INTRODUCTION:

Cefexime (Fig 1) is a third generation cephalosporin antibiotic. It acts by binding to specific penicillin binding protein located in the bacterial cell wall thereby inhibiting bacterial cell wall synthesis. Cell wall lysis is then mediated by the bacterial autolytic enzymes. N-acetyl cysteine (NAC) (Fig B.1) contains a thiol group and acts as the precursor of the natural antioxidant glutathione which plays a crucial role in the antioxidant defense by acting as a free radicle scavenger¹. NAC also acts as a mucolytic by depolymerizing the mucin glycoprotein oligomers and by hydrolyzing the disulfide bonds that link the mucin monomers, thereby reducing the sputum viscosity². Smoking is the leading reason for chronic obstructive pulmonary disease (COPD). As per the 2004 data from the World health organization (WHO), 64 million people in the world suffer from COPD and about 3 million people have died due to the disease. It is predicted by the WHO that COPD will be the third leading cause of death in the world by 2030³.

The antibiotic mechanism of cefexime combined with the ability of NAC to depolymerize mucin makes the fixed dose combination of cefexime and NAC (2:3) a boon to patients with COPD and chronic bronchitis. Cefexime is official in United states Pharmacopoeia (USP), British Pharmacopoeia (BP), European Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP)^4-7. Several methods have been reported for the simultaneous estimation of cefexime along with other drugs like azithromycin, potassium clavunate, linezolid etc^8-12. Acetyl cysteine is the derivative of cysteine with an acetyl group attached to the amino group. There are many chromatographic methods like RP-HPLC, LC-UV-MS and ion pair chromatography for the assay of NAC in bulk and formulation ^13-19. NAC is also official in EP, BP, JP, and USP^4-7. Simultaneous estimation of NAC has been reported along with other drugs like, clomiphene citrate and L-arginine^20,21. Currently there are no reported analytical methods for the simultaneous estimation of cefexime with NAC.

In light of the current lack of reported literature for the fixed dose combination it was seen fit to develop a simple, isocratic RP-HPLC method for the estimation of cefexime and NAC in tablet dosage form and to validate the performance of the developed analytical method as per ICH Q2 (R1)²² .

MATERIALS AND METHODS:

NAC (97%) were purchased from Sigma Aldrich, Bangalore, India. Cefexime was provided as a gift sample from Ce-Chem Pharmaceuticals, Pvt. Ltd, Bengaluru. The in-house milliq water (milli Q) was used. Methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from Finar Limited, Gujarat. Potassium dihydrogen phosphate (AR grade), Orthophosphoric acid (AR), were purchased from Loba Chemie, Mumbai, India. Mucomelt Forte tablets (cefexime: NAC::2:3) were procured from Apollo Pharmacy, Pune. The liquid chromatography method development and validation was carried out on a Shimadzu LC-20 prominance which is equipped with a Shimadzu LC-20AD prominance pump, Shimadzu SPD-M10 diode array detector, Shimadzu SIL-20AC HT auto sampler and a Shimadzu CTO-10AS column compartment was used. The data were collected on a PC equipped with LC solutions (version 1.25). SPSS was usedas the statistical software.

Preparation of Solutions:

Mobile phase:

The mobile phase was 25mM potassium dihydrogen phosphate solution adjusted to pH 2.75 using orthophosphoric acid and acetonitrile in the ratio 90:10v/v.

Diluent:

A mixture of water: ACN is the ratio 90:10v/v.

System suitability solution; A mixture of 10µg/ml cefexime and 100 µg/mL solution of NAC prepared in the diluents.

Test solution:

A tablet solution equivalent to 50 µg/mL solution of NAC and cefexime.

Optimization of chromatographic conditions:

The initial phase was to develop and optimize the analytical method. Literature was studied in depth to set the initial chromatographic condition. The first step was to select a suitable detection wavelength and this was achieved by scanning a 10 µg/ml solution of cefexime and NAC in methanol in the range of 200-400 nm. Column is the heart of chromatography. Literature widely reports the use of C₁₈ column for both the drugs. Gracesmart C₁₈ (250×4.6µm, 5µ) and Acclaim 120 C₁₈ (150×4.6µm, 3µ) were used for the trials. Buffer selection was done based on the pKa value of the drugs. The pka value of cefexime is 2.10, (-COOH of cephexin group), 3.45 (-COOH of side chain), 2.92 (amino group of thiazol) and NAC is 3.24 (carboxylic acid moiety), 9.52 (-SH group) as per Henderson hesselbach equation pH should be within pKa ±1. Hence trials were performed between pH 2.75-3.5(Fig 2).

Fig 2: Chromatograms representing the influence of pH on the retention time of cefexime and N-acetyl cysteine

A flow rate of 1mL/min was selected and an isocratic mode of delivery was used. Acetonitrile: water was used as the diluent.

