Development and Validation of Stability-Indicating RP-HPLC Method for Determination of Metoprolol Succinate and Olmesartan Medoxomil in Bulk and in Formulation

 

Anamika Singh¹, Jaya Dwivedi¹, Santosh V. Gandhi²*

¹Department of Chemistry, Banasthali University, Banasthali, Rajasthan- 304022

²Department of Quality Assurance, AISSMS College of Pharmacy, Kennedy Road, Near RTO, Pune – 411001, Maharashtra, India.

 

ABSTRACT:

An accurate, precise, robust and economic stability-indicating RP HPLC method for determination of Metoprolol succinate and Olmesartan medoxomil in bulk and in formulation has been developed and validated. Separation was carried out on HiQSil C18 column (250 x 4.6 mm, 5 µm) at flow rate of 1 ml/min using Methanol: 10 mM o-phosphoric acid buffer (50: 50 v/v) and detection at 216 nm. On a stabilized chromatographic condition Metoprolol succinate and Olmesartan medoxomil gave acceptable peak at RT 3.66 ± 0.04 min and 18.14 ± 0.05 min, respectively. The calibration curve for Metoprolol succinate and Olmesartan medoxomil was found to be linear in the range 1-25 µg/ml and 1-10 µg/ml, respectively. The method was validated in accordance with international conference on harmonization (ICH) guidelines and prescribed stress conditions. The drugs were subjected to stress condition of hydrolysis (acid, base, neutral), oxidation, photolysis and thermal degradation. The proposed method can be applicable for the routine determination of Metoprolol succinate and Olmesartan medoxomil in bulk and formulation.

 

 

INTRODUCTION:

Metoprolol succinate (METO), chemically, (RS)-1-(Isopropyl amino)-3-[4 (2methoxyethyl) phenoxy] propan2-ol (Figure 1) is used as an antihypertensive¹. Olmesartan medoxomil (OLME), 4-(2-hydroxypropan-2-yl)-2-propyl-1-({4-[2-(1H-1,2,3,4-tetrazol-5-yl)phenyl] phenyl} methyl)-1H-imidazole-5-carboxylic acid (Figure 2) is widely used in the treatment of hypertension².

 

Figure 1: Structure of Metoprolol succinate

 

Figure 2: Structure of Olmesartan medoxomil

 

The literature survey reveals that several UV-VIS Spectrophotometric^3-9 , HPLC^{10- 23} and HPTLC ^24-26 methods have been reported for the analysis of METO and OLME as a single drug or in combination with other drugs in pharmaceutical dosage form.

 

No reports were found for stability-indicating HPLC method for simultaneous determination of METO and OLME in tablet dosage form. This paper describes simple, precise, accurate and sensitive HPLC method development and validation as well as stability study (hydrolysis, oxidation, photo-degradation and thermal degradation) as per International Conference on Harmonization       Guidelines. ^27,28

 

EXPERIMENTAL:

Reagents and chemicals:

METO and OLME were supplied as gift sample by Cadila Healthcare Ltd., Ahmadabad, India. The brand of tablets Olsar-M (Manufactured by- Unichem Pvt. Ltd., Pune, India) labeled to contain 50 mg of Metoprolol succinate and 20 mg of Olmesartan medoxomil were procured from local market. Methanol (HPLC grade) was obtained from S. D. Fine Chem. Ltd (Mumbai, India). HPLC grade water is collected at college using ELGA water purification system and potassium hydrogen phosphate, sodium hydroxide, o- phosphoric acid (all are AR grade) were purchased from S. D. Fine chem. Limited (Mumbai, India).

 

Instrumentation and chromatographic conditions:

HPLC system used was JASCO system equipped with Model PU 2080 Plus pump, Rheodyne sample injection port (20 μl), MD 2010 PDA detector and Borwin- PDA software (version 1.5). Separation was carried out on HiQSil C₁₈ column (250 x 4.6 mm, 5 µm) at flow rate of 1 ml/min using Methanol: 10 mM o-phosphoric acid buffer (50: 50 v/v) and detection at 216 nm.

 

Preparation of standard stock solution:

Standard stock solution of METO and OLME were prepared separately by dissolving 10 mg of each drug in 10 ml of methanol to get concentration of 1000 µg/ml. Further dilutions were prepared in mobile phase to get appropriate dilution.

 

Selection of detection wavelength:

From the standard stock solution further dilutions were made using methanol and scanned over the range of 200 - 400 nm and the spectra was obtained. It was observed that both the drug showed considerable absorbance at 216 nm (Figure 3).

