NOVEL SIMULTANEOUS SEPARATION AND QUANTITATIVE DETERMINATION OF FOUR SARTANS IN PRESENCE OF HYDROCHLOROTHIAZIDE BY ISOCRATIC RP-HPLC

NOVEL SIMULTANEOUS SEPARATION AND
QUANTITATIVE DETERMINATION OF FOUR SARTANS
IN PRESENCE OF HYDROCHLOROTHIAZIDE BY
ISOCRATIC RP-HPLC
Under the guidance of:
Dr. Panchumarthy Ravisankar M. Pharm., Ph.D.
Professor & HOD
Dept. of Pharmaceutical Analysis and Quality Assurance
Vignan Pharmacy College
Vadlamudi.
Presentation by:
K. Manjusha (12AB1R0043)
V. Laya Sri (12AB1R0044)
B. Vijaya Kumar (12AB1R0009)
K. Rajyalakshmi (12AB1R0037)
K. Avinash kumar (12AB1R0039)

CONTENTS:
 AIM AND OBJECTIVE
 INTRODUCTION
 DRUGS PROFILE
 LITERATURE SURVEY
 PLAN OF WORK
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
 REFERENCES
Vignan Pharmacy College,Vadlamudi. 2

AIM:
The core AIM of the present study is to develop a novel, rapid,
precise and accurate RP-HPLC method for simultaneous
separation and quantification of Hydrochlorothiazide along with
four sartans Telmisartan(TELM), Losartan(LOSA),
Olmesartan(OLME) and Valsartan(VALS).
To develop Novel methods for separation and quantification of
all the above said drugs on single chromatographic system
without any minor changes in detection wavelength and mobile
phase composition.

To develop rapid, sensitive and economical analytical methods
based on spectrophotometric & RP-HPLC techniques for estimation
and separtion of Hydrochlorothiazide with Telmisartan, Losartan,
Olmesartan and Valsartan in pharmaceutical dosage forms.
 To develop method with shorter run time and better sensitivity.
 Reducing the solvent consumption to make it more eco-friendly.
To validate the method for different parameters like Accuracy,
Precision, Linearity, specificity, Robustness according to
International Conference on Harmonization ICH Q2(R1)
guidelines[5] .
To apply the developed RP-HPLC method in the analysis of
pharmaceutical formulations.

INTRODUCTION
HPLC is a versatile tool for the qualitative and quantitative analysis
of drugs and pharmaceuticals, chemical and biological materials has
become indispensable in pharmacokinetics studies.
 Of the many instrumental methods available for quantification RP -
HPLC is selected as a tool for developing novel analytical procedure.
 Method development is done for the products as follows:
- New products
- Existing products
 Alternate methods for existing products are developed for better
precision and ruggedness.
Method development:

Drug Name : HYDROCHLOROTHIAZIDE
Structure :
IUPAC : 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-
benzothiadiazine-7-sulfonamide
Category : Anti hypertensive
Molecular Formula : C7H8ClN3O4S2
Molecular Weight : 297.74 g/mol
Brand names : Apo-Hydro, Aquazide H, Hydrodiuril,
HydroSaluric, Hydrochlorot, Microzide,
Esidrex, and Oretic

Drug name : VALSARTAN
Structure :
IUPAC Name :(S)-3-methyl-2-(N-{[2'-(2H-1,2,3,4-tetrazol-5-
yl)biphenyl-4-yl]methyl}pentanamido)butanoic acid
Molecular weight :435.519 g/mol
Molecular formula : C24H29N5O3
Brand names : Diovan, Valent -H
Vignan Pharmacy College,Vadlamudi.
8

Drug name : LOSARTAN
Structure :
IUPAC Name :(2-butyl-4-chloro-1-{[2'-(1H-tetrazol-5-yl)biphenyl-
4-yl]methyl}-1H-imidazol-5-yl)methanol
Molecular weight :422.91 g/mol
Molecular formula :C22H23ClN6O
Brand names : Cozaar, Losagard, Zargo-H
9

