Development and validation of method for simultaneous estimation of hydrochlorthiazide and candesarten Using UV and HPLC

1. “Development and Validation of RP-HPLC method for
Simultaneous Estimation of Candesartan and Hydrochlorothiazide
in Combined Tablet Dosage Form”
Presented By:
Ramdarshan Parashar
0212PY11MP11
Guru Ramdas Khalsa Institute of Science & Technology
(Pharmacy)
Supervised By:
Mr. Rajesh Shukla
Asst.Prof , G.R.K.I.S.T. (Pharmacy)

2. INTRODUCTION
 Pharmaceutical analysis comprises the procedures necessary to
determine the identity, strength, quality and purity of substances of therapeutic
importance.
 Quality is important in every product or service, but it is vital in medicines as it
involves life.
 Analytical Chemistry split into two:
 Qualitative Analysis
 Quantitative Analysis
 Analytical techniques that are generally used for drug analysis are
 Chromatographic methods,
 Spectral methods,`
 Biological and Microbiological methods.
2

3. INTRODUCTION (cont..)
 CHROMATOGRAPHY –
Chromatography is essentially a group of techniques for the separation of the
compounds of mixtures by their continuous distribution between two phases, one of
which is moving over the other. Species in the sample undergo repeated interactions
(partitions) between the mobile phase and stationary phase.
 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY –
HPLC is a form of liquid chromatography used to separate compounds that are
dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a
pump, an injector, a separation column, and a detector.
 RP-HPLC
 NP-HPLC
3

4. INTRODUCTION(cont..)
Fig1 :- Schematic Diagram of RP-HPLC
4
 Reserve Phase HPLC –
Reverse phase chromatography refers to the use of a polar eluent with a non polar
stationary phase in contrast to normal phase chromatography, where a polar stationary
phase is employed with a non-polar mobile phase.

5. INTRODUCTION(cont..)
5
 Validation of Analytical Method –
Validation of an analytical method is the process by which it is established, by
laboratory studies, that the performance characteristics of the method meet the
requirements for the intended analytical application.
Validation is documented evidence, which provide a high degree of assurance
for specific method.
Typical analytical characteristics used in method validation.
 Linearity and Range
Accuracy
Precision
Limit of detection
Limit of quantitation
Ruggedness
Robustness

6. LITERATURE REVIEW
• T. MARY SUDHA et al (2012)9
, developed and validated A Simple, accurate reverse
phase high-performance liquid chromatographic method for simultaneous estimation
of Hydrochlorothiazide in bulk and pharmaceutical dosage forms.
• Chromatography was carried out by using phenomenex Luna C-18, column having
(250 x 4.6mm) internal diameter
• Mobile Phase- phosphate buffer: methanol: acetonitrile, 30:50:20 (V/V/V)
• Final pH 3.5 ± 0.1, as mobile phase.
• Detection was at 225nm
• Analytical validation- parameters such as linearity, precision, accuracy, and specificity,
limit of detection (LOD) and limit of quantification (LOQ) was done.
• The calibration curve was found to be linear for each analyte in the desired
concentration range.
• The percentage of recovery was found to be 99.80 and 99.65 for Candesartan and
Hydrochlorothiazide respectively.
• The proposed method is highly sensitive, precise and accurate, which was evident
from the LOD value of 0.038 and 0.006 μg/ml for Candesartan and
Hydrochlorothiazide respectively
• 6

7. LITERATURE REVIEW
R Revathi et al (2011)developed and validate a dissolution test for the quality control
of candesartan cilexetiltablets, labeled as containing 8 mg of active pharmaceutical
ingredient (API)
MODE-reverse phase – highperformance liquid chromatography
• Mobile phase-0.02M mono potassium phosphate: acetonitrile: triethylaminein the
ratio of 40:60:02
• pH- 6 with phosphoric acid flow rate - 1 ml/min.
• Validation specificity, linearity, accuracy, precision and solution stability as per
ICH guidelines to meetrequirements for a global regulatory filing.
MARIUSZ STOLARCZYK et al (2008) developed a method to determine enalapril,
hydrochlorothiazide,candesartan and walsartan in complex antihypertensive
drugs.
• The method was of high sensitivity;
• the LOD accuracy for enalapril was 2.81 μg/mL-1, 0.56 μg/mL-1 for candesartan,
4.02 μg/mL-1 for walsartan and ranged from 0.31 μg/mL-1 to 1.78 μg◊mL-1 for
hydrochlorothiazide, depending on preparation under investigation.
7

