CHAPTER – 4 SIMULTANEOUS DETERMINATION...

CHAPTER – 4

SIMULTANEOUS DETERMINATION OF RELATED SUBSTANCES OF

TELMISARTAN AND HYDROCHLOROTHIAZIDE IN TABLET

DOSAGE FORM BY USING REVERSE PHASE HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

CHAPTER - 4

SPP SPTM, SVKM’s NMIMS, Mumbai 17

CHAPTER – 4

Simultaneous determination of related substances of Telmisartan and Hydrochlorothiazide in tablet dosage form by using reverse phase high

performance liquid chromatography 4.0 INTRODUCTION The objective was to develop a single method for simultaneous determination of related

substances of Telmisartan and Hydrochlorothiazide in a tablet dosage form of two

strengths (80mg-12.5mg and 40mg-12,5mg). The method was validated as per ICH

guidelines Q2 (R1).

4.1 Drug Profile 4.1.1 Telmisartan Telmisartan, 4-[(1,4-dimethyl-2-propyl[2,6- bi-1H-benzimidazol]-1-yl) methyl-[1,1-

biphenyl]-2-carboxylic acid, is a potent, long-lasting, nonpeptide antagonist of the

angiotensin II type-1 (AT1) receptor that is indicated for the treatment of essential

hypertension. It selectively and insurmountably inhibits stimulation of the AT1 receptor

by angiotensin II without affecting other receptor systems involved in cardiovascular

regulation. In clinical studies, Telmisartan shows comparable antihypertensive activity

to members of other major antihypertensive classes, such as angiotensin-converting

enzyme (ACE) inhibitors, beta-blockers and calcium channel blockers. Telmisartan

(TE) is widely used in the treatment of hypertension.

Telmisartan is official in USP and EP General Name : Telmisartan Chemical Structure :

CHAPTER - 4


Chemical Name : 4′-[[4-Methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-

propyl- 1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxylic acid

Molecular Formula : C33H30N4O2 Molecular Weight : 514.6 CAS Number : 144701-48-4 Description : White or slightly yellowish, crystalline powder Solubility : Practically insoluble in water, slightly soluble in methanol, sparingly soluble in methylene chloride. It dissolves in 1 M sodium hydroxide Drug Category : Angiotensin II (AT1) receptor antagonist (Antihypertensive) Listed Impurities in Monograph (EP/BP): Impurities A, B, C and D are specified impurities.

Impurity A. 4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1H-benzimidazole

Impurity B. 4′-[[7-methyl-5-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1H- benzimidazol-1-yl]methyl]biphenyl-2-carboxylic acid

CHAPTER - 4


Impurity C. 1,1-dimethylethyl 4′-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2- propyl-1Hbenzimidazol-1-yl]methyl]biphenyl-2-carboxylate

Impurity D. Unidentified Impurity

Impurity E. 1-[(2′-carboxybiphenyl-4-yl)methyl]-4-methyl-2-propyl-1H-enzimidazol- 6-carboxylic acid

CHAPTER - 4


Impurity F. 4′-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1H- benzimidazol-1-yl]methyl]biphenyl-2-carboxamide

Impurity G. 4′-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2-propyl-1H-benzimidazol-1-yl]methyl]biphenyl-2-carbonitrile

Impurity F. 1,1-dimethylethyl 4′-(bromomethyl)biphenyl-2-carboxylate

The EP impurity identification mixture when injected in the EP API monograph method

was expected to generate the following chromatogram. The chromatogram given below

is obtained from EP.

CHAPTER - 4


4.1.2 Hydrochlorothiazide Hydrochlorothiazide (HCTZ) (6-chloro-3, 4-dihydro-2H-1, 2, 4-benzo-thiadiazine-7-

sulfonamide 1,1-dioxide) is a widely prescribed diuretic. It is indicated for the treatment

of edema, control of essential hypertension and management of diabetes insipidus. [4]

Hydrochlorothiazide, a thiazide diuretic, is also used to treat mild to moderate

hypertension, usually in combination with other antihypertensive agents with different

mechanisms of action. [5] Blood pressure control is often inadequate using monotherapy.

Combination therapy can simplify dosing regimens, improve compliance, and decrease

side effects.

CHAPTER - 4


Hydrochlorothiazide is official in USP and EP/ BP General Name : Hydrochlorothiazide (HCTZ) Chemical Structure :

Chemical Name : 6-Chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7- sulphonamide 1,1-dioxide

Molecular Formula : C7H8ClN3O4S2 Molecular Weight : 297.7 CAS Number : 58-93-5 Description : White or almost white, crystalline powder Solubility : Very slightly soluble in water, soluble in acetone, sparingly soluble in ethanol (96 per cent). It dissolves in dilute solutions of alkali hydroxides Drug Category : Thiazide diuretic Listed Impurities in Monograph (EP/BP):

Impurities A, B and C are specified impurities.

Impurity A. chlorothiazide

CHAPTER - 4


Impurity B. 4-amino-6-chlorobenzene-1,3-disulphonamide (salamide)

Impurity C. 6-chloro-N-[(6-chloro-7-sulphamoyl-2,3-dihydro-4H-1,2,4-benzothiadiazin- 4-yl 1,1- dioxide)methyl]-3,4-dihydro-2H-1,2,4-benzothiadiazine-7- sulphonamide 1,1-dioxide

CHAPTER - 4


4.2 LITERATURE SURVEY

The literature survey reveals that, Telmisartan and Hydrochlorothiazide are reported in

British Pharmacopoeia. [6, 7] There have been several publications describing analytical

methods for the determination of HCTZ and TE individually or with other drugs as

combination.

Although there are a few papers published on simultaneous determination of TE and

HCTZ in formulation most of them deal with the assay of each constituent. Several

methods are reported for the determination of TE like Spectrophotometric [7] and HPLC. [9-11] The other methods available in the literature are based on Linear Sweep

polarography, [12] LC–MS. [13] Articles on the determination of HCTZ in combination

with other drugs by HPLC are also reported in literature. [14, 15]

However the exhaustive literature survey revealed that none of the most recognized

pharmacopoeias or any journals includes these drugs in combination for the

simultaneous determination of related substances of TE and HCTZ and the information

regarding the stability of the drugs is not available@. The Regulatory agencies

recommend the use of stability indicating analytical methods (SIAMs) [16] for the

analysis of stability samples [17]. This requires stress studies (in order to generate the

potential related impurities under stressed conditions), method development and

validation .With the advent of the International Conference on Harmonization (ICH)

guidelines [18], requirements for the establishment of SIMs have become more clearly

mandated. The production of the potential impurities in a drug product generally takes

place under various environmental conditions like exposure to light, heat, hydrolysis or

oxidation. Hence Stress testing can help identify degradation products and provide

important information about intrinsic stability of the drug product.

Several methods have been studied earlier for simultaneous determination of

Telmisartan and Hydrochlorothiazide, but there is no report on method for related

substances of these drugs in combination. So the aim of our study is to develop simple,

fast, accurate and specific reversed phase High Performance Liquid Chromatographic

method for simultaneous determination of related substances of Telmisartan and

Hydrochlorothiazide in tablet dosage form.

---------------------------------------------------------------------------------------------------------- @ This search was performed at the start and during the course of the study. A draft monograph has been published in USP Forum Vol

36(3). The Monograph is now official as per USP 34, from December 2011. A comparison of methods is given in the next section.

CHAPTER - 4


Table 4.2.1: Related Substances – Pharmacopeia methods for Drug Substance

Telmisartan HCTZ

BP 2012 USP 35 BP 2012 USP 35

Method HPLC HPLC HPLC HPLC

Column C18

125mm x 4.0mm; 5µ; 10nm

poresize

C18

125mm x 4.0mm; 5µ; 10nm

poresize

C18

100mm x 4.6mm; 5µ;

C18

50mm x 4.6mm; 3.5µ

Column Temp 40°C 40°C Not Mentioned 35°C

Mobile Phase Mobile phase A: 2.0 g of

potassium dihydrogen

phosphate and 3.8 g of

sodium pentanesulphonate

monohydrate in 1000ml

water, pH 3.0 adjusted with

dilute

Phosphoric acid.

—mobile phase B:

methanol : acetonitrile

(20:80 V/V)

Solution A: 2.0 g of

monobasic potassium

phosphate and 3.8 g of

sodium 1-pentanesulfonate

in 1 L of water. pH adjusted

with 1 M phosphoric acid to

3.0.

Solution B: Acetonitrile and

methanol (4:1)

—mobile phase A: to 940

ml of phosphate buffer

solution pH 3.2 add 60.0 ml

of methanol and 10.0 ml of

tetrahydrofuran and mixed;

—mobile phase B: to a

mixture of 500 ml of

methanol and 500 ml of

phosphate buffer solution

pH 3.2 50.0 ml of

tetrahydrofuran was added

and mixed

Solution A— A mixture of

acetonitrile and methanol

(3:1) was prepared and

degassed.

Solution B— A solution of

anhydrous formic acid in

water (5 in 1000) was

prepared and degassed.