Drug name : OLMESARTAN
Structure :
IUPAC Name : 2,3-dihydroxy-2,2-butenyl-4(1-hydroxy-1-
methylethyl)-2-propyl-1-[p-(o-1H-tetrazol-5-
ylphenyl)benzyl]imidazole5-carboxylate2,2-carbonate
Molecular weight : 558.59
Brand names : Olmicep-H, Olmax
10
N
NN
HN
Olmesartan
N
N OH
H3C
O

Drug name : TELMISARTAN
Structure :
IUPAC Name : 2-(4-{[4-Methyl-6-(1-methyl-1H-1,3-benzodiazol-
2-yl)-2-propyl-1H-1,3-benzodiazol-1-yl]methyl}
phenyl)benzoic acid
Molecular weight : 514.617 g/mol
Brand names : Telmisat-H, Cresar, Micardis

S.NO AUTHORS TITLE STUDY
JOURNAL
NAME
YEAR,
VOLUME,
ISSUE &
PG.NO
1
Reem Youssef*,
AdnanHbash,
Ahmad Hassan
Development and
Validation of RP-
HPLC Method for
the Estimation and
Separation of
Valsartan, Losartan
and Irbesartan in
Bulk and
Pharmaceutical
Formulation.
Separation of 3
sartans using
c18 column and
its validation
according to
ICH guidelines.
International
Journal of
Pharmaceutial
Sciences
Review and
Research.
(IJPSRR)
Jan – Feb 2014,
24(2), 311-314.
2
Ganipisetty
Lakshmi
Aswini*,
D.Dachinamoor
thy,
J.V.L.N.Seshagi
ri Rao
A Sensitive RP-
HPLC Method
Development and
Validation for the
Simultaneous
Estimation of
Losartan Potassium
and
Hydrochlorothiazide
.
Validation and
estimation
using mobile
phase of
acetonitrile and
phosphate
buffer.
International
Journal for
Pharmaceutica
l
Research
Scholars.
(IJPRS)
2014, V-3, I-2,
408-416.
13

14
S. NO
AUTHORS
TITLE STUDY
JOURNAL
NAME
YEAR,VOL
UME,
ISSUE &
PG.NO
3
T. M.
Kalyankar*,
S. J. Wadher,
S. S. Pekamwar
N.G. Doiphode
Development and
Validation of RP-
HPLC method for
Estimation of
Hydrochlorothiazide
and Irbesartan in
Pharmaceutical
Preparation.
Analysis using C18
column using methanol
as solvent.
International
Journal of
PharmTech
Research
CODEN
(USA):
IJPRIF
Jan-March
2014,
Vol.6, Issue
No.1,
330-336.
4
T. Gopala
Swamy,
K. Nagaraju,
A. Lakshmana
Rao
RP-HPLC Method
for the Simultaneous
Estimation of
Telmisartan
and
Hydrochlorothiazide
in Pharmaceutical
Dosage Form.
RP-HPLC method was
found to be
rapid,precise,accurate,
Selective.
International
Journal of
Drug
Developmen
t &
Research.
(IJDDR)
October-
December
2011,Vol. 3
,Issue 4,
362-368.

S.NO AUTHORS TITLE STUDY
JOURNAL
NAME
YEAR,VO
LUME,
ISSUE &
PG.NO
5
Lakshmanarao. A,
Bhaskhara Raju .V
Simultaneous
Estimation Of
Valsartan And
Hydrochlorothiazide
In Tablets By RP-
HPLC Method.
Column having
isocratic mode with
mobile phase of
acetonitrile and
phosphate buffer.
IJPIR Jul-sep 2011,
Volume 01,
Issue-03,
170-174.
6
Hany Mohammed
Hafez*, Abdullah
Ahmed
Elshanawane
Quantitative
Determination of
three Angiotensin-II-
receptor Antagonists
in Presence of
Hydrochlorothiazide
by RP-HPLC in their
Tablet Preparations.
C18 column , phosphate
buffer is used.
Iranian
Journal of
Pharmaceuti
cal
Research.
2013, 12 (4),
635-643.
15

PLAN OF WORK:
Plan of RP-HPLC technique:
 Literature collection regarding various aspects like
physicochemical properties of the drugs
 Procurement of drug reference standards, reagents, solvents,
other chemicals and formulations.
 Establishment of initial chromatographic conditions.
 Optimization of the chromatographic conditions to achieve the
system suitability goals and validating the method according to
ICH guidelines.
 Applying the optimized method for estimation of selected drug
candidates in tablets and pharmaceutical dosage forms.

