novel simultaneous separation and quantitative...

Adv J Pharm Life sci Res, 2015 3;4:1-18 ISSN 2454 3535 (On-line)

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Advanced Journal of Pharmacie and Life science Research 1

Novel Simultaneous Separation and Quantitative Determination of Telmisartan, Losartan, Olmesartan and Irbesartan in Presence of Hydrochlorothiazide by Isocratic RP-HPLC

Panchumarthy Ravisankar*1, C. Vineela1, O. Sai Koushik1, P. Srinivasa Babu1

1Panchumarthy Ravisankar, Department of Pharmaceutical Analysis and Quality Assurance, Vignan Pharmacy College, Vadlamudi, Guntur (Dist) – 522 213, A.P state, India. 1C. Vineela, Vignan Pharmacy College, Vadlamudi, Guntur – 522 213, A.P state, India. 1O. Sai Koushik, Vignan Pharmacy College, Vadlamudi, Guntur – 522 213, A.P state, India. 1P. Srinivasa Babu, Vignan Pharmacy College, Vadlamudi, Guntur – 522 213, A.P state, India.

Abstract:

For the first time simple, selective, sensitive, rapid isocratic RP-HPLC method was developed for the separation and quantitative development Telmisartan (TEL), Losartan (LOS), Olmesartan (OLM) and Irbesartan (IRB) are angiotensin II-receptor antagonists which are used in the treatment of hypertension alone or in combination with other drugs mainly Hydrochlorothiazide (HCTZ). The most important advantage of developed method was that the five separate drugs can be determined on a single chromatographic system without modifications in detection wavelength and mobile phase by RP-HPLC. RP-HPLC method was developed by using Welchrom C18 column (4.6 X 250 mm, 5 µm) as stationary phase with the mobile phase comprising of phosphate buffer pH-3.3 and acetonitrile in the portion of 50:50 v/v. Isocratic elution at a flow rate of 1ml/min was employed. The detection was performed with Shimadzu SPD-20A prominence UV-Vis detector set at 232 nm. Total run time was 8 minutes and the elution window of only two minutes. The proposed method was statistically validated in terms of linearity, precision, accuracy, specificity, robustness and ruggedness. The optimized method proved to be specific, robust and accurate for the quality control of four above said angiotensin-II receptor blockers alone or in presence of HCTZ with TEL, HCTZ with LOS, HCTZ with OML and HCTZ with IRB in bulk drug and pharmaceutical formulations.

Key words: Telmisartan, Losartan, Olmesartan, Irbesartan, Hydrochlorothiazide

Corresponding author Dr. Panchumarthy Ravisankar M.Pharm., Ph.D.

Flat no. 501, Door no.4-1-16,

Sapthagiri Sesha Sai Sadan,

Andhra Pradesh, India.

INTRODUCTION Angiotensin antagonists are the first major innovation in essential hypertension management as a first-line treatment. Anti-hypertensive agents are largest drug class and hold a major share of drug market, as hypertension is a major cause of health

problems. The estimated market sales of anti-hypertensive agents are $29.6 billion in 2014. For providing safe, effective drug formulations to consumers direly needed. So innovative analytical methods are necessary for controlling

their quality and quantity of drugs. HCTZ acts as both RAS and sympathetic nerve system there by creating greater sensitivity to angiotensin receptor blockage. Thus HCTZ is a good choice for use in combination with an ARB. ACE inhibitors are having major drawback of cough, when compared to ARBs. All the available ARBs are in fixed-dose combination with HCTZ


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Literature survey revealed that there were few analytical methods have been reported for the estimation of above titled drugs individually, binary or in combination with some other drugs in biological samples as well as pharmaceutical dosage forms by spectrophotometry [1-3], TLC-densitometry and spectrofluorimetric method [4], Capillary electrophoresis [5-7], HPLC [8-20], RP-HPTLC [21], LC-MS [22-25], LC-MS/MS [26-29], MSC for the online solid - phase extraction [30]. Thus we have decided to develop an efficient new method that could determine all these four drugs individually and also even binary combinations ( HCTZ with TEL, HCTZ with LOS, HCTZ with OML and HCTZ with IRB) without changing the detection wave length and chromatographic conditions. By keeping all the above main points in view, it is worthwhile to develop a method which is simple, specific, precise, accurate, sensitive, economical and fast. The most important advantage of developed method was that the five separate drugs individually or in binary combination can be determined on a single chromatographic system without modifications in detection wavelength and mobile phase composition by RP-HPLC. Chemical Structures of HCTZ, TEL, LOS, OML and IRB used for the present study are depicted in Figure1

