chapter vi development and validation of an uplc method...

Chapter VI

Development and validation of an UPLC method for

fast, sensitive and simultaneous determination of

sartans in active pharmaceutical ingredients and its

residues on stainless steel surface in formulation area

Chapter VI

194

Introduction

Angiotensin II receptor blockers (ARBs) represent a class of effective and well

tolerated orally active antihypertensive drugs[1,2]. AT1-receptor antagonists or sartans, are

a group of pharmaceuticals which modulate the renin-angiotensin-aldosterone system.

Their main uses are in the treatment of hypertension (high blood pressure), diabetic

nephropathy (kidney damage due to diabetes) and congestive heart failure. Angiotensin II

receptor blockers are primarily used for the treatment of hypertension where the patient is

intolerant of ACE inhibitor therapy. They do not inhibit the breakdown of bradykinin or

other kinins, and are thus only rarely associated with the persistent dry cough

and/or angioedema that limit ACE inhibitor therapy. More recently, they have been used

for the treatment of heart failure in patients intolerant of ACE inhibitor therapy,

particularly Candesartan. Irbesartan and Losartan have trial data showing benefit in

hypertensive patients with type II diabetes, and may delay the progression of diabetic

nephropathy. Candesartan is used experimentally in preventive treatment of

migraine[3][4]. Lisinopril has been found less often effective than Candesartan at

preventing migraine.[5] Activation of AT1 receptors leads to vasoconstriction, stimulation

of the release of catecholamines and antidiuretic hormone and promote growth of

vascular and cardiac muscle[2]. AT1 receptor blockers antagonize all those effects.

Losartan was the first drug of this class marketed, shortly followed by Valsartan,

Irbesartan, Telmisartan, Candesartan and others[2]. Most of the ARBs are tetrazole

derivatives that exclude telmisartan which contains two imidazole rings (Fig: 6.1). ARBS

are generally comes under class II drug of BCS classification (low solubility and high

permeability). But Valsartan belongs to the BCS class III drug classified as low

permeability and high solubility drug. Due to its poor solubility, most of the sartans are

difficult to remove from production equipment.

In the pharmaceutical industry, an important step in the manufacture of

pharmaceutical products is the cleaning of equipment and surface[3]. Inadequate cleaning

of a pharmaceutical manufacturing plant or inadequate purging of the individual pieces of

equipment used in multi-product manufacturing or equipment not dedicated to individual

Chapter VI

195

products may lead to contamination of the next batch of pharmaceutics manufactured

using the same equipment. Challenges for cleaning validation are encountered especially

when developing sensitive analytical methods capable of detecting traces of active

pharmaceutical ingredients that are likely to remain on the surface of the pharmaceutical

equipment after cleaning. A method's inability to detect some residuals could mean that

either the method is not sensitive enough to the residue in question or the sampling

procedure is inadequate[4]. To validate the cleaning procedure there must be sensitive and

reproducible analytical method. The aim of this study was to demonstrate the

applicability of UPLC by developing a simple, accurate, precise method to determine the

residues of four sartans in support of cleaning validation.

Valsartan

Chemical Formula: C24H29N5O3

CAS number : 137862-53-4

Molecular weight : 435.519

IUPAC Name : (S)-3-methyl-2-(N-{[2'-(2H-1,2,3,4-tetrazol-5-yl)biphenyl-4-

yl]methyl}pentanamido)butanoic acid

Chapter VI

196

Candesartan

Chemical Formula : C24H20N6O3

CAS number : 139481-59-7


IUPAC Name : 2-ethoxy-1-({4-[2-(2H-1,2,3,4-tetrazol-5-

yl)phenyl]phenyl}methyl)-1H- 1,3-benzodiazole-6-carboxylic

acid

Irbesartan

Chemical Formula: C25H28N6O

CAS number : 138402-11-6


IUPAC Name : 2-butyl-3-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)-

1,3-diazaspiro[4.4]non-1-en-4-one

Chapter VI

197

Telmisartan

Chemical Formula: C33H30N4O2

CAS number : 144701-48-4


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

Fig: 6.1 Structure of sartans

Literature review

Several reported methods are available for determinations of assay of both these

drugs individually or simultaneously with other drugs but have not come across any

simultaneous determination method for determination of all four sartans. This experiment

aims to achieve very short run times which have not yet been reported.