17

Table 1. Commercial brand names of TELMI, LOS , VAL, IRBE and HYDRO
combinations used for the present study.
Brand name Formulation Labeled amount (mg) Manufacturer
Telmisat-H
(40 mg)
Tablets
Telmisartan- 40 mg
Hydrochlorothiazide- 12.5 mg
Biocon laboratories
Zargo-H
(50 mg) Tablets
Losartan- 50 mg
Biocon laboratories
Olmicep-H
(40mg) Tablets
Olmesartan –40 mg
Hydrochlorothiazide – 12.5 mg
Cipla Ltd., Mumbai, India.
Valent-H
(80 mg)
Tablets
Valsartan- 80 mg
Lupin Laboratories, Hyderabad.
18

Table 2. Materials used in the present study
S.No. Materials Procured from
1. Telmisartan Aristo Pharmaceuticals Pvt.Ltd., Bombay.
2. Losartan Hetero Labs Ltd., Hyderabad
3.
Olmesartan Hetero Labs Ltd., Hyderabad
4. Valsartan Anant Pharmaceuticals, Kamal, Haryana.
5. Acetonitrile HPLC grade Thermo Fisher Scientific India Pvt. Ltd., Mumbai.
6. Water HPLC grade Merck Specialties Pvt. Ltd., Mumbai.
7. Methanol HPLC grade Merck Specialties Pvt. Ltd., Mumbai.
8. Dipotassium hydrogen phosphate Thermo Fisher Scientific India Pvt. Ltd., Mumbai.
9. Potassium dihydrogen phosphate Glaxo Smith Kline Pharmaceuticals Ltd., Mumbai.
19

Table 3. Instruments used in the present study
S.No. Instrument Name of the company and model
1. HPLC
Shimadzu LC-20AT Prominence Liquid Chromatograph
with Shimadzu SPD-20A Prominence UV-Vis detector,
Welchrom C18 Column (4.6 X 250 mm, 5μm), with
Rheodyne manual loop injector (20 μL) and Spinchrom
data acquisition software.
2.
UV-Vis
spectrophotometer
UV-Visible Spectrophotometer ELICO – SL - 210 with
Spectra treats(TM) software.
3. Weighing balance Essae vibra AJ (0.001g), Essae-Teraoka Ltd.
4. pH meter Elico LI120 pH meter, Elico India Ltd.
5. Ultrasonicator Ultrasonic bath sonicator, PCI ltd., Mumbai.
6. Vacuum pump Single Stage Vacuum Pump.

Preparation of reagents and standards:
 Preparation of phosphate buffer pH 3.3
Phosphate buffer with 10 mM was prepared by dissolving 6.056 g KH2PO4 in
445 mL of HPLC grade water. To this said solution 55 mL of 0.1M H3PO4 was added to
adjust the pH 3.3.
 Preparation of mobile phase:
The above stated prepared phosphate buffer (pH 3.3) acetonitrile were mixed
completely in the proportion of 50: 50 v/v and was filtered through 0.45 µm nylon
membrane filter and degassed by sonication.
 Preparation of standard stock solution:
Stock standard solutions containing (1.25, 0.4, 0.5, 0.4, 0.8 mg/mL) of
Hydrochlorothiazide, Telmisartan, Losartan, Olmesartan and Valsartan respectively
were prepared by dissolving (12.5, 40, 50, 40, 80 mg) of each in methanol in 100 mL
volumetric flask respectively. It was then sonicated for 15 minutes and the final
volume of solutions was made up to 100 mL with methanol to get stock standard
solutions. Vignan Pharmacy College,Vadlamudi. 21