8. S. S. Qutabet al (2007)developed simple, sensitive, and inexpensive
high-performance liquid chromatographic method for simultaneous
determination of hydrochlorothiazide and candesartan cilexetil in
pharmaceutical formulations.
• Chromatographic separation was achieved
• Phenyl-2 column
• 25:75:0.2 mixture of 0.02 M potassium dihydrogen phosphate,
methanol, and triethyl-amine, as mobile phase
• final pH 6.0 ± 0.1
• Detection was at 271 nm.
• Response was a linear function of concentration in the range 5–45
μg mL−1
for hydrochlorothiazide and 12–56 μg mL−1
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.
8

9. DRUG PROFILE
 Name of Drug -: Candesarten
 Chemical Name -: 2-ethoxy-1-({4-[2-(2H-1,2,3,4-tetrazol-5-
yl)phenyl]phenyl}methyl)-1H-1,3-benzodiazole-6-carboxylic acid
 Molecular Formula-: C24H20N6O3
 Mole. Weight-: 440.45 g/mol
 Description -: White to cream colored
crystalline powder.
 Melting Point -: 155 to 1570
C
 Solubility-: Soluble in Methanol, Ethanol and Acetone
 Category-: Angiotensin-receptor blocker (ARB)
CANDESARTEN
9

10. DRUG PROFILE
 Name of Drug -: Hydrochlorthiazide
 Chemical Name -: 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-
sulfonamide
 Molecular Formula-: C7H8ClN3O4S2
 Mole. Weight-: 297.74 g/mol
 Description -: White to cream colored
crystalline powder.
 Melting Point -: 241o
C to 245o
C
 Solubility-: Soluble in Methanol, Ethanol and Acetone and insoluble in water.
 Category-: Thiazide class of diuretics.
HYDROCHLORTHIAZIDE
10

11. AIM & OBJECTIVE
 The objective of the present work is to develop simple, accurate, rapid, and
validated analytical methods for simultaneous estimation of Candesartan
and Hydrochlorothiazide in marketed formulation by HPLC methods.
 It also planned to validate the developed methods as per ICH guidelines.
11

12. PLAN OF WORK
 Procurement of the drug
 Authentication or identification of the Candesartan and Hydrochlorothiazide
Solubility study
Melting point
Forier Transform Infrared Absorption Spectroscopy (FT-IR)
 Method development by RP-HPLC
Selection of column
Selection of wavelength
Selection of Mobile phase
 Method Optimization
Effect of ratio of mobile phase
Effect of pH of mobile phase
12

13. PLAN OF WORK
Effect of temperature
Effect of flow rate
 Determination of Candesartan and Hydrochlorthiazide by developed method
 Method Validation
Linearity
Precision
Accuracy
Robustness
Limit of detection
Limit of quantification
13

14. EXPERIMENTAL
 PROCUREMENT OF THE DRUG
14
1 Candesartan Macleod’s Pharmaceutical Ltd.
2 Hydrochlorothiazide Kalindi Medicure Pvt. Ltd.
3 Marketed Formulation Atacand HCT, Takeda Pharmaceutical
Company,
Batch no. KTY0125
Candesartan 32 mg
Hydrochlorothiazide 12.5 mg

15. EXPERIMENTAL (cont..)
 Authentication or identification of the candesartan and
hydrochlorthiazide
Table : Solubility study of candesartan in different solvent:
15
S. No. Solvent Solubility
1 Water Insoluble
2 0.1 N HCl Insoluble
3 Methanol Freely Soluble
4 Ethanol Freely Soluble
5 Acetone Freely Soluble
6 Methanol: water (80:20) Sparingly Soluble

16. EXPERIMENTAL (cont..)
Table : Solubility study of Hydrochlorthiazide in different solvent:
16
S. No. Solvent Solubility
1 Water Insoluble
2 0.1 N HCl Insoluble
3 Methanol Freely soluble
4 Ethanol Soluble
5 Acetone Soluble
6 50 Mm KH2PO4: ACN(30:70) Soluble

