Chapter-6

Simultaneous determination of Losartan Potassium, Atenolol and Hydrochlorothiazide in pharmaceutical preparations by stability-indicating ultra performance liquid chromatography

___________________________________________

6.1 Introduction of Losartan potassium, Atenolol and

Hydrochlorathiazide and survey of analytical methods

Losartan is an angiotensin II receptor (type AT1) antagonist. Losartan

prevents the constriction (narrowing) of blood vessels (veins and arteries).

Losartan is a non-peptide molecule, is chemically described as 2-butyl-4-

chloro-1-[p-(o-1H-tetrazol-5-ylphenyl)benzyl]imidazole-5-methanol mono

potassium salt. Losartan potassium is a white to off-white free-flowing

crystalline powder with a molecular weight of 461.01. It is freely soluble

in water, soluble in alcohols, and slightly soluble in common organic

solvents, such as acetonitrile and methyl ethyl ketone. It is available as

tablets for oral administration containing 25 mg, 50 mg and 100 mg of

Losartan potassium. The empirical formula of Losartan potassium is

C22H22ClKN6O.

Atenolol is in a class of drugs called beta-blockers. Beta-blockers

affect the heart and circulatory system (arteries and veins). Atenolol is

used to lower blood pressure, lower heart rate, reduce chest pain

(angina) and to reduce the risk of recurrent heart attacks. It is chemically

described as 4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]

benzeneacetamide. It is freely soluble in 1N HCl and less soluble in

chloroform. Atenolol is available as 25, 50 and 100 mg tablets for oral

administration. The empirical formula of Atenolol is C14H22N2O3. The

molecular weight of Atenolol is 266.

Hydrochlorothiazide is a thiazide diuretic. It decreases the amount of

fluid in the body by increasing the amount of salt and water lost in the

urine. Hydrochlorothiazide is used to lower blood pressure and to

decrease edema (swelling), it is chemically described as 6-chloro-3,4-

dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide. It is a

white, or practically white, crystalline powder which is slightly soluble in

water, but freely soluble in sodium hydroxide solution. It is supplied as

12.5 mg capsules for oral use. The empirical formula of

Hydrochlorothiazide is C7H8ClN3O4S2. The molecular weight of

Hydrochlorothiazide is 297.94.

Fig: 6.1.F1: Chemical structures of Losartan potassium, Atenolol

and Hydrochlorothiazide

(c) Losartan potassium

2-butyl-4-chloro-1-[p-(o-1H-tetrazol-5-ylphenyl)benzyl]imidazole-5-

methanol mono potassium salt

Molecular formula: C22H22ClKN6O

Molecular weight: 461.01

(d) Atenolol

Molecular formula: C14H22N2O3

Molecular weight: 266

(e) Hydrochlorothiazide

Molecular formula: C7H8ClN3O4S2

Molecular weight: 297.94

In combination, these are available as 50/50/12.5 mg (Repalol H

Tablets) combined tablets with 50 mg of Losartan potassium, 50 mg of

Atenolol and 12.5 mg of Hydrochlorothiazide. Repalol H is Brand name for

Losartan potassium, Atenolol and Hydrochlorothiazide combined tablets and

it contains 50 mg of Losartan potassium per tablet, which is an angiotensin II

receptor (type AT1) antagonist, plus 50 mg of Atenolol as a beta-blockers plus

12.5 mg of Hydrochlorothiazide as a thiazide

diuretic.

Literature survey reveals that a wide variety of spectrophotometric

and chromatographic methods [1-6] were reported for determination

Losartan potassium in pharmaceutical preparations in combination with

other drugs. Spectrophotometric and chromatographic methods have

been reported for determination of Atenolol , in combination with other

drugs, in bulk and pharmaceutical preparations [7-10]. A variety of

methods have been used for determination of Hydrochlorothiazide [11-

17]. An HPTLC method has been reported for simultaneous quantitative

determination of Losartan potassium, Atenolol and Hydrochlorothiazide

in combined tablets [18]. So far no liquid chromatography method has

been reported for simultaneous quantitative determination of Losartan

potassium, Atenolol and Hydrochlorothiazide in the combined dosage

forms.

The aim of the present study was to develop a suitable stability-

indicating LC assay method for simultaneous determination of Losartan

potassium, Atenolol and Hydrochlorothiazide in combined dosage forms.

In this chapter we described a stability indicating ultra performance

liquid chromatographic (UPLC) method for the simultaneous

determination of Losartan potassium, Atenolol and Hydrochlorothiazide

in combined dosage forms along with analytical method validation as per

ICH guidelines.