Preparation of sample solution:
A composite of ten Telmisat-H, Zargo-H, Olmecip-H and Valent-H
tablets are prepared by grinding them to a fine, uniform size powder,
triturated using mortar and pestle. After calculating the average tablet
weight, amounts of powder equivalent to HCTZ, TELM, LOSA, OLME
and VALS respectively are prepared by dissolving (12.5, 40, 50, 40, 80
mg) respectively of each type of tablets were accurately weighed and
transferred separately to 100 mL volumetric flasks respectively.
Solutions were sonicated for 15 min and the solutions were then filtered
through 0.45 µm nylon membrane filters. Aliquots of appropriate volume
(10 mL) were transferred to 100 mL calibrated flasks and diluted to
volume with mobile phase to furnish the mentioned concentration above.

RESULTS:
Method Development:
 Selection of a common detection wavelength:
 The ultra violet spectrum of four Sartans and hydrochlorothiazide were
scanned individually in the region between 200 - 400 nm. The UV
overlain spectra of these five drugs showed that they absorbed at 238 nm.
So this wavelength was selected for the determination of sartans.
Figure 1 shows UV overlain spectra of four sartans with HCTZ.

Figure 1. UV overlain spectra of four sartans and HCTZ.

Method optimization:
 Numerous trials were performed to obtain the better separations.
 From all trials, eventually better reproducibility of the results and good
resolution, good peak shape, short runtime, minimal peak tailing were well
identified when mobile phase consisting of phosphate buffer mixture
properly adjusted to pH 3.3, acetonitrile in the proportion of 50:50 v/v with
satisfactory results.
Table 4. Optimized chromatographic conditions
25Vignan Pharmacy College,Vadlamudi.
Parameter Chromatographic conditions for 4 sartans & Hydrochlorothiazide
Instrument Shimadzu LC-20AT Prominence liquid chromatograph
Column Welchrom C18 column (4.6 X 250 mm, 5 µm)
Detector Shimadzu SPD-20A prominence UV-VIS detector
Mobile phase 10 mM phosphate buffer (pH 3.3) : Acetonitrile 50:50, v/v
Flow rate 1mL/min
wave length UV at 238 nm
Run time 8 minutes
Temperature Ambient temperature (25 0C)
Injection volume 20 µL
Name of the drugs HCTZ TELM LOSA OLME VALS
Retention time (minutes) 3.300 3.780 4.467 5.247 5.570
Th.Pl (Efficiency) 12,312 12,468 14,715 15,250 17,188
Theoretical plates per meter 246247 247368 294308 305005 343756
Resolution - 3.776 4.862 4.931 2.008
Tailing factor 1.194 1.116 1.182 1.160 1.154

Figure 2. Typical chromatogram showing the HCTZ, TELM, LOSA, OLME AND VALS
in a synthetic mixture.

Method validation:
 The developed analytical method was validated in pursuance of the guide
lines of ICH.
SPECIFICITY:
 The purpose of this study is to determine the effect of excipients and other
additives that are usually present in the formulations. The test results obtained
were compared with the results of standard drug.
27
Table 5. Results of specificity
Result: The present study shows that the ingredients are not interfering with the developed method.
Name HCTZ TELM HCTZ LOSA HCTZ OLME HCTZ VALS
Mobile
phase
No peaks No
peaks
No peaks No peaks No peaks No
peaks
No peaks No peaks
Placebo No peaks No
peaks
No peaks No peaks No peaks No
peaks
No peaks No peaks
Individual
Separate
standard
solutions.
Peak for HCTZ and
TELM at 3.300
min. and 3.780 min.
respectively.
Peak for HCTZ and
LOSA at 3.300 min.
and 4.467 min.
respectively.
Peak for HCTZ and
OLME at 3.300 min.
and 5.247 min.
respectively.
Peak for HCTZ and
VALS at 3.300 min.
and 5.570 min.
respectively.

 The linearity of the method was studied by analyzing different
concentration of the drugs. According to ICH recommendations at
least five concentrations are to be used. In this study five
Concentrations were chosen, in the ranges of 2.5-12.5, 8-40, 10-50,
8-40, 16-80 µg/mL. Calibration curve is constructed by plotting
concentration on X-axis and the peak areas on Y-axis.
 The least square analysis method was adopted for achieving slope,
intercept and correlation coefficient, regression data values.

