1. INTRODUCTION:

Olmesartan medoxomil(OLM) and Indapamide (IND) combination is used in cardiac disease condition like Hypertension. Olmesartan medoxomil is a new orally active Angiotensin II type 1 receptor antagonist used as an anti-hypertensive agent[1]. It is a prodrug and is rapidly de-esterified during absorption to form olmesartan, the active metabolite [2]. Olmesartan medoxomil [Figure 1] (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‑carboxylate, cyclic‑2,3‑carbonate.

Indapamide is an orally administered diuretic and anti‑hypertensive drug. Its molecule contains both a polar sulfamoyl chlorobenzamide moiety and a lipid soluble methyl‑indoline moiety[3] Indapamide is chemically 3-(aminosulfonyl)-4-chloro-N-(2, 3-dihydro-2-methyl-1H-indol-1-yl) benzamide [Figure 2] It differs chemically from thiazide in a way that it does not possess the thiazide ring system and contains only one sulfonamide group. It is used for hypertension and also for oedema, including that associated with heart failure. Currently most commonly prescribed medicines for hypertension are Angiotensin receptor blockers and diuretics. Monotherapy with oral anti‑hypertensive agents is not sufficient to achieve target blood pressure levels and henceforth, a combination tablet formulation is beneficial in terms of its convenience and patient compliance. The present drug combination has promising anti‑hypertensive effect. The clinical and pharmaceutical analysis of this drug requires effective analytical procedures for quality control and pharmacodynamics and pharmacokinetic studies as well as stability study Several HPLC assay method of Indapamide bulk and in tablet dosage form are available[4-5-6-7]. The objective of the present study was to develop an accurate, specific and repeatable stability indicating HPLC for simultaneous determination of Olmesartan medoxomil and Indapamide from Tablet dosage form. Olmesartan medoxomil is official in BP 2013 and Indapamide is official in IP,BP,EP,JP[8-9-10-11] The method was validated as per International Conference on Harmonization (ICH) guidelines[12]

Figure1. Chemical Structure of Olmesartan medoxomil

Figure2. Chemical Structure of Indapamide

MATERIALS AND METHODS:

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

· Indapamide raw material was received as gift sample from Torrent research centre, Ahmedabad.

· Marketed formulation OLMY-D 20 (20:1.5) and OLMY-D 40 (40:1.5) From Biocon

· Acetonitrile (FINAR) Gradient grade

· Hydrochloric acid (MERCK)AR grade

· Sodium hydroxide (MERCK)AR grade

· HPLC grade Water

· H₂O₂(MERCK) AR grade

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

INSTRUMENTS:

Chromatographic analysis was carried out on

· Operation-semi automatic,

· Pump-single pump

· Model- SPD 10 A-LC 10 AT

· Company-Shimadzu, Japan

· Software-Winchrome software

· Semi micro analytical balance (Sartorius CD2250, Germany) was used for weighing purpose.

· HPLC water was obtained using Arium^®611VF(Sartorius).

· Magnetic stirrer (1 MLH, Remi) was used for mixing purpose.

· pH tutor (313927, Eutech Instruments) was used for pH measurement.

· Sonications of solutions were done using Ultrasonic cleaner (D 120/1H, Trans-O-Sonic).

· Column used was Inert Phenomenex C₁₈ (250mm×4.6mm i.d.) 5μm

· Nylon membrane filters (0.22 µm, 47 mm D)

· All volumetric glass wares used were calibrated.

EXPERIMENTAL WORK AND CONDITION:

Selection of mobile phase:

From literature survey I choose mobile phase Sodium perchlorate and Triethyl amine buffer: Acetonitrile (60:40 v/v) with pH 3 but Splitting was observed and No peak resolve between two peak then I changed ratio and mobile phase [Acetonitrile: Phosphate Buffer (70:30 v/v)] but Less resolution and splitting. After many trails I choose mobile phase [0.02 M Na₂HPO₄ : Acetonitrile (55:45 v/v)] with pH 7 Because Olmesartan medoxomil having pKa 4.30 and Indapamide having pKa 8.85. Both drugs were separated with sharp peak and retention time of Olmesartan medoxomil was 4.42 min and retention time of Indapamide was 7.58 min.

