Application of simultaneous Equation Method for the Determination of Azithromycin and Cefixime Trihydrate in Tablet Formulation

 

Jagdish V. Manwar¹, Bhagwat U. Nagargoje², Vishal C. Gurumukhi¹, Dipti G. Ratnaparkhi¹,Poonam P. Warade¹, Dipak D. Kumbhar¹*, R.L. Bakal¹, Rahul S. Manmode³

¹ KYDSCT College of Pharmacy, Sakegaon (Bhusawal) 425 201, Dist. Jalgaon, MS, India

² Maharashtra Institute of Pharmacy, MIT Campus, Kothrud, Pune 411038, MS, India

³ Genzyme Corporation, 500 Soldiers Field Road, Allston 02134, MA, USA

 

ABSTRACT:

A simple, accurate, and precise uv-spectrophotometric method has been developed for the simultaneous estimation of azithromycin (AZI) and cefixime trihydrate (CEFI) in tablet formulation. The method was based on employing simultaneous equation method for the analysis of both drugs. AZI and CEFI have shown absorbance maxima at 222 and 289 nm in methanol, respectively. The linearity was obeyed in the concentration range of 10-50µg/ml for both drugs, with a significantly high correlation coefficient (r² = 0.999). The limits of detection for AZI and CEFI were 0.81 and 1.52 µg/ml, respectively, and the limits of quantitation for AZI and CEFI were 2.40 and 4.60 µg/ml, respectively. The suitability of the developed method for quantitative determination of drugs was proved by validation. The method was successfully used to analyze a tablet formulation.

 

 

1. INTRODUCTION:

There are number of analytical methods for the determination of drugs from bulk and various formulations like tablets, capsules, injections, etc. These methods include Uv-spectrophotometry^1-3, HPLC^4-8, UPLC^9-10, Gas chromatography^11-12, etc. Azithromycin (AZI) is a chemically (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-11-{[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-}oxy}-1-oxa-6-azacyclopentadec-13-yl-2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranoside (Fig. 1).¹³  It is an azalide category of drug, which is subclass of the macrolide antibiotic.^14-16  It is mainly used in upper respiratory tract infections.¹⁷

 

Fig. 1: Structure of azithromycine.

 

Cefixime trihydrate (CEFI) is chemically (6R,7R)- 7-{[(2Z)-2-(2-amino- 1,3-thiazol-4-yl)-2-methoxyimino-acetyl]amino}-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid (Fig. 2). CEFI is a third-generation cephalosporin antibiotic.¹⁸ It is used in treatment of lower respiratory tract, urinary tract, and other soft tissues infections.^19-20 AZI and CEFI are official in Indian Pharmacopoeia and United States Pharmacopoeia.^13,21

 

Fig. 2: Structure of cefixime trihydrate.

 

Combination of AZI sodium and CEFI is available in the market which is used to treat or prevent bacterial infections especially throat infection^22-23. Literature survey revealed only two Uv-spectrophotometric methods were reported for simultaneous estimation of AZI and CEFI in bulk drug and Pharmaceutical dosage forms.^24-25 Nevertheless, there was no report of simultaneous equation method for the simultaneous determination of AZI and CEFI in combined dosage form. Thus, the prime objective of present work was to develop new uv-spectrophotometric method for the simultaneous determination of AZI and CEFI in combined dosage forms. After examining the overlain spectra of both the drugs, simultaneous equation method was applied for the simultaneous determination both the drugs in tablet dosage forms.

 

2.THEORY:

Simultaneous equation method method is used where a sample contain two absorbing drugs (X and Y) each of this absorbs at the λ_max of each other i.e. λ₁ and λ₂, it may be possible to determine both the drugs by the technique of simultaneous equation method provided that certain criteria apply. The information required is (i) absorptivity of X at λ₁ and λ₂ and a_x1 and a_x2 respectively, (ii) absorptivity of Y at λ₁ and a_y1 and a_y2 respectively, (iii) absorbance of the diluted sample at λ₁ and λ₂, A₁ and A₂ respectively. Let C_x and C_y be the concentration of X and Y respectively in the diluted sample. Two equations are constructed based upon the fact that at λ₁ and λ₂ the absorbance of the mixture is the sum of the individual absorbance of X and Y.