Linearity method
HCTZ LOSA TELM OLME VALS
Conc.
μg/mL
Peak area,
mV.s.
Conc.
μg/mL
Peak area,
mV.s.
Conc.
μg/mL
Peak area,
mV.s.
Conc.
μg/mL
Peak area,
mV.s.
Conc.
μg/mL
Peak area,
mV.s.
0 0 0 0 0 0 0 0 0 0
2.5 217.93 10 209.972 8 205.794 8 175.56 16 234.722
5 435.79 20 416.85 16 410.63 16 382.42 32 466.872
7.5 669.786 30 642.32 24 628.47 24 605.81 48 706.7
10 889.52 40 862.97 32 853.23 32 839.64 64 928.68
12.5 1092.803 50 1080.64 40 1068.11 40 1053.92 80 1168.004
Table 6. Results relating to Linearity data

Table 7. Regression analysis data relating to HCTZ, TELM, LOSA, OLME and VALS
30
Parameter HCTZ LOSA TELM OLME VALS
Detection wavelength(λmax) UV at 238 nm UV at 238 nm UV at 238 nm UV at 238 nm
UV at 238 nm
Linearity range (µg/mL) 2.5-12.5 µg/mL 8-40 µg/mL 10-50 µg/mL 8-40 µg/mL
16-80 µg/mL
Regression equation (Y =aX + b)
Y = 88.146 X +
0.0586
Y = 27.099 X -
6.5173
Y = 21.431 X -
8.5685
Y = 13.36 X -
25.10
Y = 14.576 X +
1.0686
Slope(a) 88.146 27.099 20.968 13.366 14.576
Intercept(b) 0.0586 6.5173 8.5685 25.101 1.0686
Standard error of slope (Sa) 0.7592 0.1537 0.24642 0.2900 0.06053
Standard error of intercept (Sb) 1.9488 0.9310 1.49213 1.4277 2.9327
Regression coefficient (R2) 0.9997 0.9998 0.9997 0.9981 0.9999
% Relative standard deviation* 1.1875 2.4993 0.5239 0.104 1.050
Percentage range of errors
0.05 significance level
0.01 significance level
1.02904
1.6138
1.15695
1.8144
0.54992
0.86242
0.12232
0.19183
0.36373
0.57042

Figure 3. Standard chromatogram relating to HCTZ
Figure 4. Standard chromatogram relating to TELM
Figure 5. Standard chromatogram relating to LOSA
31
CHROMATOGRAMS

Figure 6. Standard chromatogram relating to OLME
Figure 7. Standard chromatogram relating to VALS
32

Figure 8: Standard chromatogram of HCTZ (2.5 μg/ml) and TELM (8 μg/ml)
Figure 9. Standard chromatogram of HCTZ (2.5 μg/ml) and LOSA (5 μg/ml).
Figure 10. Standard chromatogram of HCTZ (2.5 μg/ml) and OLME (8 μg/ml).

Fig. 11. Standard chromatogram of HCTZ (2.5 µg/ml) and VALS (16 µg/ml)

Fig. 12. Sample chromatogram of HCTZ (2.5 µg/mL) and TELM (8 µg/mL)
Fig. 13. Sample chromatogram of HCTZ (2.5 µg/mL) and LOSA
(5 µg/mL)

Fig. 14. Sample chromatogram of HCTZ (2.5 µg/mL) and OLME (8 µg/mL)
Fig. 15. Sample chromatogram of HCTZ (2.5 µg/mL) and VAL (16µg/mL)

Figure 16. Linearity plot pertaining to
HCTZ
Figure 17. Linearity plot
pertaining to LOSA
Figure 18. Linearity plot pertaining to TELM
37
y = 88.146x + 0.0586
R² = 0.9997
0
200
400
600
800
1000
1200
0 5 10 15
peakarea,mV.s
Concentration, µg/mL.
y = 27.099x - 6.5173
R² = 0.9998
-200
0
200
400
600
800
1000
1200
0 10 20 30 40 50
peakarea,mV.s
y = 21.431x - 8.0585
R² = 0.9997
-200
0
200
400
600
800
1000
1200
0 20 40 60
peakarea,mV.s

VALS
OLME
y = 13.366x - 25.101
R² = 0.9981
-200
0
200
400
600
800
1000
1200
0 20 40 60 80 100
PeakareamV.S
Concentration µg/mL.
y = 14.576x + 1.0686
R² = 0.9999
0
200
400
600
800
1000
1200
1400
0 20 40 60 80 100
peakarea,mV.s
concentration, µg/mL.