A few methods have been reported for determination of Candesartan- Ganesh, et

al. reported - RP- HPLC method development and validation of Candesartan Cilexetil in Bulk and

Their Pharmaceutical dosage forms[8]. Kamalakkannan, et al. reported - Analytical method

development and validation for Candesartan Cilexetil as bulk drug and in pharmaceutical

dosage forms by HPLC[9]. Subba Rao, et al. reported - A stability-indicating LC method

for Candesartan cilexetil”[10].Naseem AC, et al. reported - Determination of Candesartan

cilexetil in tablet dosage forms and dissolution testing samples by first derivative UV

spectrophotometric method[11]. İncilay Süslü, et al. reported - Square-wave adsorptive

Chapter VI

198

stripping voltammetric determination of candesartan cilexetil in pharmaceutical

formulations[12]. Süslü I, et al. reported - Voltammetric determination of candesartan

cilexetil in its Cu (II) complex and application to pharmaceutical formulations[13].

A few methods have been reported for determination of Irbesartan- Kamepall, et

al. reported - RP-HPLC-DAD Method for Determination of Irbesartan in Bulk and

Tablets Exposed to Forced Conditions[14]. Kishanta, et al. reported - Method

Development, Validation and Stability Study of Irbesartan in Bulk and Pharmaceutical

Dosage Form by UV-Spectrophotometric Method[15]. Gupta, et al. reported -

Electrochemical determination of antihypertensive drug Irbesartan in pharmaceuticals[16].

Hanaa, et al. reported - Voltammetry of Irbesartan Drug in Pharmaceutical Formulations

and Human Blood, Quantification and Pharmacokinetic Studies[17]. Dhanawade, et al.

reported - Derivative Spectrophotometric Method for Estimation of Irbesartan in Bulk

Drug and Dosage form[18].

A few methods have been reported for determination of Telmisartan-Sujana K, et

al. reported - Stability indicating RP HPLC method for the determination of Telmisartan

in pure and pharmaceutical formulation[19]. Phani Kishore, et al. reported - Development and

validation of stability indicating HPLC method for the estimation of Telmisartan related

substances in tablets formulation[20]. Sahu, et al. reported - Comparative Study of Forced

Degradation Behavior of Telmisartan by UPLC and HPLC and Development of

Validated Stability Indicating Assay Method According to ICH Guidelines[21]. Sagar, et al

reported - Development of UV Spectrophotometric Method of Telmisartan in Tablet

Formulation[22]. Ajit Pandey, et al reported - UV-Spectrophotometric Method for

estimation of Telmisartan in Bulk and Tablet Dosage Form[23]. MS Palled, et al reported -

Difference spectrophotometric determination of telmisartan in tablet dosage forms[24].

A few methods have been reported for determination of Valsartan - Bhatia M. et

al. reported - Determination and validation of valsartan and its degradation products by

isocratic HPLC[25]. Vinzuda D.U., et al. reported - RP-HPLC Method for Determination

of Valsartan in Tablet Dosage Form[26]. G Thanusha, et al. reported - Validated RP-

Chapter VI

199

HPLC Method for the Quantitative Estimation of Valsartan in Bulk and a Pharmaceutical

Dosage Forms[27]. K.R.Gupta, et al. reported - UV-Spectrophotometric methods for

estimation of Valsartan in bulk and tablet dosage form[28]. Habib IH, et al. reported -

Stripping voltammetric determination of valsartan in bulk and pharmaceutical

products[29].

