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6.1 Drug profile

6.1.1 Olmesartan

Olmesartan is indicated for the treatment of hypertension. It may be used

alone or in combination with other antihypertensive agents [1]. It is used for the

treatment of patients with high blood pressure according to the drug label [2].

Olmesartan is in a class of medications called angiotensin II receptor antagonists.

Fig: 6.a. Structure of Olmesartan

IUPAC name : 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

Molecular Formula : C29H30N6O6

Molecular mass : 558.585 g/mol

Drug Bank accession number : DB00275 (APRD00223)

CAS number : 144689-63-4

Half life : 13 hrs

Therapeutic category : Anti hypertensive drug

Solubility : Soluble in Methanol, Slightly in Water

Route : Oral

137

Mechanism of action:

It works by blocking the action of certain natural substances that

tighten the blood vessels, allowing the blood to flow more smoothly and the heart to

pump more efficiently. Angiotensin II is formed from angiotensin I in a reaction

catalyzed by angiotensin converting enzyme. Angiotensin II is the principle presser

agent of the renin-angiotensin system, with effects that include vasoconstriction,

stimulation of synthesis and release of aldosterone, cardiac stimulation and renal

reabsorption of sodium. Olmesartan blocks the vasoconstrictor effects of angiotensin

II by selectively blocking the binding of angiotensin II to the AT1 receptor in vascular

smooth muscle. Its action is, therefore, independent of the pathways for angiotensin II

synthesis. Olmesartan has more than a 12,500-fold greater affinity for the

AT1 receptor than for the AT2 receptor.

Adverse effects:

General side effects of Olmesartan are hives, difficulty breathing, swelling of

your face, lips, tongue/ throat. Several severe side effects are feeling like might pass

out, urinating less than usual or not at all, chest pain, fast heart rate, swelling in hands,

serious side effects of Olmesartan may include: dizziness, joint or muscle pain, back

pain, stomach pain, nausea, diarrhea, mild itching or skin rash, or weakness [3].

6.1.2 Hydrochlorothiazide:

Hydrochlorothiazide is a prototype drug of thiazide diuretics [4]. .it is

antihypertensive agent it increases the urination and reduces the amount of water and

sodium retained by the body. The drug is in the class of benzothiazide.

Hydrochlorothiazide is a 'water pill’ used to treat high blood pressure and fluid

retention caused by various conditions, including heart disease [5]. It causes the

kidneys to get rid of unneeded water and salt from the body into the urine. This

reduces the volume of the blood, decreasing blood return to the heart and thus cardiac

output and, by other mechanisms, is believed to lower peripheral vascular resistance.

138

Fig: 6.b. Structure of Hydrochlorothiazide

IUPAC name : 6-chloro-1,1-dioxo-3,4-dihydro-2H-1$l^{6},2,4-

benzothiadiazine-7-sulfonamide

Molecular formula : C7H8ClN3O4S2

Molecular mass : 297.74 g/mol

Drug Bank accession number : DB00999 (APRD00092)

CAS number : 58-93-5

Half life : 5.6 – 14.8 hrs

Therapeutic category : Anti hypertensive drug

Solubility : Soluble in Methanol, Slightly in Water

Route : Oral

Excretion : Primarily excreted unchanged in urine

Mechanism of action:

Hydrochlorothiazide belongs to the thiazide class of diuretics. It reduces blood

volume by acting on the kidneys to reduce sodium reabsorption in the distal

convoluted tubule. The major site of action is in the nephron appears on an electro

neutral Na+, Cl- co-transporter by competing the chloride site on the transporter [6.7]. It

is also used to treat the hypertension, congestive heart failure, symptomatic edema,

diabetes insipidus, renal tubular acidosis, and the prevention of kidney stones,

hypercalciuria, Dent's disease and Méniere's disease, osteoporosis [8, 9].

139

Adverse effects:

The drug dosage is increases; the following serious side effects occurs they are,

kidney disease, liver disease, glaucoma, asthma, allergies, diabetes, an allergy to sulfa

drugs, penicillin. The drug hydrochlorothiazide is more toxic in nature. The most

common side effects are muscle weakness, dizziness, cramps, thirst, stomach pain,

nausea, vomiting, diarrhea, loss of appetite, headache, hair loss, sore throat with fever,

unusual bleeding or bruising, severe skin rash with peeling skin, difficulty breathing

or swallowing[10].

The list of available brand names, label claim details and Manufacturer

company names of Olmesartan, Hydrochlorothiazide are shown in the table 6.1.