N

NCH3

CH3

N

N

CH3

HO

TEL

NNN

HN

OML

N

N OH

H3C

O

NNN

HNN N

O

IRB

SNH

HN

O O

Cl

SO O

H2N

HCTZ

NNN

HNN N

ClOH

LOS

Figure 1.Chemical Structures of HCTZ, TEL, LOS, OML and IRB

MATERIALS AND METHODS

The above said standard drugs were gifted from

Hetero Labs Ltd., Hyderabad India. All other

chemicals and solvents were of the highest

grade. HPLC grade acetonitrile and

triethylamine were obtained from Merck

pharmaceuticals private Ltd., Mumbai, India.

Methanol and water utilized were of HPLC

grade and purchased from Merck specialties

private Ltd., Mumbai, India. Commercial tablets

of above said formulation was procured from

local pharmacy

PREPARATION OF PH 3.3 PHOSPHATE BUFFER:

A 10 mM phosphate buffer was prepared by dissolving 6.056 g of potassium dihydrogen orthophosphate in 445 ml of HPLC grade water. To this 55 ml of 0.1 M phosphoric acid was added and pH was adjusted to 3.3.

PREPARATION OF MOBILE PHASE:

The above prepared buffer and acetonitrile were mixed in the proportion of 50: 50 v/v and was filtered through 0.45 µm nylon membrane filter and degassed by sonication. PREPARATION OF STOCK STANDARD SOLUTIONS: Stock standard solutions containing (1.25, 0.4, 0.5, 0.8, 1.5 mg/ml) of Hydrochlorothiazide, Telmisartan, Losartan, Olmesartan, Irbesartan, respectively were prepared by dissolving (12.5, 40, 50, 80, 150 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.

PREPARATION OF CALIBRATION PLOT

(working standard solutions)


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Each solution (n=5) was injected in triplicate and chromatographed under the mentioned conditions above. Linear relationships were obtained when average drug standard peak area were plotted against the corresponding concentrations for each drug. Regression equation was computed.

SAMPLE PREPARATION

A composite of ten Cresar-H, Olmax-H, Hyzaar, Irovel-H tablet, were 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, TEL, LOS, OML, IRB, respectively were prepared by dissolving (12.5, 40, 50, 80, 150 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.

Table 1. System suitability and column performance parameters.

Parameter Chromatographic conditions for four 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 232 nm Run time 8 minutes

Temperature Ambient temperature (25 0C)

Injection volume 20 µL

Name of the drugs HCTZ TEL LOS OML IRB

Retention time (minutes)

3.297 3.777 4.463 5.243 5.563

Th.Pl (Efficiency) 11,196 11,379 13,625 15,231 16,058

Resolution - 3.615 4.675 4.844 2.057

Tailing factor 1.194 1.150 1.182 1.100 1.154

OPTIMIZATION AND METHOD DEVELOPMENT:

A series of trials were conducted for optimization of mobile phase in order to get

proper optimized HPLC conditions. In the first instance several mobile phase trials were done such as commonly used mixture of solvents methanol: water, acetonitrile: HPLC grade water, methanol: acetonitrile: water in different


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ratio and adjusting pH to obtain required separations. Finally after reviewing the results, a mobile phase consisting of phosphate buffer mixture properly adjusted to pH 3.3, acetonitrile in the proportion of 50:50 v/v which full-fill all the criteria of system suitability and also obtain a better separation conditions which result better resolution, good peak shape, short run time, minimal peak tailing and reproducibility results were identified. So this mobile phase was selected for the present investigation. The stationary phase made up of Welchrom C18 column with 4.6 X 250 mm, 5 µm were observed and they are found to be utmost suitable for separation of four Sartans along with HCTZ. The ultra violet spectrum of four Sartans scanned individually in the region between 200 - 400 nm. The UV overlain spectra of these four drugs showed that they absorbed at 232 nm. So this wavelength was selected for the determination of sartans. Figure 2 shows UV overlain spectra of four sartans with HCTZ. A

model chromatogram which shows how the peaks are well separated for the 4 sartans along with HCTZ is presented in Figure 3. The retention times for HCTZ, TEL, LOS, OML and IRB were found to be 3.297, 3.777, 4.463, 5.243, 5.563 minutes respectively. System suitability and column performance parameters are shown in Table 1.