Buffer preparation:

Accurately weighed 2.83 gm Na₂HPO₄ was dissolved in to 1000 ml water, than pH was adjusted to 7 with ortho‑phosphoric acid.

A) Preparation of standard stock solution (1000 μg/mL):

10 mg of bulk drug was weighed accurately and transferred into a clean, dry 10 mL volumetric flask, dissolved in 1ml of Acetonitrile and volume was adjusted to 10 mL with mobile phase and further 0.1 ml dilute up to 10 ml with mobile phase to get a concentration of 10 μg/mL.Standard of Olmesartan medoxomil was injected and eluted on 4.42 min[Figure 3] , Standard of Indapamide was injected and eluted on 7.58 min[Figure 4] And mixture was injected and having good resolution between olmesartan medoxomil and Indapamide.[Figure 5]

B) Preparation of sample stock solution (1000 μg/mL):

Twenty tablets were powdered and sample weighed equivalent to 10 mg of olmesartan medoxomil and indapamide and transferred into a 10 mL volumetric flask separately, dissolved in 1 ml Acetonitrile and further diluted with mobile phase up to 10 ml, sonicated for 15 min, filtered through a Whatman filter paper ≠ 42 and volume was adjusted to 10 mL with Diluent to get a concentration of 10 μg/mL

Figure 3.standard olmesartan medoxomil (10 μg/mL)

Figure 4.standard Indapamide (10 μg/mL)

Figure 5.standard olmesartan medoxomil and medoxomil (10 μg/ml)

Figure 6 Linearity of Olmesartan medoxomil (50-250 μg/ml) and Indapamide (10-50 μg/ml)

Stability Indicating Property (Forced Degradation):

A. Acid Induced Degradation Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 0.1 N HCl in it. Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken at 0min, 10min, 30min, 1 Hr, 2 Hr and 3Hr. Blank solution was also injected without API.

B. Base Induced Degradation Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 0.1 N NaOH in it. Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken at 0min, 10min, 30min, 1 Hr, 2 Hr and 3Hr .Blank solution was also injected without API.

C. Hydrogen Peroxide Induced Degradation: Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml 3% H₂O₂ in in it.Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken at 0min, 10min, 30min, 1 Hr, 2 Hr and 3Hr .Blank solution was also injected without API.

D. water reflux : Accurately weighed 10mg bulk drug was dissolved in 1 ml Acetonitrile and add 10 ml distill water in it. Adjust pH 7.Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded. Samples were taken at 0min, 10min, 30min, 1 Hr, 2 Hr and 3Hr .Blank solution was also injected without API.

E. Thermal degradation : : Accurately weighed 10mg bulk drug was kept in a hot air oven for 3 h at a temperature of 60 °C, then made up with mobile phase. For further dilution, 1 mg sample was added to a 10 ml volumetric flask individually, and for tablet degradation, Average weight of 5 tablet powder was kept in hot air oven in same condition.10mg powder added to a 10 ml flask and made up with mobile phase and further dilute 0.1 ml in 10 ml mobile phase

F. Photochemical Degradation: 10 mg of drug was exposed to UV light for 3 hr, then made up with mobile phase up to 10ml. For further dilution, 1 ml of sample was added to a 10 ml volumetric flask individually, for tablet degradation Average weight of 5 tablet powder was exposed to UV light for 3 hr.10mg powder added to a 10 ml flask and made up with mobile phase and further dilute 0.1 ml in 10 ml mobile phase

Table 1:Percent degradation of olmesartan medoxomil and Indapamide retention time and area of degradation product

