INTRODUCTION:

Hydrochlorothiazide¹:

Chemical name :

2H-1,2,4-Benzothiadiazine-7-sulfonamide, 6-chloro-3,4-dihydro-, 1,1-dioxide

Molecular formula:C₇H₈ClN₃O₄S₂

Molecular weight: 297.739

Description:

Hydrochlorothiazide isWhite to off-white crystalline powder.

State: solid

Melting Point: 266-268^oC

Therapeutic category:

Diuretic agent: Benzothiadiazide

Solubility:

Freely soluble in sodium hydroxide solution, in n-butylamine and in dimethylformamide sparingly soluble in methanol; insoluble in dilute mineral acids

Mechanism of action:

Hydrochlorothiazide, a thiazide diuretic, inhibits water reabsorption in the nephron by inhibiting the sodium-chloride symporter (SLC12A3) in the distal convoluted tubule, which is responsible for 5% of total sodium reabsorption. Normally, the sodium-chloride symporter transports sodium and chloride from the lumen into the epithelial cell lining the distal convoluted tubule. The energy for this is provided by a sodium gradient established by sodium-potassium ATPases on the basolateral membrane. Once sodium has entered the cell, it is transported out into the basolateralinterstitium via the sodium-potassium ATPase, causing an increase in the osmolarity of the interstitium, thereby establishing an osmotic gradient for water reabsorption. By blocking the sodium-chloride symporter, hydrochlorothiazide effectively reduces the osmotic gradient and water reabsorption throughout the nephron.

Metabolism

Hydrochlorothiazide is not metabolized.

Route of elimination

Hydrochlorothiazide is not metabolized but is eliminated rapidly by the kidney. Hydrochlorothiazide crosses the placental but not the blood-brain barrier and is excreted in breast milk

Half life

5.6 And 14.8 hours

Valsartan²:

Chemical name:

N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl)

[1,1'-biphenyl]-4-yl] methyl]-L-valline.

Molecular formula: C₂₄H₂₉N₅O₃.

Molecular weight: 435.5

Description:

Valsartan is a white to practically white fine powder.

State: solid

Therapeutic category:

valsartan is a nonpeptide, orally active, and specific angiotensin II receptor blocker acting on the AT1 receptor subtype

Half-life: 6hrs

Mechanism of action:

Valsartan is an Angiotensin receptor blocker that selectively inhibits the binding of angiotensin II to Angiotensin1, which is found in many tissues such as vascular smooth muscle and the adrenal glands. This effectively inhibits the AT1-mediated vasoconstrictive and aldosterone-secreting effects of angiotensin II and results in a decrease in vascular resistance and blood pressure. Valsartan is selective for AT1 and has virtually no affinity for AT2. Inhibition of aldosterone secretion may inhibit sodium and water reabsorption in the kidneys while decreasing potassium excretion. The primary metabolite of valsartan, valeryl 4-hydroxy valsartan, has no pharmacological activity.

After through literature survey^6-9, the present method was developed.This method aims to develope a simple, sensitive, precise and validated specific Reverse Phase High Performance Liquid Chromatographic method for the determination of Valsartan and Hydrochlorothiazide in bulk and tablet dosage form as per ICH guidelines^3-5

MATERIAL AND METHODS:

Materials and Reagent:

All the materials and reagents used were of Analytical grade supplied from Rankem. Valsartan and HCTZ was gifted by Aurobindo Pharma Ltd. Formulation, Diovan tablets, was procured from local market.

Dosage form:

Procured from local market containing combination of Valsartan (320 mg) and HCTZ (25mg).

Chromatographic conditions:

Stationary Phase (Column)	Hypersil BDS C₁₈(150X4.6 mm),5μ,
Mobile Phase(Gradient)	M.P-A:Water:Acetonitrile:OPA(950:5:1) M.P-B: Acetonitrile: Water: OPA (950:5:1)
Flow Rate	1.5 ml/min
Run time	15 min
Column temperature	25ºC
Volume of injection loop	10µl
Detection wavelength	225 nm

Gradient programme:

Time(min)

MP-A

MP-B

0.01

5.00

8.00

10.00

10.10

15.0

Mobile Phase: (Gradient programme)

M.P-A: Water, acetonitrile and OPA in the ratio of 950:5:1. Solvents were sonicated to degas.