S.NO Brand Name Available form Label claim

In mg

Manufacturer

Olmesartan formulations:

1 OLMAT Tablet 20, 40 Micro Carsyon

2 OLMEZEST Tablet 10, 20 Sun

3 OLMY Tablet 10, 20, 40 Zydus

Hydrochlorothiazide formulations:

1 AQUAZIDE Tablet 12.5, 25mg SUN

2 BPZIDE Tablet 12.5, 25mg Stadmed

3 HYDRIDE Tablet 12.5, 25mg East west

4 HYZIDE Tablet 12.5, 25mg Zydus

Olmesartan and Hydrochlorothiazide combined formulations:

1 OLMAT-H Tablet OL-20, H-12.5 Micro carsyon

2 OLMETOR-H Tablet OL-20, H-12.5 Torrent

3 OLMY-H Tablet OL-40, H-12.5 Zydus

Table: 6.1: Formulations of Olmesartan and Hydrochlorothiazide

140

6.2 Review of Literature

Chaitanya prasad MK et al [11] described a Reversed phase high performance

liquid chromatographic method (RP-HPLC) and validated for the estimation of

Olmesartan medoxomil in bulk and formulation. Selected mobile phase was a

combination of phosphate buffer with pH adjusted at 2.8 and acetonitrile (35:65%

v/v) and wavelength selected was 250 nm. Retention time of Olmesartan medoxomil

was 2.591 min. Linearity of the method was found to be 50-150 µg/ml, with the

regression coefficient of 0.9993. Quantification was done by calculating area of the

peak and the limit of detection and limit of quantitation were 0.02µg/ml and

0.09µg/ml, respectively. There was no significant difference in the intraday and inter

day analysis of Olmesartan medoxomil determined for three different concentrations

using this method. This method can be applied for the determination of Olmesartan

medoxomil in quality control of formulation without interference of the excipients.

Sharma RN et al [12] developed and validated a simple, sensitive and precise

RP-HPLC-DAD method for the determination of olmesartan medoxomil (AT-II

receptor blocker) in the presence of its degradation products. Olmesartan medoxomil

and all the degradation products were resolved on a C -18 column with the mobile

phase composed of methanol, acetonitrile and water (60:15:25, V/V/V, pH 3.5 by

ortho phosphoric acid) at 260 nm using a photodiode array detector. The method was

linear over the concentration range of 1-18 µg/ml and precise with RSD < 1 % in

intraday and interday study. Excellent recoveries of 99.3 ± 0.9 to 100.8 ± 1.2% proved

the accuracy of the method. Developed method was specific, as indicated by

chromatographic resolution > 2.0 for each peak and sensitive with LOD 0.03 µg/ml

and LOQ 0.1 µg/ml. The method was used to study the drug degradation behavior

under forced conditions. Four degradation products (DP-I, II, III, IV) were formed

during the degradation study in 0.1 mol/lit Hcl solution, whereas only DP-I, II and III

were formed in water, 0.01 mol/lit NaoH solution and 3% H2O2 solution. No

significant thermal or photolytic degradation was observed in solid drug. The method

was applied successfully for the assay of Olmesartan medoxomil in the tablet dosage

form.

Kumanan Raghunathan et al [13] developed an accurate, precise, specific, and

reproducible and stability indicating HPLC method for the estimation of Olmesartan

141

medoxomil (OLM) in presence of its degradation products and related impurities for

assessment of purity of bulk drug and stability of its dosage forms. The

chromatographic separation was achieved on a column Luna C18 (Alkyl reversed

phases) using acetonitrile: 0.05M KH2PO4 (50: 50 v/v), pH-4.5 mobile phase, flow

rate was 1.0 ml/ min. The method was validated according to the regulatory

guidelines with respect to precision, accuracy, linearity and limit of detection (LOD)

and limit of quantification (LOQ). Results of estimation of OLM in tablet formulation

were accurate and precise with standard deviation < 2. All the validation parameters

were within acceptance range.

B. Raja et al [14] developed a simple reversed phase HPLC method for the

simultaneous determination of Olmesartan medoxomil in combination with

hydrochlorothiazide. The method was based on reversed phase liquid chromatography

using axterra symmetry C18 column (150 × 4.6 mm, 5μ) with UV detection at 230

nm. The mobile phase consisting of acetonitrile and potassium dihydrogen phosphate

buffer adjusted to pH 2.5 in a ratio of (45:55, v/v) and at a flow rate of 0.7 ml/min.

The method was linear over the concentration range for Olmesartan medoxomil 20-60

μg/ml and for hydrochlorothiazide 20-60 μg/ml. The recoveries of Olmesartan

medoxomil and hydrochlorothiazide were found to be in the range of 98.0-102.0%

and 98.0-102.0% respectively. The method was validated and was successfully

employed for the analysis of pharmaceutical formulations containing Olmesartan

medoxomil and hydrochlorothiazide in combined tablet dosage form.

D. J. Kalena et al [15] described a simple, precise, rapid, efficient and

reproducible reverse Phase high performance liquid chromatography (RP-HPLC)

method for the simultaneous estimation of AT and OLM present in its tablet dosage

forms. Chromatographic separations were carried out isocratically at 30°C ± 0.5°C on

a Kromasil C18 Column (5 μm, 250mm x 4.60mm) with a mobile phase composed of

Methanol: Acetonitrile: Water (pH 3.65) in the ratio of 50:27:23 % v/v at a flow

rate of 1.0 ml/min. Detection was carried out using a UV detector at 260 nm. The

retention times for AT and OLM were 5.3 ± 0.5 min and 3.4 ± 0.5 min respectively.