Sr. No.	Condition		Degradation products	Retention time(Min) and Area of impurity			% Degradation at 60 ˚c
				Retention time(Min) and Area of impurity			Olmesartan	Indapamide
				Retention time(Min)		Area of impurity peak	Olmesartan	Indapamide
	Untreated stock Solution (10μg/ml)		-	Olmesartan (Area)	Indapamide (Area)		-	-
	Untreated stock Solution (10μg/ml)		-	4.42(182415)	7.58(245142)		-	-
1	Acid hydrolysis		IMP B	-	1.12	4729	70.41%	29.73%
1	Acid hydrolysis		IMP I	2.42	-	188517	70.41%	29.73%
			IMP II		4.08	-	7607
			OLME	4.42	-	41582
			IMP A	-	5.43	5620
			INDA	-	7.63	170800
2	Base hydrolysis		IMP III	2.38	-	230436	100%	17.72%
2	Base hydrolysis		IMP IV	2.86	-	1246	100%	17.72%
			IMP C		-	3.14	75331
			IMP D		-	3.45	31923
			IMP V		3.87	-	78778
			IMP F		-	5.14	1433
			INDA		-	7.28	249826
			IMP E	-	9.05	1220
3		Oxidation	IMP H	-	2.20	4673	86.18%	50.50%
3		Oxidation	IMP VI	2.37	-	200793	86.18%	50.50%
			IMP G		-	2.85	113381
			IMP I		-	3.79	1202
			OLME		4.40	-	10048
			IMP VII		5.59	-	19805
			INDA	-	6.39	118889
4		Neutral	IMP VIII	2.61	-	180852	28.68%	20.22%
4		Neutral	IMP J	-	3.01	998	28.68%	20.22%
			OLME		4.70	-	165092
			INDA		-	6.55	193750
5		Thermal	IMP IX	2.86	-	3654	9.30%	3.87%
5		Thermal	OLME	4.73	-	3256759	9.30%	3.87%
			INDA		-	7.58	1654378
6		Photolytic	OLME	4.40		3356759	3.82%	3.82%
6		Photolytic	INDA		7.25	1754378	3.82%	3.82%

ACID DEGRADATION (MIXTURE) – AFTER 2 Hr AT 60˚C

§ Accurately weighed 10mg bulk drug (Olmesartan medoxomil and Indapamide) weere dissolved in 1 ml Acetonitrile and add 10 ml 0.1 N HCl in it separately.

§ Then this solution is kept at 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded.

§ Samples were taken after 3Hr and injected and chromatogram was recorded.[figure 7]

Figure 7 : Acid Degradations (Mixture) – after 2 Hr AT 60˚C

BASE DEGRADATION (MIXTURE) – AFTER 3 hr AT 60˚C

§ Accurately weighed 10mg bulk drug (Olmesartan medoxomil and Indapamide) were dissolved in 1 ml Acetonitrile and add 10 ml 0.1 N NaOH in it separately.

§ Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded.

§ Samples were taken after 3Hr and injected and chromatogram was recorded.[figure 8]

Figure 8: Base Degradation (Mixture) – after 3 Hr AT 60˚8

OXIDATION DEGRADATION (MIXTURE) – AFTER 2 Hr AT 60˚C

§ Accurately weighed 10mg bulk drug (Olmesartan medoxomil and Indapamide) were dissolved in 1 ml Acetonitrile and add 10 ml H₂O₂ in it separately.

§ Then this solution is kept At 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded.

§ Samples were taken after 3Hr and injected and chromatogram was recorded.[figure 10]

Figure 9: Oxidation Degradation (Mixture) – after 3 Hr AT 60˚C

WATER REFLUX (MIXTURE) – AFTER 2 Hr AT 60˚C

· Accurately weighed 10mg drug dissolve in 1 ml Acetonitrile then volume is made by Distilled Water up to 10 ml (1000 ppm).

· Then this solution is kept at 60˚C and pipette out 0.1 ml and dilute with mobile phase up to 10 ml and chromatogram was recorded.