6.2 Development of a new stability-indicating UPLC method for

simultaneous determination of Losartan potassium, Atenolol, and

Hydrochlorothiazide in combined dosage forms

6.2.1 Materials

Repolol H tablets (contains 50 mg of Losartan potassium, 50 mg of

Atenolol, 12.5 mg of Hydrochlorothiazide) were purchased from Sun

pharmaceutical industries Ltd, India and standards of Losartan

potassium, Atenolol and Hydrochlorothiazide were supplied by Dr.

Reddy’s laboratories limited, Hyderabad, India. The HPLC grade

acetonitrile, analytical grade triethyl amine and ortho phosphoric acid

were purchased from Merck, Germany. Water was prepared by using

Millipore Milli.Q Plus water purification instrument.

6.2.2 Instrumentation and Chromatographic Conditions

The UPLC system used consists of a binary solvent manager, a

sample manager and a UV detector. Zorbax C-18, 50 mm x 4.6 mm i.d

with 1.8 µm particles column was used as stationary phase. Mobile

phase contains a mixture of water: acetonitrile: triethyl amine: ortho

phosphoric acid (60:40:0.1:0.1, v/v/v/v), respectively. The mobile phase

was pumped at 0.7 mL min-1. The eluents were monitored at 225 nm.

The injection volume for samples and standards was 5 µL.

6.2.3 Sample preparation

A standard solution containing 500 µg mL-1 of Losartan potassium,

500 µg mL-1 of Atenolol and 125 µg mL-1 of Hydrochlorothiazide were

prepared by dissolving in diluent (mixture of acetonitrile: water (40:60,

v/v)). Working solutions of 50 µg mL-1 of Losartan potassium, 50 µg mL-1

of Atenolol, 12.5 µg mL-1 of Hydrochlorothiazide were prepared from

above stock solution in mobile phase for assay determination.

6.2.4 Preparation of Tablets Sample Solution

Twenty Repalol H tablets (contains 50 mg of Losartan potassium, 50

mg of Atenolol, 12.5 mg of Hydrochlorothiazide) were weighed and

transferred into a clean and dry mortar, grinded well. Then an equivalent

to 50 mg of Losartan potassium (50 mg of Losartan potassium, 50 mg of

Atenolol, 12.5 mg of Hydrochlorothiazide) was transferred to a 100 mL

volumetric flask, 70 mL of diluents was added and sonicated for 15 min

and diluted to 100 ml (0.5 mg mL-1 of Losartan potassium, 0.5 mg mL-1of

Atenolol, 0.125 mg mL-1 of Hydrochlorothiazide). About 5 ml of supernant

solution was taken and diluted to 50 mL with mobile phase. This was

filtered using 0.45 µ (Nylon 66- membrane) filter.

6.2.5 Generation of stress samples

One lot of Repolol H tablets were selected for stress testing. From the

ICH Stability guideline: “stress testing is likely to be carried out on a

single batch”. Different kinds of stress degradation conditions (like heat,

humidity, acid, base, oxidative and light) were performed on one lot of

Losartan potassium, Atenolol and Hydrochlorothiazide tablets based on

the guidance available from ICH Stability Guideline (Q1AR2). The details

of the stress conditions applied are as follows:

a) Acid hydrolysis: Drug solution in 0.1N HCl at 70° C for 4 h.

b) Base hydrolysis: Drug solution in 0.1N NaOH at 70°C for 4 h.

c) Study in Neutral solution: Drug solution dissolved in water was heated

at 70°C for 3 h

d) Oxidative stress: Drug solution in 5% hydrogen peroxide at room

temperature for 4 h.

e) Thermal stress: Tablets were subjected to dry heat at 80°C for 24 h.

f) Photolytic degradation: The photo degradation was carried out by

exposing the Losartan potassium, Atenolol and Hydrochlorothiazide

tablets samples in solid state to light providing an overall illumination of

not less than 1.2 million lux hours and an integrated near ultraviolet

energy of not less than 200 W h/m2.

6.3 Method development and optimization of chromatographic

conditions

Forced degradation studies were performed to develop a stability

indicating UPLC method for the simultaneous quantitative determination

of Losartan potassium, Atenolol and Hydrochlorothiazide in combined

pharmaceutical dosage forms. Stressed samples obtained during forced

degradation studies were utilized in the UPLC method development.

