INTRODUCTION:

Cefprozil (CEF), (6R, 7R)-7-((R)-2-amino-2-(p-hydroxy-phenyl) acetamido)-8-oxo-3-propenyl-5-thia-1-azabicyclo (4.2.0) oct-2-ene-2-carboxylic acid, is a semi synthetic, second generation, orally administered cephalosporin. It is active against a wide range of aerobic Gram-positive and Gram-negative bacteria, as well as certain anaerobic organisms. Cefprozil molecule (Fig. 1) exhibits three kinds of isomerism. The first is isomerism Z and E (cis and trans) exists in 9:1 ratio respectively, due to the presence of an asymmetric carbon atom in the propenyl group at position 3 of the dihydrothiazine ring, where as isomerism Δ² and Δ³ results from the presence of double bond in the dihydrothiazine ring of a cephalosporin molecule. The predominant Z form is much more active against Gram-negative organisms.

The third kind of isomerism is connected with the presence of 7-(R-amino-(4-hydroxyphenyl) acetylamino group (optical isomerism)^1-7.

Cefprozil is applied in therapy as tablets containing 250 mg and 500 mg of Cefprozil. The form of oral suspension dosage for pediatric purposes is also available as granules in multi-dose bottles. One 5 mL dosage of suspension contains 125 mg or 250 mg of Cefprozil.

Several LC and spectrophotometric methods have been reported for estimation of two isomers of Cefprozil in substance and in its pharmaceutical formulations^{8 - 10}. However no validated stability indicating LC method has been reported for the separation and estimation of Impurities (Fig. 2) of Cefprozil ie, 2-Amino-2-(p-Hydroxyphenyl)-acetic acid (A), (6R, 7R)-7-[(R)-2-Amino-2-(p-hydroxyphenyl)-acetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (B), (6R, 7R)-7-Amino-8-oxo-3-propenyl-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid (C), (6R, 7R)-7-Amino-8-oxo-3-propenyl-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid (D) and (6R,7R)-7-[(R)-2-Amino-2-(p-methoxyphenyl)-acetamido]-8-oxo-3-propenyl-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (E) in its powder for Oral suspension pharmaceutical dosage forms. It was essential to develop a Chromatographic method for estimation of Impurities of Cefprozil in its pharmaceutical dosage forms. The authors felt it necessary to develop a stability indicating method for separation and quantification of impurities and known degradation products in Cefprozil powder for oral suspension. The method was validated in accordance with International Conference on Harmonization (ICH) and United States Food and Drug Administration (USFDA) guidelines ^11-14.

CA and CB

7ADCA

Cephalexin

Fig 1: Chemical structures of Impurities and Cephalexin

Fig. 2. Chromatogram obtained from Cefprozil drug product spiked with its related impurities

The novelty and advantages of the method is its capaity to:

· Separate impurities with resolution suitable for quantification;

· Identify without reference material; and

· Quantify the individual impurities using response factors against drug substance.

The other merit of the method is that by using sub-2 µm column the LOD and LOQ of impurities were achieved below 0.014 and 0.037 % (w/w) respectively proving the high sensitivity. Hence the method can be used to quantify the impurities at very low concentrations.

EXPERIMENTAL:

Chemicals and Reagents: Cefprozil drug substance and Impurities were obtained from Orchid Chemicals and pharmaceutical Ltd., Chennai, Cefprozil powder for oral suspension drug product was manufactured by Ranbaxy Laboratories Ltd., Dewas, India. Ammonium dihydrogen orthophosphate was purchased from Spectrochem, Mumbai, India and the all reagents used were of HPLC grade and procured from Rankem, Mumbai, India.

Instrumentation: The LC system used for method development and validation was a Waters Alliance 2695 separation module with thermostatic compartment and photodiode-array detector (PDA) with Empower software .A photo stability chamber (Newtronic, Mumbai, India) was used for light degradation study.

