SPECTROPHOTOMETRIC METHOD FOR ESTIMATION OF MEROPENEM AND SULBACTAM SODIUM IN COMBINED DOSAGE FORM BY FIRST ORDER DERIVATIVE METHOD

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Accuracy (recovery study)
The accuracy of the method was determined by calculating recovery of MERO and SULby the standard addition method. Known amounts of standard solutions of MERO and SULwere added at 50, 100 and 150 % level to prequantified sample solutions of MERO and SUL. The amounts of MERO and SULwere estimated by applying obtained values to the respective regression line equations. The experiment was repeated for three times.

Limit of detection and Limit of quantification
The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines19.
LOD = 3.3 × σ/S

LOQ = 10 × σ/S

Where, σ = the standard deviation of the response
S = slope of the calibration curve

Analysis of MERO and SULfrom Combined dosage form
Powder equivalent to 10mg of MERO and 5mg SULwas transferred to 100.0 ml volumetric flask, 0.1 M KOHadded, sonicated for 20 minutes and volume was made-up to the mark with 0.1 M KOH. The solution was then filtered through a Whatman filter paper (No.41). The filtrate was further diluted with 0.1 M KOHto obtain 10μg/ml of MEROand 5μg/ml of SUL. From the derivative spectra, the absorbance at 333 nm and 252 nm were noted for the estimation of MERO and SUL, respectively. From these absorbance values, the concentrations of MERO and SUL were determined using calibration graph. The analysis procedure was repeated six times with mixture.

RESULTS
Zero-order absorption spectra of MERO and SUL showed overlapping peaks that interfere with the simultaneous determination of this formulation (Figure 1). Derivative spectroscopy, based on a mathematical transformation of the spectra zero-order curve into the derivative spectra, allows a fast, sensitive and precise resolution of a multicomponent mixture and overcomes the problem of overlapping of a multicomponent system. Derivative spectroscopy on the basis of zero-crossing measurements involves measurement of the absolute value of the total derivative spectrum at an abscissa value corresponding to the zero-crossing wavelength of the derivative spectra of individual components, which should be only a function of the concentration of other component. The spectroscopic parameters including derivative order, wavelength and Δλ values should be optimized to obtain maximum resolution, sensitivity and reproducibility. In this study first derivative technique (D1) traced with Δλ= 8 nm was used to resolve the spectral overlapping. The optimums D1 values without interference for MERO and SUL were 333 and 252 nm, respectively (figure 4).

The linearityof the method was established from first-derivative spectra by measurement of the absorbance of standard mixture solutions containing varying concentrations of each compound. The calibration curves were constructed by plotting the D1 value against MEROand SULconcentration at the zero-crossing wavelength of SUL(333nm) and MERO(252nm), respectively.

Linear correlation was obtained between absorbances and concentrations of MEROand SULin the concentration ranges of 5-70 µg/ml and 2-21µg/ml, respectively. The linearity of the calibration curve was validated by the high values of correlation coefficient of regression. Relative standard deviation was less than 2 %, which indicates that proposed method is repeatable. The low % RSD values of interday (0.92– 1.02and 0.17 – 0.27 for MEROat 333nm and SULat 252nm, respectively) and intraday (0.91– 1.81and 0.18 – 0.29for MEROat 333and SULat 252nm, respectively). Low % RSD values for MEROand SUL, reveal that the proposed method is precise. LOD and LOQ values for MEROwere found to be 1.71 and 5.17 µg/ml at 333nm. LOD and LOQ values for SULwere found to be 0.09 and 0.27 µg/ml at 252nm.These data show that method is sensitive for the determination of MEROand SUL. The regression analysis data and summary of validation parameters for the proposed method is summarized in Table 1.

The recovery experiment was performed by the standard addition method. The mean recoveries were 100.55 ± 1.25 and 99.63 ± 0.26 for MERO and SUL, respectively (Table 2). The resultsof recovery studies indicate that the proposed method is highly accurate. The proposed validated method was successfully applied to determine MERO and SUL in their combined dosage form. The results obtained for MERO and SUL were comparable with the corresponding labeled amounts (Table 3). No interference of the excipients with the absorbance of interest appeared; hence the proposed method is applicable for the routine simultaneous estimation of MERO and SUL in pharmaceutical dosage forms.

DISCUSSION
The proposed spectrophotometric method was found to be simple, sensitive, accurate and precise for determination of MERO and SUL in combined dosage form. The method utilizes easily available and cheap solvent for analysis of MERO and SUL hence the method was also economic for estimation of MERO and SUL from combined dosage form. The common excipients and other additives in the injection dosage formdo not interfere in the analysis of MERO and SUL in method; hence it can be conveniently adopted for routine quality control analysis of the drugs in combined pharmaceutical formulation.

ACKNOWLEDGEMENT
The authors are thankful to Montage Laboratories, Himmatnagar, Gujarat, India for providing gift sample of MERO and SUL for carry out the research work. The authors are highly thankful to S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Ganpat Vidyanagar – 384012, Mehsana, Gujarat, India for providing all the facilities to carry out the research work.

Table 1: Regression analysis data and summary of validation parameters for MERO and SUL

Parameters

MERO

SUL

Wavelength  (nm)

333

252

Beer’s law limit (µg /ml)

5-70

2-21

Regression equation

(y = a + bc)

             Slope (b)

Intercept (a)

y = 0.00008x + 0.00048

0.00008

0.00048

y = 0.00163x - 0.00234

0.00163

0.00234

Correlation coefficient (R2)

0.9989

0.9983

LOD (µg/ml)

1.71

0.09

LOQ (µg /ml)

5.17

0.27

Repeatability (% RSD,

n = 6)

1.58

0.28

Precision (% RSD, n = 3)

Interday

Intraday

0.92-1.02

0.91-1.81

0.17-0.27

0.18-0.29

Accuracy ± S. D.

(% Recovery, n = 3)

100.55±1.26

99.63±0.26

Assay ± S. D. (n = 6)

99.42±0.68

100.08±0.55

RSD = Relative standard deviation. LOD = Limit of detection. LOQ = Limit of quantification S. D. is standard deviation

Table 2: Recovery data of MEROand SUL

For MERO: 100μg/ml sample solution, 100 μg/ml standard stock solution

For SUL: 50 μg/ml sample solution, 100 μg/ml standard stock solution

Drug

Amount taken (µg/ml)

Amount added (%)

% Recovery ± S. D. (n = 3)


MERO


100

100

100

50

100

150

100.56±1.27

100.63±1.25

100.48±1.25


SUL


50

50

50

50

100

150

99.52±0.47

99.33±0.16

100.04±0.15

S. D. = Standard deviation.        n = Number of determinations

Table 3: Analysis of MEROand SULin combined dosage form

Label claim (mg)

Amount found (mg)

% Label claim ± S. D.

(n = 6)

MERO

SUL

MERO

SUL

MERO

SUL

100

50

99.42

50.04

99.42± 0.68

100.08 ± 0.55

 S. D. = Standard deviation.        n = Number of determinations.

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