6.3.1 Selection of wavelength

All the three drugs Losartan potassium, Atenolol and

Hydrochlorothiazide spectrums were collected using Water PDA system.

All the three drugs were having UV maxima at around 225 nm (Fig:

6.2.F1). Hence detection at 225 nm was selected for method development

purpose.

Fig: 6.3.F1: Typical UV spectrums of Losartan potassium, Atenolol

and Hydrochlorothiazide.

Wavelength (nm)

6.3.2 Column Selection

Three different C18 (Inertsil ODS-4 50X2.1 mm, 2 microns, Aquity

BEH C18, 50 x 2.1 mm; 1.7 microns and Zorbax C-18, 50 x 4.6 mm, 1.8

microns) Columns, 2 different C8 Columns (Inertsil C8-4 50X2.1 mm, 2

microns and Aquity BEH C8, 50 x 2.1 mm; 1.7 microns and Zorbax SB-

CN, 50 x 4.6 mm, 1.8 microns) were used for method development. The

stationary phase was found to be a great influence on the retention time,

resolution and peak shape.

The main target of the chromatographic method is to get the

separation for closely eluting degradation products, mainly the

degradation product at 1.26 RRT (with respect to Atenolol), which is

eluting between Atenolol and Hydrochlorothiazide. When Aquity BEH

C18, 50 x 2.1 mm; 1.7 microns, Zorbax C-18, 50 x 4.6 mm, 1.8 microns

and Inertsil ODS-4 50X2.1 mm, 2 microns columns were used in initial

experiment with water: Acetonitrile, triethylamine and orthophosphoric

acid in the ratio 60: 40: 0.1: 0.1 (v/v/v/v), as mobile phase and 0.7 ml

min-1 as flow rate. For Inertsil and Aquity BEH columns, the separation

of 1.26 RRT degradation product was poor from Atenolol and

Hydrochlorothiazide.

For Zorbax C-18, 50 x 4.6 mm, 1.8 microns column, the tailing of all

three drugs Losartan, Atenolol and Hydrochlorothiazide was less than

1.2, resolution between these three drugs Losartan, Atenolol and

Hydrochlorothiazide was more than 3 and the separation of degradation

product at RRT 1.26 (with respect to Atenolol) was found to be good (USP

resolution was more than 2.0). Hence Zorbax C-18, 50 x 4.6 mm, 1.8

microns column was chosen for further method development. On the

other hand, when Zorbax SB-CN, 50 x 4.6 mm, 1.8 microns column was

used, the anylyte Atenolol was eluted in void.

Table 6.3.T1: Experimental results on different stationary phases

Column USP Tailing

of Losartan

USP Tailing of

Atenolol

USP Tailing of

Hydrochloro

thiazide

USP resolution

betwwen

Atenolol and 1.26

degradant

Inertisil ODS-4 (50 x 2.1 mm) with 2 µm

particles

1.3 1.2 1.3 < 1.0

Aquity BEH C18 (50

x 2.1 mm) with 1.7 µm particles

1.3 1.2 1.1 < 1.0

Zorbax SB-CN (50 x

4.6 mm) with 1.8 µm particles

1.3 * 1.2

Atenolol peak

is eluted in

void

Inertisil C8 (50 x 2.1

mm) with 2 µm

particles

1.2 1.3 1.2 < 1.0

Aquity BEH C8 (50

x 2.1 mm) with 1.7

µm particles

1.2 1.2 1.1 < 1.0

Zorbax C-18 (50 x 4.6 mm) with 1.8

µm particles

1.1 1.2 1.1 > 2.0

* Atenolol was eluted in void

6.3.3 Effect of Organic solvent

When methanol was used as a solvent in the mobile phase (water:

methanol: triethylamine: ortho phosphoric acid in the ratio 60: 40: 0.1:

0.1 (v/v/v/v)) the retention time of Losartan is around 5 min due to the

interaction of Losartan with column and the tailing factor for Losartan

and Atenolol is also high (>1.4). To improve the retention time of

Losartan, the strength of solvent was increased by replacing methanol

with acetonitrile. Different mixtures of water and acetonitrile were

applied to improve the retention time and symmetry of the drugs

Losartan, Atenolol and Hydrochlorothiazide . When acetonitrile content

was high (water: Acetonitrile: triethylamine: ortho phosphoric acid in the

ratio 40: 60: 0.1: 0.1 (v/v/v/v)), the resolution between Atenolol and

degradation product at 1.26 RRT was poor (<1.0).

