A NEW VALIDATED THIRD ORDER DERIVATIVE SPECTROSCOPIC METHOD FOR SIMULTANEOUS ESTIMATION OF METOPROLOL SUCCINATE AND RAMIPRIL IN TABLET DOSAGE FORM

Objective: The objective of the present work is to develop and validate a new UV derivative spectrophotometric method for simultaneous estimation of metoprolol succinate and ramipril in methanol: water (50:50v/v). Methods: “Zero crossing technique” was chosen for quantitative determination. The zero-crossing points (ZCP’s) were found to be 209 nm where metoprolol succinate was quantified and 211 nm where ramipril was quantified. This method was then subjected to accuracy, linearity, sensitivity and reproducibility according to ICH guidelines to ensure and confirm its validity. Results: The method was found to be obeying Beer’s law in the range of 10-50 µg/ml and 5-25 µg/ml for metoprolol succinate and ramipril, respectively. The % recoveries were observed between the range of 99.2-100.2 for metoprolol succinate and 99.57-99.86 for ramipril. The intra-day and inter-day results showed reproducibility. Conclusion: It can be concluded that the developed third-order UV derivative spectroscopic method for the simultaneous determination of metoprolol succinate and ramiprilcan be recommended for routine quantitative analysis.

Literature survey revealed that metoprolol succinate and ramipril can be estimated either individually or combinedly by different methods like HPLC [5][6][7], LC [8], RP-UPLC [9], UV-Spectroscopy [10,11]. However, simultaneous estimation of these drugs by derivative spectrophotometric method was not reported till date. Hence a third order derivative spectrophotometric method was developed for the simultaneous estimation of these drugs for the first time. The developed UV derivative method was validated according to the ICH guidelines.

Chemicals and reagents
Analytically pure metoprolol succinate and ramipril from Yarrow chemicals, Mumbai were used. PROLOMET R 50 tablets (in the dose of 50 mg metoprolol succinate and 5 mg ramipril) manufactured by Sun Pharma were procured and used for the investigation.

Preparation of ramipril standard stock solution
25 mg of ramipril was accurately weighed and transferred into a 25 ml volumetric flask and made up with methanol and water in the ratio of 50:50 v/v to get 1000 µg/ml solution. From the above stock solution different working standards in the range of 5-25 µg/ml were prepared.

Method development
For quantitative estimation, UV derivative spectroscopic method using zero crossing technique was chosen [12]. The working standard solutions containing 30 μg/ml of metoprolol succinate and 15 µg/ml of ramipril were scanned in the wavelength range of 200-400 nm using methanol and water solvent (50:50 v/v) as reference in derivative mode at bandwidth of 2 nm and a scan speed of 400 nm/min. It showed wavelength maxima at 215 nm for metoprolol succinate ( fig. 3) and 210 nm for ramipril ( fig. 4). The isobestic point was found to be 216 nm as shown in (fig. 5). The obtained zero order spectra were converted to first order ( fig. 6), second order ( fig. 7) and third order spectrum ( fig. 8).

Method validation
The developed method was validated in terms of linearity, accuracy, intra-day and inter-day precision studies, detection limit and quantification limit according to ICH guidelines [13,14]. For the third order derivative method, 10-50 μg/ml of metoprolol succinate and 5-25 μg/ml of ramipril were scanned at the working wavelengths of 209 nm and 211 nm for metoprolol succinate and ramipril respectively. Calibration curves were constructed by plotting concentration against the absorbance values and correlation coefficients were calculated from the regression line equations.

Accuracy
To check the accuracy of the developed method, recovery studies were carried out by applying standard addition method. A known amount of standard metoprolol succinate and ramipril corresponding to 80, 100 and 120% of the label claim was added to pre-analyzed sample of the tablet. The recovery studies were carried out at each level three times and the average amount was calculated.

Precision
The precision of the developed method was carried out by intermediate precision. The intraday and interday precisions of the third order derivative method were determined by analyzing responses in triplicate on the same day and on 3 different days over a period of 1 w of metoprolol succinate (50 µg/ml) and ramipril (5 µg/ml).