Precision:
 The repeatability and intermediate precision experiments were conducted
by determining the intra - day and inter - day precision of the method for
HCTZ with TELM, HCTZ with LOSA, HCTZ with OLME and HCTZ with
VALS.
 The intraday precision was done by repeating the assay thrice for the 3
levels in the same day, under the same experimental conditions
Inter - day precision was done by taking over the assay on 3 different days,
three times on the each day for the 3 concentration levels respectively.
The results of precision study were stated in terms of % RSD which was
calculated and results were within the acceptable criteria of NMT 2.0. The
results of precision study are presented in table 8.

Accuracy/Recovery studies:
 The accuracy of the method was assessed by standard addition method.
 Recovery tests were carried out by analyzing mixtures of HCTZ
with TELM, HCTZ with LOSA, HCTZ with OLME and HCTZ with
VALS with different compositions.
Known amounts of standards drugs were added to a pre-analyzed sample
at 3 different levels 80 %, 100 % and 120 % and the mixed standard
solutions were analyzed in triplicate at every level as per suggested
method.
The percent of individual combination of mixtures recovery and % RSD
results of accuracy data is shown in table 8.

Robustness:
The robustness of developed analytical method was proven by the analysis of HCTZ
with TELM, HCTZ with LOSA, HCTZ with OLME and HCTZ with VALS under
different experimental conditions such as making deliberate changes in
chromatographic conditions like flow rate (± 0.2 ml/min), detection wavelength (±5 nm)
and mobile phase composition (±5 %). All results were well within acceptable limits.
LOD and LOQ:
Limit of Detection is the lowest concentration in a sample that can be detected, but
not necessarily quantified under the stated experimental conditions.
Limit of quantification is the lowest concentration of analyte in a sample that can be
determined with acceptable precision and accuracy.
LOD and LOQ were calculated using following formula LOD = 3.3σ/S and
LOQ = 10σ/S, where SD = standard deviation of response (peak area) and S = slope of
the calibration curve. The LOD and LOQ results are shown in the following table.
41

Formulation Telmisat – H
(40mg)
Zargo – H
(50mg)
Olmecip
(40mg)
Valent - H
Composition TELM HCTZ LOSA HCTZ OLME HCTZ VALS HCTZ
Linearity
(μg/mL)
8 - 40 2.5 –
12.5
5 - 50 2.5 –
12.5
8 – 40 2.5 –
12.5
16 - 80 2.5 –
12.5
LOD & LOQ
(μg/mL)
0.570 &
1.889
0.295 &
0.982
0.736 &
2.43
0.299 &
0.986
1.195 &
3.958
0.292 &
0.983
0.116 &
0.352
0.296 &
0.980
Assay ± SD
(n = 6)
99.95 ±
1.13
98.16 ±
0.38
99.85 ±
1.16
98.21 ±
0.45
99.90 ±
1.10
99.19 ±
0.16
100.12 ±
0.15
99.59 ±
0.12
Mean % recovery 100.02±
0.68
100.01 ±
0.15
100.06 ±
0.20
99.95 ±
0.09
100.41 ±
0.35
99.93 ±
0.11
100.05±
0.14
99.96 ±
0.15
Precision Intra
day
(n=6)
0.125 0.131 0.346 0.129 0.435 0.171 0.312 0.213
Inter
day
(n=3)
0.165 0.321 0.326 0.434 0.657 0.322 0.319 0.435
Table 8. Summary of various validation parameters