To the best of the author‘s knowledge no method is available in the literature for

simultaneous determination of sartans (Candesartan, Telmisartan, Irbesartan and

Valsartan) and their impurities by UPLC. The current chapter thus describes a unique and

novel method for simultaneous determination of these drugs using UPLC.

Materials and methods

Reagents and Chemicals

HPLC gradient grade ACN and methanol from Merck (Mumbai, India) has been

used. Potassium dihydrogen phosphate (AR grade), ortho phosphoric acid and

triethylamine Solution from Merck have been used. Demineralized water was further

purified in the laboratory by filtering through an ultrapure Milli-Q (Millipore, Milford,

MA, USA). The drug substances, standards and impurities required for this work were

obtained from Dr Reddy‘s laboratories ltd.

Instrumentation and liquid chromatographic conditions

Chromatographic separation was carried out on a Waters Aquity UPLC with

photodiode array detector. The output signal was monitored and processed using

Empower 2 software. The mobile phase buffer consisted of 0.01M of potassium

dihydrogen phosphate pH adjusted to 2 with diluted ortho phosphoric acid as mobile

phase A and aceotnitrile as mobile phase B. The chromatographic separation was

performed in gradient mode (min/%B, 0/30, 1.2/46, 2.2/80, 2.5/90, 2.7/30, 3.0/30) The

chromatographic separation was carried out in used Kinetex XB C18 (50 mm X 2.1 mm,

and 1.7 µm particle size) at flow rate of volume 0.8 ml/min with 1.0 µl injection volume.

Chapter VI

200

The column temperature was at 25°C. UV detection was performed at λmax 225 nm. All

the glassware used for the following experimentation is of class A grade to obtain

maximum precision.

Standard preparation for assay for active pharmaceutical ingredient

Telmisartan standard stock solution: Accurately weighed and transferred

Telmisartan working std in 20 ml volumetric flask, dissolved in 10 ml of 0.1N NaOH,

added 5ml of acetonitrile and sonicated for 5 minutes and e made up to the volume with

water to get final concentration of 3000µg/mL.

Accurately weighed and transferred Candesartan, Irbesartan, Valsartan working standard

in 100ml volumetric flask, dissolved in 60 ml of acetonitrile, to this added 10 ml of

Telmisartan standard stock solution, and sonicated for 5 minutes and made up to volume

with water to get final concentration of 100µg/mL of Candesartan, Irbesartan, Valsartan

and 300µg/mL of Telmisartan and filtered through 0.22 µm filter.

Sample preparation for assay for active pharmaceutical ingredient

Accurately weighed and transferred Candesartan, Irbesartan, Valsartan and

Telmisartan active pharmaceutical ingredient in 100ml volumetric flask, dissolved in 60

ml of acetonitrile and sonicated for 5 minutes made up to volume with water to get final

concentration of 100µg/mL of Candesartan, Irbesartan, Valsartan and 300µg/mL of

Telmisartan and filtered through 0.22 µm filter.

Cleaning standard and sample preparation

The stock solution of standard was prepared by accurately weighing Candesartan,

Irbesartan, Valsartan and Telmisartan working standard and transferring to a 100 mL

volumetric flask, dissolved in 60 ml of acetonitrile and volume made up to the mark with

water to get final concentration of 20µg/mL. The selected surfaces (10 cm×10 cm) of

stainless steel, previously cleaned and dried, were sprayed with 1ml of cleaning stock

solution, for the positive swab control at all concentration levels, and the solvent was

Chapter VI

201

allowed to evaporate. The surface was wiped in one direction with wet tex swab soaked

with extraction solution (4 ml water and 6 ml of methanol) was pipette into swab tube.

The background control sample was prepared from the extraction solvent. The negative

swab control was prepared in the same way as the samples, using swabs, which had not

been in contact with the test surface. Subsequently, the tubes were placed in an ultrasonic

bath for 10 min and the solutions were analyzed by UPLC.

