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A PRESENTATION ON :DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF LOSARTAN POTASSIUM, HYDROCHLOROTHIAZIDE, AND THEIR DEGRADATIONPRODUCTS

PRESENTED BY:

NITIN P. KANWALE

{M. PHARMACY: Quality Assurance Techniques}

GUIDED BY:

Dr. M. H. BELE

N.D.M.V.P. Samaj’s College of Pharmacy, Gangapur Road, Nasik – 422 002

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DRUG PROFILE

LOSARTAN

HYDROCHLOROTHIAZIDE

STUCTURE

CHEMICAL NAME

(2-butyl-4-chloro-1-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-1H-imidazol-5-yl)methanol

6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide

MOA Angiotensin II receptor antagonist

Diuretics

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DRUG NAME

IMPURITY DEGRADATES

LOSARTAN

HCTZ

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INTRODUCTION:

RESEARCH QUESTION ?

As it possible to develop a HPLC method for simultaneous determination of losartan, hydrochlorothiazide and their degradation product by using a single sample preparation ?

Losartan potassium is the active ingredient in tablets COZAAR and

is combined in the product HYZAAR.

WHY COMBINATION ?

Studies have shown an increase in Losartan efficacy when a low dose of hydrochlorothiazide (HCTZ) is administered concomitantly due to stimulation of the renin–angiotensin system.

GOAL OF WORK :

To develop and validate a single high-performance liquid chromatography method selective for the eight main components of tablets HYZAAR®.

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NAME OBTAINED BY

Losartan potassium tablets and Losartan potassium/ HCTZ tablets

DuPont Pharmaceuticals Company

Losartan, HCTZ, DSA, and CTZ reference standards

Merck

NaH2PO4 ·H2O, KH2PO4, Na2HPO4, phosphoric acid 85% solution, and acetonitrile.

Fisher Scientific

CHROMATOGRAPHIC EQUIPMENT

EQUIPMENT OBTAINED BY

Thermo Separations HPLC system

Riviera Beach, FL, USA

C8 columns (150×3.9 mm 5 µm particle size)

Waters Corporation,USA

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MOBILE PHASE : (Both the Losartan tabletsand the Losartan/HCTZ tablets)

Mobile Phase A : phosphate buffer solution of KH2PO4 and Na2HPO4

(pH 7.0, 0.02 M): Acetonitrile in the ratios of 85:15(v/v) and 93:7(v/v).

Mobile Phase B: 100% Acetonitrile

CONDITIONS:For Losartan tablets: the profile is a linear gradient from Mobile Phase A–Mobile Phase B (80:20, v/v) to Mobile Phase A–Mobile Phase B (40:60, v/v) over 10 min. The mobile phase composition is ramped back to Mobile Phase A Mobile Phase B (80:20, v/v) for equilibration prior to the next injection for a total run time of 15 min. Mobile phase flow rate: 1ml/min. Ambient column temperature Injection volume: 10 µl. UV detection at: 250 nm.

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For Losartan/HCTZ tablets:

The two-step gradient profile begins with a 12 min linear gradient from 100% Mobile Phase A to Mobile Phase A–Mobile Phase B (92:8, v/v). This is followed by a linear ramp over the next 16 min to Mobile Phase A–Mobile Phase B (38:62, v/v). Finally, the composition is returned to 100% Mobile Phase A over 2 min followed by a 5 min equilibration for a total run time of 35 min. Mobile phase flow rate: 1ml/min. Column temperature: 35ºC. Injection volume: 20 µl. UV detection at: 280 nm.

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STANDADRD AND SAMPLE SOLUTION PREPARATION:

Losartan tablets: 1) 100% working std. solution for losartan tablets:

Losartan potassium reference std. dissolve in mobile phase A to get 0.25 mg/ml conc. diluated by mobile phase A

1% degraded level std. 0.1% sensitivity solution.2) System suitability solution:

containing 0.2-0.5% of losartan degrades, compound E and F was produced by acid stressing under accelerated heating condition of 105ºC for 1-2 hrs.

3) Sample losartan tablet solution: tablet dissolved in mobile phase A Diluted by mobile phase A Sonication and shaking losartan final conc. Of 0.25 mg/ml.