17. EXPERIMENTAL (cont..)
 Melting points by melting point apparatus:
17

18. EXPERIMENTAL (cont..)
1818
ANALYTICAL METHOD DEVELOPMENT OF CANDESARTAN AND
HYDROCHLOROTHIAZIDE BY HPLC:-
Mobile Phase Selection:-
Mobile phase Ratio Flow rate Retention time Conclusion
CANDE HCZ
Methanol 100 1.00 ml/min. 11.67 Not Found Not Suitable
Methanol: water 50:50 1.00 ml/min. 8.56 Tailing Not Suitable
Acetonitrile :
Methanol
60 : 40 1.00 ml/min Not found Tailing Not Suitable
50 Mm KH2PO4:
ACN
50:50 1.00 ml/min 7.216 Tailing Not Suitable
50 Mm
KH2PO4(pH4
with OPA): ACN
30:70 1.00 ml/min 8.701 5.471 Most Suitable

19. EXPERIMENTAL (cont..)
1919
Selection of wavelength

20. EXPERIMENTAL (cont..)
2020
Selection of Separation Variable:-
Variable Condition
Column C18
Dimension. 250mm x 4.60mm
Particle Size 5µ
Bonded Phase Octadecyl silane (C18)
Mobile Phase 50 mM KH2PO4 (pH4 with
OPA): Acetonitrile
Acetonitrile 30
50 mM KH2PO4 (pH4.0 with OPA) 70

21. EXPERIMENTAL (cont..)
2121
Selection of Separation Variable:-
Flow rate 1.0 ml/min
Temperature Ambient
Sample Size 20µl
Detection wavelength 260 nm
Retention time
Candesartan
Hydrochlorothiazide
8.701 + 0.3 min
5.471 + 0.3 min

22. EXPERIMENTAL (cont..)
22
Preparation of Standard Stock Solution:-
10 mg of Candesartan and 10 mg of Hydrochlorothiazide was
weighed accurately and transferred to separate 10 ml volumetric flask,
and the volume was adjusted to the mark with the mobile phase (50
mM KH2PO4 (pH4.0 with OPA): Acetonitrile in the ratio of 30:70 v/v)
to give a stock solution of 1000mg/ml.
Preparation of Working Standard Solution:- From stock
solutions of Candesartan 1 ml was taken and diluted up to 10 ml.
from this solution 0.8, 1.6, 2.4, 3.2 and 4.0 ml solutions were
transferred to 10 ml volumetric flasks and make up the volume up to
10 ml with mobile phase, gives standard drug solution of 8, 16, 24,
32, and 40 mg/ ml concentration and From stock solutions of
Hydrochlorothiazide 1 ml was taken and diluted up to 10 ml. from this
solution 0.5, 1.0, 1.5, 2.0 and 2.5 ml solutions were transferred to 10
ml volumetric flasks and make up the volume up to 10 ml with mobile
phase, gives standard drug solution of 5, 10, 15, 20 and 25 mg/ ml
concentration.

23. EXPERIMENTAL (cont..)
23
 Preparation of the Calibration Curves of the Drug:-
Each of the standard drug solutions were injected 3 times and the
mean peak area of drug was calculated and plotted against the
concentration of the drug.
Calibration Graph of Candesartan

24. EXPERIMENTAL (cont..)
24
Calibration Graph of Hydrochlorothiazide

25. EXPERIMENTAL (cont..)
25
Chromatogram of
Candesartan
Chromatogram of Hydrochlorothiazide

26. EXPERIMENTAL (cont..)
26
System Suitability Parameters:-
Result of System Suitability Parameters for Candesartan
System suitability
Parameter →
RT AUC Theoretical plates Tailing factor
Rep-1
8.701+0.2 min 5214.365 2558 1.22
Rep-2
8.700+0.3 min 5245.145 2585 1.24
Rep-3
8.702+0.4 min 5241.363 2581 1.23
Mean
8.701+0.3min 5233.624 2574 1.20
S.D.
8.0008+0.3min 13.70565 1.45 0.015
System suitability
Parameter →
RT AUC Theoretical plates Tailing factor
Rep-1
5.470+0.4min 6574.236 2590 1.10
Rep-2
5.471+0.3min 6547.236 2592 1.12
Rep-3
5.472+0.2min 6589.236 2590 1.11
Mean
5.470+0.3min 6570.236 2591 1.10
S.D.
8.0008+0.3min 17.37815 1.45 0.01
Result of System Suitability Parameters for Hydrochlorothiazide