When the acetonitrile content was decreased to 30%, the RT of

Losartan was about 5.5 min. When water: Acetonitrile: triethylamine:

ortho phosphoric acid in the ratio 40: 60: 0.1: 0.1 (v/v/v/v) was used as

the mobile phase, the separation and retention times of Losartan,

Atenolol and Hydrochlorothiazide were satisfactory (Resolution was more

than 3, symmetry of Losartan, Atenolol and Hydrochlorothiazide peaks

was less than 1.2 and Retention time of Losartan, Atenolol and

Hydrochlorothiazide peaks were 2.3, 0.6 and 0.9 min, respectively.

6.3.4 Effect of pH

The method was optimized to separate major degradation products

formed under varies stress conditions. Losartan potassium (pKa = 4.9) is

acetic compound where as Atenolol (pKa = 9.4) and Hydrochlorothiazide

(pKa = 7.9, 9.2) are basic compounds. The main target of the

chromatographic method was to get the separation for closely eluting

degradation products, mainly for the degradation product at 0.76 min,

which was eluting very closely to the Atenolol. At lower pH (pH 2.0)

Losartan was retaining in column for longer time (4.5 min) and also

tailing of Atenolol was more (1.6). At higher pH 7.0, the resolution

between Atenolol and Hydrochlorothiazide peaks was poor. Hence to

have a better separation and symmetry of the three drugs and also to

reduce the retention of Losartan, triethyl amine and ortho phosphoric

were added to mobile phase containing water and acetonitrile in the ratio

60: 40 (v/v). The resulting pH of the mobile phase was about 3.5. Hence

water: acetonitrile, triethylamine and ortho phosphoric acid in the ratio

60: 40: 0.1: 0.1 (v/v/v/v) was used as mobile phase.

6.3.5 Optimized chromatographic conditions for the simultaneous

determination of Losartan potassium, Atenolol and

Hydrochlorothiazide

Column: Zorbax C-18 (50 X 4.6 mm,

with 1.8 µm particle size

Mobile phase: Water: acetonitrile, triethylamine and ortho

phosphoric acid in the ratio 60: 40: 0.1: 0.1

(v/v/v/v)

Flow rate: 0.7 ml min-1

Column temperature: 25 °C

Wavelength: 225 nm

Injection volume: 5 µL

Run time: 3 min

Diluent: Acetonitrile: water (40:60, v/v)

Retention time: Losartan is about 2.3 min, Atenolol is about

0.6 min and Hydrochlorothiazide is about 0.9

min

The interference from excipients Starch, lactose anhydrous, colloidal

silicon dioxide, polyethylene glycol and opadry blue was also checked by

injecting sample solutions of excipients. There was no interference of

excipients with Losartan, Atenolol and Hydrochlorothiazide peaks.

6.4 Degradation behavior:

UPLC studies on Losartan potassium, Atenolol and

Hydrochlorothiazide tablets under different stress conditions suggested

the following degradation behavior.

6.4.1 Degradation in acidic solution

Losartan potassium, Atenolol and Hydrochlorothiazide drugs were

exposed to 0.1 N HCl at room temperature for 48 h. In 1 N HCl at room

temperature after 48 h, no major degradation was observed. When more

stressed condition was applied (0.1N HCl, 4 hours reflux at 70°C), no

degradation of the drugs was observed (Fig: 6.4.F1 (a) to Fig: 6.4.F1 (d)).

The drugs were stable to acidic condition.