Method validation:

Specificity:

Specificity is the ability to assess unambiguously the analyte in the presence of components which may be expected to be present. Standards (10 µg/ml of Cefexime and 100 µg/mL of NAC) and tablet formulation (10 µg/mL) were analyzed and the purity of the peaks was assessed by PDA detector

System suitability:

System suitability test is carried out to confirm that the analytical system is functioning properly and can give accurate and precise results. Standard preparations of NAC (100µg/mL) and cefexime (10µg/mL) were injected six times and checked for the system suitability parameters like resolution, tailing factor, peak purity, and theoretical plates.

Linearity:

It is the ability of the method to produce detector responses that are directly proportional to the analyte concentration. Linearity was determined for NAC and cefexime from 10-90 µg/ml by injecting each solution three times. The mean area at each level was calculated and was plotted against concentration.

Precision:

It is the measure of the degree of repeatability of an analytical method under regular operation. System precision, method precision, intermediate precision of the method was assessed. System precision (injection repeatability) was evaluated by chromatographing six replicate injections of the standard solution mixture. Method precision was performed by chromatographing on a single day, six aliquots of homogeneous samples (100%), each of which was independently prepared. Intermediate precision was performed by chromatographing six aliquots of samples on different days.

Accuracy:

The method accuracy was determined at three concentration levels with triplicates at each level. The test concertation was spiked with standard solutions at 80%, 100% and 120% level. The individual % recovery, mean % recovery, % RSD was calculated. The % recovery was calculated by the following formula:

% Recovery =

Robustness:

It is the capacity of the method to remain unaffected by small thoughtful variations in method parameters. The robustness of the method was evaluated by varying method parameters like pH, flow rate, column temperature and wavelength. The student’s t test was used to evaluate the significant influence of these variations on the method performance at p=0.05.The percent RSD was calculated in all cases.

Assay of marketed formulation:

10 tablets were accurately weighed and the average weight was calculated. The tablets were then crushed and an amount of powder equivalent to 200 mg of cefexime was transferred into 25 ml volumetric flask. 15 ml of methanol was added to this and sonicated for 30 min with intermediate shaking. The sonicate was maintained at 25˚C. The volume was further made up with methanol and filtered through whatman filter paper. 1 ml of the above solution was diluted to 10 ml by the diluent to reach a concentration of 100µg/ml. 5 ml of the above solution was further diluted with diluent to get a final concentration of 50 µg/ml. The resultant solution was transferred to HPLC vials and injected six times.

RESULTS:

Optimized Chromatographic conditions: The chromatographic separation was achieved on Acclaim 120 C18 (150 × 4.6 mm), 3µm column from Thermo scientific. The mobile phase consists of 25 mM phosphate buffer (pH 2.7±0.05): Acetonitrile (90:10 v/v). An isocratic program was followed for 13 minutes. The flow rate was 1mL/min and the sample injection volume was 20µL. The detection wavelength was set at 220 nm and column oven temperature at 40˚C. (Fig 3b)

Results of validation of method: The method was validated to show amenability with regulatory requirements for analytical methods. The guideline as per the International Conference on Harmonization (ICH) for validation of analytical procedures: text and methodology: Q2(R1) was followed. The peak purity index was less than the single point threshold (Fig 4) in all the cases indicating that the peaks are pure and the method is specific. The system suitability parameters like tailing factor, and theoretical plates (Table 1) were found to be within acceptable limits.

Table 1: system suitability data

Parameter	System suitability		Acceptance criteria
Parameter	NAC	Cefexime	Acceptance criteria
Tailing factor	0.98	0.65	NMT 2.0%
Theoretical plates	4231	7304	NLT 2000
%RSD of six injections	0.11	0.78	NMT 2.0%

(n=6)

The method was found to be linear from 10µg/mL to 90µg/mL. The regression coefficient was found to be not less than 0.999. The calibration graph i.e. slope, regression coefficient and Y-intercept for both the drugs are depicted in Fig 3. The percent relative standard deviation of system precision, method precision, intermediate precision was found to be less than 2% confirming the method to be precise (Table 4).

The accuracy of the method at 80%, 100% and 120% level was found to be within 98-102%w/w (Table 5). To assess the influence of temperature on the method the elution volume of the peaks was calculated. From the student’s t test there was no significant difference found from the optimum set condition indicating the method to be robust in nature (Table 6).

Table 4: System precision and intermediate precision

Precision	Percent Relative standard deviation
Precision	NAC	Cefexieme
System prescision	0.23	0.18
Intermediate presicion	0.31	0.41
Method prescision	0.62	0.14

(n=6)

The validated method was applied for the simultaneous quantification of these drugs in the marketed formulation and the percent assay of NAC was found to be 99.60% and Cefexime was found to be 100.76 % (Table 7) thus clearly indicating it to be between 90-110% of label claim.