CHAPTER - 4


Gradient Time

(min)

%A %B

0 70 30

3 70 30

28 20 80

Re-equilibrate to initial

conditions

Time

(min)

%A %B

0 70 30

2 70 30

27 20 80

32 20 80

32.1 70 30

37 70 30

Time

(min)

%A %B

0 100 0

17 55 45

30 55 45

35 100 0

50 100 0

Time

(min)

%A %B

0 3 97

5 3 97

14 36 64

18 3 97

20 3 97

Flow Rate 1ml/min 1ml/min 0.8ml/min 1ml/min

Wavelength 230nm 230nm 224nm 275nm

Inj Vol (µL) 10 10 10 10

CHAPTER - 4


Table 4.2.2: Related Substances – Pharmacopeia methods for Drug Product

Telmisartan HCTZ Telmisartan-HCTZ

BP 2012 USP 35 BP 2012 USP 35 USP 35

Method Not Available HPLC HPLC HPLC HPLC

Column Not Applicable C18

40mm x 4.0mm; 5µ

C18

100mm x 4.6mm; 3µ

C18

250mm x 4.6mm;

C8

125mm x 4.0mm; 5µ

Column Temp Not Applicable 40°C Ambient Not Mentioned 40°C

Mobile Phase Not Applicable Buffer - 2.0 g/L

ammonium

dihydrogen

phosphate was

prepared. Adjusted

the pH with 1 M

phosphoric acid to

a 3.0

Mobile Phase:

Methanol and Buffer

(7:3)

--mobile phase A: A

mixture of 10

volumes of

tetrahydrofuran, 60

volumes of methanol

and 940 volumes of

phosphate buffer

solution pH 3.2 was

prepared.

--mobile phase B: A

mixture of 50

volumes of

A mixture of 0.1 M

monobasic sodium

phosphate and

acetonitrile (9:1) was

prepared and

degassed. The pH

adjusted with

phosphoric acid to

3.0 ± 0.1

Buffer: 2.0 g/L of

ammonium

dihydrogen

phosphate was

prepared. The pH

was adjusted with

phosphoric acid to

3.0.

Solution A: Methanol

and acetonitrile (1:1)

was mixed.

CHAPTER - 4


tetrahydrofuran, 500

volumes of methanol

and 500 volumes of

phosphate buffer

solution pH 3.2 was

prepared.

Gradient Not Applicable Isocratic (Run time

not mentioned)

Isocratic (Run time

not mentioned)

Time

(min)

%A %B

0 85 15

3.50 85 15

3.51 45 55

7.70 45 55

7.71 20 80

12.0 20 80

12.1 85 15

15.5 85 15

Flow Rate Not Applicable 0.7ml/min 0.8ml/min 2ml/min 1.2ml/min

Wavelength Not Applicable 298nm 224nm 254nm 270-Telmisartan

298- HCTZ

Inj Vol (µL) Not Applicable 5 20 20 10

CHAPTER - 4


4.3 PRESENT WORK AND DISCUSSION 4.3.1. Selection of Chromatographic Method Official methods and methods published in literature for Telmisartan and

Hydrochlorothiazide were based on reverse phase chromatographic (RPC) separation.

So, all development was conducted using reverse phase chromatography. Reverse Phase

chromatography is a choice because of its ease of handling and robust nature.

4.3.2 Selection of Stationary Phase

USP, EP monograph and reported HPLC methods of Telmisartan or

hydrochlorothiazide recommended use of C18 column for the purpose of determination

of related impurities and assay. So C18 columns were preferred as stationary phase. The

EP impurity mixture was used to understand the separation of Telmisartan impurities.

Since EP API method was based on Kromasil C18 column, this column was given

higher preference over other C18 brands to ensure similarity of elution to EP

monograph.

4.3.3 Selection of Wavelength for Analysis

The optimum wavelength selected was 270 nm which represents the wavelength where

all impurities have suitable responses in order to permit simultaneous determination of

related impurities of Telmisartan and HCTZ. The overlain UV spectrum for HCTZ, TE

and their related substances were recorded. All overlaid components showed desirable

absorbance at 270nm. Hence 270nm was selected for method development. 270nm is

also the wavelength which has an absorption maximum for hydrochlorothiazide and an

absorption minimum for Telmisartan. Thus at this wavelength, minor changes will not

affect peak areas and thus the final result. This will in effect produce a robust method

with respect to wavelength.

CHAPTER - 4


Figure: 4.3.3.1 Overlaid UV spectrum of Telmisartan and Hydrochlorothiazide

4.3.4 Selection and Optimization of Mobile Phase From literature survey, it was observed that Hydrochlorothiazide is poorly retained on

C18 stationary phase whereas Telmisartan is strongly retained. Thus a gradient elution

program was selected over isocratic elution. A high aqueous percentage was used in the

initial period to retain the hydrochlorothiazide and its impurities while a higher organic

was used to elute the Telmisartan and its impurities in the later period.

Various gradient trials were taken. It was observed that there was inadequate separation

between Hydrochlorothiazide and its related impurities. Additionally they were eluting

very close to the dead volume. To overcome this problem, initial trials were conducted

with high percentage of aqueous phase. It led to longer retention time for Telmisartan

and its impurities. Further trials were taken by using cationic ion pairing agent (Sodium

salt of hexane sulphonic acid). This not only served to retain the Hydrochlorothiazide

better, it also showed a better separation of the Telmisartan impurity peaks. The run

time was found considerably less as compared to previous trials. The use of ion pairing

reagent permitted usage of higher percentage of organic phase during the start of the

CHAPTER - 4


gradient. This facilitated earlier elution of Telmisartan and its impurities with desirable

separation.

The above developed method was tried for separation of known impurities and

unknown impurities generated by forced degradation.

The major challenge faced during the development of a single method for simultaneous

determination of related substances of more than one API is not only the separation of

impurities from each other and the main peaks but also to identify the source of

unknown impurities. It is of critical importance to allot an unknown impurity to the

correct API component since the estimation of impurities is based on the label claim of

the API in the finished product. The limits of the unknown impurities are in turn based

on daily doses of the individual API. A wrong identification can pass an out of

specifications (OOS) batch or erroneously reject a correct batch.

This critical determination was handled in the following manner by analysing three

placebo preparations apart from the sample.

1. Placebo which has all the excipient except for the APIs

2. Placebo which has all excipient and Telmisartan API spiked in the same

quantitative composition as in the finished product. This is the placebo for

Hydrochlorothiazide part.

3. Placebo which has all excipient and Hydrochlorothiazide API spiked in the same

quantitative composition as in the finished product. This is the placebo for

Telmisartan part.

The forced degradation studies were performed on all the above preparations in addition

to the finished product to determine the source of the unknown impurities.

The next challenge was in selection of common diluent for both the drugs in the tablet.

Initially 0.1M Ethanolic sodium hydroxide was used as a common diluent for the tablet,

since Telmisartan is soluble is basic alcoholic diluents. Though this diluent was suitable

for dissolving both the drug, it was observed that in this diluent Hydrochlorothiazide

degraded rapidly. This problem was overcome by using a weaker (0.01M) Ethanolic

sodium hydroxide solution.

Initial gradient trials were conducted using buffer (pH = 3) containing ion pair reagent

as majority of the methods reported in the literature used the same strategy. Sodium

Hexane sulphonate, monohydrate was chosen due to its easy availability in pure form

and stability. Purity of ion pair reagent used is very critical for method development and

CHAPTER - 4


further routine analysis. The ion paring capacity of the reagent directly depends on its

purity.

Initial trials showed a slight fronting on the main peaks, probably due to action of the

ion paring reagent. This was overcome by increasing the column temperature to 40°C.

Various gradient elution programs were tried to obtain desirable results. The final

optimized chromatographic conditions are given below.

Optimized Chromatographic Conditions:

Chromatographic condition for related substances:

Instruments/Equipment : HPLC, Make – Waters, Alliance, 2695 Separation

Module, (UV/PDA), or equivalent.

Analytical Balance, Make –Mettler Toledo, Model-

XS205DU, or equivalent.

Column : Kromasil C18, 250 x 4.6 mm, 5µm or equivalent

Flow rate : 1.0 ml/minute

Column temperature : 40°C

Wavelength : 270 nm

Sample temperature : 20°C

Injection volume : 10 µl

Run time for sample : 45 minutes

Gradient Program:

Time in minutes Mobile Phase A % Mobile Phase B %

0.01 94 6

15 94 6

35 20 80

40 20 80

42 94 6

45 94 6

Mobile Phase A : 2.0 gm of Potassium dihydrogen phosphate anhydrous and 1.04 gm of Sodium 1- Hexane sulphonic acid monohydrate, in 1000ml of water; pH 3.0 with ortho phosphoric acid Mobile Phase B : A mixture of Acetonitrile: Methanol in the ratio 80:20 v/v

CHAPTER - 4


4.4 FORCED DEGRADATION STUDIES

The forced degradation studies were carried out to achieve adequate degradation of the

individual drugs. They were carried out on the higher strength tablets (80mg-12.5mg)

and chromatographed along with a non-stressed sample (control). The role of placebo

degradation is a significant part of method development for multi-drug formulation.

4.4.1 Telmisartan Degradation

4.4.1.1 Hydrolytic conditions: acid-, base-induced degradation.

Acid Degradation

10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml volumetric

flask. 10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with intermittent

shaking till the tablets disintegrated. The solution was cooled to room temperature. About

10 ml of diluent C was added. 5 ml of 5N Hydrochloric acid was added and the solution

was heated in the oven at 70°C for 24 hours. The solution was then cooled and neutralized

with same volume and same strength alkali and made up the volume with diluent-D. Further

3 ml was diluted to 25 ml with diluent-D and filtered through 0.45 Nylon filter.