The linearity range for AT and OLM were found to be 10-60 μg/ml and 20-120μg/ml

with correlation coefficient of 0.996 and 0.999 respectively. The %recovery of the

proposed method was found in the range of 98.36-100.91 for AT and 99.27-100.99

for OLM. The relative standard deviations for three replicate measurements in three

142

concentrations of standard solution were always less than 2%. The results of the study

showed that the proposed RP- HPLC method was simple, rapid, precise and accurate,

which may be useful for the routine estimation of AT and OM in bulk drug and in its

pharmaceutical dosage form.

Bahiamoussa et al [16] described reversed phase high performance liquid

chromatography (RP-HPLC) and high performance thin layer chromatography

(HPTLC), densitometry methods as a stability indicating assays of olmesartan

medoxomil in presence of its acid or alkaline induced degradation products.

Olmesartan medoxomil and its degradation products were analyzed by HPLC

equipped with UV-Variable wave length detector at 257 nm where quantitation was

achieved by isocratic elution on Agilent, Exclipse XDB- C18 column with mobile

phase composed of acetonitrile: methanol: water: glacial acetic acid (40:35:25:0.1

v/v/v/v) at flow rate 1ml/min. HPTLC was performed on aluminum packed Nano

silica gel 60 F254TLC plates as stationary phase with significant difference in Rƒ

values between olmesartan medoxomil and its degradates using chloroform:

methanol: formic acid (8:1.5:0.5 v/v/v) as mobile phase. Densitometric evaluation of

intact drug was carried out at 260 nm. The calibration curve of olmesartan medoxomil

in bulk form was linear from 0.5- 10 µg/ ml and 0.05- 1 mg/ml with mean percentage

accuracy 99.97 ± 1.085 % and 100.35 ± 1.060 % for HPLC and HPTLC methods,

respectively. The two proposed methods were successfully applied for the

determination of olmesartan medoxomil in drug substance and in drug product.

Methods validation was tested for linearity; accuracy; precision; selectivity and

robustness, according to USP guidelines.

Md. Arif Hossen et al [17] developed a simple, sensitive and specific liquid

chromatography (RP-HPLC) method and validated for the quantification of

hydrochlorothiazide and losartan potassium in tablet dosage form. A shim-pack CLC-

ODS column (250 mm X 4.6 mm, 5µ and a mobile phase constituting 0.025 M

phosphoric acid solution: acetonitrile (60:40 v/v, pH 3.0 adjusted with 80%

phosphoric acid) were used. The flow rate was 1.5 ml/min and detection was carried

by using ultraviolet (UV) detector at a wavelength of 254 nm. The retention times of

hydrochlorothiazide and losartan potassium were 3.748 min and 8.790 min,

respectively. The peaks of hydrochlorothiazide and losartan potassium were well

separated (resolution 22.17). The calibration curves were linear over the concentration

143

range of 80% to 120% (r2 > 0.999 for both the drugs). The proposed method was

accurate with 100.165% recovery for hydrochlorothiazide and 100.422% recovery for

losartan potassium and precise (% RSD < 0.5). The proposed method was

successfully applied for the estimation of hydrochlorothiazide and losartan potassium

in market products (three brands) and potency was found within limit. Therefore, this

method can be a convenient and efficient option for the analysis of

hydrochlorothiazide and losartan potassium in tablet dosage form.

Kullai Reddy Ulavapally et al [18] developed a simple and accurate RP-HPLC

method for the simultaneous estimation of Hydrochlorothiazide, Amlodipine besylate

and Valsartan by using C18 column 150 x 4.6 mm, 5μm with a simple gradient

elution(0-4min, sol-A:80-55; 4-8min- sol-A:55-40; 8-11min- sol-A:40-30; 11-13min-

sol-A:30-80 and 13-16min- sol-A:80-80). Mobile phase comprising of sol-A (pH

3.00±0.05 of 0.01M Potassium dihydrogen phosphate) and sol-B (Acetonitrile).

Flow rate was 1.00 ml/min and the detection was monitored out by UV detector at

237nm. The retention times for Hydrochlorothiazide, Amlodipine besylate and

Valsartan were found 4.5, 6.0 and 10.6 minutes. The proposed method has permitted

the quantification of Hydrochlorothiazide, Amlodipine besylate and Valsartan over

linearity in the range of 5-75 µg/ml and applicable for bulk and all type of

pharmaceutical dosage forms.

S. S. Qutab et al [19] described a simple, sensitive, and inexpensive high-

performance liquid-chromatographic method for simultaneous determination of

hydrochlorothiazide and candesartan cilexetil in pharmaceutical formulations.

