Bioanalytical  Method  Development  and Validation  for Simultaneous  Estimation  of  Paracetamol  and  Cefixime  by  using  RP-HPLC  in  Rabbit  Plasma

Introduction

Paracetamol (Figure 1) is chemically N-(4-hydoxyphenyl) ethanamide and it was a  non-steroidal anti-inflammatory drug having potent antipyretic, analgesic and anti-inflammatory action^1-2. Paracetamol was used for the pains and high temperature fevers³.  Paracetamol acts by selectively inhibiting the prostaglandin biosynthesis. The bioavailability of the  Paracetamol  was  80% ⁴.

Figure 1: Structure of Paracetamol   Click here to View figure

 

Cefixime (Figure 2) is chemically (6R,7R)-7-((2-(2-amino-1,3-thiazol-4-yl)-2-(carboxymethoxyimino)acetyl)amino)-3-ethenyl-8-oxo-5-thia-1-azabicyclo(4.2.0)octo-2-ene-2-carboxylic acid and it was a third generation cephalosporin antibiotic used for the several  bacterial infections^5-6. Cefixime acts by binding to specific penicillin binding protein leads to  inhibiting bacterial cell wall synthesis and the bioavailability was 40% to 50%⁷.

Figure 2: Structure of Cefixime   Click here to View figure

 

Cefaclor (Figure 3) is chemically 3-chloro-7-D-(2-phenylglycinamido)-3-cephem-4-carboxylic acid monohydrate and it was a semisynthetic cephalosporin antibiotic for oral administration. Cefaclor is used to reduce the development of drug-resistant bacteria.

Figure 3: Structure  of Cefaclor   Click here to View figure

 

Literature  survey  revealed  that  few analytical methods have been reported for  estimation of Paracetamol and Cefixime individually or in combination with other drugs. The  reported methods are Spectrophotometric^8-10, HPLC^11-14, HPTLC^15-16 and LC-MS^17-18. Although no method was reported for the simultaneous estimation of Paracetamol and  Cefixime in combined pharmaceutical formulations.

The  present  study was aimed to develop a simple, sensitive, rapid, precise and  accurate and validated the bionalytical method¹⁹ for simultaneous estimation of Paracetamol and Cefixime according to ICH guidelines²⁰ by using high  performance liquid chromatography in rabbit plasma.

Experimental

Chemicals, reagents, standards and samples

Blank  rabbit  plasma,  pure  samples  of  drugs  like  Paracetamol, Cefixime and Cefaclor was obtained from Spectrum Pharma Research Solutions, Hyderabad, India. Analytical grade of Orthophosporic  acid was obtained from SD Fine Chemicals Ltd., Mumbai, India. HPLC grade of Acetonitrile and Water was obtained from Qualigens Fine Chemicals,  Mumbai, India.

Instrumentation

The analysis was performed by using a chromatographic system Water  2695  series  HPLC  comprised  of  vacuum  degas,  auto  injector,  dual  gradient  pump  with  photo  diode  array  detector. The HPLC system was equipped with Empower 2 software.

Chromatographic  conditions

Paracetamol and Cefixime was analysed in ODS C18 column (250mmx4.6mm, 5µm particle size) for the chromatographic separation. The mobile phase was composed of Phosphate buffer (p^H 6.4) and Acetonitrile (80:20, v/v) and was used as diluent. Filtered through 0.45µm nylon membrane filter under vacuum filtration and pumped at a temperature of 30⁰C, at flow rate of 1ml/min with UV detection wavelength at 245nm. Injection volume was 10μl. The run time was 8min and the retention time of Paracetamol and Cefixime was found to be 4.608min and 5.914 min respectvily.

Preparation  of  stock  solutions

Primary stock solutions of Paracetamol and Cefixime were prepared individually by  dissolving 10mg of each in 25ml volumetric flask in diluent. Series  of  working  solutions  of  Paracetamol and Cefixime were prepared by the suitable dilution of the stock solutions with  same diluent to achieve the concentration range of 10-100μg/ml for Paracetamol and 5-50μg/ml for Cefixime.

Procedure

The  standard  solutions  were  prepared  by  dilution  of  the  stock  solution  with  diluent to obtain a concentration  range 10-100μg/ml for Paracetamol and 5-50μg/ml for  Cefixime. Triplicate of 10μl injections were prepared for each concentration and  chromatographed under the conditions reported above. The peak area of each concentration  was plotted against the corresponding concentration for getting the calibration curve and  the regression equation was computed.

Preparation  of  spiked  plasma  sample

250µl of rabbit plasma, 50µl of internal  standard, 10µl of Paracetamol and 10µl of  Cefixime were pipetted into 10ml centrifuge tube and to it 2ml of Acetonitrile was added.  The mixture was mixed shortly, standing for 5min at room temperature,  then vortex for 3 min, finally the mixture was centrifuged at 3200rpm for 10min. After the centrifugation, 10 μl of the supernantant layer was collected and injected into HPLC.

Calibration  and  linearity 

Calibration curves were plotted in the concentration range of 10-100μg/ml and 5-50 μg/ml for  Paracetamol and Cefixime respectively of required concentrations in the measured samples. Triplicate of 10μl injections were constructed for each working standard solution. The  peak  area for each concentration was measured and subsequently to draw against the each  concentration to obtain the calibration curve.