Placebos were treated similarly.

Base Degradation

10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml volumetric flask.

10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with intermittent shaking till

the tablets disintegrated. The solution was cooled to room temperature. About 10 ml of diluent

C was added. 5 ml of 5N Sodium Hydroxide was added and the solution was heated in the oven

at 70°C for 24 hours. The solution was then cooled and neutralized with same volume and same

strength acid and made up the volume with diluent-D. Further 3 ml was diluted to 25 ml with

diluent-D and filtered through 0.45 Nylon filter.


4.4.1.2 Oxidative condition: hydrogen peroxide-induced degradation.



CHAPTER - 4



C was added. 5 ml of 50 % Hydrogen peroxide was added. The solution was heated in the oven

at 70°C for 24 hours. The solution was then cooled and made up the volume with diluent-D


4.4.1.3 Thermal degradation.

10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml

volumetric flask. 10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with

intermittent shaking till the tablets disintegrate. The solution was cooled at room

temperature and 10 ml of diluent C was added. The solution was heated in the oven at

70°C for 24 hours, cooled and volume was made up the with diluent-D. Further 3 ml of

this solution was diluted to 25 ml with diluent-D


4.4.1.4 Photolytic degradation.

As per guidelines for photostability testing of new drug substances and products,

samples

should be exposed to light providing an overall illumination of not less than 1.2 million

lx hours and an integrated near ultraviolet energy of not less than 200Wh/m2 to allow

direct comparisons to be made between the drug substance and drug product. [19]

For photo stability testing 10 tablets of Telmisartan + Hydrochlorothiazide were

transferred into each of 100 ml clear glass, 100 ml flask covered with aluminium foil

and 100 ml amber colour volumetric flask. 10 ml of diluent-B was added and

sonnicated for 5 to 10 minutes with intermittent shaking till the tablets disintegrated.

The solution was cooled at room temperature and 10 ml of diluent C was added. These

flasks were kept under UV and white light for 1.2 million lux hours in photo stability

chamber/ 200Wh m-2. After study the sample was cooled and diluted upto the mark

with diluent-D. Further 3 ml of this solution was diluted to 25 ml with diluent-D and

filtered through 0.45μ Nylon filter.


CHAPTER - 4


4.4.2 Hydrochlorothiazide Degradation

4.4.2.1 Hydrolytic conditions: acid-, base-induced degradation.

Acid Degradation

10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml volumetric

flask. 10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with intermittent

shaking till the tablets disintegrated. The solution was cooled to room temperature. About

10 ml of diluent C was added. 5 ml of 5N Hydrochloric acid was added and the solution

was heated in the oven at 70°C for 1 hour. The solution was then cooled and neutralized

with same volume and same strength alkali and made up the volume with diluent-D. Further

3 ml was diluted to 25 ml with diluent-D and filtered through 0.45 Nylon filter.


Base Degradation




C was added. 5 ml of 5N sodium hydroxide was added and the solution was heated in the oven

at 70°C for 6 hours. The solution was then cooled and neutralized with same volume and same

strength acid and made up the volume with diluent-D. Further 3 ml was diluted to 25 ml with

diluent-D and filtered through 0.45 Nylon filter.


4.4.2.2 Oxidative condition: hydrogen peroxide-induced degradation.




C was added. 5 ml of 50 % Hydrogen peroxide was added. The solution was heated in the oven

at 70°C for 6 hours. The solution was then cooled and made up the volume with diluent-D

4.4.2.3 Thermal degradation.

10 tablets of Telmisartan + Hydrochlorothiazide were transferred into 100 ml

volumetric flask. 10 ml of diluent-B was added and sonnicated for 5 to 10 minutes with

intermittent shaking till the tablets disintegrate. The solution was cooled at room

temperature and 10 ml of diluent C was added. The solution was heated in the oven at

CHAPTER - 4


70°C for 6 hours, cooled and volume was made up with diluent-D. Further 3 ml of this

solution was diluted to 25 ml with diluent-D

4.4.2.4 Photolytic degradation.

As per guidelines for photostability testing of new drug substances and products,

samples should be exposed to light providing an overall illumination of not less than 1.2

million lx hours and an integrated near ultraviolet energy of not less than 200Wh/m2 to

allow direct comparisons to be made between the drug substance and drug product. [19]

For photo stability testing 10 tablets of Telmisartan + Hydrochlorothiazide were

transferred into each of 100 ml clear glass, 100 ml flask covered with aluminium foil

and 100 ml amber colour volumetric flask. 10 ml of diluent-B was added and

sonnicated for 5 to 10 minutes with intermittent shaking till the tablets disintegrated.

The solution was cooled at room temperature and 10 ml of diluent C was added. These

flasks were kept under UV and white light for 1.2 million lux hours in photo stability

chamber/ 200Wh m-2. After study the sample was cooled and diluted upto the mark

with diluent-D. Further 3 ml of this solution was diluted to 25 ml with diluent-D and

filtered through 0.45μ Nylon filter.


4.4.3 Observations in forced degradation studies.

It was observed that overall Telmisartan is a much stable molecule than

Hydrochlorothiazide under the harsh conditions. Hydrochlorothiazide degraded in most

conditions. The chromatograms are given in the below figures.

CHAPTER - 4


Figure-4.4.1: Chromatogram of Blank.

Figure-4.4.2: Chromatogram of Placebo without Telmisartan and Hydrochlorothiazide.

Figure-4.4.3: Chromatogram of Placebo with Telmisartan.

CHAPTER - 4


Figure-4.4.4: Chromatogram of Placebo with Hydrochlorothiazide.

Figure-4.4.5: Chromatogram of Standard solution.

Figure-4.4.6: Chromatogram of Control Sample solution.

CHAPTER - 4


Figure-4.4.7: Chromatogram of Impurities mixture with Telmisartan and Hydrochlorothiazide.

Name Retention Time USP Resolution

HCTZ-IMP-B 10.674 HCTZ-IMP-A 14.909 6.23

HYDROCHLOROTHIAZIDE 17.918 3.72 TELMISARTAN IMP-A 27.582 20.43

HCTZ-IMP-C 27.812 2.05 TELMISARTAN IMP-E 31.130 26.65 TELMISARTAN IMP-F 31.497 2.73 TELMISARTAN IMP-B 32.911 10.35

TELMISARTAN 34.704 11.92 TELMISARTAN IMP-C 36.157 9.53

Figure-4.4.8: Chromatogram of sample in Acid for Telmisartan.

CHAPTER - 4


Figure-4.4.9: Chromatogram of sample in Acid for Hydrochlorothiazide.

Figure-4.4.10: Chromatogram of sample in Base for Telmisartan.

Figure-4.4.11: Chromatogram of sample in Base for Hydrochlorothiazide.

CHAPTER - 4


Figure-4.4.12.1: Chromatogram of sample in Peroxide for Telmisartan.

Figure-4.4.12.2: Chromatogram of sample in Peroxide for Telmisartan (Zoom Scale from 20 to 40 mintues)

Figure-4.4.13.1: Chromatogram of sample in Peroxide for Hydrochlorothiazide.

CHAPTER - 4


Figure-4.4.13.2: Chromatogram of sample in Peroxide for Hydrochlorothiazide (Zoom Scale from 20 to 40 mintues)

Figure-4.4.14: Chromatogram of sample Heat for Telmisartan.

Figure-4.4.15: Chromatogram of sample Heat for Hydrochlorothiazide.

CHAPTER - 4


Figure-4.4.16: Chromatogram of sample in Photolytic study.

Telmisartan

The major degradation was observed under acidic and oxidative stress conditions. There

was no significant degradation observed under other stress conditions.

Under acidic stress conditions one degradant was generated and eluted at RRT of about

1.05. Since Telmisartan is a relatively stable molecule further investigation was carried

out to find out the nature of this degradant. The sample was treated under same stress

conditions by replacing the methanol in the diluents with Acetonitrile. This particular

degradant was not generated under this stress condition. This study confirmed that the

observed peak was actually an ester formed in situ due to interaction between methanol

and Telmisartan. Please refer to Figures 4.4.17 and 4.4.18 respectively.

Based on the above facts, the following impurity formation pathway can be predicted:

N

N

CH3

N

N

CH3

CH3

O

OH

Methanol

Hydrochloric

acid

N

N

CH3

N

N

CH3

CH3

O

O CH3

Telmisartan Methyl ester of Telmisartan

CHAPTER - 4


Figure-4.4.17: Degradation of Telmisartan under Acidic condition with diluents containing

Acetonitrile in place of methanol.

Under oxidative conditions none of the individual degradant generated was significant

to perform degradant qualification.

Hydrochlorothiazide

Hydrochlorothiazide, unlike Telmisartan, degraded under most of the stress conditions.

A common degradant formed under all the stress conditions was Impurity B, i.e. 4-

Amino-6-chloro-1,3-benzenedisulfonamide.

Peak Observed when methanol is present in Diluents

Peak is not observed when Acetonitrile is present in Diluents instead of Methanol

CHAPTER - 4


This is a known and qualified impurity as per USP/ EP

A short summary of the observation is given in the table 4.4.1.