· Samples were taken after 3Hr and injected and chromatogram was recorded.[figure 10]

Figure 10 : Water reflux (Mixture) – after 2 Hr AT 60˚C

THERMAL DEGRADATION (MIXTURE) – AFTER 2 Hr AT 60˚C

§ OlmyD-20 tablets (10 tablets) were taken and triturated and powder was placed in a cleaned Petridish and put it into the oven at 60˚C for 3 hours. The equivalent Weight 350 mg (Tablet powder equivalent to 200 mg olmesartan medoxomil and 15 mg indapamide. i.e., 10 tablets’ powder was added in the flask for assay of tablets.) was transferred into 250 ml volumetric flask. Then add about 100.0 ml Mobile phase was added and sonicated for 30 min with intermittent shaking. Then volume was made up to 250ml with Mobile phase. Then 2.5 ml of standard stock solution was diluted to 10 ml with Mobile phase to make final standard concentration of olmesartan medoxomil (200 ppm) and indapamide (15 ppm) respectively and chromatogram was recorded.

§ Similar way 100 mg of bulk drug was taken in a cleaned Petridish and was put it into the oven at 60˚C for 3 hour

§ Accurately weight 10 mg were taken and dilute with Mobile phase and Filtered it then dilute 0.1 ml to 10 ml mobile phase to obtained 10 μg/ml concentration of both drugs.

Figure 11: Thermal Degradation (Mixture) – after 2 Hr AT 60˚C

PHOTOLYTIC DEGRADATION (MIXTURE) – AFTER 2 Hr AT 60˚C

OlmyD-20 tablets(10 tablets) were taken and triturated and powder was placed in a cleaned Petridish and exposed to UV light for 3 hours. The equivalent Weight 350 mg (Tablet powder equivalent to 200 mg olmesartan medoxomil and 15 mg indapamide. i.e., 10 tablets’ powder was added in the flask for assay of tablets.) was transferred into 250 ml volumetric flask. Then add about 100.0 ml Diluent was added and sonicated for 30 min with intermittent shaking. Then volume was made up to 250ml with Mobile phase. Then 2.5 ml of standard stock solution was diluted to 10 ml with Mobile phase to make final standard concentration of olmesartan medoxomil (200 ppm) and indapamide (15 ppm) respectively. and chromatogram was recorded.

Similar way 100 mg of bulk drug was taken in a cleaned Petridish and was exposed to UV light for 3 hour

Accurately weight 10 mg were taken and dilute with Mobile phase and Filtered it then dilute 0.1 ml to 10 ml mobile phase to obtained 10 μg/ml concentration of both drugs.

Figure 12 :Photolytic Degradation (Mixture) – after 2 Hr AT 60˚C

Table 2: DEGRADATION SUMMARY

Sr. No.	CONDITION	% DEGRADATION at 60 ^˚C
Sr. No.	CONDITION	OLMESARTAN	INDAPAMIDE
1	Acid hydrolysis	70.41%	29.73%
2	Base hydrolysis	100%	17.72%
3	Oxidation	86.18%	50.50%
4	Water reflux	28.68%	20.22%
5	Thermal	9.30%	3.87%
6	Photolytic	3.82%	3.82%

METHOD VALIDATION[13]

5.4.5. VALIDATION OF DEVELOPED RP-HPLC METHOD

The RP-HPLC method has been developed 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. with retention time of olmesartan medoxomil and Indapamide at 4.72min and 7.52 min . As the HPLC method has been developed, the validation using various parameters was performed to ensure that the performance characteristic of the method meets the requirements for the intended analytical applications. Validation was carried out with respect to various parameters, as required under ICH guideline Q2 (R1). The developed method validated with respect to parameters such as linearity, precision, accuracy, LOD,LOQ robustness, and solution stability.

The parameters performed for method validation work:

· System suitability

· Specificity

· Linearity and Range

· Accuracy

· Precision (Intraday Precision)

· Limit of Detection and Limit of Quantification

5.4.5.1 SYSTEM SUITABILITY

Purpose

This test ensures that the analytical system is working properly and can give accurate and precise results.