Method validation

Linearity

Calibration curves showed the relation between the concentrations of drugs versus  peak area were established. Results manifest linear relationship in the range of 10-100μg/ml   and 5-50μg/ml for Paracetamol and Cefixime. In two drugs run from which linear regression equation was measured by following ICH guidelines shown  in Figure 4 and Figure 5.

Figure 4: Calibration curve of Paracetamol   Click here to View figure

 

Figure 5: Calibration curve of Cefixime Click here to View figure

 

Precision and accuracy

Results from the validation data from rabbit plasma were within the acceptable limits. Within batch precision method was measured to range from 6.21 to 8.96% for Paracetamol  and 3.16 to 7.73% for Cefixime. The accuracy of the method was measured in the range of  96.33 to 106.0% for Paracetamol and 96.00 to 105.67% for Cefixime shown in Table 1.

Table 1: Precision and accuracy 

Within batch	LLOQ QC		LQC		MQC		HQC
	Paracetamol	Cefixime	Paracetamol	Cefixime	Paracetamol	Cefixime	Paracetamol	Cefixime
Mean	106	105.67	103.65	96	100.37	96.33	96.33	102.67
S.D (+/-)	0.013	0.008	0.05	0.006	0.089	0.037	0.037	0.053
C.V (%)	6.21	3.16	8.05	3.16	8.96	7.73	7.73	5.80

 

Recovery

The analyte recovery for the Paracetamol and Cefixime were measured to be 50.63% and 51.74% respectively. The internal standard for Cefaclor was found to 57.20%  shown  in  Table  2.

Table 2: Recovery studies

	Paracetamol	Cefixime	Cefaclor
Mean	50.63	51.74	57.20
S.D (+/-)	1.98	1.42	0.509
C.V  (%)	3.93	2.75	8.17

 

Stability studies

Long term stability

The concentration of sample of stability ranged between 99.27 to 104.86% for  Paracetamol and 93.08 to 98.71% for Cefixime of the utility value shown in Table 3.

Table 3: Long term stability studies

	LQC		HQC
	Paracetamol	Cefixime	Paracetamol	Cefixime
Mean	99.27	98.71	104. 86	93.08
S.D (+/-)	0.049	0.018	0.170	0.085
C.V (%)	8.41	8.82	9.09	9.63

 

Freeze-thaw stability

The sample concentration of freeze-thaw stability was measured to be 97.83 to 104.83% for Paracetamol and 100.97 to 104.60% for Cefixime of utility concentraton  showing the stability of the drug above three freeze-thaw cycles shown in Table 4.

Table 4: Freeze-thaw stability studies

	LQC		HQC
	Paracetamol	Cefixime	Paracetamol	Cefixime
Mean	104.8	104.6	97.83	100.97
S.D (+/-)	0.035	0.012	0.185	0.056
C.V (%)	5.57	6.19	9.84	5.83

 

Bench top stability

The drug was developed to be stable for at least 4 hours on bench top at room  temperature. The freshly spiked calibration standards against back calculated concentration  was within a range of 94.02 to 107.54% for Paracetamol and 88.92 to 103.54% for Cefixime  of the utility concentration shown in Table 5.

Table 5: Bench top stability studies   

	LQC		HQC
	Paracetamol	Cefixime	Paracetamol	Cefixime
Mean	107.54	103.54	94.02	88.92
S.D (+/-)	0.0143	0.062	0.009	0.169
C.V (%)	6.91	10.36	10.40	9.97

 

Sample stability 

The concentration of sample of stability was measured range of 99.46 to 99.94% at  2-8⁰C and 98.73 to 101.38% at room temperature for Paracetamol and Cefixime,  respectively shown in Table 6.

Table 6: Sample studies

	LQC		HQC
	Paracetamol	Cefixime	Paracetamol	Cefixime
Mean	99.46	98.73	99.94	101.38
S.D (+/-)	0.018	0.018	0.994	0.958
N	3	3	3	3

 

Reults  and  Discussion

Different stationary phases were tried with various columns,  poor  and  bend  peakes  were  observed  with  different columns while C18 column gave satisfactory resolution and  free from tailing. Several  mobile  phases  used  like  Methanol  with  Acetonitrile  were  found to separate the compounds doubtful and Potassium dihydrogen phosphate buffer with  Acetonitrile lead to good separation. The present study objective was acquired using mobile  phase be composed of Phosphate buffer: Acetonitrile in the ratio of 80:20, v/v with  the p^H adjusted to 6.4±0.2 with Orthophosphoric acid. The mobile phase composition is optimized under described conditions, free from tailing, peaks are well defined, resolved, the  tailing factors were < 2 for all peaks. The retension times for Paracetamol was 4.608 min  and for Cefixime was 5.914min respectvily, at flow rate of 1ml/min. The optimum wavelength  for detection was 245nm at where much better detector responses for the two drugs were  obtained. The chromatograms of rabbit plasma blank and drugs Cefaclor, Paracetamol and Cefixime were shown in Figure  6 & 7 respectively.

Figure 6: Chromatogram of rabbit plasma blank   Click here to View figure

 

Figure 7: Chromatogram of Cefaclor, Paracetamol and Cefixime in spiked rabbit plasma   Click here to View figure

 

Acknowledgements

The authors are thankful to A.K.R.G. College of Pharmacy, Nallajerla, India for providing necessary facilities for carryout the research work. The authors are thankful to Spectrum Pharma Research solutions, Hyderabad, India for  providing the pure drugs like Paracetamol, Cefixime and Cefaclor.

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