Table: 4.4.1 Samples injected under different stress conditions

Condition % Degradation % Degradation

Telmisartan Hydrochlorothiazide

Acidic 6.65% 20.90%

Basic Not Significant 29.86%

Oxidation 6.18% 20.11%

Thermal Not Significant 22.25%

Photolytic Not Significant 13.18% (with

respect to control)

CHAPTER - 4


4.5 EXPERIMENTAL WORK 4.5.1 Instrumentation

Equipment Make Model

HPLC Waters 2695Alliance Separation

Module, (PDA/UV

Detector) 2996/2487

Column AKZO

NOBEL

Kromasil C-18,

250 x 4.6 mm, 5µm

pH meter Thermo

Electron

Corp.

Orion-4star 1117000

Analytical

Balance

Mettler

Toledo

XS205DU

Micro Balance Mettler

Toledo

UMX-2

Ultrasonnicator Spectralab -

Photostability

Chamber

Thermolab 400litr

Water Bath Spectralab

4.5.2 Chemicals and Reagents

Name Grade Manufacturer

Potassium dihydrogen phosphate GR Merck Sodium 1- Hexane sulphonic acid monohydrate HPLC Alfa Aesar Methanol HPLC, Gradient grade Merck Acetonitrile HPLC, Gradient grade Rankem Monobasic sodium phosphate GR Merck

Ortho-Phosphoric acid GR Merck Sodium Hydroxide GR Merck Hydrochloric acid GR Merck 50% Hydrogen peroxide GR Merck Water HPLC milli-Q In-house

CHAPTER - 4


4.5.3 Working Standard

Standard Lot .No. Potency (as is) %

Telmisartan TE0010108 98.7

Hydrochlorothiazide HCT/60914 99.5

HCTZ-Impurity-B CRD/HCTZ/01/30/01 99.5

4.5.4 Solution Preparation Preparation of Diluent

Diluent – A: 0.1 N alcoholic NaOH

Weigh accurately and transfer 4 gm of Sodium hydroxide in 1000 ml volumetric flask

add 40 ml water sonnicate to dissolve and make up to mark with methanol.

Diluent – B: 0.005 N alcoholic NaOH

Dilute 50 ml of Diluent A to 1000 ml volumetric flask and make up to mark with

Methanol.

Diluent – C

Prepare a mixture of Buffer: Acetonitrile in the ratio 70:30 v/v.

Diluent – D

Prepare a mixture of Methanol: Water in the ratio 50:50 v/v.

Diluent – E

Prepare a mixture of Acetonitrile: Water in the ratio 50:50 v/v.

Preparation of Mobile Phase

Mobile Phase A: Buffer (pH – 3.0)

Weigh accurately 2.0 gm of Potassium dihydrogen phosphate anhydrous and 1.04 gm

of Sodium 1- Hexane sulphonic acid monohydrate, transfer into 1000ml of water.

Adjust the pH 3.0 with ortho phosphoric acid.

Mobile Phase B

Prepare a mixture of Acetonitrile: Methanol in the ratio 80:20 v/v. Mix and degas.

CHAPTER - 4


Preparation of Reference Solution – Hydrochlorothiazide

Weigh accurately and transfer about 1.0 mg of Hydrochlorothiazide Impurity B in 10

ml volumetric flask and dilute to volume with Diluent E.

Preparation of Impurity Mixture Solution

Take 150 µl of Impurity B and 1850 µl of Hydrochlorothiazide API.

Preparation of Standard solution

Preparation of Standard Stock Solution - Telmisartan

Weigh accurately about 40.0 mg of Telmisartan working standard and transfer into a

200ml volumetric flask. Add about 70 ml of Diluent - A, sonnicate to dissolve and

make up the volume up to the mark with Diluent - A. Further dilute this solution 5 ml

to 50 ml with Diluent – D.

Preparation of Standard Stock Solution - Hydrochlorothiazide

Weigh accurately about 60.0 mg of Hydrochlorothiazide working standard and

transfer into a 200 ml volumetric flask. Add about 70 ml of in Diluent -E, sonnicate to

dissolve and make up the volume up to the mark with Diluent E. Further dilute this

solution 5 ml to 50 ml with Diluent E.

Preparation of Standard Solution

Take 15 ml Standard stock solution of Telmisartan and 5 ml Standard stock solution

of Hydrochlorothiazide in 100 ml volumetric flask, make the volume with Diluent -D.

Preparation of Sample solution

For 80 – 12.5 mg

Weigh accurately 10 tablets and transfer in 100 ml volumetric flask. Add 10 ml of

Diluent-B, sonnicate for 5 to 10 minutes with intermittent shaking till the tablets

disintegrate. Cool the solution at room temperature, add 10 ml of Diluent C and make

up the volume with Diluent-D. Further dilute 3 ml of this solution to 25 ml with

Diluent -D. Filter through 0.45 µ Nylon filter and inject.

CHAPTER - 4


For 40 – 12.5 mg

Weigh accurately 10 tablets and transfer in 50 ml volumetric flask. Add 5 ml of

Diluent - B, sonnicate for 5 to 10 minutes with intermittent shaking till the tablets

disintegrate. Cool the solution at room temperature, add 5 ml of Diluent C and make

up the volume with Diluent-D. Further dilute 3 ml of this solution to 25 ml with

Diluent -D. Filter through 0.45 µ Nylon filter and inject.

Preparation of Placebo solution

For Telmisartan

Weigh Placebo sample equivalent to 10 tablets and transfer in 100 ml volumetric

flask. Add 10 ml of Diluent - B, sonnicate for 5 to 10 minutes with intermittent

shaking till the placebo disperses. Cool the solution at room temperature, add 10

ml of Diluent C and make up the volume with Diluent-D. Further dilute 3 ml of this

solution to 25 ml with Diluent -D. Filter through 0.45 µ Nylon filter and inject.

For Hydrochlorothiazide

Weigh Placebo sample equivalent to 10 tablets and transfer in 100ml volumetric flask.

Add 10 ml of Diluent - B, sonnicate for 5 to 10 minutes with intermittent shaking

till the placebo disperses. Cool the solution at room temperature, add 10 ml of Diluent

C and make up the volume with Diluent-D. Further dilute 3 ml of this solution to 25

ml with Diluent -D. Filter through 0.45 µ Nylon filter and inject.

For Placebo without Telmisartan + Hydrochlorothiazide

Weigh Placebo sample equivalent to 10 tablets and transfer in 100ml volumetric flask.

Add 10 ml of Diluent - B, sonnicate for 5 to 10 minutes with intermittent shaking till

the placebo disperses. Cool the solution at room temperature, add 10 ml of Diluent C

and make up the volume with Diluent-D. Further dilute 3 ml of this solution to 25 ml

with Diluent -D. Filter through 0.45 µ Nylon filter and inject.

Procedure

Separately inject equal volumes of Blank (Diluent), Placebo solution and sample

solution and measure the area counts for the impurity peaks. The retention time of

Telmisartan Peak is about 34.85 minutes. The retention time of Hydrochlorothiazide

Peak is about 17.93 minutes.

CHAPTER - 4


When the chromatograms are recorded under the prescribed conditions, the

approximate relative retention times, in the chromatogram of Impurity mixture

preparation are:

Name Relative retention

time

Relative response

factor

Impurity B About 0.6 0.74

Evaluation of System Suitability

Inject the Standard solution six times into the chromatograph and record the

chromatogram. The relative standard deviation for six replicate injections of standard

solution should not be more than 5.0 %.The system suitability has to be ensured at the

beginning of the exercise as well as at the end of the analysis. For a lengthy sample

sequence system suitability need to be ensured during the analysis as well.

Calculation

Calculate the % Impurities as follows:

For Telmisartan (80-12.5 mg)

AT Std. Wt. 5 15 100 25 P 100 1

% Impurity = ------ x ------------ x ------ x ------ x --------- x ------ x ------- x ------- x ------

AS 200 50 100 10 3 100 LC RRF

For Telmisartan (40-12.5 mg)

AT Std. Wt. 5 15 50 25 P 100 1

% Impurity = ------ x ------------ x ------- x ------ x --------- x ------ x ----- x ------- x ------

AS 200 50 100 10 3 100 LC RRF

Where,

AT : Peak area of Impurity in the chromatogram of sample solution.

AS : Average area count of Telmisartan peak in the chromatogram of

standard solution.

Std.wt : Weight of Telmisartan taken in mg

P : Percent potency of Telmisartan working standard on as such basis.

CHAPTER - 4


LC : Label claim of Telmisartan in mg

RRF : Relative response factor

Note: For impurities where RRF value is not mentioned, take it as 1.

For Hydrochlorothiazide (80-12.5 mg)

AT Std.Wt. 5 5 100 25 P 100 1

% Impurity = ------ x --------- x ------- x ------ x ------- x ------ x -----x ------- x -----

AS 200 50 100 10 3 100 LC RRF

For Hydrochlorothiazide (40-12.5 mg)

AT Std.Wt. 5 5 50 25 P 100 1

% Impurity = ------ x --------- x ------- x ------ x ------- x ------ x -----x ------- x -------

AS 200 50 100 10 3 100 LC RRF

Where ,

AT : Peak area of Impurity in the chromatogram of sample solution.