Preparation of Solutions:

Preparation of Mobile phase-A:

2.3 g of Ammonium dihydrogen orthophosphate was dissolved in 1000 mL of HPLC grade water in a suitable container and filtered through 0.2 µm nylon membrane filter and degassed prior to use.

Preparation of Mobile phase-B:

A mixture of mobile phase-A and Methanol in the ratio 20:80 (v/v) was prepared and filtered through 0.2 µm nylon membrane filter and degassed prior to use.

Diluent: A mixture of Water and Methanol in the ratio 90:10 (v/v) respectively was used as diluent.

Preparation of Standard solution (10 µg mL^-1) : About 50 mg of Cefprozil working standard was accurately weighed and transferred into a 100 mL volumetric flask, dissolved in about 70 mL of diluent and made up to volume with diluent and mixed well. 5 ml of this solution was further diluted to 50 mL with diluent. Further 5 mL of the resulting solution was pipetted out into a 25 mL volumetric flask and made up to volume with diluent and mixed well. The final solution was filtered through 0.45 µm nylon membrane filter prior to use.

Preparation of Test solution: Cefprozil powder for oral suspension bottle was reconstituted with specified amount of water, the resulting suspension equivalent to 100 mg of Cefprozil was accurately weighed and transferred in to a 100 mL volumetric flask, about 70 mL of diluent was added, sonicated for 30 minutes with intermediate shaking and made up to volume with diluent and mixed well. The solution was filtered through 0.45 µm nylon membrane filter prior to use.

Chromatographic conditions:

Compounds were separated on a Peerless HT C18 (100 x 4.6 mm, 1.8 µm packing) column at a flow rate of 0.6 mL/min and at column temperature 25ºC. The sample injection volume was 10 µL. The Diode array detector was used to record the signal at a wavelength of 290 nm. The Mobile phase elution was in gradient mode.

RESULTS AND DISCUSSION:

Method Development:

The chromatographic conditions were adjusted to provide the best separation between peaks. Keeping the aim of best separation with shorter run time, it was decided to use sub-2 µm particle size column. Accordingly column chemistry, solvent strength in the mobile phase and flow rate were varied. The mobile phase conditions were optimized so that impurity and analyte peaks were free from interference of excipients and solvent. Other criteria, for example injection run time and sensitivity, extraction of drug from formulation matrix were also considered. After trying columns of different brands of C18 stationary phases, the final choice giving satisfactory resolution between the peaks of interest and the runtime with the 10 cm x 4.6 mm i.d., 1.8 µm particle, Peerless HT C18 reversed-phase column. Trials were conducted to achieve the better selectivity for substances C and B by changing the composition of mobile phase A, prepared by dissolving 2.3 g of ammonium dihydrogen orthophosphate in 1000 mL of water, and mobile phase B, prepared by mixing of mobile phase A and methanol in the ratio 20 : 80 (v/v) respectively, with gradient mode elution. The flow rate was established by testing the effect of different flow rates on peak area and resolution and 0.6 mL min^-1 was found to be optimum. All experiments were performed at ambient column oven temperature. The mobile phase gradient elution was as below:

Time (minutes)	% Mobile phase-A	%Mobile phase-B
0	100	0
3	100	0
10	90	10
25	85	15
35	55	45
40	50	50
45	50	50
45.5	100	0
53	100	0

Impurity pathways: Substances A, C and D are the starting materials used in the synthesis of Cefprozil; C is also formed in the alkaline degradation study. Impurity B is formed when Impurity A is coupled with another starting material in the synthesis process; it is also formed in the alkaline and oxidation degradation studies. Impurity E is formed when Impurity C is coupled with another starting material used in the synthesis process; it is also formed in the in alkaline degradation study.

METHOD VALIDATION: ^11-14

The proposed method was validated for Precision, Limits of Detection (LOD) and Limits of Quatitation (LOQ), Specificity, Linearity, Accuracy, Ruggedness, Robustness and solution stability as per FDA and ICH guidelines.