Results and Discussion

Acceptance limit calculation

Careful examination of the vessel for trace residues is vital to the pharmaceutical

manufacturing process as residues can contaminate subsequent products. The maximum

allowable carryover (MACO) is the acceptable transferred amount from the previous to

the following product. The MACO is determined based on the therapeutic dose, toxicity

and generally 10 ppm criterion. Once the maximum allowable residue limit in the

subsequent product was determined, the next step was the determination of the residue

limit in terms of the contamination level of active ingredient per surface area of

equipment. The total surface area of the equipment in direct contact with the product was

accounted for in the calculation. The limit per surface area was calculated from the

equipment surface area and the most stringent maximum allowable carryover. The 0.1%

dose limit criterion is justified by the principle that an active pharmaceutical ingredient

(API) at a concentration of 1/1000 of its lowest therapeutic dose will not produce any

adverse effects on human health. The calculated limits per surface area (LSA) in the case

of sartans were 2 µg/swab pro 100 cm2. A stainless steel surface area of 10 cm×10 cm

was chosen for practical reasons.

Optimization of the sample treatment for cleaning method validation

The stainless steel plates were spiked with required quantities of Candesartan, Irbesartan,

Telmisartan, valsartan and surface was wiped with tex swab soaked with of different

volume of water and methanol as extraction solvent placed into tubes and the tubes were

sonicated for different times (5 and 20 min) and the solutions were analyzed by UPLC.

Chapter VI

202

The optimum conditions were achieved with 40% water and 60% methanol as the

extracting solvent and sonication time of 10 min. This technique was applied in the

subsequent work.

Method development

The main objective was to develop a single UPLC method that can be used for the

estimation of Candesartan, Irbesartan, Telmisartan, Valsartan in active pharmaceutical

ingredient, in cleaning samples and in dissolution samples.

For analysis, the combination of 0.1% ortho phosphoric acid, 0.01M KH2PO4 (pH

2.5), 0.01M KH2PO4 (pH 2.0) with acetonitrile is tried as the mobile phase. The amount

of buffer was varied from 30% to 70% and flow rate varied from 0.3 ml min−1 to 1.0 ml

min−1.

The sufficient separation, tailing factor and plate number were achieved with the

proposed mobile phase (acetonitrile and 0.01M KH2PO4 (pH 2.0)) at flow rate 0.8 ml

min−1. Wavelength 225nm was selected for detection because the all drug have a

sufficient absorption and low quantities can be detected correctly. Furthermore, the

calibration curve obtained at 225nm showed good linearity. Regarding the

chromatographic procedure, different columns, BEH C18, (50X2.1mm, 1.7µ) Zorbax SB

C8 (50x4.6mm, 1.8µ) Kinetix XB-C-18 (50 x 2.1, 1.7µ) were evaluated but the Kinetix

XB-C-18 (50 x 2.1, 1.7µ) was preferred to improve the peak symmetry, plate number

and resolution. The column temperature was varied from 25 to 40oC but the analysis at

25oC was preferred to improve the peak symmetry, plate number and resolution. The

injection volume varied between 1µl to 3 µl, at 1 µl peak symmetry was good with good

plate count and resolution. On the basis of solubility of all sartans as well as stability,

difuent was selected.

Method robustness evaluation Factorial design

As the final method is selected against method attributes, it is highly likely

that the selected method is reliable and will remain operational over the lifetime

Chapter VI

203

of product. Therefore, the evaluation of method robustness is mainly for the method verif

ication and finalization. A risk‐based approach based on the QbD principles set

out in ICH Q8 and Q9 was applied to the evaluation of method robustness and

ruggedness. Structured methodologies for risk assessment, such as Design of experiment

can be implemented to identify the potential risk of the due to a small change of method

parameters column temperature (25 and 30°C), Mobile phase Buffer pH and flow rate

were simultaneously evaluated to assess the effects of these parameters on each of the

four response variables. Optimization of the analytical method was tested applying 23 full

factorial designs. The experimental domain of the selected factors is shown in Table: 6.1.