Methodology

An alliquote of 1 mL (1000 μg/mL) standard stock solution of Bamifylline HCl (Sec:5.3.1.3.1) was transferred into a 10 mL volumetric flask and the volume adjusted with diluents to get a concentration of 100 μg/mL. 20 µl of this standard solution (50 μg/mL) solution was injected in to HPLC system. Retention time, peak area, theoretical plates and tailing factor in chromatogram for Bamifylline HCl was observed and are presented in Figure 5.12 and in Table 5.17.

Acceptance Criteria

· Tailing factors for Bamifylline HCl should be NMT 2.0 in standard solution\ preparation.

· Theoretical plates of Bamifylline HCl peak should NLT 2000 in standard solution preparation.

5.4.5.3 CALIBRATION CURVE (LINEARITY)

[A] LINEARITY

Purpose

The linearity of the analytical method is its ability to elicit test results which are directly proportional to the concentration of the analyte in the sample.

Methodology

The portions of 0.5 mL, 1 mL, 1.5 mL, 2.0 mL, and 2.5 mL of 1000 µg/mL of standard stock solution of Olmesartan medoxomil (Sec: 5.3.1.3.1) were transferred separately to a series of 10 mL of volumetric flasks and volume was adjusted to 10 mL with Mobile phase to obtain the concentrations of 50 μg/mL, 100 μg/mL, 150 μg/Ml, 200 μg/mL and 250 μg/mL respectively.and 1 mL, 2 mL, 3 mL, 4 mL and 5 mL of 100 µg/mL of standard stock solution of Indapamide (Sec: 5.3.1.3.1) were transferred separately to a series of 10 mL of volumetric flasks and volume was adjusted to 10 mL with Mobile phase to obtain the concentrations of 10 μg/mL, 20 μg/mL, 30 μg/Ml, 40 μg/mL and 50 μg/mL respectively Calibration curve was constructed by plotting peak areas v/s concentrations of Olmesartan medoxomil and Indapamide.. The results are presented in Table 5.130 and Figure 5.117, 5.118.

Acceptance Criteria

The regression coefficient (r²) should be ideally 1.0 or value as lose as possible to 1.0

5.4.5.4 ACCURACY (% RECOVERY)

Purpose

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. The accuracy of the method was determined by calculating recovery of Olmesartan medoxomil and Indapamide by the standard addition method.

Methodology

Solution I: 2.5 mL of sample stock solution(200 μg/mL ) of Olmesartan medoxomil and 1 ml of sample stock solution(200 μg/mL) of Indapamide (Sec: 5.3.1.3.2) and 0.4 mL of standard stock solutions(1000 μg/mL) of Olmesartan medoxomil (Sec: 5.3.1.3.1) and 0.8 ml of sample stock solution(100 μg/mL) of Indapamide were transferred into 10 mL volumetric flask and the volume was adjusted to 10 mL with Mobile phase.

Solution II: 2.5 mL of sample stock solution(200 μg/mL ) of Olmesartan medoxomil and 1 ml of sample stock solution(200 μg/mL) of Indapamide (Sec: 5.3.1.3.2) and 0.5 mL of standard stock solutions(1000 μg/mL) of Olmesartan medoxomil (Sec: 5.3.1.3.1) and 1 ml of sample stock solution(100 μg/mL) of Indapamide were transferred into 10 mL volumetric flask and the volume was adjusted to 10 mL with Mobile phase.

Solution III: Similarly 2.5 mL of sample stock solution(200 μg/mL ) of Olmesartan medoxomil and 1 ml of sample stock solution(200 μg/mL) of Indapamide (Sec: 5.3.1.3.2) and 0.6 mL of standard stock solutions(1000 μg/mL) of Olmesartan medoxomil (Sec: 5.3.1.3.1) and 1.2 ml of sample stock solution(100 μg/mL) of Indapamide were transferred into 10 mL volumetric flask and the volume was adjusted to 10 mL with Mobile phase._{Accuracy was calculated using the following equation:}

_{% Recovery =}

_[(Sp_{iked Area – Unspiked
Area) / Standard Area] x 100}

Acceptance Criteria

The % recovery at each level should be between 98.0%-102.0%l.

5.4.5.5 PRECISION

Purpose

The precision of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.