Fig: 6.4.F1 (a): Typical UPLC chromatogram of acid hydrolysis

Fig: 6.4.F1 (b): Peak purity plot of Acid hydrolysis for Atenolol

Purity Angle Purity

Threshold Purity Flag

Peak Purity

0.227 0.287 No Pass

Fig: 6.4.F1 (c): Peak purity plot of Acid hydrolysis for

Hydrochlorothiazide

Purity Angle Purity

Threshold Purity Flag

Peak Purity

0.097 0.254 No Pass

Fig: 6.4.F1 (d): Peak purity plot of Acid hydrolysis for Losartan

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.029 0.252 No Pass

6.4.2 Degradation in Basic solution

Losartan potassium, Atenolol and Hydrochlorothiazide drugs were

exposed to 0.1 N NaOH at room temperature for 48 h. In 0.1 N NaOH at

room temperature after 48 h, no major degradation was observed. When

more stressed condition was applied (0.1N NaOH, 4 hours reflux at

70°C), major degradation products observed at 0.76 RT (~1.26%) and at

1.72 RT (~3%) (Fig: 6.4.F2 (a) to Fig: 6.4.F2 (d)).

Fig: 6.4.F2 (a): Typical UPLC chromatogram of alkali hydrolysis

Fig: 6.4.F2 (b): Peak purity plot of Alkali hydrolysis for Atenolol

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.924 1.544 No pass

Fig: 6.4.F2 (c): Peak purity plot of Alkali hydrolysis for

Hydrochlorothiazide

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.151 1.467 No pass

Fig: 6.4.F2 (d): Peak purity plot of Alkali hydrolysis for Losartan

Purity Angle Purity

Threshold Purity Flag

Peak Purity

0.104 0.912 No pass

6.4.3 Degradation in peroxide (Oxidative degradation)

Losartan potassium, Atenolol and Hydrochlorothiazide drugs were

exposed to 1 % peroxide at room temperature for 6 h. In 1 % peroxide at

room temperature after 6 h, no major degradation was observed. When

more stressed condition was applied (5 % peroxide, 4 hours reflux at

room temperature), major degradation products observed (~0.4%) at 1.11

RT, (1.0%) Atenolol 3.628 RT and (1.5%) at 4.172 RT (Fig: 6.4.F3 (a) to

Fig: 6.4.F3 (d))(The peak next to Atenolol is from hydrogen peroxide).

Fig: 6.4.F3 (a): Typical UPLC chromatogram of oxidative degradation

Fig: 6.4.F3 (b): Peak purity plot of Oxidative degradation for Atenolol

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.231 4.653 No pass

Fig: 6.4.F3 (c): Peak purity plot of Oxidation for Hydrochlorothiazide

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

1.589 3.208 No pass

Fig: 6.4.F3 (d): Peak purity plot for Oxidation for Losartan

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.088 4.417 No pass

6.4.4 Degradation in Neutral (Water) solution

No major degradation products were observed after 48 h at room

temperature. When more stressed condition was applied (3 hours reflux

at 70 ºC), minor degradation product observed (~1.1%) at 0.74 RT (Fig:

6.4.F4 (a) to Fig: 6.4.F4 (d)).

Fig: 6.4.F4 (a): Typical UPLC chromatogram of Water degradation

Fig: 6.4.F4 (b): Peak purity plot of water degradation for Atenolol

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.610 0.720 No pass

Fig: 6.4.F4 (c): Peak purity plot of water degradation for

Hydrochlorothiazide

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.123 0.412 No Pass

Fig: 6.4.F4 (c): Peak purity plot of water degradation for Losartan

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.023 0.263 No Pass

6.4.5 Photolytic conditions:

The drugs were stable to the effect of photolysis. When the drugs

powder was exposed to light for an overall illumination of 1.2 million lux

hours and an integrated near ultraviolet energy of 200-watt

hours/square meter (w/mhr), no degradation of the drugs were observed

(Fig: 6.4.F5 (a) to Fig: 6.4.F5 (d)).

Fig: 6.4.F5 (a): Typical UPLC chromatogram of photolytic

degradation

Fig: 6.4.F5 (b): Peak purity plot of photolytic degradation for

Atenolol

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.413 0.743 No Pass

Fig: 6.4.F5 (c): Peak purity plot of photolytic degradation for

Hydrochlorothiazide

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.337 0.389 No Pass

Fig: 6.4.F5 (d): Peak purity plot of photolytic degradation for

Losartan

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.019 0.261 No Pass

6.4.6 Thermal Degradation:

Minor degradation was observed (~0.9%) at 0.74 RT when drugs

powder was exposed to dry heat at 80°C for 24 h (Fig: 6.4.F6 (a) to Fig:

6.4.F6 (d)).

Fig: 6.4.F6 (a): Typical UPLC chromatogram of thermal degradation

Fig: 6.4.F6 (b): Peak purity plot of thermal degradation for Atenolol

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.540 0.637 No Pass

Fig: 6.4.F6 (c): Peak purity plot of thermal degradation for

Hydrochlorothiazide

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.091 0.381 No pass

Fig: 6.4.F6 (d): Peak purity plot of thermal degradation for Losartan

Purity Angle Purity

Threshold

Purity

Flag Peak Purity

0.026 0.267 No pass

Peak purity test performed for the Losartan, Atenolol and

Hydrochlorothiazide peaks using photodiode array (PDA) detector, data

confirmed the purity of the peaks for all the stressed samples.