 

Preparation of sample solution (Tablet Formulation Analysis):

Twenty tablets (Uni Chem Pvt. Ltd., Pune, India) each containing 50 mg of METO and 20 mg of OLME was weighed and powdered. Powder equivalent to 10 mg of METO and 4 mg of OLME was transferred to 10 ml volumetric flask and diluted with methanol. It was sonicated for 10 mins and filtered. Then the volume was made to 10 ml (1000 µg/ml of METO and 400 µg/ml of OLME) with methanol. Further dilutions were made with mobile phase to get the final concentration of 10 µg/ml of METO and 4 µg/ml of OLME. Sample solutions were injected and the contents of drugs in tablet were determined by the proposed method using the calibration curve.

 

 

Figure 3: Overlaid UV-Vis Spectra of METO (10 µg/ml) and OLME (10 µg/ml)

 

Figure 4: Chromatogram of METO (10 µg/ml) and OLME (10 µg/ml)

Stress degradation studies of bulk drug:

Stability studies were carried out to provide evidence on how the quality of drug varies under the influence of variety of environmental conditions like hydrolysis, oxidation, temperature, etc. OLME as well as mixture of METO with OLME was treated in similar manner to METO.

 

Alkaline treatment:

1 ml working standard solution of METO was mixed with 1 ml of 0.1 N methanolic NaOH and 8 ml of methanol. The solution was kept for 15 min in dark place. The 1 ml of resulting solution was diluted with mobile phase to 10 ml and then was injected (10 μg/ml).

 

Acid treatment:

1 ml working standard solution of METO was mixed with 1 ml of 0.1 N methanolic HCl and 8 ml of methanol. The solution was kept for 15 min in dark place. The 1 ml of resulting solution was diluted with mobile phase to 10 ml and then was injected (10 μg/ml).

 

Neutral hydrolysis:

1 ml working standard solution of METO was mixed with 1 ml water and 8 ml of methanol. The solution was kept for 15 min in dark place. The 1 ml of resulting solution was diluted with mobile phase to 10 ml and then was injected (10 μg/ml).

 

Oxidation degradation:

1 ml working standard solution of METO was mixed with 1 ml 30 % solution of H₂O₂ and 8 ml of methanol. The solution was kept for 15 min in dark place. The 1 ml of resulting solution was diluted to 10 ml with mobile phase and then was injected into the system (10 µg/ml). 

 

Degradation under dry heat:

Dry heat studies were performed by keeping drug sample as METO and OLME as single in oven (100⁰ C) for a period of 1 hour. Samples were withdrawn after 1 hr, dissolved in methanol and further diluted in mobile phase to get 10 µg/ml as final concentration and were injected.

 

Photo-degradation:

Photolytic studies were also carried out by exposure of drug as mixture to UV light up to 200 watt hours/square meter and subsequently to cool fluorescent light to achieve an illumination of 1.2 million Lux. Hr. Sample was weighed, dissolved and diluted with mobile phase to get 10 µg/ml as final concentration and were injected.

 

RESULT AND DISCUSSION:

Optimization of chromatographic conditions:

The primary target in developing this stability indicating HPLC method is to achieve the resolution between METO, OLME and its degradation products. To achieve the separation, we used a stationary phase C-18 column and mobile phase Methanol: 10 mM o-phosphoric acid buffer (50: 50 v/v). The tailing factor obtained was less than two and retention time was 3.66 ± 0.04 min and 18.14 ± 0.05 min for METO and OLME, respectively (Figure 4). Forced degradation study showed the method is highly specific and no degradation products were eluted at retention time of drugs.

 

Result of forced degradation studies:

Degradation was observed for METO and OLME samples during stress conditions like base, acid, neutral, oxidation, and dry heat except under photodegradation. Summary of stress degradation results are given in Table 1. Although degradation found under above mentioned conditions, the degradation peaks were observed only for OLME under acid, neutral, oxidation stress studies. Peak purity results greater than 990 indicate that METO and OLME peaks are homogeneous in all stress conditions tested. The Figure 5 shows degradation behavior of drugs in mixture under different stress conditions tested. The unaffected assay of METO and OLME in the tablet confirms the stability indicating power of the method.

 

 

Table 1 Summary of stress degradation results

 

 

Validation of Analytical Method:

Linearity:

The linearity of the responses of the drugs was verified at six concentration levels, ranging from 1-25 μg/ml for METO and 1- 10 μg/ml for OLME, respectively. The calibration graph was obtained by plotting peak area versus the concentration and data was treated by least-squares linear regression analysis. (Figure 6 and Figure 7)

 

Precision:

The precision of the method was demonstrated by intra-day and inter-day variation studies. In the intra-day studies, 3 replicates of 3 different concentrations of METO (1, 10, 20 µg/ml) and of OLME (1, 4, 8 µg/ml) were analyzed in a day and percentage RSD was calculated. For the inter day variation studies, 3 different concentrations were analyzed on 3 consecutive days and percentage RSD were calculated. For intraday precision % RSD found to be 0.44 for METO and 0.36 % for OLME. For inter-day precision % RSD found to be 0.40 for METO and 0.20 % for OLME.