Chromatographic separation was achieved on a Phenyl-2 column with a 25:75:0.2

mixture of 0.02 M potassium dihydrogen phosphate, methanol, and triethylamine,

final pH 6.0 ± 0.1, as mobile phase. Detection was at 271 nm. Response was a linear

function of concentration in the range 5–45 µg/ml for hydrochlorothiazide and 12–56

µg/ml for candesartan cilexetil; the correlation coefficients were 0.9993 and 0.9991,

respectively. Total elution time for the two components was less than 5 min.

Meyyanathan sn, rajan s et al [20] developed a simple, selective, rapid, precise

and economical reverse phase high pressure liquid chromatographic method for the

simultaneous estimation of nebivolol and hydrochlorthiazide from pharmaceutical

formulation. Phenomenexgeminic (18) (25 cm×4.6 mm i.d., 5 μ) column with a

144

mobile phase consisting of acetonitrile: 50mm ammonium acetate (adjusted to pH 3.5

using orthophosphoric acid) (70:30 v/v) at a flow rate of 1.0 ml/min was used.

Detection was carried out at 254 nm. Probenecid was used as an internal standard.

The retention times of probenecid, nebivolol and hydrochlorthiazide were 13.05, 3.32

and 4.25 min, respectively. The developed method was validated in terms of accuracy,

precision, linearity, limit of detection, limit of quantification and solution stability.

The proposed method can be used for the estimation of these drugs in combined

dosage forms.

Gandhimathi M et al [21] described a simple, precise and rapid HPLC method

and validated for the estimation of quinapril and hydrochlorothiazide simultaneously

in combined dosage form. The mobile phase used was a mixture of 0.1% v/v

triethylamine (pH 3.5), containing 1 mm of hexane sulphonic acid: acetonitrile

(30:70% v/v). The detection of quinapril and hydrochlorothiazide was carried out on

photo diode array detector at 220 nm. Results of the analysis were validated

statistically and by recovery studies. The proposed method can be successfully used to

determine the drug contents of marketed formulation.

Gong Q et al [22] described a HPLC method on C18 column using methanol-

0.1% phosphoric acid (20: 80) as mobile phase, and the detection wave length was

327 nm, the flow rate was 1.0 ml/min and the temperature of column was 400C.In the

HPLC method, the calibration curve for chlorogenic acid, hydrochlorothiazide were

linear in the range of 0.049 6-0.496 (r2 = 0.999 5) and 1.002-10.02 (r2 = 0.999 8). The

average recovery for chlorogenic acid, hydrochlorothiazide was 101.0% and 100.1%.

RSD were 2.0% and 1.4% (n=9), respectively. The method was convenient, precise

and reliable for determining the content of chlorogenic acid and hydrochlorothiazide.

Jain ps et al [23] developed and validated a simple, specific, accurate and

precise stability-indicating reversed-phase high-performance liquid chromatographic

method for simultaneous estimation of olmesartan medoxomile (olme), amlodipine

besylate (amlo) and hydrochlorothiazide (hctz) in tablet dosage form. The method was

developed using an RP C18 base deactivated silica column (250 × 4.6 mm, 5 µm)

with a mobile phase consisting of triethylamine (pH 3.0) adjusted with

orthophosphoric acid (a) and acetonitrile (b), with a timed gradient program of t/%b:

0/30, 7/70, 8/30, 10/30 with a flow rate of 1.4 ml/min. Ultraviolet detection was used

145

at 236 nm. The retention times for olme, amlo and hctz were found to be 6.72, 4.28

and 2.30 min respectively. The proposed method was validated for precision,

accuracy, linearity, range, robustness, ruggedness and force degradation study. The

calibration curves of olme, amlo and hctz were linear over the range of 50-150, 12.5-

37.5 and 31-93 µg/ml, respectively. The method was found to be sensitive. The limits

of detection of olme, amlo and hctz were determined 0.19, 0.16 and 0.22 µg/ml and

limits of quantification of olme, amlo and hctz were determined 0.57, 0.49 and

0.66µg/ml, respectively. Forced degradation study was performed according to

international conference on harmonization guidelines.

Carlucci g et al [24] developed an HPLC method with dad detection and

validated for the simultaneous determination of zofenopril and hydrochlorothiazide in

tablets. The separation was carried out through a gradient elution using an agilent

lichrospher C18 column (250x4.0 mm id, 5 microm) and a mobile phase consisting of

(a) water-tfa (99.9:0.1 v/v) and (b) acetonitrile-tfa (99.1:0.1 v/v) delivered at a flow-

rate of 1.0 ml/min. 8-chlorotheophylline was used as internal standard. Calibration

curves were found to be linear for the two drugs over the concentration ranges of 5.0-

40 and 1.0-20 µg/ml for zofenopril and hydrochlorothiazide, respectively. Linearity,

precision, accuracy, specificity and robustness were determined in order to validate

the proposed method, which was further applied to the analysis of commercial tablets.

