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

ABOUT AUTHORS:
Patel Sannil R*, Patel Satish A
Department of Quality Assurance,
Shree S. K. Patel College of Pharmaceutical Education & Research,
Ganpat University, Ganpat Vidyanagar – 384012, Mehsana, Gujarat, India.
*srpatel2200@gmail.com

ABSTRACT
The present manuscript describes simple, sensitive, rapid, accurate, precise and economical First order derivative spectrophotometry method for the simultaneous determination of Meropenem and Sulbactam Sodium in bulk and combined dosage form. The absorbance values at 333nm and 252 nm of first derivative spectrum was used for the estimation of Meropenem and Sulbactam Sodium, respectively without mutual interference. This method obeyed beer’s law in the concentration range of 5-70 μg/ml for Meropenem and 2-21 μg/ml for Meropenem. The method was successfully applied to pharmaceutical combined dosage form because no interference from the excipients was found. The suitability of this method for the quantitative determination of Meropenem and Sulbactam Sodiumwas proved by validation. The proposed method was found to be simple and sensitive for the routine quality control analysis of Meropenem and Sulbactam Sodium in bulk and combined dosage form. The results of analysis have been validated statistically and by recovery studies.

REFERENCE ID: PHARMATUTOR-ART-1781

INTRODUCTION
Meropenem, chemically 3-[5-(dimethylcarbamoyl) pyrrolidin-2-yl] sulfanyl-6-(1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylic acid, is an anti-bacterial agent for systemic use¹. The bactericidal activity of Meropenem results from the inhibition of cell wall synthesis. Meropenem readily penetrates the cell wall of most gram-positive and gram-negative bacteria to reach penicillin-binding- protein (PBP) targets. It is official in Indian Pharmacopoeia (IP)². Literature survey reveals UV and HPLC methods for the estimation of Meropenem in pharmaceutical formulations^5-8. Sulbactam is (R)-3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid 4, 4-dioxide is a beta-lactamase inhibitor. Sulbactam is an irreversible inhibitor of beta-lactamase; it binds the enzyme and does not allow it to interact with the antibiotic. It is official in United States Pharmacopoeia (USP)³. Literature survey reveals UV and HPLC methods for the estimation of Sulbactam with other drugs in pharmaceutical formulations^9-18. Literature survey also reveals spectrophotometric method based simultaneous equations for simultaneous estimation of Meropenem and Sulbactam in mixture by using 0.1M NaOH as a solvent⁴. The present communication describes simple, sensitive, rapid, accurate, precise and cost effective spectrophotometric method based on First order derivative spectrophotometry method for simultaneous estimation of both drugs in their combined dosage form by using 0.1M KOH as a solvent .

MATERIALS AND METHODS
Apparatus
A shimadzu model 1700 (Japan) double beam UV/Visible spectrophotometer with spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cell was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe 2.0 system software. A Sartorius CP224S analytical balance (Gottingen, Germany), an ultrasonic bath (Frontline FS 4, Mumbai, India) was used in the study.

Reagents and Materials
Meropenem (MERO) and Sulbactam Sodium (SUL) were kindly supplied as a gift samples from Montage Laboratories, Himmatnagar, Gujarat, India. The pharmaceutical formulation containing 1000 mg MERO and 500 mg SUL was kindly purchased from local market. Potassium Hydroxide (S.D. Fine Chem Ltd., Mumbai) and Whatman filter paper no. 41 (Millipore, USA) were used in the study.

Preparation of 0.1M Potassium Hydroxide Solution
An accurately weighed 5.611 gm Potassium Hydroxide Pallets were dissolved in 1000 ml distilled water.

Preparation of standard stock solutions
An accurately weighed quantity of standard MERO (10 mg) and SUL (10 mg) powder were transferred to 100 ml separate volumetric flasks and dissolved in 0.1 M KOH. The flasks were sonicated for 5 minutes and volumes were made up to mark with 0.1 M KOH to give a solution containing 100 μg/ml each of MERO and SUL.

Methodology
This method is based on First order derivative spectroscopy to overcome spectral interference from other drug. Zero order spectrums of both the drugs were converted to First order derivative spectra with the help of spectra manager software.

It was observed that MERO showed dA/dλ at 333 nm in contrast to SUL that has considerable dA/dλ at this wavelength. Further, MERO has zero dA/dλ at 252 nm while at this wavelength SUL has significant dA/dλ. Therefore wavelengths 333 nm and 252 nm were employed for the determination of MERO and SUL respectively without interference of other drug. The calibration curves were plotted at these two wavelengths of concentrations against dA/dλ separately. Eight working standard solutions having concentration 5, 10, 20, 30, 40, 50, 60, 70 μg/ml for MERO and 2, 3, 6, 9, 12, 15, 18, 21 μg/ml for SUL were prepared in 0.1 M KOH from the mixture and the absorbances at 333 nm (zero crossing point for SUL) and 252 nm (zero crossing point for MERO) were measured and the calibration curves were plotted at these wavelengths.

Validation of the proposed method
The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines¹⁹

Linearity (calibration curve)
The calibration curves were plotted over a concentration range of 5-70 µg/ml for MERO and 2-21 µg/ml for SUL. Accurately measured standard solutions of MERO (0.5, 1, 2, 3, 4, 5, 6, 7ml) and SUL (0.2, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1ml) were transferred to a series of 10 ml of volumetric flasks and diluted to the mark with 0.1 M KOH. The absorbances of the derivatised spectra were measured at 333 nm and 252 nm for MERO and SUL respectively against 0.1 M KOH as blank. Six replicate analysis were carried out. Absorbance Vs concentration were plotted to obtain the calibration graph. Both drugs obey the Beer‘s law with the above concentration range with R² value of 0.9989 and 0.9983 for MERO and SUL, respectively.

Method precision (repeatability)
The precision of the instrument was checked by repeated scanning and measurement of absorbance of solutions (n = 6) for 20 µg/ml MERO and SUL 9 µg/mlwithout changing the parameter of the proposed spectrophotometry method.

Intermediate precision (reproducibility)
The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of standard solutions of MERO and SUL(20, 30, 40 µg/ml for MEROand 9, 12, 15 µg/ml for SUL). The result was reported in terms of relative standard deviation (% RSD).

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 D₁value 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

150

100.56±1.27

100.63±1.25

100.48±1.25

SUL

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

99.42

50.04

99.42± 0.68

100.08 ± 0.55

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

Figure 1: Chemical structure of Meropenem(MERO)

Figure 2: Chemical structure of Sulbactam Sodium(SUL)

Figure 3: Zero order overlain spectra of MERO 10µg/ml and SUL 10µg/ml in 0.1 M KOH

Figure 4: Overlain First Order derivative spectra ofMERO and SUL.

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