Methodology

Intraday precision

The portions of 0.5 mL, 1.0 mL and 1.5 mL of 1000 µg/mL of standard stock solution of Olmesartan medoxomil (Sec: 5.3.1.3.1) was transferred to a separate 10 mL of volumetric flasks and volume adjusted up to 10 mL with Mobile phase to obtain a concentrations of 50 μg/mL, 100 μg/mL and 150 μg/mL for Olmesartan medoxomil and The portions of 1 mL, 2 mL and 3 mL of 100 µg/mL of standard stock solution of Indapamide (Sec: 5.3.1.3.1) was transferred to a separate 10 mL of volumetric flasks and volume adjusted up to 10 mL with Mobile phase to obtain a concentrations of 10 μg/mL, 20 μg/mL and 30 μg/mL for Indapamide.20 μl of each of these standard solutions of Olmesartan medoxomil and Indapamide were injected under the operating chromatographic conditions into system three times on the same day and chromatograms were recorded. The results are presented in terms of % relative standard deviation (% RSD) in Table 5.132.

Interday Precision:

The portions of 0.5 mL, 1.0 mL and 1.5 mL of 1000 µg/mL of standard stock solution of Olmesartan medoxomil (Sec: 5.3.1.3.1) was transferred to a separate 10 mL of volumetric flasks and volume adjusted up to 10 mL with Mobile phase to obtain a concentrations of 50 μg/mL, 100 μg/mL and 150 μg/mL for Olmesartan medoxomil and The portions of 1 mL, 2 mL and 3 mL of 100 µg/mL of standard stock solution of Indapamide (Sec: 5.3.1.3.1) was transferred to a separate 10 mL of volumetric flasks and volume adjusted up to 10 mL with Mobile phase to obtain a concentrations of 10 μg/mL, 20 μg/mL and 30 μg/mL for Indapamide.20 μl of each of these standard solutions of Olmesartan medoxomil and Indapamide were injected under the operating chromatographic conditions into system three times on the three consecutive days and chromatograms were recorded. The results are presented in terms of % relative standard deviation (% RSD) in Table 5.132.

Acceptance Criteria

The % RSD of the absorbance for μl of each of these standard solutions of obtained should be NMT 1.0%.

5.4.5.6 LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION

Purpose

LOD and the LOQ of the drug were calculated using the following equations as per _{International Conference on Harmonization (ICH) guidelines.}

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

_W_h_e_r_e,

σ = Standard deviation of the response

S = Slope of calibration curve.

The results of LOD and LOQ are presented in Table 5.134.

5.4.5.7. ROBUSTNESS

Robustness was measured by changing the pH of mobile phase, Ratio of Mobile phase and flow rate. The of mobile phase was set ±2 ºC, The , Ratio of Mobile phase was set ±5ml and Flow rate was set ±2 mL/Min. solution of both the drugs was injected three times.The results are presented in Table no. 5.135.

5.4.6 RESULTS AND DISCUSSION

5.4.6.1 SYSTEM SUITABILITY

It includes various parameters like theoretical plates, tailing factor, system precision and capacity factor. Various system suitability parameters for the developed method are shown in Table 5.130.

Table 3: System suitability parameters for HPLC Method

Sr. No.	System suitability parameter	Values obtained		Acceptance limit according to FDA guidelines
Sr. No.	System suitability parameter	Olme	Inda	Acceptance limit according to FDA guidelines
1	Theoretical Plates (N)	8398	73829	>2000
2	Retention time	4.79 min	7.59 min	-
3	Tailing Factor (T)	1.24	1.45	≤2

5.4.6.3 LINEARITY

The linearity of the response of olmesartan medoxomil was found to be between 50-250 μg/mL concentration and indapamide was found to be 10-50 μg/mL. The calibration graphs were obtained by plotting the peak area versus the concentration. Regression coefficient was found to be 0.998 for both Olmesartan medoxomil and indapamide.The linearity of the analytical method is its ability to elicit test results which are directly proportional to the concentration of the analyte in the sample.