The proposed method was simple and rapid, and gives accurate and precise results.

Hplc-dad method for the simultaneous determination of zofenopril and

hydrochlorothiazide in oral pharmaceutical formulations.

Singh Brijesh et al [25] described a simple, sensitive and rapid reverse phase

HPLC method for the simultaneous analysis of metoprolol succinate and

hydrochlorothiazide in a solid dosage form. The drugs were analysed by a reverse

phase C18 column using 50mm di-sodium hydrogen phosphate: methanol: acetonitrile

in a ratio of 52.5:22.5:25.0 as mobile phase. The flow rate was 1 ml/min and the

compounds were detected by a UV-detector at 222 nm at a column temperature of 24

± 2 ºc. The method was statistically validated for linearity and accuracy. The

retention time and drug content of metoprolol succinate and hydrochlorothiazide were

5.38 min, 96.05 % and 3.04 min., 97.64 %, respectively. The study shows that the

developed method was simple and accurate and that it would be suitable for the

146

simultaneous determination of metoprolol succinate and hydrochlorothiazide in

pharmaceutical formulations.

Psrchnp et al [26] developed a simple, fast and precise reverse phase, isocratic

HPLC method for the separation and quantification of telmisartan and

hydrochlorothiazide in pharmaceutical dosage form. The quantification was carried

out using prontosil C18-EPS 4.6x150mm, 3µm enhanced polar selectivity column and

mobile phase comprised of potassium dihydrogen phosphate buffer pH adjusted to 3.2

± 0.5 with orthophosphoric acid and acetonitrile in proportion of ratio 55:45 and

degassed under ultrasonication. The flow rate was 0.8ml/min and the effluent was

monitored at 271nm. The retention time of telmisartan and hydrochlorothiazide were

5.01±0.5 and 2.94±0.5 respectively. The method was validated in terms of linearity,

precision, accuracy, specificity, limit of detection and limit of quantification.

Linearity of telmisartan and hydrochlorothiazide were in the range of 15.01 to

75.05µg/ml and 5.02 to 25.10µg/ml respectively. The percentage recoveries of both

the drugs were 100.8% and 99.5% for telmisartan and hydrochlorothiazide

respectively from the tablet formulation. The proposed method was suitable for

simultaneous determination of telmisartan and hydrochlorothiazide in pharmaceutical

dosage form.

147

6.3. Material and Methods

6.3.1. Instrumentation:

To develop a High Pressure Liquid Chromatographic method for quantitative

estimation of Olmesartan and Hydrochlorothiazide, an isocratic PEAK HPLC

instrument with Chromosil C18 column (250 mm x 4.6 mm, 5μ) was used. The

instrument was equipped with, Rheodyne manual sample injector with a 20 μl loop

for the injection of sample. PEAK LC software was used. UV 2301

Spectrophotometer was used to determine the wavelength of maximum absorbance.

Standard and sample drugs were weighed by using Denver electronic analytical

balance (SI-234) and pH of the mobile phase was adjusted by using Systronics digital

pH meter.

6.3.2 Chemicals and Solvents:

The drug samples, Olmesartan and Hydrochlorothiazide working standards were

obtained as gift sample by Ranbaxy Pvt. Ltd, Hyderabad, AP, India. The

pharmaceutical formulation was procured from local market. Methanol, Acetonitrile

TEA and water used were HPLC grade and were purchased from Merck Specialties

Private Limited, Mumbai, India. Orthophosphoric acid and remaining buffer solutions

used were AR Grade and purchased from Merck Specialties Private Limited,

Mumbai, India.

6.3.3 Preparation of standard stock solution:

Olmesartan and Hydrochlorothiazide (1mg/ml) standard stock solutions were

prepared using methanol as a solvent. Aliquots of mixed standard solutions of

Olmesartan and Hydrochlorothiazide were diluted in mobile phase to get a final

concentration of 40-100µg/ml.

6.3.4 Preparation of sample solution:

Pharmaceutical dosage form containing 8 mg of Olmesartan and 12.5 mg of

Hydrochlorothiazide was weighed and dissolved in 25 ml of methanol and sonicated

for 15 min. Using methanol the volume was made up to 50 ml and filtered through

0.45μ membrane filter. The final mixed sample solution corresponding to 40µg/ml of

Olmesartan and 60.25µg/ml of Hydrochlorothiazide was prepared.

148

6.4. Method Development

For developing the method, a systematic study of the effect of various factors

was undertaken by varying one parameter at a time and keeping all other conditions

constant. Method development consists of selecting the appropriate wave length and

choice of stationary and mobile phases. The following studies were conducted for this

purpose.