LOD and LOQ:

The LOD and LOQ values were determined by injecting A, B, C, D and E impurity solutions prepared in diluent, individually at the lowest concentrations at which Signal-to-Ratio is about 3 and 10 respectively. Six different samples were prepared by spiking all the impurities at LOQ level to prove the Precision at LOQ. The RSD (%) of the % impurity was calculated for six replicates at the predicted LOQ level. RSD for six replicates at the LOQ level was <10%. The recovery of impurities at LOQ level was found to be from 95 to 115%. The results are given in Table 1.

Table 1. Method validation Analytical data of proposed method

Parameter		A	B	C	D	E
LOD	Concentration	0.098	0.053	0.016	0.020	0.028
LOD	% Impurity	0.013	0.005	0.002	0.002	0.003
LOQ	Concentration	0.293	0.160	0.048	0.061	0.083
	% Impurity	0.037	0.017	0.004	0.005	0.008
	Precision (% RSD)	7.2	1.8	5.9	6.3	6.3
Linearity of Detector Response	Correlation co-efficient	0.99	0.99	0.98	0.99	0.99
Precision (% RSD)		1.3	0.4	6.1	1.1	1.8
Intermediate Precision (% RSD)		0.9	0.7	0.8	0.7	1.5
Accuracy (%Recovery)		L1 - 115% L2 - 103% L3 - 98%	L1 - 109% L2 - 98% L3 - 96%	L1 - 96% L2 - 105% L3 - 111%	L1 - 106% L2 - 93% L3 - 92%	L1 - 108% L2 - 102% L3 - 100%
Linearity of Method (Correlation co-efficient)		0.99	0.99	0.98	0.99	0.99

PRECISION OF TEST METHOD: The precision of the LC instrument (System precision) was evaluated by calculating RSD (%) for the area from ten replicate injections of standard solution to prove the consistency of the output signal produced by the LC system. The RSD 0.4 % shows the good precision of the instrument. Method precision was established by spiking the impurities at target concentration level in the test solution containing the drug product. Six spiked samples were prepared and injected to prove the precision of the method. The % R.S.D values of all individual impurities were found to be satisfactory for all the six analytical measurements. This was also repeated on different day to determine inter-day precision. A different scientist established intermediate precision on a different chromatographic system. The small %RSD values presented in Table 1 prove the high precision of the proposed method.

LINEARITY OF DETECTOR RESPONSE : Linearity of detector response (LDR) was established by plotting a graph to concentration versus area of Cefprozil impurities and determining the correlation coefficient. A series of solutions of impurities in the concentration ranging from about LOQ level to about 150% of the target concentration were prepared and injected onto the HPLC system. The correlation coefficient values proving that the detector response was found to be linear from LOQ to 150% of target concentration. The results are summarized in Table 1.

ACCURACY: The accuracy of the developed method was determined by spiking method. Different concentration levels ranging from LOQ level (L1) to 125 % level (L3) covering 100% level (L2) as target concentration of impurities were prepared by spiking in Test solution. At each level three solutions were prepared. The % recovery was found to be good and the values are presented in Table 1.

LINEARITY OF METHOD: Linearity of the method was checked by plotting calibration curves between the amount µg mL ^-1 of component added in spiked solution versus the amount µg mL ^-1 of component found (recovered). The slope, intercept and correlation coefficient were derived from least-square regression method. The values were presented in Table 1. It was found that the responses of impurities were linear from 50% level to 125 % of target concentration. The correlation coefficient values of impurities indicate the best linearity of the method.

SPECIFICITY:

Placebo interference and Forced degradation study:

A study to demonstrate the Placebo interference was conducted. The placebo samples were prepared in triplicates by following the procedure same as test solution. The possibility of excipient interference in the analysis was also studied. Chromatograms of Placebo samples showed no peaks at the retention time of Cefprozil and the Impurity peaks. This indicates that excipients used in the formulation do not interfere in the estimation of impurities.