6.5 Analytical method Validation, results and discussion

The developed and optimized UPLC method was taken up for method

validation. The analytical method validation was carried out in

accordance with ICH guidelines [19-21].

6.5.1 System Suitability Test

Standard containing Losartan potassium, Atenolol and

Hydrochlorothiazide was injected into UPLC system for 5 times and the

system suitability results were tabulated in Table 6.5.T1.

Table 6.5.T1: System Suitability results

Compound

(n=5)

USP Tailing

factor (T) USP Resolution

% RSD for 5

replicate injections

Atenolol

1.2 - 0.5%

Hydrochloro

thiazide

1.1 5.7 0.2%

Losartan 1.2 19.8 0.2%

n = Number of determinations

6.5.2 Precision

Assay method precision study was evaluated by carrying out six

independent assays of Losartan potassium, Atenolol and

Hydrochlorothiazide test sample against qualified reference standards

and RSD of six consecutive assays was 0.4%, 0.4% and 0.5% for

Losartan potassium, Atenolol and Hydrochlorothiazide respectively for

repeatability (Table 6.5.T2) and 1.0%, 0.8% and 0.8% for Losartan

potassium, Atenolol and Hydrochlorothiazide respectively for

intermediate precision (Table 6.5.T3). Results showed insignificant

variation in measured response, which demonstrated that the method

was repeatable with RSDs below 1.0 %.

Table 6.5.T2: Precision results of the assay method (Repeatability)

Preparation

% Assay

Losartan

potassium Atenolol

Hydrochlorothia

zide

1 99.2 99.4 99.3

2 98.5 99.1 98.1

3 98.8 99.1 98.8

4 98.6 98.9 98.4

5 98.9 98.4 98.8

6 99.4 98.9 99.5

Average 98.9 99.0 98.8

SD 0.36 0.35 0.51

% RSD 0.4 0.4 0.5

95% Confidence

interval of mean 98.6 to 99.2 98.7 to 99.3 98.4 to 99.2

Table 6.5.T3: Results of repeatability and Intermediate precision

S.No

Parameter

Variation

% RSD

for Assay

LK ATN HCT

1

Repeatability

(inter-day)

(a) Analyst-1

(b) Waters

Acquity UPLC

system with PDA detector.

(c) Day-1

0.4 0.4 0.5

2

Intermediate

precision

(intra-day)

(a) Analyst-2

(b) Waters

Acquity UPLC system with

TUV detector.

(c) Day-2

1.0 0.8 0.8

LK = Losartan potassium, ATN = Atenolol, HCT = Hydrochlorothiazide

6.5.3 Linearity

The linearity of the assay method was established by injecting test

sample from 50% to 150% of all the three drugs Losartan potassium,

Atenolol and Hydrochlorothiazide assay concentrations (i.e. 50 µg mL-1 of

Losartan potassium, 50 µg mL-1 of Atenolol and 12.5 µg mL-1 of

Hydrochlorothiazide). Each solution injected into UPLC and from that

calculated the correlation coefficient (Table 6.5.T4 to Table 6.5.T6). The

slope and Y-Intercept of the Losartan peak in the linearity study was

found to be 26538 and -13476, respectively, the slope and Y-Intercept of

the Atenolol peak in the linearity study was found to be 12541 and -

4094, respectively and The slope and Y-Intercept of the

Hydrochlorothiazide peak in the linearity study was found to be 43100

and -14825, respectively.

Calibration curve obtained by least square regression analysis

between average peak area and the concentration showed (Fig: 6.5.F1 to

Fig: 6.5.F3) linear relationship with a regression coefficient of 0.999. The

best fit linear equation obtained was Y =26538 Con - 13476 for Losartan

potassium, Y =12541 Con - 4094 for Atenolol and Y=43100 Con - 14825

for Hydrochlorothiazide. Analysis of residuals indicated that the

residuals were normally distributed (Table 6.5.T7 to Table 6.5.T9) around

the mean with uniform variance across all concentrations suggesting the

homoscedastic nature of data.