 

At  λ₁   A₁ = a_x1 bc_x + a_y1 bc_y ………(1)

At   λ₂    A₂ = a_x2 bc_x + a_y2 bc_y ……….(2)

 

Rearrange eq. (2)

c_y = A₂ - a_x2c_x / a_y2

 

Substituting for c_y in eq. (1), and rearranging gives

 

c_x = A₂a_y1 – A₁a_y2 / a_x2a_y1 – a_x1ay2

and

c_y = A₁a_x2 – A₂a_x1 / a_x2a_y1 – a_x1a_y2

 

Criteria for obtaining maximum precision, based upon absorbance ratios, have been suggested that place limit on the relative concentrations of the components of the mixture. The criteria are that the ratios,

 

A₂/A₁/ a_x2/ a_x1

and

a_y2/a_y1/ A₂/A₁

 

For the precise determination of Y and X respectively, these ratios should lie outside the range of 0.1-20. These criteria are satisfied only when the λ_max of the two components are reasonably dissimilar. An additional criterion is that the two components must not interact chemically, thereby negating the initial assumption that the total absorbance is the sum of individual absorbance.

 

3.MATERIALS AND METHODS:

3.1. Instrument:

A Shimadzu UV-Visible spectrophotometer model 1600 with matched pair of quartz cell (1.0 cm path) was used for absorption measurements.

 

3.2. Chemical and reagents:

Pure drug sample of AZI and CEFI were obtained as a gift sample from Ajanta pharmaceutical Ltd, Mumbai. Methanol AR grade of Rankem Ltd., New Delhi was used. Drug product samples (tablets) were obtained from local market. Tablet use for analysis was HYFEN-AZ  manufactured by Hetro labs (Hyderabad, India) containing AZI 250 mg and CEFI 200 mg.

 

3.3. Selection of common solvent:

Methanol was selected as common solvent for studying spectral characteristics of the selected drugs.

 

3.4. Preparation of standard stock solutions:

Stock standard solutions of AZI and CEFI were separately prepared by dissolving 1000 mg in 1000 ml volumetric flask containing 30 mL methanol, shaken for 30min and the volume was made up to the mark with methanol (1000 µg/ml of each drug).

 

3.5. Selection of analytical wavelength:

Appropriate volume, 10 ml of AZI and 10 ml CEFI standard stock solution was transferred to two separate 100 ml volumetric flasks and the volume was adjusted to mark with methanol to get concentration 10µg/ml and 10µg/ml, respectively. The solutions were scanned separately in the UV-region i.e. 200-400nm.

3.6. Linearity and Range: Selection of Concentration

In order to select the final concentration of each drug, a separate calibration graph for AZI and CEFI was plotted as absorbance versus concentration by measuring the absorbance of each solution at 289 and 222 nm and regression equations were established. For this purpose, a series of dilutions (10, 20, 30, 40 and 50 μg/ml) were made separately for each drug using standard stock solution. From calibration graph, final concentration for AZI and CEFI were 50μg/ml and 40μg/ml, respectively. The limit of detection and limit of quantitation were measured on the basis of signal-to-noise ratios.

 

3.7. Determination of Absorptivities:

 (A (1%, 1 cm)). The absorbance (a) of selected concentration for both drugs was taken at selected wavelength. These absorbance values were then divided by concentration in gm/l to get absorptivities A (1%, 1 cm).

 

3.8. Methodology and Simultaneous Equation:

For each drug, the absorptivity value A (1%, 1 cm) was determined at both the wavelengths for each drug. A set of two simultaneous equations was formed as:

 

c_x = A₂a_y1 – A₁a_y2 / a_x2a_y1 – a_x1ay₂    

and

c_y = A₁a_y2 – A₂a_y1 / a_x2a_y1 – a_x1a_y2

 

where,

A₁ and A₂ are absorbances of mixture at 289 nm and 222 nm;

a_x1 and a_y1 are absoptivities of AZI and CEFI respectively at 289 nm;

a_x2 and a_y2 are absoptivities of AZI and CEFI respectively at 222 nm;

 

3.9. Calculation of Concentration:

The values c_x and c_y were calculated by putting the values of A₁ and A₂ to solve the simultaneous equations 1 and 2.

 

3.10. Assay of Tablet Formulation:

Twenty tablets of marketed combination were weighed accurately and crushed to fine powder. A quantity of tablet powder equivalent to 50 mg of AZI was accuratly weigheed and transferred to 100 ml volumetric flask. To this flask, about 20-30 ml of methanol was added. The flask was shaken for 30 min and then sonicated for 30 min. After complete dissolution, final volume was made to 100 ml with the same solvent. The solution was filtered and was further diluted to get a final concentration of about 50µg/ml of AZI and 40µg/ml of CEFI. The response of sample solutions was measured at 222 nm and 289 nm against blank. The content of AZI and CEFI in tablet dosage form was calculated using two framed simultaneous equations and derivative method.

 

4. VALIDATION OF METHOD:

The method was validated according to the ICH guidelines.²⁶ Parameters studied for validation were accuracy, precision, linearity, LOD, LOQ and specificity for the analyte.