Sensitivity
The LOD and LOQ values were calculated from the linearity data using Microsoft excel 542010 version. Standard deviation of the analytical response and the slope of the calibration curve method was followed according to the equations, LOD = 3.3σ/S and LOQ = 10σ/S, where σ is the standard deviation of the sample and S is the slope.

Assay of commercial brand
Twenty tablets of PROLOMET R 50 were taken, weighed and finely powdered. The weight equivalent to the labelled claim (55 mg) was taken, 20 ml of solvent (methanol and water 50:50v/v) was added in a 100 ml volumetric flask and sonicated in an ultrasonic bath for 15 min. This solution was then made upto 100 ml using the same solvent, filtered through a 0.45 μm filter and this filtrate was used to prepare sample solution (55 µg/ml). The mixture solution was scanned at the wavelengths of metoprolol succinate (209 nm) and ramipril (211 nm) using a UV-Visible spectrophotometer. These absorbance values were then substituted in the regression equations to calculate the concentration.

RESULTS AND DISCUSSION
Few chromatographic methods have been mentioned in the literature to date for the simultaneous estimation of metoprolol succinate and ramipril in their binary mixtures [6,8,15]. Also one spectrophotometric method, based on simultaneous equation has been reported for combination of metoprolol succinate, atorvastatin calcium and ramipril [16]. However, no spectrophotometric method has been reported for metoprolol succinate and ramipril in their fixed dose comination.

Third order derivative method
From the zero order overlain UV spectra of Metoprolol succinate (30 µg/ml) and ramipril (15 µg/ml), it was found that the spectra are overlapping each other, exhibiting the complexity in measuring these drugs by direct UV absorption method in a binary mixture. The UV derivative method has advantage that it removes the spectral interference from one of the two drugs while estimating the other drug at zero crossing point. The first order spectra did not show any zero crossing points (ZCP), while the second order spectra was not informative enough to carry out further experimentation. Hence in the present investigation, third order derivative spectrophotometric techniques was described. The third order spectrum showed zero crossing points (ZCP) at 211 nm for metoprolol succinate and 209 nm for ramipril. Hence working wave lengths of 209 nm for metoprolol succinate and 211 nm for ramipril were selected.

Validity of the method
The linearity was found in the range of 10-50 µg/ml and 5-25 µg/ml for metoprolol succinate and ramipril. The calibration curves were shown in ( fig. 9 and fig. 10) and the correlation coefficient (r 2 ) value was found to be 0.998 and 0.997, respectively. The r 2 values for both the drugs were found to be less than or equal to 1. Hence the developed method was found to be linear. The % recoveries were found to be 99.2%, 100.02% and 99.18% for metoprolol succinate and 99.5%, 99.4% and 99.81% for ramipril respectively. As the % recovery values were found to be between 98%-102%, the developed method can be considered to be accurate. The intraday % RSD was found to be 0.1008 for metoprolol succinate and 0.424 for ramipril. The inter-day % RSD was found to be 0.02693 for metoprolol succinate and 0.754 for ramipril. As the % RSD values were found to be 2%, the developed method can be considered to be precise. Detection limit was found to be 3.18 µg/ml for metoprolol succinate and 1.56 µg/ml for ramipril. Quantification limit was found to be 9.62 µg/ml for metoprolol succinate and 4.68 µg/ml for ramipril. The assay of the commercial brand was carried out and the recovery was found to 101.6% for metoprolol succinate and 98.8% for ramipril. The results of the validation are summarized in table 1, 2, 3, 4 and 5.

CONCLUSION
A simple, precise, economical and sensitive UV derivative spectroscopic method was developed for the simultaneous estimation of metoprolol succinate and ramipril. The developed method was validated according to the ICH guidelines and was applied to the pharmaceutical dosage form. The result of the assay obtained was found to be within the acceptable limits. Hence, the developed method was said to be effective for routine analysis of metoprolol succinate and ramipril in tablet dosage form.