Forced Degradation Studies of Olmesartan Medoxomil and Characterization of Its Major Degradation Products by LC-MS/MS, NMR, IR and TLC

Rucha A Patel¹*_, Meghna P. Patel¹, Hasumati A. Raj¹, Nehal Shah²

¹Department of Quality Assurance, Shree Dhanvantry Pharmacy College, Kim, Surat, Gujarat, India

²Dharmaj Degree of Pharmacy, Dharmaj, Anand, Gujarat, India

*Corresponding Author E-mail: ruchajigar6114@gmail.com

ABSTRACT:

The proposed study describes an isocratic reversed phase HPLC method for investigation of degradation products of Olmesartan medoxomil API under different stress conditions (acid hydrolysis degradation). Separation of Olmesartan medoxomil and its degradation products was achieved on symmetry C₁₈(150 mm × 4.6 mm, 5 μ) column using a Acetonitrile: 0.02 M Na₂HPO₄(45:55 v/v) mobile phase and pH 7 adjusted with ortho phosphoric acid. Isocratic elution mode at a flow rate of 1.0 ml/min at Room temperature with a load of 20μl Injection volume. The detection was carried out at 240 nm followed by Base hydrolysis Structures of the degradation products were studied using a Triple Quadrapole Mass Spectrometer. A separate gradient LCMS method was developed for this purpose Depending on the degradation type, possible chemical reactions were predicted and supported by fragmentation data obtained from LC-MS/NMR. One major products were obtained in solid form using Rotavap and were then analyzed by NMR, IR and TLC to confirm their structural details.

KEYWORDS: Olmesartan medoxomil, Forced degradation studies, Degradation products, LC, LCMS,NMR, IR and TLC

INTRODUCTION:

Olmesartan medoxomil(a prodrug, which is hydrolyzed in body active Olmesartan during absorption from the gastrointestinal tract) is chemically, 2, 3-dihydroxy‑2-butenyl 4-(1-hydroxy-1-methylethyl)-2-propyl-1-[p-(o‑1H-tetrazol-5-ylphenyl)benzyl] imidazole‑5‑carboxy late, cyclic‑2,3‑carbonate is new orally active Angiotensin II type 1 receptor antagonist used as an anti-hypertensive agent^[1].Olmesartan medoxomil widen Peripheral blood vessels , This is the main mechanism by which the pressure in the blood vessels is lowered and Blocking the actions of Angiotensin II also reduces the action of aldosterone on the kidneys^[2].

The result of this is an increase in the amount of fluid removed from the blood by the kidneys. This decreases the amount of fluid in the blood vessels, which also lessens the resistance and pressure in the blood vessels. Forced degradation is a process whereby the natural degradation rate of a drug product or drug substance is accelerated by the application of an additional stress. Forced Degradation Studies (FDS) or stress testing form a very important part of the drug development strategy in pharmaceutical industries under the guidelines of International Conference on Harmonization (ICH) and is carried out under more severe conditions than accelerated conditions (ICH guidelines, 2003). ^[3]These studies serve to give information on a drug’s inherent stability and assist in the validation of analytical methods to be used in stability studies. It also helps in determining the degradation products and in estimating the tentative degradation pathway of the drug. Identification and qualification of these Degradation Products (DPs) is quite essential since it can cause undesirable side effects in patients, at times these side effects could also be fatal. FDS is also very effective for optimizing the formulation, packaging and storage conditions of the drug product and hence even though ICH and FDA ask to include this study at Phase III of the clinical trial, it is recommended to start it as early as possible For the proposed study, Olmesartan medoxomil (Figure 1) API was subjected to degradation under Base hydrolysis, Generally, a drug molecule is subjected to a maximum of 70% degradation which is enough to get the relevant information regarding its degradation and likely degradation products However, more harsh conditions were used in the present study to achieve complete or near to complete degradation of the drug substance so as to study the degradation products formed after maximum degradation. Thus, the main purpose of this study was to identify and characterize the DPs formed after complete or maximum degradation of OLM, under stress conditions using LC-MS/NMR technique. An isocratic LC method was developed for separation of Olmesartan medoxomil and its degradation products. Simultaneously, a LC-MS/MS method was also developed to study the structural details of the DPs. The isocratic LC method was also used on preparative LC in order to isolate the major degradation products. The fractions of DPs collected was processed through rotavap to evaporate the aqueous phase followed by neutralization to obtain the DPs in solid form. NMR and IR spectra of these DPs were then obtained as a supporting data to MS results.

Fig.1. Structure of Olmesartan Medoxomil

MATERIALS AND METHODS:

Olmesartan medoxomil raw material was received as gift sample from Cadila Healthcare Limited, Ankleshwar.

Acetonitrile (Finar) Gradient grade

Hydrochloric acid (Merck) AR grade

Sodium hydroxide (Merck) AR grade

HPLC grade Water

Disodium hydrogen Phosphate (RANKEM) LR grade were used for development purpose.

Sample Preparation:

Alkali Degradation:

100 mg of Olmesartan medoxomil API was weighed and dissolved in 1.0 mL of Methanol, to this 10 mL of 2M NaOH was added. The solution was kept in water bath at 60°C for 2 h; it was then cooled at room temperature and neutralized with 2 M HCl (stock solution).From the above stock solution, 0.1 mL was taken in a 25 mL volumetric flask and volume was made up with methanol. This was analyzed on HPLC. One major product was obtained in solid form using Rotavap and were then analyzed by NMR, IR and TLC to confirm their structural details.