6.4.1 Detection wavelength:

The spectrum of diluted solutions of the Olmesartan and Hydrochlorothiazide

in methanol was recorded. The absorption spectrum of Olmesartan and

Hydrochlorothiazide obtained by scanning the sample separately on UV

spectrophotometer in UV region (200-400nm) in spectrum mode showed that the drug

has maximum absorbance at 259nm. Analysis was carried out by adjusting the UV

detector of the HPLC system at 259nm.

6.4.2 Choice of stationary phase:

Preliminary development trials have performed with octadecyl columns with

different types, configurations and from different manufacturers. Finally the expected

separation and shapes of peak was succeeded Analytical column Inertsil ODS C-18

column with 250 x 4.6mm internal diameter and 5µm particle size.

6.4.3 Selection of the mobile phase:

Several systematic trials were performed to optimize the mobile phase.

Different solvents like Methanol, Water and Acetonitrile in different ratios and

different pH values of the mobile phase ratios by using different buffer solutions in

order to get sharp peak and base line separation of the components and without

interference of the excipients. Satisfactory peak symmetry, resolved and free from

tailing was obtained in mobile phase Methanol: Acetonitrile: TEA (46:50:04 V/V/) in

isocratic condition.

6.4.4 Selection of the mobile phase flow rate:

Flow rates of the mobile phase were changed from 0.5 – 1.2 ml/min for

optimum separation. A minimum flow rate as well as minimum run time gives the

maximum saving on the usage of solvents. It was found from the experiments that

1ml/min flow rate was ideal for the successful elution of the analyte.

149

6.4.5. Optimization of HPLC Method:

All drugs were subjected to chromatographic analysis using mobile phases of

differing pH, flow rate using under mentioned chromatographic conditions. The

changes in the retention time of all drugs were noted as a function of changing mobile

phase, pH, flow rate, strength and selectivity. Initially the mobile phase taken for this

combination analysis was methanol: Acetonitrile: TEA (46:50:04 V/V/) and the flow

rate is 1.0ml/min, the wavelength was fixed at 259nm, 20 micro liters of the sample

was injected. The percentage RSD for precision and accuracy of the method was

found to be less than 2%. The method was validated as per the ICH guidelines.

API Concentration Olmesartan - 70µg/ml Hydrochlorothiazide- 70µg/ml

Mobile Phase Methanol: Acetonitrile: TEA(46:50:04 V/V/)

Wavelength 259nm

Column C18 Column

Pump mode Isocratic

Diluents Mobile phase

Injection volume 20µl

Mobile phase pH 6.8

Concentration 70µg/ml

Retention Time Olmesartan -1.5 Hydrochlorothiazide - 3.5

Run Time 10min

Area Olmesartan -317192 Hydrochlorothiazide - 350243

Theoretical Plates Olmesartan -6017 Hydrochlorothiazide - 32262

Tailing Factor Olmesartan -1.41 Hydrochlorothiazide - 1.39

Pump Pressure Ambient

Table 6.2: Optimized chromatographic conditions for Olmesartan and

Hydrochlorothiazide

150

Figure 6.c: chromatogram of blank solution

Figure 6.d: standard chromatogram of Olmesartan and Hydrochlorothiazide

151

Figure 6.e: standard chromatogram of Hydrochlorothiazide

Figure 6.f: standard chromatogram of Olmesartan

152

6.5. Validation of the Proposed Method

The proposed method was validated as per ICH guidelines. The parameters studied

for validation were specificity, linearity, precision, accuracy, robustness, system

suitability, limit of detection and limit of quantification.

6.5.1 Linearity and range:

The mixed standard stock solution 100µg/ml of Olmesartan and 100µg/ml of

hydrochlorothiazide were further diluted to get Olmesartan and Hydrochlorothiazide

concentration in the range of 40 to 100 µg/ml. Linearity of the method was studied by

injecting six concentrations of the drug prepared in mobile phase into the LC system

keeping the injection volume constant. The peak areas were plotted against the

corresponding concentrations to obtain the calibration Curves indicates that the

response was linear over the concentration range studied with correlation coefficient

(r2) value, slope and intercept values were also shown in the given table 6.3.

S.NO Concentration

µg/ml

Olmesartan Hydrochlorothiazide

1 40 127605 146522

2 50 163545 180158

3 60 191473 215992

4 70 221862 246402

5 80 256579 276930

6 90 286424 313269

7 100 317192 350243

Calibration

range:

40-100

µg/ml

Slope:

Intercept:

C.C:

3172.797

1251.173

0.9998

3459.117

4318.556

0.9995

Table: 6.3 Linearity results of Olmesartan and hydrochlorothiazide

153

Fig 6.g: Calibration curve of Olmesartan

\

Fig 6.h: Calibration curve of Hydrochlorothiazide

6.5.2 Precision:

The precision of the method was verified by repeatability and intermediate precision

studies. Repeatability was calculated from six replicate injections of freshly prepared

Olmesartan and Hydrochlorothiazide combined test solutions at a concentration range

of 70µg/ml on the same day. The experiment was repeated by assaying freshly

prepared solution at the same concentration additionally on two consecutive days to

determine intermediate precision. Peak areas were determined and % of RSD was

calculated.