Table 4: Concentration and Area of Olmesartan medoxomil and Indapamide.

Sr. no	Concentration (μg/ml)		Peak Area ± SD (n=6)
	Olmesartan Medoxomil	Indapamide	Olmesartan Medoxomil	Indapamide
1	50	10	900265±24	482722±5586
2	100	20	1774723±738	865436±221
3	150	30	2607065±493	1297231±3580
4	200	40	3490396±220	1787321±1101
5	250	50	4492569±427	2219382±105

Figure 13. Linearity graph of Olmesartan medoxomil and Indapamide

Figure 14: Calibration curve of Olmesartan medoxomil and Indapamide

[B] PRECISION:

Method precision (intra-day precision) was evaluated by carrying out three independent measurements of standard drug solution at three times on the same day .The results obtained for Mean % Relative Standard Deviation (RSD)values obtained for three concentrations (50 μg/mL, 100 μg/mL and 150 μg/mL)of olmesartan medoxomil and Indapamide(10 μg/mL, 20 μg/mL and 30 μg/mL) at intraday precision were 0.23% and 0.28% for olmesartan medoxomil and Indapamide respectively and at interday precision were 0.58% and 0.64% for olmesartan medoxomil and Indapamide respectively (Table 5.131). All the data are within the acceptance criteria of 1%

INTRADAY:

Table 5: Intraday precision of Olmesartan medoxomil and Indapamide

Conc.(µg/ml)		Peak Area ± SD (n=3)		%RSD		Mean % RSD
OLME	INDA	OLME	INDA	OLME	INDA	OLME	INDA
50	10	901417±648	485271±670	0.07%	0.13%	0.23%	0.28%
100	20	1772761±2697	866080±544	0.15%	0.06%
150	30	2606748±581	1295498±3205	0.02%	0.23%

INTERDAY:

Table 6: Interday precision of Olmesartan medoxomil and Indapamide

Conc.(µg/ml)		Peak Area ± SD (n=3)		%RSD		Mean % RSD
OLME	INDA	OLME	INDA	OLME	INDA	OLME	INDA
50	10	903950±1053	508178±2913	0.11%	0.57%	0.58%	0.64%
100	20	1794179±6767	876320±114	0.37%	0.01%
150	30	2619641±7180	130313±1284	0.27%	0.75%

C] ACCURACY (RECOVERY STUDY)

Accuracy expresses the closeness of agreement between the values which are accepted either as a conventional true value or an accepted reference value and the value found practically recovery was performed by preparing concentration of 50 μg/mL of Olmesartan medoxomil and 10 μg/mL of Indapamide sample solution. Three samples were prepared for each recovery level of 80%, 100% and 120% spiking of standard solution. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The accuracy was calculated as the percentage of the drug recovered from the formulation matrix.

_{% Recovery =}

_[(Sp_{iked Area – Unspiked
Area) / Standard Area] x 100}

Table 7:Accuracy of Olmesartan medoxomil and Indapamide

Conc (μg/ml )		% Spiking	Total Conc		Peak Area		Recovered Amt.		%Recovery		Mean % Recovery
OLME	INDA		OLME	INDA	OLME	INDA	OLME	INDA	OLME	INDA	OLME	INDA
50	10	-	50	10	889175	442680	49.98	10.06	99.96%	100.62%	99.92%	100.35%
50	10	80%	90	18	1600922	792100	89.98	18.02	99.97%	100.11%
50	10	100%	100	20	1774720	873055	99.75	19.86	99.75%	99.32%
50	10	120%	110	22	1957582	967900	110.03	22.02	100.02%	100.09%

[D] LOD and LOQ

The peak area of ten solutions containing 50 µg/mL were measured at 240 nm and calculated according to equation of LOD [3.3 x MSD/ slope] and LOQ [10 x MSD/ slope].