AS : Average area count of Hydrochlorothiazide peak in the

chromatogram of standard solution.

Std.wt : Weight of Hydrochlorothiazide taken in mg

P : Percent potency of Hydrochlorothiazide working standard on as

such basis.

LC : Label claim of Hydrochlorothiazide in mg

RRF : Relative response factor

Note: For impurities where RRF value is not mentioned, take it as 1.

CHAPTER - 4


4.6 VALIDATION OF THE DEVELOPED METHOD 4.6.1 Validation Parameters and Acceptance Criteria The forced degradation studies showed only one major degradant originating due to hydrochlorothiazide. This was 4-Amino-6-chloro-1,3-benzenedisulfonamide or Impurity B. None of the known impurities due to Telmisartan were generated during forced degradation. Thus during validation, only this impurity was identified as “known”, the rest were included under “unknown” or “unspecified” impurities whose limits are governed by ICH guidelines. In the specificity study the Telmisartan impurity mixture (obtained from EP) was injected to confirm separation of individual impurities.

Table 4.6.1: The validation summary

Sr.No. Parameters Acceptance

criteria

Result obtained

1.0 System suitability

% RSD for Standard

solution.

USP Tailing Factor

USP Plates

NMT 5.0 %

NMT 2.0

NLT 500000 for

Telmisartan and

10000 for HCTZ.

Telmisartan HCTZ

0.60

1.03

740969

2.63

0.95

17221

2.0

2.1

Specificity

Identification

Results should be

comparable with

respect to the

retention time and

relative retention

time.

Component RT RRT

Telmisartan 34.716 -

HCTZ 17.871 -

Impurities mixture

HCTZ-IMP-B 10.674 0.60*

HCTZ-IMP-A 14.909 0.83*

HCTZ 17.918 -

HCTZ-IMP-C 27.812 1.55*

TELMI-IMP-A 27.582 0.79°

TELMI-IMP-E 31.130 0.90°

TELMI-IMP-F 31.497 0.91°

TELMI-IMP-B 32.911 0.95°

TELMISARTAN 34.704 -

TELMI-IMP-C 36.157 1.04°

2.2

Interference

No interference

from blank and

placebo to main

component and

impurities

Complies

CHAPTER - 4


2.3

Peak purity

Purity angle

should be less

than purity

threshold.

Standard peak

should be pure.

Telmisartan HCTZ

Purity angle 0.472 2.449

Purity

Threshold 1.288 3.538

XX*= RRT with respect to Hydrochlorothiazide. XX°= RRT with respect to Telmisartan.

Table 4.6.1: The validation summary (continued)

Sr.No. Parameters Acceptance

criteria

Result obtained

2.4

Forced degradation

The peak due to

known impurities

should be pure as

shown on the

PDA.

Telmisartan

Condition % Total impurities

Control 0.04

Acid 6.65

Base 0.03

Peroxide 6.18

Heat 0.05

Photolytic 0.12

Hydrochlorothiazide

Control 0.43

Acid 20.90

Base 29.86

Peroxide 20.11

Heat 22.25

Photolytic 12.81

3.0 Limit of Detection

% RSD for LOD:

between 10% and

33%

Component % RSD Conc. (%)

HCTZ-IMP-B 25.37 0.007

HCTZ 15.68 0.020

Telmisartan 17.26 0.010

4.0 Limit of Quantitation % RSD for LOQ:

NMT 10%

HCTZ-IMP-B 7.36 0.027

HCTZ 5.43 0.060

Telmisartan 5.42 0.031

CHAPTER - 4



Sr.No. Parameters Acceptance criteria Result obtained

5.0 Linearity

Response should be

Linear Response is linear

Correlation coefficient

should not be less than

0.99.

HCTZ-IMP-B 0.9998

HCTZ 1.0000

Telmisartan 0.9940

% Limit of Y-

Intercept should be

within ± 10.0% of the

corresponding Y-co-

ordinate of the

working level.

HCTZ-IMP-B -1.11

HCTZ -1.53

Telmisartan 0.68

6.0 Accuracy

(Recovery)

At LOQ Level mean

recovery should be in

the range 75.0 % to

125.0 %.

% Mean Recovery

mg HCTZ

IMP-B HCTZ Telmisartan

80 97.8 100.6 100.4

40 87.7 106.4 106.4

Mean recovery should

be in the range of

80.0%- 120.0%.

80 102.8 98.1 100.3

40 96.1 97.8 102.0

CHAPTER - 4



Sr.

No

Parameters Acceptance criteria Result obtained

7.0

7.1

7.2

System Precision

System suitability

% RSD for Standard solution.

USP Tailing Factor

USP Plates

NMT 5.0 %

NMT 2.0

NLT 500000 for

Telmisartan and 10000

for HCTZ.

Telmisartan HCTZ

0.60

1.03

740969

2.63

0.95

17221

Method Precision

RSD for % Impurity content.

NMT 10.0%.

Component % RSD

Telmisartan

Strength 80/

12.5 40/ 12.5

Single max

imp 0.00 0.00

HCTZ

HCTZ-

IMP-B 4.43 2.00

Single max

imp 7.91 6.20

7.3 Intermediate Precision

(Ruggedness)

System suitability

% RSD for Standard solution.

USP Tailing Factor

USP Plates

RSD for % Impurity content.

RSD for pooled result

( Analyst-I and II )

NMT 5.0 %

NMT 2.0

NLT 500000 for

Telmisartan and 10000

for HCTZ.

NMT 10.0%.

NMT 10.0%.

Telmisartan HCTZ

0.97

0.94

765481

0.78

0.95

14693

Component % RSD

Telmisartan

Strength 80/

12.5 40/ 12.5

Single max 0.00 0.00

CHAPTER - 4


imp

Pooled result

Single max

imp 0.00 0.00

HCTZ

HCTZ-

IMP-B 2.03 0.52

Single max

imp 6.20 5.96

Pooled result

HCTZ-

IMP-B 4.49 2.93

Single max

imp 7.07 6.14


Sr.

No

Parameters Acceptance criteria Result obtained

8.0 Stability in analytical

solution

The % Difference of Initial and

after 24 hours result should be

0.05.

Telmisartan HCTZ

Sample and

Standard

solutions are

stable for at

least upto

24hours at

20°C

Standard solutions

are stable for at

least upto 24hours

at 20°C and

Sample Solutions

are stable upto

6hours at 20°C

9.0 Filter compatibility

% Difference for impurity content

of Centrifugate and filtered should

be within 0.05.

Complies Complies

10.

0

Robustness

Change in Flow rate

(± 0.1 ml/min)

Change in wavelength

There should be no significant

change in system suitability

parameters.


No significant change


CHAPTER - 4


(265 and 272 nm)

Change in Buffer pH

(± 0.2)

Column oven

temperature (± 5°C)


parameters.



parameters.



parameters.



4.6.2 System Suitability Single injection of Blank (Diluent) and six replicate injections of standard solution were

made on the system to demonstrate that the instrument is suitable for carrying out analytical

work. The data obtained is summarized in Table. 4.6.2.1

Table 4.6.2.1: System suitability

Standard Solution


USP Tailing 1.03 0.95

USP Plates 740969 17221

Area

69682 55157

68722 53993

69827 53093

69291 51298

69544 52004

69027 52601

Mean 69349 53024

SD 418.95 1392.25

%RSD 0.60 2.63

CHAPTER - 4


4.6.3 Specificity Peak Purity:

Blank (diluent), Placebo without Telmisartan and Hydrochlorothiazide, Placebo with

Telmisartan, Placebo with Hydrochlorothiazide, Standard solution, individual impurities,

mixture of all impurities with sample and sample solution were injected.

The data demonstrate that there is no interference in blank, placebo, Telmisartan,

Hydrochlorothiazide, Known and unknown impurities peaks. All peaks are well resolved.

The data obtained is summarized in Table 4.6.3.1, 4.6.3.2 and 4.6.3.3.

Table 4.6.3.1: Specificity (Identification and Interference)

Component Retention

time (min)

RRT Peak Purity

Telmisartan 34.716 - Pass

Hydrochlorothiazide 17.871 - Pass

Impurities mixture

HCTZ-IMP-B 10.674 0.60* Pass

HCTZ-IMP-A 14.909 0.83* Pass

Hydrochlorothiazide 17.918 -

HCTZ-IMP-C 27.812 1.55* Pass

TELMI-IMP-A 27.582 0.79° Pass

TELMI-IMP-E 31.130 0.90° Pass

TELMI-IMP-F 31.497 0.91° Pass

TELMI-IMP-B 32.911 0.95° Pass

Telmisartan 34.704 -

TELMI-IMP-C 36.157 1.04° Pass

XX*= RRT with respect to Hydrochlorothiazide. XX°= RRT with respect to Telmisartan.