27. EXPERIMENTAL (cont..)
27
Assay of Tablet Formulation:-

28. EXPERIMENTAL (cont..)
28
 Validation of Developed Method:-
Linearity:-
Linearity of Candesartan
Replicates
Concentration
(µg/ml)
Mean AUC Response Ratio
Rep-1 8 5233.624 654.203
Rep-2 16 10463.27 653.9544
Rep-3 24 14691.92 612.1633
Rep-4 32 20564.98 642.6556
Rep-5
40 25465.28 636.632
Mean
639.9217
S.D. 17.24599
R.S.D. 2.695017

29. EXPERIMENTAL (cont..)
29
 Validation of Developed Method:-
Linearity:-
Linearity of Hydrochlorothiazide
Replicates
Concentration
(µg/ml)
Mean AUC Response Ration
Rep-1 5 3270.279 654.0558
Rep-2 10 6570.236 657.0236
Rep-3 15 9257.464 617.1643
Rep-4 20 12450.7 622.535
Rep-5 25 15502.46 620.0984
Mean 634.1754
S.D. 19.62338
R.S.D. 3.094315

30. EXPERIMENTAL (cont..)
30
Accuarcy:-
Level of
Recovery (%)
Drug % Recovery Standard
Deviation*
% RSD
80
CANDE
100.28 0.955 0.953
HCZ
99.7 0.608 0.61
100
CANDE
100 0.167 0.167
HCZ
99.625 0.331 0.332
120
CANDE
99.967 0.208 0.208
HCZ
99.861 0.127 0.127
Statistical Validation of Recovery Studies
*Denotes average of three determinations.

31. EXPERIMENTAL (cont..)
31
 Precision:-
(A) Repeatability:-
Drug Label
claim
Mg/ml
Amount
found*
mg/ml
Label
claim
(%)
S.D. % RSD
CANDE 32 31.99
99.97
0.155 0.159
HCZ 12.5 12.48
99.84
0.154 0.156

32. EXPERIMENTAL (cont..)
32
(B) Intermediate Precision- (Inter-day and Intra-day Precision):-
Intra-day Precision Inter-day Precision
% Label Claim % Label Claim
CANDE HCZ CANDE HCZ
After 1hr 99.90 98.90 First day 97.00 96.00
After 2hr 99.45 98.50 Second day 96.50 95.50
After 3hr 99.10 98.10 Third day 95.00 94.50
After 4hr 99.00 98.00
After 5hr 98.95 97.80
After 6hr 98.80 97.50
Mean
99.2 98.13
Mean
96.16 95.33
SD
0.37 0.45
SD
0.84 0.62
% RSD
0.37 0.46
% RSD
0.88 0.65

33. EXPERIMENTAL (cont..)
33
(C) Analyst to Analyst:-
Anal
yst
Label claim
mg/ml
Amount
found*
mg/ml
Label claim
(%)
S.D. % RSD
CAN
DE
HCZ CAN
DE
HCZ CAN
DE
HCZ CAN
DE
HCZ CAN
DE
HCZ
1 32 12.5 31.95 12.45
99.84 99.60
0.112 0.098 0.110 0.115
2 32 12.5 31.99 12.48
99.97 99.84
0.212 0.211 0.225 0.220

34. EXPERIMENTAL (cont..)
34
 Robustness:
Compound % RSD in Normal Changed Condition n= 6
Temperature - 5 o
C + 5 o
C
CANDE 0.52 0.68 0.27
HCZ 0.51 0.56 0.23
Flow rate (-10%) (+10%)
CANDE 0.45 0.56 0.65
HCZ 0.33 0.52 0.54
Mobile phase ratio - 2 % + 2 %
CANDE 0.34 0.89 0.54
HCZ 0.54 0.67 0.65