[A] LC Analysis^[^4]:

Chromatographic analysis was carried out on semi automatic, Pump-single pump Model- SPD 10 A-LC 10 AT Company-Shimadzu, Japan, Software-Winchrome software Analysis was done using Phenomenex C18 column (250 mm x 4.6 mm, 5 μ). The mobile phase comprised of 0.02 M Na₂HPO₄ : Acetonitrile (55:45 v/v) with pH 7. The flow rate was maintained at 1.0 mL/min, injection volume was 20 μL at room temperature. Run time for the analysis was kept 10 min. The chromatograms were monitored at 242 nm.

Figure 2: Base Degradation blank

Figure 3: Olmesartan medoxomil standard

Figure 4: Base Degradation – after 3 Hr At 60˚C

Table 1: Olmesartan medoxomil degradation by HPLC

Retention Time (min.)	Peak description	Area	% Degradation
6.46	2N NaOH	17735	98.00%
4.42	Olmesartan standard	182415	98.00%
2.37	IMP A	178778
2.80	IMP-B	2661

Figure 6 Mass analysis report Standard Olmesartan medoxomil

Figure 7: Mass analysis report of degraded product of Olmesartan medoxomil

Calculated value of degraded product of olmesartan medoxomil 444.5

Observed value of degraded product of olmesartan medoxomil 445.6(n+1)

Figure 8: NMR report of Standard of Olmesartan medoxomil

[D] NMR Analysis:

The NMR experiment was performed on a Bruker instrument working at 400 MHz for 1H. CDCl3 was used

Figure 9: NMR report of degraded product of Olmesartan medoxomil

Table 2: Value of Function group of Olmesartan medoxomil by NMR

δ VALUE(DMSO )	GROUP
1.53	2H of CH2
1.55	6H of CH3
2.51	2H of CH2
3.35	-OH
5.6	2H of CH2
6.93-7.69	8H of Ar-H
8.33	1H of NH

[E] IR ANALYSIS:

IR analysis was performed on Shimadzu’s FTIR

Figure 10: Olmesartan medoxomil degradation by IR

Table 3: Value of Function group of Olmesartan medoxomil by IR

GROUP	OBSERVED FREQUENCY(cm^-1)		STANDARD RANGE(cm^-1)
-CH₂-	764.80		772
C-O-OR	1136.11		1124
-C-O		1259.56	1240
C=C	1476.56		1600-1400
-C=O	1832.44		1700-1600
-CH₃	2875.00		2872
=CH-	2972.33		2962
Ar-H	3039.91		3050-3000
-NH2	3290.67		3500-3300

[F] UV SCANE:

Olmesartan medoxomil and test solution (Base Degradation – after 3 Hr At 60˚C) was scane in UV at 242nm.

Figure 11: Standard Olmesartan medoxomil(40-50-60 μg/ml)

Figure 12: Standard Olmesartan medoxomil in basic hydrolysis (40-50-60 μg/ml l)

RESULTS:

Olmesartan medoxomil was found to degrade to different extent under various Alkali Degradation .The percentage of degradation obtained is depicted in Table 1 and the respective chromatograms are shown in Figure 4.From TLC it was conformed that there was some changes in Structure of olmesartan medoxomil due to stress condition in figure 5. The degradation products was subjected to Mass study to elucidate their structural details. The molecular scan is provided in Figure .6 and figure 7.NMR and IR analysis of the major degradation products were performed, results of these analyses are provided in Table 2 and 3, respectively.

DISCUSSION:

The objective of this study was to investigate the degradation products of OLME under Alkali conditions. NMR and IR analysis of these degradation products were performed. IR and NMR interpretations are given in Table 2 and Table 3, respectively. A separate gradient LC method (Table 1) was developed for analyzing the degradation products on LC-MS/MS. This method was quite fast in comparison to the isocratic LC method which was employed for separation of OLME and its degradation products. The molecular weight of OLME is 558.5851 g/mol and that of the protonated ion in the positive ion mode is 445.6. Structural elucidation of the degradation products and the tentative degradation pathway of OLME were also predicted based on the product ions formed and their respective fragmentation pattern (Figure 4).The major functional groups of the degradation products were also indicated in IR figure 9.8 and table 9.3 and NMR analysis. Data obtained from these analyses supported the structural elucidation done based on the MS data (Table 4 and 5).

CONCLUSION:

An isocratic LC method was optimized for the separation of OLME and its degradation products Olmesartan medoxomil was subjected to Alkali stress conditions. It was found to degrade the most under basic conditions A fast LC-MS/MS method was developed to identify and characterize the degradation products formed under different stress condition. One major degradation product from each of the stress conditions was isolated using preparative LC technique. Structure elucidation of the major degradation products was done using LC-MS/MS data and it was supported by NMR and IR results. A tentative degradation pathway of OLM was also predicted under different chemical stress conditions. In Olmesartan medoxomil break down of ester linkage occurred and medoxomil salt was removed.

ACKNOWLEDGMENT:

The authors are thankful to Oxygen Research center Chemical laboratory, Ahmedabad, Gujarat, India. We are also grateful to the Director of oxygen Shree Dhanvantry college of Pharmacy and Research Center, Surat, Gujarat, India for providing all the research related facilities, required to accomplish the present research work.

REFERENCES:

1. Tripathi KD. Essentials of medical pharmacology; 6th Edn; Jaypee Brothers Medical Publishers (P) Ltd, 2008, pp 539-542.

2. Goyal R.K. Derasari and Gandhi's Element of pharmacology; 12thEdn; B. S. Shah Prakashan, Ahmadabad , 2011, pp 457-466.

3. International Conference on Harmonization, Harmonized Tripartite Guideline, Stability Testing of New Drug Substances and Products (Revision 2), ICH Q1A(R2), 2003.

4. Sethi PD and Sethi R; “HPLC: Quantitative Analysis of Pharmaceutical Formulation, 15th Edn; Published by CBS,2007.