Table 6.5.T4: Linearity results for Losartan Potassium

Concentration (µg mL-1) Peak area

25.08 655974

37.62 982227

50.16 1326884

60.19 1562134

75.24 1994476

Correlation Coefficient(r) 0.999

Limit 0.999

Slope 26538

Intercept -13476

Fig: 6.5.F1: Linearity plot for Losartan potassium

Table 6.5.T5: Linearity results of Atenolol

Concentration (µg mL-1) Peak area

25.05 308526

37.58 470454

50.10 629814

60.12 737250

75.15 943523

Correlation Coefficient(r) 0.999

Limit 0.999

Slope 12541

Intercept -4094

Fig: 6.5.F2: Linearity plot for Assay Method for Atenolol

Table 6.5.T6: Linearity results of Hydrochlorothiazide

Concentration (µg mL-1) Peak area

6.26 258150

9.38 383995

12.51 524652

14.39 607085

18.77 793996

Correlation Coefficient(r) 0.999

Limit 0.999

Slope 43100

Intercept -14825

Fig: 6.5.F3: Linearity plot for Hydrochlorothiazide

Table 6.5.T7: Residual summary of Linearity results for Losartan

Concentration in µg/ml

Mean area

Response

achieved

Response calculated

thru Trend

line equation

Residual (Response

practical -

Response theoretical)

Residual square

25.08 655974 652087.98 -3886 15101166.4

37.62 982227 984874.53 2648 7009402.6

50.16 1326884 1317661.08 -9223 85062305.3

60.19 1562134 1583837.24 21703 471030662.1

75.24 1994476 1983234.18 -11242 126378600.4

Residual sum of squares 704582136.7

Trend line equation y = 26538x – 13476

Table 6.5.T8: Residual summary of Linearity results for Atenolol

Concentration

in µg/ml

Mean area

Response achieved

Response calculated

thru Trend

line equation

Residual

(Response

practical -Response

theoretical)

Residual

square

25.05 308526 310026.17 1500 2250507.0

37.58 470454 467167.80 -3286 10799120.3

50.10 629814 624184.02 -5630 31696723.2

60.12 737250 749847.15 12597 158688298.7

75.15 943523 938341.86 -5181 26844186.7

Residual sum of squares 230278835.8

Trend line equation y = 12541x – 4094

Table 6.5.T9: Residual summary of Linearity results for Hydrochlorothiazide

Concentratio

n in µg/ml

Mean area Response

achieved

Response

calculated

thru Trend line equation

Residual

(Response practical -

Response

theoretical)

Residual

square

6.26 25815

0 254933.96 -3216

10342939

.0

9.38 38399

5 389382.79 5388

29028330

.7

12.51 52465

2 524262.56 -389 151664.3

14.39 60708

5 605276.60 -1808

3270300.

7

18.77 79399

6 794022.09 26 680.6

Residual sum of squares 42793915

.2

Trend line equation y = 43100x – 14825

6.5.4 Accuracy

Accuracy of the method was established by injecting three

preparations of test sample using tablets at 50%, 100% and 150% of

analyte concentration (i.e. 500 µg mL-1 of Losartan potassium, 500 µg

mL-1 of Atenolol and 125 µg mL-1 of Hydrochlorothiazide). Each solution

was injected thrice (n=3) into UPLC and the mean peak area was

calculated. % Assay of test solution was determined against three

injections (n=3) of qualified Losartan potassium, Atenolol and

Hydrochlorothiazide standards (Table 6.5.T8). The method showed

consistent and high absolute recoveries at all three concentration (50,

100 and 150%) levels with mean absolute recovery ranging from 99.1 to

100.5% for Losartan potassium, from 99.0 to 100.8% for Atenolol and

from 99.1 to 100.5% for Hydrochlorothiazide. The obtained absolute

recoveries were normally distributed around the mean with uniform RSD

values.

Table 6.5.T10: Recovery from Drug product

S.No

Concent

ration

(%)

Mean recovery (%)

(n = 3)

% RSD

LK ATN HCT LK ATN HCTZ

1 50 100.5 100.8 100.5 0.44 0.15 0.12

2 100 99.1 99.0 99.1 0.96 0.91 0.74

3 150 100.0 100.1 99.6 0.40 0.17 0.41

LK= Losartan potassium, ATN= Atenolol , HCT= Hydrochlorothiazide

6.5.5 Solution state stability

The solution state stability of Losartan potassium, Atenolol and

Hydrochlorothiazide in diluent in the assay method was carried out by

leaving the test solutions of sample in tightly capped volumetric flasks at

room temperature for 24 h. The same sample solution were assayed for

every six hours interval up to the study period, each time freshly

prepared reference standard was used to estimate the assay of test

sample. The % RSD of assay of Losartan potassium, Atenolol and

Hydrochlorothiazide during solution stability experiments was within

0.5% for all the three drugs. Hence sample solutions are stable for at

least 48 hours in the developed method.