Figure 2. UV overlain spectra of the four sartans & HCTZ

Figure 3. A typical chromatogram showing the separation of HCTZ, TEL, LOS, OML, IRB in a synthetic mixture


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METHOD VALIDATION

The developed method of analysis was validated as per the ICH Q2 (R1) [ 31] guidelines for the parameters like system suitability, specificity, linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantitation (LOQ).

SYSTEM SUITABILITY (SST):

At first allow the HPLC system to stabilize for forty minutes. SST was carried out to verify the parameters such as resolution (NLT 2.0), tailing factor (NMT 1.5), and theoretical plate count (NLT 3000). If system suitability parameters are met, then inject sample preparation in duplicate and record the chromatograms and the system

suitability and column performance parameters are shown in Table 1. SPECIFICITY: This method was conducted by assessing whether excipients present in the pharmaceutical formulations interfered with the analysis or not. A placebo for each tablet was mixed by adding the respective excipients in mobile phase without the drug. Infact drug to excipients ratio utilized were similar to that in the commercial formulations and solutions were prepared by above said sample preparation procedure. The commonly used tablet excipients like HPMC, PEG, purified talc, microcrystalline cellulose and titanium dioxide in tablet formulations. The mixture was filtered through 0.45µm membrane filters before injection. The specificity study table is shown in Table 2.

Table 2. Results of specificity.

Name HCTZ TEL HCTZ LOS HCTZ OML HCTZ IRB

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 at

3.297 min. and

3.777 min. for

HCTZ and TEL

respectively.

Peak for HCTZ at

3.297 min. and

4.463 min. for

HCTZ and LOS

respectively.

Peak for HCTZ at

3.297 min. and

5.243 min. for

HCTZ and OLM

respectively.

Peak for HCTZ at 3.297

min. and 5.563 min. for

HCTZ and IRB

respectively.

LINEARITY:

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, 5-50, 8-40, 16-80, 30-150 µg/ml. The

linearity of peak area responses versus concentrations was demonstrated by linear least square regression analysis. The linear regression equations were Y = 88.146 X + 0.0586 (r2= 0.9997), Y = 27.099 X - 6.5173(r2= 0.9998), Y = 21.431 X - 8.5685 (r2 =0.9997), Y = 53.466 X -


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25.101 (r2= 0.9981), Y = 7.9362 X + 13.668 (r2= 0.999) for HCTZ, TEL, LOS, OML and IRB respectively. Where Y is the peak area of

standard solution and X is the drug concentration

Figure 4. Standard chromatogram of Hydrochlorothiazide.

Figure 5. Standard chromatogram of Telmisartan.


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Figure 6. Standard chromatogram of Losartan.

Figure 7. Standard chromatogram of Olmesartan.

Figure 8. Standard chromatogram of Irbesartan.


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Figure 9. Standard chromatogram of HCTZ and TEL.

Figure 10. Standard chromatogram of HCTZ and LOS.

Figure 11. Standard chromatogram of HCTZ and OML.


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Figure 12. Standard chromatogram of HCTZ and IRB.

PRECISION:

The repeatability and intermediate precision

experiments were conducted by determining the

inter - day and inter - day precision of the

method for HCTZ with TEL, HCTZ with LOS,

HCTZ with OML and HCTZ with IRB. The

intraday precision was done by repeating the

assay thrice for the 3 levels in the same day,

under the same experimental conditions and

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. The percent relative

standard deviation (% RSD) was calculated

which is within the acceptable criteria of not

more than 2.0. The results of precision study are

presented in Tables 2.

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 TEL, HCTZ with LOS, HCTZ with OML

and HCTZ with IRB 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 2.

ROBUSTNESS:

The robustness of developed analytical

method was proven by the analysis of with TEL,

HCTZ with LOS, HCTZ with OML and HCTZ

with IRB 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.


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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. The limit of

quantitation is the lowest concentration of

analyte in a sample that can be determined with

acceptable precision and accuracy. Limit of

detection and limit of quantitation 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 Table 2.

Stability of Analytical Solution

Generally stability of analytical solution was

evaluated by monitoring the peak area response.

Standard stock solutions were analyzed after

one, two and three days after at 50 C and for a

day at room temperature. These solutions were

found to be stable for three days at 50 C and for

a day at room temperature also.