Formulation TELMISAT – H
(40mg)
ZARGO – H
(50mg)
OLMECIP – H
(40mg)
VALENT – H
(80 mg)
Robustness, % Assay ± % RSD
Flow rate
(± 2
mL/min)
0.8
mL/min
99.81 ±
1.05
97.94 ±
0.46
99.98 ±
1.04
98.93 ±
0.49
98.70 ±
1.02
98.96 ±
0.25
99.91
± 0.29
100.96 ±
0.23
1.2
mL/min
100.12±
0.97
99.54 ±
0.23
98.76 ±
1.32
98.04 ±
0.36
100.03 ±
1.38
99.97 ±
0.35
100.5
4 ±
0.77
98.74 ±
0.71
Detection
wave
Length
(± 5 nm)
233 nm 99.97 ±
1.46
99.43 ±
0.29
98.42±
1.46
97.95 ±
0.98
100.21 ±
1.37
99.96 ±
0.84
100.4
5 ±
0.97
99.98 ±
0.54
243nm 97.98 ±
1.09
98.04 ±
0.76
100.05
± 1.03
98.94 ±
1.08
98.79 ±
0.37
98.76 ±
1.09
99.94
± 1.43
98.47 ±
1.11
Mobile
phase
compos -
ition (±
5%)
45:55
v/v
98.74 ±
o.79
99.42 ±
0.99
99.93 ±
1.42
97.92 ±
0.68
98.91 ±
0.94
100.07
± 0.93
99.01
± 0.61
98.76 ±
0.37
55:45
v/v
99.96 ±
1.2
97.81 ±
0.27
98.54 ±
0.86
98.86 ±
0.30
98.12 ±
1.57
98.72 ±
1.09
99.49
± 0.74
100.03 ±
1.18

CONCLUSION:
• Statistical analysis lucidly proves that it is feasible for analysis of said ARB’s in their
formulations in single run without changing the mobile phase composition and
chromatographic conditions.
• This method was very fast, cost effective, precise, accurate sensitive, highly efficient and
robust than the existing adopted methods. It is noticed that the method is free from
interferences of the excipients and additives utilized in the preparation of above stated ARB’s
after validation.
• This new RP-HPLC method satisfactorily separated all the above said combination drugs
with short retention time (eight minutes and elution window of only two minutes), optimum
peak shape, good separation and best reproducible results. Hence, it is apt to conclude that it
is successfully feasible for the application of routine analysis of said ARB’s agents
individually or binary combinations of HCTZ with TELM, HCTZ with LOSA, HCTZ with
OLME and HCTZ with VALS in quality control laboratories for usual analysis.
44

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1. Development and Validation of RP-HPLC Method for the Estimation and
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Formulation, Youssef ,IJPSRR , Jan – Feb 2014, 24(2), 311-314.
2. Quantitative Determination of three Angiotensin-II-receptor Antagonists in
Presence of Hydrochlorothiazide by RP-HPLC in their Tablet Preparations. Hany
Mohammed Hafez, Abdullah Ahmed, Elshanawane, Lobna Mohammed Abdelaziz,
Iranian Journal of Pharmceutical Research, 2013; 12(4): 635–643.
3. RP-HPLC Method for the Simultaneous Estimation of Telmisartan and
Hydrochlorothiazide in Pharmaceutical Dosage Form, Gopala Swamy, IJDDR,
2011,Vol. 3 ,Issue 4,362-368.
4. RP-HPLC method for simultaneous estimation of Telmisartan and
Hydrochlorothiazide in tablet dosage form. Wankhede SB, Tajne MR, Gupta KR,
Wadodkar SG, Indian Journal of Pharmaceutical Sciences, 2007; 298-300.
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 We Heart fully thank our guide Dr. P. Ravisankar sir .
I also thank Hetero Labs Limited unit-III, Jeedimetla,
Hyderabad for providing gift sample of pure drugs.
ACKNOWLEDGEMENT

49Vignan Pharmacy College,Vadlamudi.

NOVEL SIMULTANEOUS SEPARATION AND QUANTITATIVE DETERMINATION OF FOUR SARTANS IN PRESENCE OF HYDROCHLOROTHIAZIDE BY ISOCRATIC RP-HPLC

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