CHAPTER - 4


Forced degradation data

Table 4.6.3.2: For Telmisartan

Condition % Impurity

Unk-1 Unk-2 Unk-3 Unk-4 Unk-5 Unk-6 Unk-7

RRT 0.79 0.82 0.83 0.85 0.86 0.87 0.88

Control

% Impurity - - - - - - -

Acid degradation

% Impurity 0.01 - - - - - -

Base degradation

% Impurity - - - - - - -

Peroxide degradation

% Impurity 0.82 0.45 1.96 0.56 0.38 0.33 0.20

Heat degradation

% Impurity 0.04 - - - - - -

Photolytic degradation

% Imp

Control

(Aluminium)

- - - - - - -

% Imp Amber

colour flask - - - - - - -

% Impurity in

Clear glass - - - - - - -

CHAPTER - 4


Table 4.6.3.2: For Telmisartan (continued)


Unk-8 Unk-9 Unk-10 Unk-11 Unk-12 Unk-13 Unk-14

RRT 0.90 0.91 0.92 0.93 0.94 0.95 0.97

Control

% Impurity - - 0.02 - - - -

Acid degradation

% Impurity - - - - - - -

Base degradation

% Impurity - - - - - - -


% Impurity 0.14 0.07 0.19 0.38 0.50 0.01 0.47

Heat degradation

% Impurity - - - - - - -


% Imp

Control

(Aluminium)

- - 0.01 - 0.02 - -

% Imp Amber

colour flask - - 0.01 - 0.02 - -

% Impurity in

Clear glass - - 0.06 - 0.03 - -

CHAPTER - 4


Table 4.6.3.2: For Telmisartan (continued)


Unk-15 Unk-16 Unk-17 Unk-18 Peak Purity

Standard

Solution

% Total

Impurity RRT 0.98 0.99 1.01 1.05

Control

% Impurity - - - 0.02 Peak Pure 0.04

Acid degradation


Base degradation



% Impurity 0.06 0.04 0.54 0.05 Peak Pure 6.18

Heat degradation



% Imp

Control

(Aluminium)

- - - 0.02 Peak Pure 0.05

% Imp Amber

colour flask - - - 0.03 Peak Pure 0.06

% Impurity in

Clear glass - - - 0.03 Peak Pure 0.12

CHAPTER - 4


Table 4.6.3.3: For Hydrochlorothiazide


Imp-B Unk

1

Unk

2

Unk

3

Unk

4

Unk

5

Unk

6

Peak

Purity

%

Total

Impuri

ty

RRT 0.59 0.65 0.69 0.75 0.80 0.90 1.53

Control

%

Impurity 0.35 - - - - - 0.08

Peak

Pure 0.43

Acid degradation

%

Impurity 20.44 - - - 0.10 0.24 0.12

Peak

Pure 20.90

Base degradation

%

Impurity 24.42 0.49 0.39 0.42 3.75 0.39 -

Peak

Pure 29.86


%

Impurity 17.63 0.17 1.83 - 0.21 0.13 0.14

Peak

Pure 20.11

Heat degradation

%

Impurity 20.71 - - - 0.24 1.30 -

Peak

Pure 22.25


% Imp

Control

(Alumini

um foil

wrapped)

11.77 - - - 0.21 1.03 0.03 Peak

Pure 13.04

% Imp

Clear

glass

flask

14.53 - - - 0.22 11.4 0.04 Peak

Pure 26.22

%

Impurity

in Amber

flask

11.71 - - - 0.09 0.98 0.03 Peak

Pure 12.81

CHAPTER - 4


4.6.4 Determination of Limit of Detection (LOD) and Limit of Quantitation (LOQ) For determining LOD and LOQ, A series of dilutions with decreasing concentrations were

injected into the system and the areas were determined. Graph of concentration vs. area

were plotted and SLOPE of the line was calculated. Also the STEYX of this line

(Correction for the residual error of the peak areas and concentration) was determined.

The data obtained are summarized in Table 4.6.4.1.

Table 4.6.4.1: Prediction Linearity

Sr.No. HCTZ-IMP-B HCTZ Telmisartan

Conc.

(ppm)

Area Conc.

(ppm)

Area Conc.

(ppm)

Area

1 0.52 12473 0.50 17219 0.51 13108

2 0.41 9774 0.40 13126 0.41 10566

3 0.31 7490 0.30 10156 0.30 8940

4 0.21 4936 0.20 6080 0.20 7401

5 0.10 2504 0.10 2801 0.10 6315

CORREL 0.9998

0.9989

0.9886

STEYX 91.129 302.212 466.990

SLOPE 23819.686 35882.000 16269.506

PREDICTED LOD

(ppm) 0.01 0.03 0.09

PREDICTED LOQ

(ppm) 0.04 0.08 0.29

PREDICTED LOD

(%) 0.008 0.019 0.010

PREDICTED LOQ

(%) 0.026 0.056 0.030

Six replicates of above predicted LOD and LOQ solution were done on system and

calculated % RSD are summarized in Table 4.6.4.2 and 4.6.4.3. A percentage RSD of

below 10 is required for LOQ whereas a percentage RSD value from 10 to 30 is required

for LOD. Both the criteria had been fulfilled.

CHAPTER - 4


Table 4.6.4.2: Precision for LOD.


Concentration (ppm) 0.010 0.030 0.100

Conc. w.r.t sample (%) 0.007 0.020 0.010

227 962 4246

535 1165 4904

457 969 4852

511 816 3949

420 1064 6218

433 763 5715

Mean 431 957 4981

SD 109.21 149.93 859.85

% RSD 25.37 15.68 17.26

Table 4.6.4.3: Precision for LOQ.


Concentration (ppm) 0.041 0.090 0.300

Conc. w.r.t sample (%) 0.027 0.060 0.031

1338 2993 8483

1438 3063 9002

1231 2889 9611

1303 2954 9372

1185 2943 9076

1213 3346 9904

Mean 1285 3031 9241

SD 94.49 164.64 500.51

% RSD 7.36 5.43 5.42

Impurities LOD LOQ

Hydrochlorothiazide and its Related Impurities

Impurity B 0.007% (0.010ppm) 0.027% (0.041ppm)

HCTZ (Unknown) 0.020% (0.030ppm) 0.060% (0.090ppm)

Telmisartan and its Related Impurities

Telmisartan (Unknown) 0.010% (0.100ppm) 0.031% (0.300ppm)

CHAPTER - 4


4.6.5 Linearity and Range The Linearity of response was determined by preparing different concentrations of standard

stock solution ranging from LOQ to 200% of the limit concentration. The data summarized

in Table 4.6.5.1, 4.6.5.2 and 4.6.5.3. The data shows that the response is found to be linear;

Correlation coefficient is more than 0.99.

Table 4.6.5.1: Linearity of HCTZ-IMPURITY-B

Level (%) Concentration w.r.t

sample (%) Area

LOQ 0.03 1285

50.7 0.51 19731

68.0 0.68 26080

102.0 1.02 39030

135.3 1.35 51620

169.3 1.69 63000

203.3 2.03 77273

406.7 4.07 157727

CORRELATION COEFFICIENT (r) 0.9998

Y-INTERCEPT -502.40

SLOPE 38643.69

MEDIAN (AREA) 45325

% LIMIT OF Y-INTERCEPT ± 10% OF MEDIAN -1.11

Figure 4.6.5.1 Linearity of HCTZ Impurity B

CHAPTER - 4


Table 4.6.5.2: Linearity of HCTZ


sample (%) Area

LOQ 0.06 3031

50.7 0.51 25697

67.3 0.67 35110

100.7 1.01 52425

134.7 1.35 69854

201.3 2.01 102554

336.0 3.36 175453

537.3 5.37 286142

1008 10.08 530695


Y-INTERCEPT -1066.00

SLOPE 52848.28

MEDIAN (AREA) 69854

% LIMIT OF Y-INTERCEPT ± 10% OF MEDIAN -1.53

Figure 4.6.5.2 Linearity of HCTZ

CHAPTER - 4


Table 4.6.5.3: Linearity of Telmisartan


sample (%) Area

LOQ 0.031 9241

51.0 0.05 13443

76.0 0.08 18009

102.0 0.11 22762

152.0 0.16 31932

203.0 0.21 41345

305.0 0.32 60045

508.0 0.53 116242


Y-INTERCEPT 184.82

SLOPE 208947.31

MEDIAN (AREA) 27347

% LIMIT OF Y-INTERCEPT ± 10% OF MEDIAN 0.68

Figure 4.6.5.3 Linearity of Telmisartan

CHAPTER - 4


4.6.6 Accuracy

Thirteen samples of dosage in form of spiked placebo with HCTZ-IMP-B,

Hydrochlorothiazide and Telmisartan at four different levels for two strengths, each level

in triplicate were prepared. Single placebo preparation (unspiked), 3x LOQ, 3x 50%, 3x

100% and 3x 200% spiked placebo of the limit concentration were prepared. From the

amount added and the amount found, the percentage recovery was calculated along with

mean recovery. The results obtained were summarized in Table 4.6.6.1 (for system

suitability of 80-12.5 strength), 4.6.6.2, 4.6.6.3, 4.6.6.4, 4.6.6.5, 4.6.6.6, 4.6.6.7 and

4.6.6.8.

