (0.5, 1.0, 2.0 mL) were used to a constant concentration of Faropenem. And the results proved that 2ml of 20% sodium carbonate solution was found to be optimum. Larger volumes had no effect on the absorbance of the colored species.

Effect of F-C reagent concentration

Several experiments were carried out to study the influence of F-C reagent concentration on the color development. It was apparent that 1mL of reagent gave maximum absorbance and 1mL of reagent in a total volume of 10mL was used throughout the work.

Reaction time and stability of the colored species

The color reaction was not instantaneous. Maximum color was developed within 15 minutes of mixing the reactants and was stable for 60 minutes.

Effect of order of addition of reactants

After fixing all other experimental variables, a few further experiments were performed to ascertain the influence of order of addition of reactants on the color development and it was found that the addition of FC followed by sodium carbonate to the aliquots of drug solution gave maximum intensity of the colour.

RESULTS AND DISCUSSIONS:

Absorption spectrum and calibration graph

Absorption spectrum of the colored complex was scanned at 400-800 nm against a reagent blank.

Figure 3 Absorption Spectrum of faropenem with F.C reagent

The reaction product shows absorption maximum at 745 nm and is shown in figure 3. Calibration graph was obtained according to the above general procedure. The linearity was checked. All the spectral characteristics like Sandell’s sensitivity, molar absorptivity, precision and accuracy were also calculated.Themeasured standard deviation (SD), relative standard deviation (RSD) are summarized in the Table.1, and results are considered satisfactory.

Table 1: Optical Characterstics and precision data

PARAMETERS	PROPOSED METHOD OBSERVATIONS
λmax( nm)	745
Beers law limit (mcg/ml)	50-300
Molar absorptivity( lt.mol^-1.cm^-1)	0.376x10³
Sand ell’s sensitivity(micrograms/cm²/0.001 Absorbance unit)	0.5875
Regression equation Slope (m)Y Intercept	0.00080.0479
Correlation coefficient (r)	0.9948
Regression coefficient (r²)	0.9870
Precision( % relative standard deviation)	0.34
Standard error of estimate	0.0098
LOD (limit of detection)	1.850
LOQ (limit of quantitation)	5.625

*y = mx +c, where X is the concentration in micrograms/ml and Y is absorbance unit

Recovery Studies: To ensure the accuracy and reproducibility of the results obtained, adding known amounts of pure drug to the previously analyzed formulated samples and these samples were reanalyzed by the proposed method and also performed recovery experiments. The percentage recoveries thus obtained were given in Table 2.

Table 2 :: Assay of faropenem in tablet formulations.

Tablet Formulation	Labelled Amount (Mg)	Amount obtained(mg)* By proposed method	%Recovery By the Proposed Method **
01	200	198.96	99.48
02	200	199.32	99.66
03	200	199.78	99.89

*Average of three determinations**After spiking the sample

Table 3:Linearity Values

Concentration	Absorbance
50	0.0851
100	0.1275
150	0.1703
200	0.2125
250	0.2519
300	0.2933

ACKNOWLEDGEMENT:

The authors are grateful to Hetero Labs Limited for the supply of Faropenem as a gift sample and to the SSJ College of Pharmacy, Hyderabad, for providing the necessary facilities to carry out the research work.

REFERENCES:

1. Critchley IA, Brown SD, Traczewski MM, Tillotson GS, JanjicN(December 2007). "National and regional assessment of antimicrobial resistance among community-acquired respiratory tract pathogens identified in a 2005-2006 U.S. Faropenem surveillance study". Antimicrob. Agents Chemother. 51 (12): 4382–9.

2. Mushtaq S, Hope R, Warner M, Livermore DM (May 2007). "Activity of faropenem against cephalosporin-resistant Enterobacteriaceae". J. Antimicrob. Chemother. 59 (5): 1025–30.

3. Milazzo I, Blandino G, Caccamo F, Musumeci R, Nicoletti G, Speciale A (March 2003). "Faropenem, a new oral penem: antibacterial activity against selected anaerobic and fastidious periodontal isolates". J. Antimicrob. Chemother. 51 (3): 721–5.

4. Shobana. Kmenon, Jayesh G. Panchal ,Ravindra V. Patel Development and validation of stability indicating LC method for the determination of Faropenem in pharmaceutical formulations. Chromatographia69 (2009): 1013-14

5. Liang Yan, Zhu Lan-Zhen, He Chun-Hui. “HPLC Determination of Faropenem in Human plasma and its bioequivalence” Central South Pharmacy Journal (2009) 8 : 593-95.

6. Sun Ya-xin, Zhao Li-mei, QiuFeng , He Xiao-jing “Determination of Faropenem for injection in human plasma and urine by high performance liquid chromatography “Central South Pharmacy Journal (2009) 8 : 465- 67.

7. Gettig JP, Crank CW, Philbrick AH (January 2008). "Faropenemmedoxomil".Ann Pharmacother 42 (1): 80–90.

Received on 23.01.2013 Modified on 10.02.2013

Research J. Pharm. and Tech. 6(4): April 2013; Page 363-365