154

Intraday precision:

Injection No. Concentration Olmesartan Hydrochlorothiazide 1

70µg/ml

221884 246202 2 221015 246589 3 221328 246397 4 221492 246571 5 221030 246056 6 221563 246959

Result: SD:

Mean: % RSD

334.1578 221385.3

0.15

319.8998 246462.3

0.154

Table 6.4: Intraday precision results of Olmesartan and hydrochlorothiazide

Interday precision:

Injection No Concentration Olmesartan Hydrochlorothiazide

1

70µg/ml

221386 246176

2 221934 246372

3 221058 246087

4 221943 246905

5 221683 246712

6 221708 246534

Result:

SD: Mean:

% RSD:

342.1191 21618.7

0.154

314.4587 246464.3

0.127

Table 6.5: Interday precision results of Olmesartan and hydrochlorothiazide

6.5.3 Limit of Detection and Limit of Quantification:

To determine the Limit of Detection (LOD) sample was dissolved by using

mobile phase and injected until peak was disappeared. After 0.05µg/ml and 0.08

µg/ml dilution Peaks were not clearly observed, based on which 0.05µg/ml and

0.08µg/ml was considered as Limit of Detection for olmesartan and

hydrochlorothiazide, respectively. Limit of Quantification was 0.15µg/ml and

0.02µg/ml for Olmesartan and hydrochlorothiazide, respectively.

155

Parameter Olmesartan (µg/ml) Hydrochlorothiazide (µg/ml)

Limit of

Quantification 0.15

0.02

Limit of Detection 0.05 0.08

Table 6.6: LOD and LOQ results of Olmesartan and hydrochlorothiazide

6.5.4 Robustness of the method:

To evaluate robustness of a HPLC method, few parameters were deliberately

varied. The parameters included variation of flow rate, percentage of mobile phase

changes, and slight variations of the wave length changes. At standard concentration

was analyzed under these experimental conditions. It was observed that there were no

marked changes in chromatograms, which demonstrated that the developed method

was robust in nature. The robustness acceptance criteria set in the validation were the

same established on system suitability test. Robustness results were shown in table

6.7.

S.NO Parameter Change Olmesartan Hydrochlorothiazide

Area %Change Area %Change

1 standard No change 221386 0 246176 0

2

Mobile

phase -1

Mobile

phase -2

Methanol,

Acetonitrile,

TEA

(40:50:10)

(35:55:10)

221453

221467

0.03

0.04

246871

246951

0.28

0.31

4 pH 6.6

7.2

221867

221553

0.22

0.075

246374

246428

0.08

0.10

6 Wave

Length

261nm

257nm 221069

221047

0.143

-0.15 246985

246854

0.33

0.275

Table 6.7: Robustness results of Olmesartan and hydrochlorothiazide

156

6.5.6 Specificity:

By comparing the results obtained by injecting standard and blank specific of

the developed method was established. It was found that no peak was observed on

blank injection and standard solution showed two sharp and well resolved peaks,

indicates that the proposed method was specific. Specificity results were shown in

table 6.8.

Condition Olmesartan Hydrochlorothiazide

Standard Blank Standard Blank

Retention Time 1.5 No peak 3.5 No peak

Theoretical

plates

6017 No peak 32282 No peak

Tailing factor 1.41 No peak 1.39 No peak

Table 6.8: Specificity results of Olmesartan and hydrochlorothiazide

6.5.7 Accuracy:

The accuracy of the method was determined by standard addition method. A

known amount of standard drug was added to the fixed amount of pre-analyzed tablet

solution. Percent recovery was calculated by comparing the area before and after the

addition of the standard drug. Recovery test was performed at 3 different

concentrations i.e. 60µg/ml, 80µg/ml, 100µg/ml. The percent recovery was calculated

and results were presented in Table. Satisfactory recoveries ranging from 98.1 to

100.2 for Olmesartan and 98.5-101.3 for hydrochlorothiazide were obtained by the

proposed method. This indicates that the proposed method was accurate. Results of

the recovery were shown in table 6.9 for Olmesartan and 6.10 for

hydrochlorothiazide.