35. EXPERIMENTAL (cont..)
35
Analysis of the formulation:-
twenty tablets taken and determine the average weight. Powder
equivalent to weight 32 mg of Candesartan was transferred to 10
ml volumetric flask and dissolved in HPLC grade methanol. The
solution was shaking vigorously for 10 mins and filtered through
Whatman filter paper no.41, then volume was made up to mark
with methanol. From the above solution 1 ml of solution was taken
and diluted to 10 ml with mobile phase to get a solution containing
100 µg/ml. From the above solution 3.2 ml of solution was taken
and diluted to 10 ml with mobile phase to get a solution containing
32 µg/ml. of Candesartan and corresponding concentration of
Hydrochlorothiazide 12.5 µg/mL. The solution contains Candesartan
and Hydrochlorothiazide in the proportions of 32:12.5. The amounts
of Candesartan and Hydrochlorothiazide calculated by extrapolating
the value of area from the calibration curve. Analysis procedure was
repeated six times with tablet formulation.

36. EXPERIMENTAL (cont..)
36
Std Conc. µg/ml CANDESARTAN HYDROCHLOROTHIAZIDE
32 12.5
Rep-1 31.99 12.5
Rep-2 31.99 12.45
Rep-3 32.00 12.48
% found *
Rep-1
99.96875 100
Rep-2
100 99.6
Rep-3
100.0313 100.241
Mean
100 99.94699
SD
0.031255 0.323754
% RSD
0.031255 0.323925
Result of Analysis for Candesartan and Hydrochlorothiazide in Tablet Formulation

37. SUMMARY
37
 A convenient and rapid RP-HPLC method was developed for estimation of Ropinirole
HCl in bulk drug and marketed formulation. All parameters are summarized below.
Table : Summary of Validation parameters

38. CONCLUTION
38
Based on the statistical data it can be concluded that the proposed RP-HPLC
method for estimation of candesartan and Hydrochlorthiazide in bulk drug and marketed
formulation is accurate, precise, and very sensitive.
The results obtain from validation parameters met ICH requirement and it is also
suitable for routine quality control analysis.

39. BIBLIOGRAPHY
39
1. Kasture A.V. (2003) Pharmaceutical Analysis- Instrumental Method.
9th
Edition, Part-II, Nirali Prakashan, Pune, pp 6, 7, 10, 49, 50, 156,
159.
2. Kemp W. (1996) Organic Spectroscopy, 3rd
Edition, Macmillan Press
Ltd, Hampshire, pp 1, 7.
3. Snyder LR. (1996) Practical HPLC Method Development. Wiley
Interscience Publishing Inc, Co., USA, pp 1, 3, 15, 631.
4. Meyer VR. (2004) Practical High Performance Liquid
Chromatography. 4th
Edition, John Wiley and Sons, pp 6-9, 87-92,
114, 228-234, 261-263.
5. Sethi P.D. (2001) High Performance Liquid Chromatography-
Quantitative Analysis of Pharmaceutical Formulations, 5th
Edition,
CBS Publication and Distributors, New Delhi, pp 5, 101, 102.
6. International Conference on the Harmonization, Draft guideline on
Validation of analytical Procedure for Pharmaceutical Availability,
Federal Register, 1994, 59, 9750.
7. International Conference on the Harmonization, Draft guideline on
Validation of analytical Procedure for Pharmaceutical Availability,
Federal Register, 1995, 60, 11260.

40. BIBLIOGRAPHY
40
9. Mariusz Stolarczyk, Ann A Maalanka, Jan Krzek and
Joannamilczarekapplicationofderivativespectrophotometryfordeterminat
ion of Enalapril, Hydrochlorothiazideand Walsartan In Complex
Pharmaceutical Preparations Vol. 65 No. 3 pp. 275n281, 2008
10.T. Mary sudha, g .subba rao, p. Vineetha, k. Spandana, sk.
Tasleem, b. Ashapaul “simulataneous estimation and
validatioocandesartan andhydrochlorthiazide in tablet dosage form by
rp-hmethod”Volume 3 September-October 2012 Pages 325-332
11. Jane Jacob*, Aghera Jonils P, Joshi Chintan KAnalytical methods for
the estimation of Candesartan in Pharmaceutical Formulations.
2011,4(11),3930-3932
12.R Revathi*1, T Ethiraj 2, Jhansi L. Marreddy1, V
GaneshanDevelopment and validation of a dissolution test for
Candesartan cilexetil intablet forms using reverse phase – High
performance liquid chromatographyVol. 2, Issue No. 2, December 2011

41. THANK YOU…!!!
41