Table 8: LOD and LOQ of Olmesartan medoxomil and Indapamide

Sr No.	Parameter	OLME	INDA
1.	S.D of the Y-intercepts of 6 calibration curve	3018	11885
2.	Mean slope of the 6 calibration curves.	17790	43950
3.	LOD = 3.3 × (SD/Slope) (μg/ml)	0.55 μg/ml	0.89 μg/ml
4.	LOQ = 10 × (SD/Slope) (μg/ml)	1.69μg/ml	2.70 μg/ml

[E] ROBUSTNESS

The %RSD for Olmesartan medoxomil and indapamide for Robustness was measured by changing the pH of mobile phase, Ratio of Mobile phase and flow rate. The of mobile phase was set ±2 ºC, The , Ratio of Mobile phase was set ±5ml and Flow rate was set ±2 mL/Min.

FLOW RATE CHANGE

Table 9:Rubustness of Olmesartan medoxomil and Indapamide

Sr. No.	Flow rate (ml/min)	Conc.(µg/ml)		Peak area ± SD( n=3 )		%RSD		Mean % RSD
Sr. No.	Flow rate (ml/min)	OLME	INDA	OLM	INDA	OLME	INDA	OLME	INDA
1	0.8	50	10	902616±1561	482882±881	0.17	0.18	0.29%	0.47%
2	1.2	50	10	905232±3887	475063±3613	0.42	0.76	0.29%	0.47%

pH CHANGE

Table 10:Rubustness of Olmesartan medoxomil and Indapamide

Sr. No.	wavelength (ml/min)	Conc.(µg/ml)		Peak area ± SD( n=3 )		%RSD		Mean % RSD
Sr. No.	wavelength (ml/min)	OLME	INDA	OLM	INDA	OLME	INDA	OLME	INDA
2	6.8	50	10	901264±1739	489929±3391	0.19%	0.69%	0.125%	0.705%
3	7.2	50	10	900731±10	505025±3643	0.06%	0.72%	0.125%	0.705%

RATIO OF MOBILE PHASE CHANGE

Table 11:Rubustness of Olmesartan medoxomil and Indapamide

Sr. No.	Ratio (µg/ml)		Peak area ± SD ( n=3 )		%RSD		Mean % RSD
Sr. No.	Acetonitrile	Buffer	OLM	INDA	OLME	INDA	OLME	INDA
1	50	50	2483623±12960	439522±1297	0.52%	0.29%	0.42%	0.30%
2	40	60	178393±581	615715±722	0.32%	0.11%	0.42%	0.30%

DETERMINATION OF ACTIVE INGREDIENTS IN TABLET FORMULATION

Table 12:Assay of Olmesartan medoxomil and Indapamide

Marketed formulation	Claim (mg)	Concentration taken (μg/ml)		Concentration found (μg/ml)		% Assay
		OLME	INDA	OLME	INDA	OLME	INDA
OlmyD 20	200:15	200	15	199.87	15.03	99.93%	100.2%
Olmy D 40	400:30	400	30	400.34	29.79	100.08%	99.33%

Times (Hr)	Area		%RSD
Times (Hr)	Olmesartan	Indapamide	Olmesartan	Indapamide
3	1854187	242536	1.34 %	0.09 %
6	1858362	242563
24	1899652	242942

Sr No.	Parameters	OLMESARTAN MEDOXOMIL	INDAPAMIDE
1.	Wavelength (nm)	240 nm
2.	Linearity range (μg/ml)	50-200 µg/ml	10-50 µg/ml
3.	Standard Regression equation	Y=17790x -3018	Y=43950x + 11885
4.	Correlation coefficient (R²)	0.998	0.998
5.	Precision (%RSD) Intraday Interday	0.23 0.58	0.28 0.64
6.	% Recovery (Accuracy, n = 3)	99.92%	100.35%
7.	LOD (μg/ml)	0.55%	0.89%
8.	LOQ (μg/ml)	1.69%	2.70%
9.	Robustness Flow rate change pH change Ratio change	0.29% 0.12% 0.42%	0.47% 0.70% 0.30%
10.	Assay (% Label claim) OlmyD 20 OlmyD 40	99.93% 100.08%	100.20% 99.33%