 

Figure 5: Chromatogram of METO and OLME in combination (10 µg/ml each) after A) alkaline hydrolysis B) acid hydrolysis C) neutral hydrolysis D) oxidation E) dry heat and F) photo degradation. [Degradation products of OLME D1 - RT 9.3 and D2 - RT 10.2]

 

Figure 6: Calibration curve for METO

 

Figure 7: Calibration curve for OLME

 

Table 2: Results of recovery tests of METO

* Average of three determinations

 

Table 3: Results of recovery tests of OLME

* Average of three determinations

 

Accuracy:

To check accuracy of the method, recovery studies were carried out by adding standard drug solution to sample solution at three different levels 50, 100 and 150 %. Basic concentration of sample chosen was 10 μg/ml of METO and 4 μg/ml of OLME from tablet solution. The drug concentrations were calculated from respective linearity equation. The results obtained are shown in Table 2 and Table 3.

 

Specificity:

The specificity of the method was ascertained by peak purity profiling studies. The peak purity values were found to be more than 990, indicating the no interference of any other peak of degradation product, impurity or matrix.

Limit of detection (LOD) and limit of quantification (LOQ):

LOD and LOQ were calculated as 3.3σ/S and 10σ/S, respectively; where σ is the standard deviation of the response (y-intercept) and S is the slope of the calibration plot. The LOD of METO and OLME were found 0.055 µg/ml and 0.178 µg/ml, respectively. The LOQ of METO and OLME were 0.167 µg/ml and 0.539 µg/ml, respectively.

 

Robustness studies:

Robustness of the method was determined by carrying out the analysis under conditions during which flow rate and quantification wavelength were altered and the effects on the area were noted. The results are shown in Table 4.

Table 4: Robustness data for METO and OLME

DRUG	% RSD Found For Robustness Study (peak area)
	Flow rate (ml/min)			Wavelength (nm)
	0.9	1.0	1.1	215	216	217
METO	0.11	0.06	0.06	0.13	0.12	0.15
OLME	0.90	0.91	0.72	0.58	0.29	0.24

 

 

CONCLUSIONS:

The developed method is stability indicating and can be used for assessing the stability of METO and OLME in bulk drug and pharmaceutical dosage form. The developed method is specific, selective, robust and precise.

 

ACKNOWLEDGEMENT:

Authors are thankful to Cadila Healthcare Ltd., Ahmadabad for providing gift sample of METO and OLME.

 

REFERENCES:

1.        http://en.wikipedia.org/wiki/ Olmesartan medoxomil accessed in march 2014

2.        http://en.wikipedia.org/wiki/ metoprolol succinate accessed in march 2014

3.        Vachhani KH and Patel SA. Development and validation of spectrophotometric method for simultaneous estimation of metoprolol succinate and olmesartan medoxomil in tablet. Journal of Applied Pharmaceutical Science. 2011; 1 (7):112-115.

4.        Chitlange SS, Talole RA, Bhusal RD and Nikumbh MB. Development and Validation of Spectrophotometric methods for simultaneous determination of Olmesartan medoxomil and Metoprolol succinate in pharmaceutical preparations. International Jouranal of Pharmacy. 2012; 2(3): 666-669.

5.        Patel DD and Patel MM. Simultaneous Estimation of Metoprolol Succinate and Olmesartan Medoxomil in Pharmaceutical Dosage Form by UV Spectroscopy. International Journal of Research in Pharmaceutical and Biomedical Sciences. 2012; 3(2):935-939.

6.        Vachhani KH and Patel SA. Development and Validation of First Order Derivative Spectrophotometric method for simultaneous estimation of Metoprolol Succinate and Olmesartan Medoxomil in tablets. Journal of pharmaceutical science and bioscientific research. 2011; 1(2): 113-117.

7.        Jadhav AS., Tarkase KN and Deshpande AP. Quantitative Determination of Metoprolol Succinate in bulk and tablet Dosage form through comparative study of UV and derivative Spectroscopy. Der Pharmacia Lettre. 2012; 4 (3):763-767.

8.        Vora BN, Parmar RR, Nayak PP, Shah DA. Development and validation of the simultaneous UV spectrophotometric method for estimation of metoprolol succinate and olmesartan medoxomil in the tablet dosage form. Pharmaceutical Methods 2012; 3 (1):44-47.

9.        Patel S, Patel D. Simultaneous Determination of Metoprolol Succinate and Chlorthalidone by UV Spectrophotometric Method. Pharmagene; 1(3): 39-43.

10.     Muralidharan S and Kumar JR. Sensitive estimation of olmesartan medoxomil tablets by RP-HPLC method. International Journal of Pharmacy & Life Sciences 2012; 3(11): 2149-2152.