For 80/12.5 mg:


Standard Solution



USP Plates 753230 14182

Area

54128 55008

53361 54571

54033 54809

53510 54702

54061 55456

53198 54297

Mean 53715 54807

SD 406.46 397.10

%RSD 0.76 0.72

CHAPTER - 4


Table 4.6.6.2: Accuracy for HCTZ-IMP-B

Level

%

Amount

added

%

Response

Amount

recovered

%

%

Recovery

Mean recovery

%

LOQ

0.031 1263 0.031 99.4

97.8 0.031 1206 0.029 94.9

0.031 1259 0.031 99.0

50 %

0.582 23787 0.580 99.7

102.8

0.582 23975 0.585 100.5

0.582 24280 0.592 101.7

100 %

1.163 48683 1.187 102.1

1.163 49539 1.208 103.9

1.163 49358 1.204 103.5

200 %

2.326 99818 2.434 104.7

2.326 99757 2.433 104.6

2.326 99575 2.428 104.4

Table 4.6.6.3: Accuracy for Hydrochlorothiazide

Level

%

Amount

added

%

Response Amount

recovered

%

%

Recovery

Mean recovery

%

LOQ

0.050 2825 0.051 102.0

100.6 0.050 2787 0.050 100.6

0.050 2747 0.050 99.1

50 %

0.495 26238 0.473 95.7

98.1

0.495 26490 0.478 96.6

0.495 26591 0.480 96.9

100 %

0.991 53521 0.966 97.5

0.991 54009 0.975 98.3

0.991 54030 0.975 98.4

200 %

1.981 108561 1.959 98.9

1.981 110176 1.988 100.4

1.981 110142 1.988 100.3

CHAPTER - 4


Table 4.6.6.4: Accuracy for Telmisartan

Level

%

Amount

added

%

Response

Amount

recovered

%

%

Recovery

Mean recovery

%

LOQ

0.031 5488 0.032 103.0

100.4 0.031 5329 0.031 100.0

0.031 5227 0.030 98.1

50 %

0.051 9348 0.054 105.3

100.3

0.051 9328 0.054 105.1

0.051 9375 0.054 105.6

100 %

0.103 17400 0.101 98.0

0.103 17647 0.102 99.4

0.103 17569 0.101 98.9

200 %

0.205 34162 0.197 96.2

0.205 34607 0.200 97.4

0.205 34469 0.199 97.1

CHAPTER - 4


For 40/12.5 mg:


Standard Solution



USP Plates 778385 12225

Area

52731 55504

52786 54547

53642 55366

53324 55582

53703 56439

54607 55032

Mean 53466 55412

SD 694.52 630.37

%RSD 1.30 1.14

Table 4.6.6.6: Accuracy for HCTZ-IMP-B

Level

%

Amount

added

%

Response

Amount

recovered

%

%

Recovery

Mean recovery

%

LOQ

0.015 2179 0.013 87.6

87.7 0.015 2188 0.013 88.0

0.015 2179 0.013 87.6

50 %

0.582 45178 0.545 93.6

96.1

0.582 42373 0.511 87.8

0.582 50621 0.610 104.9

100 %

1.163 92948 1.121 96.4

1.163 91496 1.103 94.9

1.163 90611 1.093 94.0

200 %

2.326 188991 2.279 98.0

2.326 187841 2.265 97.4

2.326 189530 2.286 98.3

CHAPTER - 4


Table 4.6.6.7: Accuracy for Hydrochlorothiazide

Level

%

Amount

added

%

Response

Amount

recovered

%

%

Recovery

Mean recovery

%

LOQ

0.025 5996 0.027 107.0

106.4 0.025 5859 0.026 104.6

0.025 6036 0.027 107.7

50 %

0.495 52358 0.467 94.4

97.8

0.495 50013 0.446 90.2

0.495 52975 0.473 95.5

100 %

0.991 109678 0.979 98.8

0.991 107620 0.960 96.9

0.991 106648 0.952 96.0

200 %

1.981 229187 2.045 103.2

1.981 228791 2.042 103.1

1.981 226977 2.026 102.3

CHAPTER - 4


Table 4.6.6.8: Accuracy for Telmisartan

Level

%

Amount

added

%

Response

Amount

recovered

%

%

Recovery

Mean recovery

%

LOQ

0.026 4734 0.027 107.1

106.4 0.026 4770 0.028 108.0

0.026 4603 0.027 104.2

50 %

0.051 9523 0.055 107.8

102.0

0.051 9110 0.053 103.1

0.051 9599 0.056 108.6

100 %

0.103 17768 0.103 100.5

0.103 17764 0.103 100.5

0.103 17521 0.102 99.1

200 %

0.205 35262 0.205 99.8

0.205 35181 0.204 99.5

0.205 34942 0.203 98.8

CHAPTER - 4


4.6.7 Precision 4.6.7.1 System Precision:

Single injection of Blank (Diluent) and six replicate injections of standard solution were

made on the system. The data obtained is summarized in Table 4.6.7.1.1. The data

demonstrated that the system was suitable.

Table 4.6.7.1.1: System precision

Standard Solution



USP Plates 740969 17221

Area

69682 55157

68722 53993

69827 53093

69291 51298

69544 52004

69027 52601

Mean 69349 53024

SD 418.95 1392.25

%RSD 0.60 2.63

CHAPTER - 4


4.6.7.2 Method Precision:

Six sample solution, were prepared and injected on the HPLC. The data obtained is

summarized in Table 4.6.7.2.1, 4.6.7.2.2, 4.6.7.2.3 and 4.6.7.2.4. All data are well within

acceptance criteria.

For 80/12.5 mg Tablets:

Table 4.6.7.2.1: Method precision (Telmisartan)

Spl. No. Unknown-1 Unknown-2 Unknown-3 % Total

impurities RRT-0.92 RRT-1.05 RRT-1.07

1 0.01 0.02 0.01 0.04

2 0.01 0.02 0.01 0.04

3 0.01 0.02 0.01 0.04

4 0.01 0.02 0.01 0.04

5 0.01 0.02 0.01 0.04

6 0.01 0.02 0.01 0.04

Mean 0.01 0.02 0.01 0.04

SD 0.000 0.000 0.000 0.000

% RSD 0.00 0.00 0.00 0.00

Table 4.6.7.2.2: Method precision (Hydrochlorothiazide)

Spl. No.

HCTZ-IMP-B Unknown-1 % Total

impurities

% Total Impurities

(Telmisartan +

HCTZ) RRT-0.60 RRT-1.51

1 0.34 0.08 0.42 0.46

2 0.35 0.08 0.43 0.47

3 0.34 0.08 0.42 0.46

4 0.34 0.09 0.43 0.47

5 0.38 0.08 0.46 0.50

6 0.35 0.07 0.42 0.46

Mean 0.35 0.08 0.43 0.47

SD 0.015 0.006 0.015 0.015

% RSD 4.43 7.91 3.60 3.30

CHAPTER - 4



Table 4.6.7.2.3: Method precision (Telmisartan)



1 0.01 0.02 0.01 0.04

2 0.01 0.02 0.01 0.04

3 0.01 0.02 0.01 0.04

4 0.01 0.02 0.01 0.04

5 0.01 0.02 0.01 0.04

6 0.01 0.02 0.01 0.04

Mean 0.01 0.02 0.01 0.04

SD 0.000 0.000 0.000 0.000

% RSD 0.00 0.00 0.00 0.00

Table 4.9.6.7.4: Method precision (Hydrochlorothiazide)

Spl. No. HCTZ-IMP-B Single max imp % Total

impurities

% Total Impurities

(Telmisartan +


1 0.75 0.08 0.95 0.99

2 0.74 0.09 0.95 0.99

3 0.74 0.09 0.95 0.99

4 0.78 0.08 0.98 1.02

5 0.76 0.08 0.96 1.00

6 0.75 0.08 0.95 0.99

Mean 0.75 0.08 0.96 1.00

SD 0.015 0.005 0.012 0.012

% RSD 2.00 6.20 1.27 1.22

CHAPTER - 4


4.6.7.3 Intermediate Precision (Ruggedness):

Same procedure of system precision and method precision was followed by another Analyst

on different instrument and on different day. The data obtained from Analyst-II are

summarized in Table 4.6.7.3.1, 4.6.7.3.2, 4.6.7.3.3, 4.6.7.3.4 and 4.6.7.3.5. Table 4.6.7.3.1

demonstrates the system was suitable for the experiment.