M.P-B: Acetonitrile, Water and in the OPA ratio of 950:5:1. Solvents were sonicated to degas.

Diluent: prepare a mixture of 500ml of Water, 500ml of Acetonitrile and 5ml of NaoH.

Preparation of stock solution-A:

50mg of Valsartan was weighed and taken into clean 50mL dry volumetric flask and small quantity of diluent was added for solubilising the drug and sonicated for about 10min and finally make up the solution with diluent.

Preparation of stock solution-B:

75mg of Hydrochlorothiazide was weighed and taken into clean 50mL dry volumetric flask and small quantity of diluent was added for solubilising the drug and sonicated for about 10min and finally make up the solution with diluent.

Preparation of working standard solution:

Transfer 10ml of standard stock solution A and 5ml of standard stock solution B into 50ml clean, dry volumetric flask, and dilute to the volume with diluent and mix. Filter through 0.45µ membrane(PVDF)

Preparation of sample drug solution for pharmaceutical formulations:

Transfer 5 tablets into 500ml clean, dry volumetric flask. Add 50ml of Water, and swirl to disperse the tablets. Then add about 250ml of diluent and sonicate at room temperature for about 25min with shaking in short intervals, and allow to cool for some time. Make up to the mark with diluent and mix and centrifuge the solution at 1000rpm for 10min. Dilute 3ml of clear supernatant solution to 50ml with diluent. Filter through 0.45µ membrane (PVDF).

METHOD VALIDATION:

The method was validated in accordance with ICH guideline Q2 (R1).

System suitability

It is defined as tests to measure the method that can generate result of acceptable accuracy and precision. The system suitability was carried out after the method development and validation was completed. For this, parameters like plate number (N), tailing factor, RSD of peak area for repetitive injections were measured

Acceptance criteria

· The % RSD for the peak area responses of Valsartan and Hydrochlorothiazide peaks from 6 replicate injections of each standard solution should be not more than 2.0%.

· The number of theoretical plates (N) for theValsartan and Hydrochlorothiazide peaks is not less than 2000.

· The Tailing factor (T) for Valsartan and Hydrochlorothiazide peak is not more than 2.0.

Linearity and Range:

The linearity of calibration curves (peak area Vs concentration) in standard solution was checked over the concentration ranges of about 50-300μg/ml and 3.7-22.5 μg/ml. Injection volumes of 10 µl of each of standard solution were injected into HPLC system to get the chromatograms. The calibration line was obtained by plotting peak area against concentration.

Acceptance criteria:

The relationship between the concentration (in %) of Valsartan and Hydrochlorothiazide and area of Valsartan and Hydrochlorothiazide should be linear in the specified range and the correlation should not be less than 0.99.

Precision:

Precision of the method was demonstrated by injecting samples 6 times and checking the closeness of these values. Intermediate precision is the agreement of complete measurements when the same method is applied many times within the same laboratory. This includes full analysis on different days, instruments, analyst, but would involve multiple preparation of samples and standards.Intermediate precision was established for the same analytical samples of concentration 0.03mg/ml. The analysis was done on different day. Six preparations of samples were prepared and injected into HPLC system. The results along with mean value for assay of valsartan and HCTZ were shown in the following table.

Acceptance criteria:

The % Relative standard deviation of Assay preparations of Valsartan and Hydrochlorothiazide should be not more than 2.0%.

Specificity:

For determining specificity of the method, a tablet dosage form was analyzed. The chromatograms were examined to determine any additional excipient peaks. Injections of the marketed product revealed the absence of extra peaks. These results demonstrated that there was no interference from other materials in the tablet formulation and therefore confirmed the specificity of the method.

The mean ± standard deviation (SD) for the area, slope, intercept and correlation coefficient of standard curves (n=3) were calculated.