Table 6.5.T11: Solution stability results of the assay method

S.No Interval

% Assay

Losartan potassium

Atenolol Hydrochlorothiazide

1 0 h 99.2 99.4 99.3

2 6 h 98.9 99.4 99.2

3 12 h 99.0 99.5 99.0

4 18 h 98.8 99.3 99.0

5 24 h 99.2 99.0 98.8

Average 99.0 99.3 99.1

% RSD 0.18 0.19 0.19

6.5.6 Robustness

To determine the robustness of the developed method experimental

conditions were purposely altered and the tailing factor, USP resolution

between Atenolol and Hydrochlorothiazide and between

Hydrochlorothiazide and Losartan and also % RSD for 5 replicate

injections of Losartan, Atenolol and Hydrochlorothiazide peaks were

evaluated. In each of the deliberately altered chromatographic condition

(flow rate 0.6 and 0.8 mL min-1, acetonitrile composition 95% and 105%

in the mobile phase, column temperature 20 ºC and 30 ºC) the tailing

factor, USP resolution between Atenolol and Hydrochlorothiazide; and

between Hydrochlorothiazide and Losartan and % RSD for 5 replicate

injections of Losartan, Atenolol and Hydrochlorothiazide peaks were

calculated and the results are tabulated in Table 6.5.T10.

Table 6.5.T12: Results of robustness study

Parameter

Temperature

(± 5 0C of set

temperature)

Flow rate

(± 0.1 mL min-1 of

the set flow)

Acetonitrile

(± 5 of set

composition)

Variation 200C 300C 0.6 0.8 95% 105%

USP

Tailing

factor (T)

LK 1.3 1.2 1.3 1.1 1.2 1.2

ATN 1.2 1.2 1.2 1.1 1.3 1.2

HCT 1.1 1.1 1.2 1.1 1.2 1.1

USP

Resolution

between

ATN

&HC

T

5.2 5.8 5.5 5.7 5.0 5.9

HCT

&LK 17.8 19.5 15.9 19.1 16.6 18.5

% RSD for 0.3 0.6 0.4 0.2 0.6 0.5

5

replicate

injections

LK

ATN 0.3 0.1 0.4 0.2 0.2 0.3

HCT 0.7 0.8 0.2 0.5 0.5 0.4

LK= Losartan potassium, ATN= Atenolol , HCT= Hydrochlorothiazide

6.6 Summary and Conclusions

A new stability- indicating UPLC method was developed for the

simultaneous estimation of Losartan potassium, Atenolol and

Hydrochlorothiazide in pharmaceutical dosage forms in the presence of

degradation products. The column employed for separation was Zorbax

XDB C-18, 50 mm x 4.6 mm, 1.8 µm column. The chromatographic

separation was arrived on isocratic elution mode. The mobile phase

consists of a mixture of acetonitrile: Water: triethyl amine: ortho

phosphoric acid (40:60:0.1:0.1, v/v/v/v). The flow rate of mobile phase

was 0.7 mL min-1 and the wavelength for detection was 225 nm. The

retention times of Losartan, Atenolol and Hydrochlorothiazide were 2.3

min, 0.6 min and 0.9 min; respectively. The total runtime was 3 min.

Losartan potassium, Atenolol and Hydrochlorothiazide were subjected to

ICH prescribed stress conditions. The method developed was successfully

employed to the simultaneous estimation of Losartan potassium,

Atenolol and Hydrochlorothiazide in pharmaceutical preparations. The

developed RP-UPLC method was validated for precision, accuracy,

linearity, robustness and ruggedness and found to be precise, accurate,

linear, robust, rugged and stability indicating. Thus the proposed method

can be employed for assessing the stability of Losartan potassium,

Atenolol and Hydrochlorothiazide for its individual dosage form and for

its combined dosage forms.

Table 6.6.T1: Summary of analytical method validation

Test

Parameter

Assay method

LK ATN HCT

Precision (RSD) 0.4 0.4 0.5

Linearity (Corre coefficient)

> 0.999 > 0.999 > 0.999

Accuracy % 99.1-100.5 99.0-100.8 99.1-100.5

Robustness

% RSD for 5 replicate injections < 2.0, USP tailing is less than 2.0 and Resolution

between pair compounds was > 5.0

Solution stability Stable for 24 hours at room temperature

Specificity

For all stress conditions, purity angle is less

than the purity threshold and there is no

purity flag observed for purity results

LK= Losartan potassium , ATN= Atenolol , HCT= Hydrochlorothiazide

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