Table: 6.1 Chromatographic conditions and the range investigated during method

optimization

Factors (chromatographic

variables)

Low High

Column temperature 25°C 30°C

Mobile phase buffer pH 2.0 3.0

Flow rate 0.6 ml\ min 0.8 ml\ min

Resolution (Rs1) between Telmisartan and Irbesartan was chosen as the response

parameters. The design matrix of 23 full factorial design methodology shows 12 treatment

combinations of a low (–) and high (+) level of the factors Table: 6.2.

Chapter VI

204

Table: 6.2 Model matrix for 23 full factorial design methodologies with response data

Runs Column

Temperature

Mobile phase

buffer pH

Flow rate Rs 1

1 30 2.0 0.6 3.33

2 30 2.0 1.0 2.75

3 30 3.0 1.0 3.09

4 30 3.0 0.6 2.7

5 25 3.0 0.6 2.29

6 25 2.0 1.0 2.81

7 25 2.0 0.6 3.3

8 30 2.0 0.8 3.11

9 25 3.0 1.0 2.91

10 30 3.0 0.8 2.93

11 25 2.0 0.8 3.08

12 25 3.0 0.8 2.86

Based on the results of these experiments, the following statistical parameters and

ANOVA equations with model graph that Resolution (Rs1) between Telmisartan and

Irbesartan was chosen as the response parameters with the most important

chromatographic conditions was derived using Design Expert 8.0.

Table : 6.3 Analysis of variance table for Resolution (Rs1) between Telmisartan andIrbesartan

Source Sum ofSquares

Mean Square F Value p-value Prob > F

Chapter VI

205

Model 0.84 0.12 9.71 0.0221

A-ColumnTemperature

0.036 0.036 2.93 0.1621

B-Mobilephase buffer

pH

0.21 0.21 17.22 0.0143

C-Flow rate 4.500E-004 4.500E-004 0.036 0.8581

AB 0.036 0.036 2.93 0.1621

AC 0.013 0.013 1.03 0.3669

BC 0.54 0.54 43.64 0.0027

ABC 2.450E-003 2.450E-003 0.20 0.6796

The Model F-value of 9.71 implies the model is significant.There is only a 2.21%

chance that a "Model F-Value" this large could occur due to noise. Values of "Prob > F"

less than 0.0500 indicate model terms are significant.

ANOVAequation

Rs1 = +2.93 +0.055 * A -0.13* B -7.500E-003 * C +0.055 * A * B 0.040 * A * C+0.26 *B * - 0.018 * A * B * C

3D surface plots:

In order to better estimate how the variables affect the response over the whole

experimental domain including any interaction, 3D surface plots for resolution between

impurity C and impurity B were constructed using Design expert 8.0.

The resolution between Telmisartan and Irbesartan (Rs1) predicted from 3D

surface plots also coincide with observed resolution value which is listed in Table: 6.2

Moreover from the above 3D surface plots, where one parameter kept constant and other

parameter can be changed and we can predict resolution by clicking at any point of the

contour plot. If we extrapolate to other two axis, it will give corresponding values of the

other two variable factors.