157

%

Recovery

Olmesartan

Target

(µg/ml)

Spiked

(µg/ml)

Total

(µg/ml)

Conc.,

Obtained

% of

Recovery

Conc.,

Obtained

50% 40 20 60 59.8 99.6 Average: 98.83

%RSD: 0.76 50% 40 20 60 59.3 98.8

50% 40 20 60 58.9 98.1

100% 40 40 80 79.2 99.0 Average: 99.52

%RSD: 0.45 100% 40 40 80 79.8 99.75

100% 40 40 80 79.9 99.8

150% 40 60 100 100.2 100.2 Average: 99.47

%RSD:0.71 150% 40 60 100 98.8 98.8

150% 40 60 100 99.4 99.4

Table 6.9: Accuracy results of Olmesartan

%

Recovery

Hydrochlorothiazide

Target

(µg/ml)

Spiked

(µg/ml)

Total

(µg/ml)

Conc.,

Obtained

% of

Recovery

Conc.,

Obtained

50% 40 20 60 59.5 99.1 Average: 98.97

%RSD: 0.42 50% 40 20 60 59.6 99.3

50% 40 20 60 59.1 98.5

100% 40 40 80 79.1 98.8 Average: 99.22

%RSD: 0.404 100% 40 40 80 79.4 99.25

100% 40 40 80 79.7 99.6

150% 40 60 100 99.7 99.7 Average: 99.90

%RSD:1.31 150% 40 60 100 101.3 101.3

150% 40 60 100 98.7 98.7

Table 6.10: Accuracy results of hydrochlorothiazide

158

6.5.8 Formulation:

From the prepared formulation solution, 20µl of the sample was injected into

HPLC system. Peak area response was compared with the standard values and the %

assay was calculated. Formulation estimation was carried out at market available

tablet of Olmesartan and Hydrochlorothiazide (CAMRI-4). Formulation result was

found to be 99.8% for Olmesartan and 99.93% for Hydrochlorothiazide. This

indicates that the proposed method can be successfully applicable for the estimation

of Olmesartan and Hydrochlorothiazide in formulations. Formulation chromatogram

was shown in figure 6.i. and table was shown in table 6.11.

Figure 6.i: Chromatogram of Olmesartan and hydrochlorothiazide

formulation

S.NO Formulation Available

form

Label

claim

Sample

concentration

Sample

estimated

%

Assay

1 Olmesartan Tablet 8mg 40 µg/ml 39.92µg/ml 99.8

2 Hydrochlorothiazide Tablet 12.5mg 60.25µg/ml 60.21µg/ml 99.93

Table 6.11: Formulation results of Olmesartan and hydrochlorothiazide

159

6.6 Results & Discussion

The results of validation studies on simultaneous estimation method developed

for Olmesartan and Hydrochlorothiazide in the current study involving Methanol:

Acetonitrile: Tri ethyl amine (TEA) (46:50:04 V/V/) were given below. Showed good

correlation coefficient (r2= 0.9998 for Olmesartan and r2 = 0.9995 for

Hydrochlorothiazide) in given concentration range 40-100 µg/ml Olmesartan and

hydrochlorothiazide. The mean values of the slope and intercept were 3172.797,

1251.173 for Olmesartan, and 3459.117, 4318.556 for Hydrochlorothiazide

respectively. The results of the repeatability and intermediate precision experiments

were above. The developed method was found to be precise as the % RSD values for

repeatability and intermediate precision studies were< 2 %, respectively as

recommended by ICH guidelines.

The recovery technique was performed to study the accuracy and

reproducibility of the proposed methods. For this, known quantities of the 40 µg/ml

solution were mixed with definite amounts of pre-analyzed formulations and the

mixtures were analyzed. The total amount of drug was determined by using the

proposed methods and the amount of added drug was calculated by the difference.

Satisfactory recoveries ranging from 98.1 to 100.2% for Olmesartan and 98.5-101.3%

for hydrochlorothiazide were obtained by the proposed method. This indicates that the

proposed method was accurate. Results of the recovery were shown in table 6.9 for

Olmesartan and 6.10 for hydrochlorothiazide. This showed that the recoveries of

Olmesartan and hydrochlorothiazide by the proposed methods were satisfactory.

Robustness of the method was confirmed by change in the optimized

chromatographic conditions and % change in each changed condition was calculated.

% change in all changed condition was found to be well acceptance criteria of less

than 2. This indicates that the proposed method is Robust.

The Limit of Detection (LOD) and Limit of Quantification (LOQ) of the

developed method were determined by injecting progressively low

concentrations of the standard solutions using the developed RP-HPLC method.

The LOD was the smallest concentration of the analyte that gives a measurable

response (signal-to-noise ratio of 3). The LOQ was the smallest concentration of

the analyte, which gives response that can be accurately quantified (signal-to-noise

160

ratio of 10). LOD and LOQ for Olmesartan and hydrochlorothiazide were found to

be 0.05 & 0.15 µg/ml, and 0.02 & 0.08µg/ml respectively. Formulation study was

carried out by using marketed formulation tablet of Olmesartan and

hydrochlorothiazide. Standard concentration was prepared and the area of the peak

response was used for the calculation of the % assay. It was found that up to 99%

accurately estimate Olmesartan and hydrochlorothiazide in pharmaceutical dosage

forms.

Thus the method developed in the present investigation is simple, sensitive,

accurate, rugged, robust, rapid and precise. Hence, the above said method can be

successfully applied for the estimation of Olmesartan and hydrochlorothiazide in

tablet dosage forms.

161

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