Acceptance criteria:

Chromatogram of blank should not show any peak at the retention time of analyte peak.

There is no interference due to blank at the retention time of analyte. Hence the method is specific.

Accuracy:

Accuracy of the method was determined by recovery experiments. To the formulation, the reference standards of the drugs were added at the levels of 50%, 100%, and 150%. The recovery studies were carried out three times and the percentage recovery and percentage relative standard deviation of the recovery for valsartan and hydrochlorothiazide was calculated.

Acceptance criteria:

The % recovery of Hydrochlorothiazide and Valsartan should lie between 98% and 102%.

Robustness:

The robustness of the method was evaluated by analyzing the system suitability standards and evaluating system suitability parameter data after varying the HPLC pump flow rate(±10%) and organic solvent content (± 5%), wavelength (±2 nm). None of the alterations caused a significant change in retention time, peak area, tailing factor and theoretical plates.

Acceptance criteria

1. The tailing factor of standard should be not more than 2.0 for Variation in flow.

2. The % RSD of Asymmetry and valsartan, HCTZ standard should be not more than 2.0 % for variation in flow.

Limit of detection and Limit of quantitation:

The limit of detection (LOD) and limit of quantitation (LOQ) for the procedure were performed on sample containing very low concentrations of analyte under the ICH guidelines. From the linearity data the limit of detection and quantitation was calculated using the following formula.

σ = standard deviation of the response

S = slope of the calibration curve of the analyte.

σ = standard deviation of the response

S = slope of the calibration curve of the analyte.

Acceptance criteria

The % Relative standard deviation of Assay preparations of Hydrochlorothiazide and Valsartan should be not more than 2.0%.

Forced Degradation:

In the force degradation study under validation of the method, drug was allowed to degrade in acidic, basic, oxidative and thermal conditions. To carry out this experiment drug was kept in different conditions and with different concentrations of these substances i.e. acid, base and H₂O₂ and also in different temperature for different periods.

Preferable, the following stress conditions are recommended for specificity study.

However stress conditions can be decided based on experimental data, or physical properties of the analyte.

· Heat: At 105ºC for about 6 hours

· Humidity: About 90% RH at 25ºC for NLT 7 days

· UV Light: NLT 7 days in UV cabinet or 200 watt hours/m2 in Photo stability chamber

· Sun light : NLT 55 hours in sunlight or 1.2 million lux hours in Photo stability chamber.

· Acid : 0.1 N HCl refluxed for 30 minutes at 60ºC

· Base: 0.1 N NaOH refluxed for 30 minutes at 60ºC

· Peroxide: 1% H₂O₂ refluxed for 30 minutes at 60ºC

· Water : Refluxed for 6 hours at 60ºC

Stress testing is different from accelerated tests because they carried out under more severe conditions. The studies normally include exposure of drug to elevated temperatures/humidity, light and oxidizing agents, as well as susceptibility to hydrolysis across a range of pH values.

From this study, one can clearly understand the intrinsic stability of the drug molecule. The results of these studies are the basis for developing.

ü Appropriate dosage forms

ü Formulations

ü Manufacturing process

RESULTS AND DISCUSSION:

The main objective of this work was to develop and validate RP-HPLC method for simultaneous estimation of valsartan and HCTZ in tablet dosage form. The method has provided adequate separation for valsartan and HCTZ from their dosage form. Separation was obtained by using Hypersil BDS C18 column (150 x 4.6 mm), 5μ at room temperature and using mobile phase-A water: ACN: OPA(950: 5: 1) and mobile phase-B ACN: water: OPA(950: 5: 1) at a flow rate of 1.5ml/min and wavelength for detection was 225nm.