APPLICATION OF PHARMACEUTICAL

PREPARATION:

The developed method was finally

successfully applied for quantitative

determination of marketed formulation such as

CRESAR-H 40 mg, Hyzaar 50 mg, Olmax-H 40

mg, Irovel-H 150 mg. Six replicate

determinations were performed. Excellent

results were obtained for each compound. The

mean assay values were in good agreement with

the label claim (Table 2).

Table 2: Summary of validation of various parameters

Formulation (tablets) Composition

Linearity (µg/mL)

LOD & LOQ

(µg/mL) Assay ± SD (n=6)

Mean % recovery ± SD (n=3)

Precision

Intra-day (n=6) (%

RSD)

Inter-day (n=3)

(% RSD)

CRESAR-H 40 mg)

TEL 8- 40 0.565 &

1.867 99.97 ± 1.12

100.09 ± 0.71

0. 127 0.167

HCTZ 2.5 - 25

0.298 & 0.980

98.14 ± 0.32 100.05 ±

0.16 0.132 0.324

Hyzaar 50 mg LOS 5- 50 0.731 & 2.414

99.88 ± 1.14 100.02 ±

0.16 0.342 0.324

HCTZ 2.5 - 25

0.297 & 0.980

98.18 ± 0.42 99.98 ± 0.12 0.127 0.432

Olmax-H (40mg) OML 16 - 80 1.198 & 3.953

99.99 ± 1.12 100.45 ±

0.40 0.432 0.654

HCTZ 2.5 - 25

0.295 & 0.980

99.15 ± 0.12 99.97 ± 0.13 0.168 0.324

Irovel-H (150mg) IRB 30 -150 1.220 & 4.026

100.15±1.30

100.47 ± 0.4 0.467 0.324

HCTZ 2.5 - 25

0.296 & 0.980

99.59 ± 0.12 99.96 ± 0.15 0.213 0.435


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RESULTS AND DISCUSSION:

The main objective of the present study is to

develop a rapid, new, simple, precise and

accurate Reversed Phase - High Pressure liquid

chromatographic methods for simultaneous

separation and quantitative determination of

HCTZ with TEL, HCTZ with LOS, HCTZ with

OML and HCTZ with IRB in combination tablet

dosage form. When developing a new method

one of the prominent goals is to achieve a

consistent reproducible separation by selecting a

highly reproducible high performance liquid

chromatography method which is essential to get

the required goal. However, trial and error

methods were taken up prior to identifying the

best operational and environmental conditions

for optimizing suitable methods for separations.

Since the all the above stated drugs are relatively

polar, a RP-HPLC method was selected for the

initial separation process. C18 column with a 4.6

mm internal diameter, 250 mm length and 5

micron particle size was preferred and a number

of trials were performed using different buffer

solutions of different pH ranges with different

compositions of mobile phases, variable flow

rate and column temperature. Finally an

optimum separation condition was achieved with

a mixture of phosphate buffer (pH-3.3) and

acetonitrile in a composition of 50:50 v/v. A

mobile phase flow rate adjusted at 1mL/min, a

common detection wavelength was set at 232

nm for all the drugs, and the column temperature

was kept at ambient conditions. After the

adjustment of such operational parameters at

their corresponding optimum values, better

chromatographic peak was obtained with

characterizes of well resolution, good symmetry

and minimal peak tailing.

The proposed method was validated as per the

ICH guidelines with regard to specificity,

linearity, precision, accuracy, robustness and

LOD and LOQ. As per the prescribed

guidelines, system suitability was conducted to

ensure the suitability of complete testing system

for the intended purpose. As a result system

suitability parameters such as resolution, number

of theoretical plates, tailing factor of the peaks

were computed for the optimized

chromatographic condition for the proposed

method. Accordingly 3.297, 3.777, 4.463, 5.243

and 5.563 minutes of retention time, 11,196,

11,379, 13,625, 15,231 and 16,058 of plate

number, 1.194, 1.150, 1.182, 1.100 and 1.154

tailing factors were obtained for HCTZ, TEL,

LOS, OML and IRB respectively. The resolution

for TEL, LOS, OML and IRB were 3.615,

4.675, 4.844 and 2.057. As all of these results

were within the acceptable limit, the method is

suitable for the intended purpose of estimation

of the above said drugs.