 

4.1. Accuracy:

To study the accuracy, reproducibility and precision of the proposed method, recovery study was carried out by addition of standard drug solutions to preanalysed sample. Recovery studies of proposed method was carried out on the basis of standard addition method at different levels (i.e. 80, 100, 120%). Tablet solution containing 50 mcg/ml of AZI and 40 mcg/ml of CEFI considered at 100% and from that three different levels i.e. 80%, 100%, 120% was prepared by using standard stock solution of each drug. Then by taking the absorbance calculate the amount added and find out percentage recovery.

 

4.2. Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ values were determined by diluting known concentrations of drug until the average responseswere approximately 3 or 10 times the standard deviationof the responses of the blank for six replicate determina-tions. The signal/noise ratios 3:1 and 10:1 were taken asthe LOD and LOQ, respectively.

 

4.3. Precision:

precision of method was checked by measuring the absorbance of standard solution for  AZI (50 µg/ml) and CEFI (40 µg/ml) repeatedly (n = 6).  

 

5. RESULTS AND DISCUSSION:

Simultaneous equation method was applied for the determination of AZI and CEFI in tablet formulation. Based on the solubility of both the drugs, methanol was selected as a common solvent in the present study. From the overlain spectra (Fig. 3), two wavelengths 222 nm (λ_max of AZI) and 289nm (λ_max of CEFI) were selected for the formation of simultaneous equation.

 

Fig. 3: The overlain Spectra of AZI and CEFI in methanol.

 

Both the drugs had broad spectral features in the range of wavelength from 200-400nm. Their spectral peaks were well separated. The peak distribution of AZI was range 200-250nm with peak maximum at the 222nm. For CEFI the peak distribution of CEFI was range 200-400nm with peak maximum at the 289nm. For calculation using simultaneous equation, we have to find wavelength where one component absorbs strongly while another absorbs weakly. Because the two drugs were well separated spectrally, one cannot use the maximum of one in the analysis as the molar absorptivity of the other component would be zero. Following the recommended procedure, calibration curves were made at selected wavelength for the standard of AZI and CEFI. The linearity and range was studied by analyzing the standard solution of AZI and CEFI separately in triplicate. The calibration curves for AZI and CEFI are shown in Fig. 4 and Fig. 5, respectively. The standard deviation of the slope value was less than 2 (see Table 1).

 

Fig. 4: Calibration curve for standard solution of AZI.

 

Fig. 5: Calibration curve for standard solution of CEFI.

 

The LOD for AZI and CEFI was found to be 0.81 µg/ml and 1.52 µg/ml, respectively. The LOQ for AZI and CEFI was found to be 2.40 µg/ml and 4.60 µg/ml, respectively (Table 1).

Table 1: Results of linearity study (n=6)

Parameters	Results
	AZI	CEFI
Linearity range (µg/ml)	10-50	10-50
Correlation coeff. (r2)	0.999	0.999
Slope	0.005	0.041
LOD (µg/ml)	0.81	1.52
LOQ (µg/ml)	2.40	4.60

 

Molar absorptivities of AZI and CEFI were obtained from the calibration curves made at selected wavelength. Absorptivity values calculated for each drug at both the wavelengths are shown in Table 2. The AZI shows higher absorptivity than CEFI. This is because of higher sensitivity of CEFI as compare to AZI that is because of more number of chromophoric groups present on CEFI. These groups –NH₂, -NH-, and  =N-O-.

 

Table 2: Estimated absorptivities for AZI and CEFI

Components	Absorptivity
	at 222nm	at 289 nm
AZI	0.57	0.92
CEFI	39.25	44.5

 

In the assay of tablet formulation, the % relative standard deviation by proposed method in tablet for AZI and CEFI were 0.0165 and 0.0165. The recovery for AZI and CEFI were found to be 99.90% to 99.97% w/w, respectively. The method was validated to ensure accuracy and reproducibility. The recovery studies and statistical data for the method were found to be satisfactory and therefore the method can be used for routine analysis. The results are shown in Table 3.

 

Table 3: Results of assay of tablet formulation and recovery study (n = 6)*

Level of Recovery	Amt. present (mg/tab)		Amt. of std. drug added		Total amt. recovered		Percent Recovery
	AZI	CEFI	AZI	CEFI	AZI	CEFI	AZI	CEFI
80%	250	200	200	160	449.50	359.87	99.88	99.96
100%	250	200	250	200	499.56	499.91	99.91	99.98
120%	250	200	300	240	549.60	439.85	99.92	99.96
		Avg.					99.90	99.97
		Std. Dev.					0.0164	0.0114
		%RSD					0.0165	0.124

*Amount in milligram (mg)

 

5. CONCLUSION:

The results confirm the method is accurate and free from any negative or positive interfering of other compounds present in formulation. Thus it concludes that proposed simultaneous equation method is suitable for the determination of AZI and CEFI from bulk drug and tablet formulation.

 

6. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 15.11.2016          Modified on 16.12.2016

Accepted on 28.12.2016        © RJPT All right reserved