Table 4.6.7.3.1: System suitability

Standard Solution



USP Plates 765481 14693

Area

59038 53876

59296 53502

60409 54612

59058 53512

59999 54133

59111 54051

Mean 59485 53948

SD 578.91 419.51

%RSD 0.97 0.78

CHAPTER - 4



Table 4.6.7.3.2: Intermediate precision (Telmisartan)



1 0.01 0.02 0.01 0.04

2 0.01 0.02 0.01 0.04

3 0.01 0.02 0.01 0.04

4 0.01 0.02 0.01 0.04

5 0.01 0.02 0.01 0.04

6 0.01 0.02 0.01 0.04

Mean 0.01 0.02 0.01 0.04

SD 0.000 0.000 0.000 0.000

% RSD 0.00 0.00 0.00 0.00

Table 4.6.7.3.3: Intermediate precision (Hydrochlorothiazide)

Spl. No. HCTZ-IMP-B Single max % Total

impurities

% Total

Impurities

(Telmisartan +


1 0.37 0.08 0.45 0.49

2 0.36 0.08 0.44 0.48

3 0.38 0.09 0.47 0.51

4 0.37 0.08 0.45 0.49

5 0.37 0.09 0.46 0.50

6 0.38 0.08 0.46 0.50

Mean 0.37 0.08 0.46 0.50

SD 0.008 0.005 0.010 0.010

% RSD 2.03 6.20 2.31 2.12

CHAPTER - 4



Table 4.6.7.3.4: Intermediate precision (Telmisartan)



1 0.01 0.02 0.01 0.04

2 0.01 0.02 0.01 0.04

3 0.01 0.02 0.01 0.04

4 0.01 0.02 0.01 0.04

5 0.01 0.02 0.01 0.04

6 0.01 0.02 0.01 0.04

Mean 0.01 0.02 0.01 0.04

SD 0.000 0.000 0.000 0.000

% RSD 0.00 0.00 0.00 0.00

Table 4.6.7.3.5: Intermediate precision (Hydrochlorothiazide)

Spl. No. HCTZ-IMP-B Single max % Total

impurities

% Total Impurities

(Telmisartan +


1 0.79 0.09 1.01 1.05

2 0.79 0.09 1.01 1.05

3 0.79 0.08 1.00 1.04

4 0.79 0.08 1.00 1.04

5 0.79 0.09 1.02 1.06

6 0.80 0.09 1.02 1.06

Mean 0.79 0.09 1.01 1.05

SD 0.004 0.005 0.009 0.009

% RSD 0.52 5.96 0.89 0.85

CHAPTER - 4


The pooled data obtained from Analyst-I and Analyst-II is summarized in Table 4.6.7.3.6 and

4.6.7.3.7. All data are within acceptance limits.

Table 4.6.7.3.6: Pooled data (80/12.5 mg Tablets)

Analyst

% Impurity of Telmisartan% Impurity of

HCTZ % Total

impuritiesUNK-1 UNK-

2 UNK-

3 HCTZ-IMP-B

Single max

RRT-0.92

RRT-1.05

RRT-1.07

RRT-0.60

RRT-1.51

I

0.01 0.02 0.01 0.34 0.08 0.46

0.01 0.02 0.01 0.35 0.08 0.47

0.01 0.02 0.01 0.34 0.08 0.46

0.01 0.02 0.01 0.34 0.09 0.47

0.01 0.02 0.01 0.38 0.08 0.50

0.01 0.02 0.01 0.35 0.07 0.46

II

0.01 0.02 0.01 0.37 0.08 0.49

0.01 0.02 0.01 0.36 0.08 0.48

0.01 0.02 0.01 0.38 0.09 0.51

0.01 0.02 0.01 0.37 0.08 0.49

0.01 0.02 0.01 0.37 0.09 0.50

0.01 0.02 0.01 0.38 0.08 0.50

Mean 0.01 0.02 0.01 0.36 0.08 0.48

SD 0.000 0.000 0.000 0.016 0.006 0.018

% RSD 0.00 0.00 0.00 4.49 7.07 3.76

CHAPTER - 4


Table 4.6.7.3.7: Pooled data (40/12.5 mg Tablets)

Analyst

% Impurity of Telmisartan% Impurity of

HCTZ % Total

impurities UNK-

1 UNK-

2 UNK-

3 HCTZ-IMP-B

Single max

RRT-0.92

RRT-1.05

RRT-1.07

RRT-0.60

RRT-0.85

I

0.01 0.02 0.01 0.75 0.08 0.99

0.01 0.02 0.01 0.74 0.09 0.99

0.01 0.02 0.01 0.74 0.09 0.99

0.01 0.02 0.01 0.78 0.08 1.02

0.01 0.02 0.01 0.76 0.08 1.00

0.01 0.02 0.01 0.75 0.08 0.99

II

0.01 0.02 0.01 0.79 0.09 1.05

0.01 0.02 0.01 0.79 0.09 1.05

0.01 0.02 0.01 0.79 0.08 1.04

0.01 0.02 0.01 0.79 0.08 1.04

0.01 0.02 0.01 0.79 0.09 1.06

0.01 0.02 0.01 0.80 0.09 1.06

Mean 0.01 0.02 0.01 0.77 0.09 1.02

SD 0.000 0.000 0.000 0.023 0.005 0.030

% RSD 0.00 0.00 0.00 2.93 6.14 2.90

CHAPTER - 4


4.6.8 Stability in Analytical solution:

The sample solution was kept at sample temperature for 24 hours were injected on to the

HPLC. The data obtained are summarized in Table 4.10.1 and 4.10.2. Sample solution was

stable for 24 hours with respect to Telmisartan while it was stable for 6 hours with respect

to Hydrochlorothiazide.

Table 4.6.8.1 Stability in analytical solution (Telmisartan)

Condition Standard

Area

Cumulative

RSD of

Standard

% Impurity

Single max imp

RRT-1.05

%

Cumulative

Difference

INITIAL 69682 - 0.02 -

6Hrs 69027 0.67 0.02 0.00

10Hrs 71511 1.83 0.02 0.00

15Hrs 72166 2.48 0.02 0.00

20Hrs 72891 3.18 0.02 0.00

24Hrs 70526 0.85 0.01 0.01

Table 4.6.8.2 Stability in analytical solution (Hydrochlorothiazide)

Condition Standard

Area

Cumulative

RSD of

Standard

% Impurity

% HCTZ-IMP-B

%

Cumulative

Difference

Single max imp

RRT-1.51

%

Cumulative

Difference

INITIAL 55157 - 0.34 - 0.08 -

6Hrs 52601 3.35 0.35 0.01 0.07 0.01

10Hrs 53964 1.55 0.41 0.07 0.07 0.01

15Hrs 53814 1.74 0.54 0.20 0.07 0.01

20Hrs 52749 3.16 0.63 0.29 0.05 0.03

24Hrs 55102 0.07 0.80 0.46 0.05 0.03

CHAPTER - 4


4.6.9 Filter Compatibility

Injection of spiked sample solution filtered through different types of filters

(Centrifuged, Glass, Nylon, PVDF and Teflon) were done on HPLC. Calculate % difference

against centrifuged sample solution. All the tested filters were found to be compatible with

the sample.

4.6.10 Robustness Small, deliberate changes in the chromatographic conditions were made and the effect of those

changes on the system suitability parameters was monitored by injecting system suitability

solutions. The data obtained are summarized in Tables 4.12.1 and 4.12.2. All results are within

acceptable limits for Telmisartan. For Hydrochlorothiazide the wavelength variation was

limited to ±2nm since at ±5nm the response of impurity B was changing.

Table 4.6.10.1 Robustness (Telmisartan)

System suitability solution

Changes in

parameters Values

Retention

Time of

Telmisartan

Tailing

Factor

USP

Plates

%

RSD

STD

area

Single max Impurity

RRT-1.05

% Cumulative

Difference

Control As per

method 34.716 1.03 740969 0.60 0.02 -

Flow

(ml/min)

0.9 35.510 0.99 707554 0.73 0.01 0.01

1.1 34.157 1.03 739536 1.26 0.02 0.00

Wavelength

(nm)

265 34.716 1.02 744020 0.42 0.02 0.00

272 34.716 1.03 723823 0.91 0.02 0.00

Temperature

°C

35 34.768 1.05 704933 0.50 0.01 0.02

45 34.763 1.01 790608 0.34 0.02 0.00

Buffer pH 2.8 34.043 0.99 792305 4.98 0.02 0.00

3.2 35.321 1.07 762886 2.39 0.01 0.01

CHAPTER - 4


Table 4.6.10.2 Robustness (Hydrochlorothiazide)

System suitability solution

Changes in

parameters Values

RT of

HCTZ

Tailing

Factor

USP

Plates

%

RSD

STD

area

HCTZ-IMP-B

RRT-0.60

Single max

Impurity

RRT-1.51

%

Cu

mu

lati

ve

Dif

fere

nce

%

Cu

mu

lati

ve

Dif

fere

nce

Control As per

method 17.871 0.95 17221 2.63 0.35 - 0.08 -

Flow

(ml/min)

0.9 19.484 0.97 23726 1.61 0.35 0.00 0.04 0.04

1.1 16.449 1.03 17994 2.04 0.40 0.05 0.05 0.03

Wavelength

(nm)

265 17.874 1.05 23045 2.75 0.39 0.04 0.06 0.02

272 17.878 1.04 22727 2.41 0.31 0.04 0.09 0.01

Temp.

°C

35 20.484 0.95 82012 0.88 0.34 0.01 0.06 0.02

45 15.126 1.02 16436 1.91 0.39 0.04 0.08 0.00

Buffer pH 2.8 17.747 0.96 16297 2.06 0.35 0.00 0.08 0.00

3.2 18.265 1.06 18625 2.80 0.38 0.03 0.07 0.01

4.6.11 CONCLUSIONS:

The method has been shown to be specific for Telmisartan + Hydrochlorothiazide

Tablets.

The method has been shown to be Linear, precise and accurate across the suitable analytical range.

Solution has been shown to be stable for at least upto 24 hours at 20°c for Telmisartan

and stable for at least upto 6 hours at 20°C for Hydrochlorothiazide. Thus sample has to be injected within 6 hours of preparation.

The method has been shown to be robust towards deliberate minor changes in the

method parameters.

Limit of detection and Limit of quantification concentration have been set.

The method can be used in quality control laboratory for release of production batches.

CHAPTER – 4 SIMULTANEOUS DETERMINATION...

Documents

Transcript of CHAPTER – 4 SIMULTANEOUS DETERMINATION...