Linearity of the proposed method was evaluated

by taking five points concentrations. All

calibration curves for 5 drugs showed linearity

over a concentration ranges in the ranges of 2.5-

12.5, 5-50, 8-40, 16-80, 30-150 µg/ml for

HCTZ, TEL, LOS, OML and IRB respectively


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and for Creshar-H 40 mg tablet, 8-40 µg/mL for

TEL and 2.5-25 µg/mL for HCTZ; for Hyzaar

50 mg tablet, 5-50 µg/mL for LOS and 2.5-25

µg/mL for HCTZ; for Olmax-H 40 mg tablet

16-80 OML µg/mL and 2.5-25 µg/mL for

HCTZ; for Iovel-H 150 mg tablet, 30-150 µg/ml

for IRB and 2.5-25 µg/mL for HCTZ. The

corresponding correlation coefficient were also

calculated from the linear regression analysis

and found to be above 0.9998 in all cases. In all

the above cases, the calculated results showed

that they were within the acceptable limits. The

said results indicate that there exist strong linear

relationship between concentrations of each drug

and their peak areas.

As stated by ICH guideline a mixture of pure drug samples combined with suitable excipients were injected to the system to test specificity of the proposed method for quantifying the all drugs individually and also combination of two drugs HCTZ with TEL, HCTZ with LOS, HCTZ with OML and HCTZ with IRB to test specificity of all these drugs. In the similar manner synthetic mixture solutions as well as blank solution with only generally utilized excipients were also injected separately. The peak response for the analyte and the blank were compared with each relevant drug. The results showed no interference due to the commonly used excipients. Therefore the method lucidly proves to be specific for estimating the above stated drugs. Precision of the method was evaluated by utilizing intra-day and inter-day studies. Different concentration levels were taken in triplicate samples and evaluated for the repeatability and intermediate precision of the method. The samples used were standard quality. For intra and inter-day precision the % RSD values for each drug were evaluated and

found RSD percentages of all above specified drugs were found less than 2 % which can be showed the present method is precise. To evaluate the accuracy of the proposed

method by combining the known amount of pure

standard drugs to pre-analyzed samples at 80 %,

100 % and 120 % (three levels) and the recovery

levels were keenly examined. The said solutions

were again prepared and analyzed in triplicate

thoroughly. The above process was followed for

all the single drugs as well as combination of


OML and HCTZ with IRB as specified above

and found % RSD was also established to be less

than 2 % for each drug. Robustness was

determined by assessing duly effecting slight

changes in chromatographic conditions such as

lambda max, mobile phase composition and

flow rate. In the chromatograms the author

found that there were no any marked changes.

The results are well within the acceptable limits.

Therefore the method found to be remaining

uninterrupted even though tiny changes effected

to the chromatographic conditions. The results

relating to limit of detection (LOD) and limit of

quantitation (LOQ) for HCTZ with TEL, HCTZ

with LOS, HCTZ with OML and HCTZ with

IRB combination of drugs were found to be

0.298 µg/mL and 0.980 µg/mL; 0.565 µg/mL

and 0.565 µg/mL, 0.298 µg/mL and 0.980

µg/mL; 0.731 µg/mL and 2.414, 0.298 µg/mL

and 0.980 µg/mL; 1.198 µg/mL and 3.953

µg/mL, 0.298 µg/mL and 0.980 µg/mL; 1.220

µg/mL and 4.026 respectively. These results

specifically show that the method has relatively


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lower LOD and LOQ values. The developed

method was eventually applied for quantification

of marketed formulation. The mean assay values

for HCTZ with TEL, HCTZ with LOS, HCTZ

with OML and HCTZ with IRB combination of

drugs were found to be 98.14 ± 0.32 and 99.97

± 1.12; 98.18 ± 0.42 and 99.88 ± 1.14; 99.15 ±

0.12 and 99.99 ± 1.12; 99.59 ± 0.12 and 100.15

± 1.30 respectively and by the developed

method all the said drugs were perfectly

recovered from the tablet dosage form. Thus the

method invented by the author was found to be

aptly suitable for determination of the

commercial formulations.

Figure 13. Sample chromatogram of HCTZ and TEL.

Figure 14. Sample chromatogram of HCTZ and LOS.


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Figure 15. Sample chromatogram of HCTZ and OML.

Figure 16. Sample chromatogram of HCTZ and IRB 10 μg/ml.

CONCLUSION: Statistical analysis lucidly proves that it is

feasible for analysis of said ARBs 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, robust and congenial than the existing

adopted methods hitherto. 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


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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


OML and HCTZ with IRB in quality control

laboratories for usual analysis.

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