Table 1: System suitability parameter

Parameter	Valsartan	HCTZ
Retention Time (min)	9.77±0.002	4.05±0.002
Theoretical Plates	199261	3872
Asymmetry	1.2	1.2
Capacity Factor	1.19	1.82

Table 2: Assay Results

Drug

Label Claim (mg/tab)

Amount Recovered

Percentage Purity (%w/w)

RSD

HCTZ

24.92

99.68

1.02

Valsartan

320

320.21

100.65

0.98

Spectra of valsartan and HCTZ:

Fig 1: UV overlap spectra of valsartan and HCTZ

Linearity results:

Table-3: Linearity of Valsartan

S.No.	Conc.(µg/ml )	Area
1	50	1198200
2	100	2391315
3	160	3862919
4	180	4290867
5	200	4781207
6	240	5783722
7	300	7172149

Table-4:Linearity of Hydrochlorothiazide

S.No.	Conc.(µg/ml )	Area
1	3.7	201894
2	7.5	408763
3	12.00	644980
4	13.5	721585
5	15.00	806539
6	18.00	964765
7	22.5	1205966

Table 5: Linearity of Valsartan and Hydrochlorothiazide

Parameter

Valsartan

Hydrochlorothiazide

Slope

Intercept

Correlation coefficient

23952

2688

0.999

53301

5730

0.999

Table: 6 Accuracy results

Recovery level		Accuracy Valsartan
Recovery level		Weight of sample taken (mg)	Area	mg added	Mg found	%Recovery
50	810		2328321	813.81	812.98	100.73
100	1615		4669278	1622.59	1630.37	99.81
150	2423		6917577	2434.39	2415.41	99.18

Table: 7 Accuracy results

Recovery level		Accuracy HCTZ
Recovery level		Weight of sample taken (mg)	Area	mg added	Mg found	%Recovery
50	63.45		417785	62.84	64.22	101.3
100	126.25		815904	125.02	125.41	100.3
150	189.10		1241987	187.25	190.91	101.4

*Mean of three readings

Observation:

The percentage mean recovery of Valsartan and Hydrochlorothiazide was 99.18% and 101.4% respectively.

Limit of Detection and Limit of Quantitation (LOD and LOQ):

Table: 8Results of LOD and LOQ

Parameter	HCTZ	Valsartan
LOD	0.22	0.636
LOQ	0.66	1.92

Observation

Test results for Hydrochlorothiazide and Valsartan of LOD and LOQ were within limits.

Robustness:

Table9: Results of Robustness study

Parameter	Mean*	SD	%RSD
Change in Flow rate	2975118	39920.69	1.34
Change Wavelength	2296156	3231.82	0.14

Observation:

The % RSD peak area was found to be within limit. So the method was robust.

Ruggedness:

Table 10: Ruggedness data of HCTZ and Valsartan

Ruggedness		HCTZ	Valsartan
% RSD (Rt)		0.05	0.24
Assay*	Analyst-1	99.89	99.16
Assay*	Analyst-2	99.83	99.66

* mean of three readings

Observation:

Test results for Hydrochlorothiazide and Valsartan are showing that the %RSD of Assay results were within limits.

FORCED DEGRADATION STUDIES:

Table-11: Valsartan

Test No	Unstressed	Acid stress	Alkali Stress	Peroxide Stress	Heat Stress	Photolytic Stress	Humidity
Average weight	320mg
No of tablets Taken	5	5	5	5	5	5	5
Area	4612457	4285791	4476946	4101242	4326679	4122146	4079961
Assay (in mg)	322.11	299.29	312.64	286.41	302.15	287.87	284.92
Assay (in %)	100.7	93.5	97.7	89.5	94.4	90.0	89.0
%Degradation	NA	7.1	2.9	11.1	6.2	10.6	11.5

Table-12: HYDROCHLOROTHIAZIDE

Test No	Unstressed	Acid stress	Alkali Stress	Peroxide stress	Heat stress	Photolytic Stress	Humidity
Average Weight	25mg
No of Tablets Taken	5	5	5	5	5	5	5
Area(Injection1)	818303	521406	714278	731787	757101	733841	741419
Assay(in mg)	25.16	16.03	21.96	22.50	23.27	22.56	22.79
Assay(in %)	100.6	64.1	87.8	90.0	93.1	90.2	91.2
%Degradation	NA	36.3	12.7	10.6	7.5	10.3	9.4