Drug product was subjected to forced degradation under acidic (0.1N HCl), Basic (0.1N NaOH), water, oxidation (1% Peroxide), Humidity, Thermal and photolytic conditions to demonstrate the interference of Degradation products from Cefprozil. Degradation products were obtained in basic, water and oxidation degradation studies only. The results from the forced degradation studies are given in Table 2. The peak-purity angle was found to be less than purity threshold for Cefprozil in all the degradation studies, which confirms that no degradation product was co-eluted with Cefprozil. This study proves the specificity and stability indicating nature of the method.

ROBUSTNESS: The robustness of the method was studied by varying to 0.2 mL min-1 in the flow rate, 5 ºC units of column temperature and 5 % in organic solvent composition in mobile phase. By changing the above chromatographic parameters the separations, sensitivity and reproducibility were not affected. The results revealed that these alterations did not have any impact on the chromatographic performance indicating the robustness of the proposed method. The results of the study are given in Table 3.

Solution stability: A study to establish the stability of Cefprozil in standard and test solutions was conducted on bench top and in refrigerator at 0 hour, 24 hours and 48 hours. The stability of Cefprozil in test solution was established by calculating the difference in % total impurities from 0 hour.

Table 2. Summary of degradation results from Forced degradation studies

Stress condition	% Impurity
Stress condition	A	B	C	D	E
Unstressed	NA	NA	NA	0.03	NA
Acid Stress	NA	NA	0.07	0.04	NA
Base Stress	NA	0.12	0.11	NA	2.94
Peroxide stress	NA	1.48	NA	0.03	NA
Water stress	NA	0.07	NA	0.04	0.08
UV stressed	NA	0.02	NA	0.05	NA
Visible Stressed	NA	0.06	NA	0.04	NA
Heat stressed	NA	0.04	NA	0.04	0.02
Humidity Stressed	NA	0.02	NA	0.04	NA

Table 3. Effect of deliberately altered chromatographic conditions on USP tangent and USP resolution between Cefprozil Z isomer and E isomer peaks.

Results		Flow (mL min^-1)			Organic strength (% Mobile phase-B)**			Column oven temperature (ºC)
Results		0.6*	0.4	0.8	100%*	95%	105%	25*	20
USP tangent for Z-isomer peak		210641	310456	70678	186518	204574	194833	181134	249581
USP resolution between Z and E-isomer peaks		10.0	8.0	10.2	10.1	10.2	10.1	9.7	9.9
% impurity	A	1.1722	1.1616	1.1653	1.0739	1.0321	1.0328	1.2316	1.1787
	B	1.0439	0.9020	1.0272	1.0214	1.0145	1.0159	1.2017	1.2120
	C	0.9754	0.9933	0.9836	0.9677	0.9605	0.9620	1.1783	1.1229
	D	0.9370	0.9242	0.9562	0.9539	0.9485	0.9478	1.1001	1.0715
	E	0.9216	0.9225	1.0003	0.9316	0.9436	0.9362	1.0030	0.9843

* Chromatographic parameters not altered. ** Composition of Methanol is altered in mobile phase-B

CONCLUSION:

A new, reversed-phase HPLC method has been developed for simultaneous estimation of impurities of Cefprozil in its powder for oral suspension formulation. The above results shown that the method is precise, linear, specific and accurate, and proving the reliability of the method. This validated stability-indicating method can be conveniently used for quality control and studies for assessment of the stability of Cefprozil powder for oral suspension formulations.

ACKNOWLEDGEMENTS:

The authors wish to thank the management of Orchid Chemicals and Pharmaceuticals Ltd., Chennai for providing facilities to carry out this work.

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Received on 06.01.2011 Modified on 24.02.2011

Asian J. Research Chem. 4(5): May, 2011; Page 810-814