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Losartan/ HCTZ Tablets:

1)100% working std. for losartan/HCTZ Tablet: Dissolving reference std. in diluent(0.02 M, NaH2PO4, pH 2.5 ) and acetonitrile (70:30 v/v) to get a conc. Of 0.4 mg/ml losartan, 0.1 mg/ml HCTZ and 0.001 mg/ml CTZ and DSA.

DILUTION

1% Degrade level std. 0.1% sensitivity std.

2) System suitability solution: Combing 100% working std. solution +stressed losartan solution as described previously (contain HCTZ, CTZ, Losartan degrades, E and F)

3) A single sample solution: by dissolving tablet in (60:40 v/v) acetonitrile and diluent with mechanical shaking for 30 min. further dilute by a diluent to yield losartan and HCTZ Conc. At 0.4 and 0.1 mg/ml resp.

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METHOD DEVELOPMENT AND OPTIMIZATION:Losartan tablets: 1) The method was developed for the simultaneous determination of

the active ingredient at the 100% level and degradation products at 0.1% levels in Losartan tablets.

2) To obtain the adequate retention time for polar compound losartan and maintaining reasonable elution time for less polar compound F and F symmetry C8 (150×3.9 mm, 5 particle size) give a best resolution and shortest retention time for active and degrade compound.

3) The interference of acetonitrile at the detection wavelength 230 nm so use of 250 nm detection wavelength.

4) Sensitivity to impurities in phosphate salt so use of fresh phosphate salt.

5) By use high conc. Of acetonitrile in mobile phase and methanol to elute active drug and degrade isocratically but the isocratic method of run less than 1 hr. for all the 3 compound are not realised so use of 15 min. gradient method developed and validated successfully to quantitation for losartan and its degrade.

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Losartan/HCTZ Tablets:

1) The mobile phase (phosphate buffer) pH was changed from 2.5 to 7.0 to optimize the Losartan retention time, while still allowing separation of CTZ, DSA, and HCTZ from the solvent front and each other.

2) Normal fluctuations in ambient laboratory temperatures affected the resolution of the HCTZ–CH2–HCTZ impurity and Losartan so column temp. Control at 35ºC.

3) Losartan is present at four times the concentration of HCTZ in the tablet formulations; therefore, a higher wavelength of 280 nm was chosen to achieve similar degree of absorbance values for the two active ingredient peaks.

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METHOD VALIDATION: 1) SELECTIVITY:

LOSARTAN TABLET:

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Losartan/HCTZ Tablets:

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2) ACCURACY:3) LINEARITY: For losartan tablets and losartan/HCTZ tablets:

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4) PRECISION: For losartan tablet: RSD 0.2% and 0.3% at 1 and 100% level respectively. For losartan/HCTZ tablets: RSD 0.1% and 0.0.2% at 100 and 1% level resp. for losartan and HCTZ.5) RUGGEDNESS: Ruggedness chemist reported retention times for all major components detected within 0.5 min. and 0.2 min. from those observed by the developing chemist for losartan and losartan/HCTZ tablets resp.

6)ROBUSTNESS: For losartan tablet: The method was found to be very robust.

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For losartan/HCTZ tablet: Effect of change in pH:

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CONCLUSION:

1)Successful method develop of HPLC for separation and quantitation of all related compounds in COZAAR and HYZAAR TABLET.

3) This method provide advantages of time saving, decrease no. of samples preparation, instrument run time, solvent and drug waste etc.

4) Losartan tablet method was found to be very robust, whereas losartan/HCTZ tablet method found temp. And pH as critical conditions.OVER VIEW:

POSITIVE POINT:

NEGATIVE POINT:

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REFERENCES:

Deanne L. Hertzog ,Jennifer F. McCafferty et al. Development and validation of a stability-indicating HPLC method for the simultaneous determination of Losartan potassium, hydrochlorothiazide, and their degradation products. Journal of pharmaceutical and biomedical analysis 2002;30:747-760.

Validation of analytical procedures: Text and methodology Q2(R1).ICH Harmonization tripartite guidelines:1994.

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