Table 5: Accuracy data

Recovery level	NAC			Cefexieme
Recovery level	% Recovery	Average % recovery	%RSD	% Recovery	Average % recovery	%RSD
80%	101.82	101.96	0.52	102.39	101.81	0.76
	102.57			100.90
	101.51			102.20
100%	101.55	101.16	0.50	101.54	100.20	1.15
	101.35			99.46
	100.58			99.6
120%	101.20	101.60	0.95	101.58	101.51	0.56
	100.89			102.12
	102.71			100.97

(n=3)

Table 6: Robustness data

Drug	Parameter	Change	Percent RSD	Calculated t value	Table t value
Cefexime	Temperature(ºC)	Low (38)	0.13	2.9	1.81
Cefexime		High(42)	0.41	3.6
NAC		Low(38)	0.18	3.3
NAC		High(42)	0.32	4.3
Cefexime	pH	Low (2.5)	0.34	151.99
Cefexime		High (2.9)	0.31	215/97
NAC		Low(2.5)	0.27	31.86
NAC		High(2.9)	0.48	30.84
Cefexime	Flow rate (ml/min)	Low (0.8)	1.20	42.92
Cefexime		High (1.2)	0.63	76.74
NAC		Low (0.8)	0.24	129.52
NAC		High (1.2)	0.23	151.09
Cefexime	Wavelength (nm)	Low(218)	0.95	22.81
Cefexime		High(222)	0.52	33.57
NAC		Low (218)	0.05	309.86
NAC		High(222)	0.10	117.83

Table 7: Assay

Injection no:	Percent Assay
Injection no:	Acetyl cysteine	Cefexime
1	98.9	100.80
2	99.64	100.80
3	99.91	100.91
4	100.57	100.60
5	98.96	100.59
6	99.65	100.90
Mean	99.60	100.76
SD	0.62	0.14
%RSD	0.62	0.13

(n=6)

DISCUSSION:

The reported literature methods of cefexime in bulk drug, tablet, capsule and in combination with other drugs employs an organic phase comprising of acetonitrile and/or methanol in the range of 20-40 % v/v. These conditions are insufficient to retain NAC on the column as it is highly polar in nature (log p is -0.66). Therefore an increase in the organic phase of the mobile phase will cause rapid decrease in the retention time of NAC and at the void volume. The pH range employed includes 2.79-8.00. The pKa of the acid group in NAC is 3.00 and above its pka NAC will ionize and elute at the void volume. NAC is an organic thiol and it shows absorption in the 200-220 nm range so at 254 nm it will show negligible absorbance.

The reported literature methods for NAC when extrapolated to cefexime were not feasible. The USP method uses 100% v/v buffer and JP 95 % v/v in the mobile phase which increases the retention of cefexime on the column. The JP method employs the mobile phase as the diluent. Cefexime is insoluble in water hence extraction of the drug form the formulation will be difficult. Also the method employs alkyl sulphonate as an ion pair reagent which is not realistic for cefexime.

The principle objective of method development was to achieve separation between N-acetyl cysteine and cefexime within a reasonable run time.290nm was found to be the lambda max for cefexime. NAC is found to give response in the range of 200nm -220nm. 220nm nm was selected as both the drugs show acceptable response and is away from the solvent cutoff of potassium dihydrogen phosphate hence reducing noise. Acclaim 120 C₁₈ was seen to give better peak shape and less run time when compared to Gracemart C₁₈as it is a short endcapped column. pH 2.75 was seen optimum to retain the both the drugs. At this pH the carboxyl functional group in both the drugs will remain unionized thus facilitating the retention of the drug on the column with suitable selectivity (Fig 2). 25mM potassium dihydrogen phosphate was seen as the optimum buffer concentration which resulted in better peak shape. 90:10v/v was seen as the optimum composition of buffer: acetonitrile. Increase in the ratio of acetonitrile was seen to cause the elution of NAC from the column as the drug is polar in nature. Temperature is known to influence the retention time of the analytes. Cefexime seems to be largely affected by temperature than NAC. The effect of column temperature was studied between 25˚-42˚C (Fig 3).

The different aspects of the performance of the method were evaluated based on ICH recommendations and the method was found to specific, linear, accurate, precise and robust.

CONCLUSION:

In the present work a simple, specific and reproducible RP-HPLC method was developed for the quantitative determination of NAC and cefexime from its fixed dose combination. The need for the development of an analytical method was identified because of the inadequate capacity of the reported HPLC methods in retaining both the drugs with adequate peak shape within a reasonable run time. The developed method shows good resolution between the two drugs. The equilibrium between the various chromatographic conditions like column temperature, pH, and organic phase content permitted the retention of both the drugs within a reasonable run time. The method has been validated as per ICH guidelines for specificity, linearity, accuracy, precision, robustness. The developed method was applied in the assay of the marketed tablet.

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Received on 16.04.2016 Modified on 21.05.2016

Research J. Pharm. and Tech. 2016; 9(7):835-842

DOI: 10.5958/0974-360X.2016.00158.X