11.     Ganduri RB, Lanka RA, Pamidi S, Peddareddigari JR and Mohammed M. New RP-HPLC Method for the Determination of Olmesartan Medoxomil in Tablet Dosage Form. Eurasian Journal of Analytical Chemistry 2010; 5(2): 145-151.

12.     Venkateswararao L, Vardhan S.V.M., Venkatrao S.V., Chintala R. Validated RP-HPLC Method for the Estimation of Metoprolol Succinate in Dosage Formulations. American Journal of Pharmtech Research 2013; 3(2), 228-234.

13.     Aqil M, Ali A, Ahad A, Sultana Y, Najmi AK, and Saha N.  A Validated HPLC Method for Estimation of Metoprolol in Human Plasma. Acta Chromatographica 2007; 19: 130-140.

14.     Rawool ND, Venkatchalam A. Analytical Method for the Simultaneous Estimation of Hydrochlorothiazide and Metoprolol Tartrate using RP HPLC. Indian Journal of Pharmaceutical Sciences 2011; 73 (1): 219-223.

15.     Chitlange SS, Imran M, Sakarkar DM. RP-HPLC method for simultaneous estimation of amlodipine and metoprolol in tablet formulation. Asian Journal of Pharmaceutics 2008; 232-234.

16.     Tsvetkova BG, Pencheva IP, Peikov PT. Development and validation of RP-HPLC method for simultaneous determination of metoprolol and aspirin in fixed dose combinations. Der Pharma Chemica 2012; 4(4):1512-1516.

17.     Rao CH.M.M.P, Rahaman SA, Prasad YR, Reddy PG. RP-HPLC method of simultaneous estimation of Amlodipine Besylate and Metoprolol in combined dosage form. International Journal of Pharma. Research & Development 2010; 2 (9): 69-76.

18.     Vachhani KH, Patel SA, Vachhani HH. Development and Validation of RP-HPLC method for the simultaneous estimation of Metoprolol succinate and Olmesartan medoxomil in tablet dosage form.  International Journal of Institutional Pharmacy and Life Sciences 2012; 2(2):390-399.

19.     Kumanan R, Reddy JM, Manasa R. Stability Indicating RP-HPLC Method Development and Validation of Olmesartan Medoxomil.  Asian J Pharm Biol Res 2011; 1(2): 79-86.

20.     Sharma RN, Pancholi SS. RP-HPLC-DAD method for determination of Olmesartan medoxomil in bulk and tablets exposed to forced conditions.  Acta Pharm 2010; 60: 13–24.

21.     Kalisetty S, Reddy ST, Reddy AM, Palnati JB, Rao DV  and Manikandan R. Stability Indicating Reverse phase Liquid chromatographic Method for the Determination of Metoprolol succinate in Pharmaceutical Dosage Forms.  Journal of Chemical and Pharmaceutical Research 2012; 4(9): 4420-4425.

22.     Patil KR, Rane VP, Sangshetti  JN,  Yeole RD, and Shinde DB. Stability Indicating LC Method for the Simultaneous Determination of Amlodipine and Olmesartan in Dosage Form. Journal of Chromatographic Science 2010; 48: 601-606.

23.     Psrchn P, Varma D, Rao A L and Dinda SC. Validated Stability Indicating HPLC Method for Simultaneous Determination of Amlodipine and Metoprolol in bulk drug and Pharmaceutical Formulation. International Journal of Research in Pharmacy and Chemistry 2012; 2(3): 876-884.

24.     Chitlange SS, Talole RA, Bhusal RD and Nikumbh  RB. Development and Validation of TLC Densitometric and HPLC Method for Determination of Olmesartan Medoxomil and Metoprolol Succinate in Combination. International Research Journal of Pharmacy. 2012; 3 (6): 171- 175.

25.     Jain PS, Patel KM, Bari SB and Surana SJ. Development and Validation of HPTLC Method for Simultaneous Determination of Amlodipine Besylate and Metoprolol Succinate in Bulk and Tablets. Indian Journal of Pharmaceutical Sciences. 2012; 152-156.

26.     Nawale PS, Shirkhedkar AA, Surana SJ, and Patil AS. Normal and Reversed-Phase HPTLC Methods for Simultaneous Estimation of Telmisartan and Metoprolol Succinate in Pharmaceutical Formulation. International Scholarly Research Network. 2012; 1-6.

27.     ICH - International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Q₂ (R1), Validation of Analytical Procedures, Methodology, 1996, London, 2005.

28.     ICH- International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Q2B (R1), Guideline on Validation of Analytical Procedure: Text and Methodology, Geneve, 2005.

 

 

Received on 07.05.2014          Modified on 05.06.2014

Accepted on 10.06.2014         © RJPT All right reserved