Chapter VI

206

Factor C (Flow rate) kept constant:

Design-Expert® SoftwareFactor Coding: ActualRs1

Design points above predicted value3.33

2.29

X1 = A: Column temperatureX2 = B: Mobile phase buffer pH

Actual FactorC: Flow rate = 0.80

2.00

2.20

2.40

2.60

2.80

3.00

25.00

26.00

27.00

28.00

29.00

30.00

2.4

2.6

2.8

3

3.2

3.4

Rs1

A: Column temperature

B: Mobile phase buffer pH

Factor B (mobile phase Buffer)kept constant:


3.33

2.29

X1 = A: Column temperatureX2 = C: Flow rate

Actual FactorB: Mobile phase buffer pH = 2.50

0.60

0.70

0.80

0.90

1.00

25.00

26.00

27.00

28.00

29.00

30.00

2.4

2.6

2.8

3

3.2

3.4

Rs1


C: Flow rate

Chapter VI

207

Factor A (column temperature) kept constant:


3.33

2.29

X1 = B: Mobile phase buffer pHX2 = C: Flow rate

Actual FactorA: Column temperature = 27.50

0.60

0.70

0.80

0.90

1.00

2.00

2.20

2.40

2.60

2.80

3.00

2.4

2.6

2.8

3

3.2

3.4

Rs1

B: Mobile phase buffer pH

C: Flow rate

Fig: 6.2 3D Surface plots for Resolution (Rs1) between Telmisartan and Irbesartan (Rs1)


Rs1 = 2.81Std # 5 Run # 6

X1 = A: Column temperature = 25.00X2 = B: Mobile phase buffer pH = 2.00X3 = C: Flow rate = 1.00

CubeRs1


B: M

obile

pha

se b

uffe

r pH

C: Flow rate

A-: 25.00 A+: 30.00B-: 2.00

B+: 3.00

C-: 0.60

C+: 1.00

3.30833

2.81833

2.37667

2.99667

3.35333

2.77333

2.71167

3.10167

Prediction 2.81833Observed 2.8195% CI Low 2.8407895% CI High 3.0192295% PI Low 2.6083195% PI High 3.25169SE Mean 0.0321347SE Pred 0.115863X1 25.00X2 2.00X3 1.00

Fig: 6.3 Cube plots for Resolution (Rs1) between Telmisartan and Irbesartan (Rs1)

Cube plots are useful for representing the effects of three factors at a time. The

resolution between Telmisartan and Irbesartan (Rs1) predicted from 3D surface plots

also coincide with observed resolution value which is listed in Table: 6.2. Moreover from

Chapter VI

208

the above cube plots, by clicking at any of given point, it will give statistical values such

as confidence interval, probability interval, standard error mean and standard error

prediction.

Method Validation

Validation is required for any new or amended method to ensure that it is capable

of giving reproducible and reliable results. Once the chromatographic conditions had

been selected, the method was validated, whereby attention was paid to the selectivity,

linearity, limit of detection, limit of quantification, precision and accuracy.

Specificity

For Assay Method

Specificity is the ability of the method to accurately measure the analyte response

in the presence of all potential sample components. Specificity is the ability of the

method to measure the analyte response in presence of its process related impurities. The

specificity of the developed HPLC method was performed by injecting blank solution and

standard solution of each sartans separately. The chromatograms of sartans were

compared with the blank chromatogram, to verify the blank interference. No peak was

observed at the retention time of Sartans (Fig: 6.8) Hence the method is specific for the

determination of sartans in active pharmaceutical ingredient.

To check the performance of the optimized LC method for the separation of

degradation products, the drug was subjected to various stress conditions. The sample

solution was employed for acidic, alkaline, thermal and oxidation degradation condition

(in 1N HCl for 30 min at 60°C), (in 1 NaOH for 30 min at 60°C) (80°C for 24 h), and (in

10% H2O2 for 30 min at 60°C). The samples have been chromatographed according to

the experimental method to demonstrate the resolution of the all four sartans from any

unknown peaks. All four sartans has chromatographic resolution more than 1.5 from

other peaks. The mains peaks were well separated from degradation products peaks;

Chapter VI

209

purity angle of main peaks was more than purity threshold that proves method specificity.

The results of degradation studies are shown in Table: 6.4.

Table: 6.4 Results of degradation studies

For Cleaning Method

The specificity of the cleaning method was checked by injecting standard

solution, sample solution, the background control sample, the negative swab control,

swabbed un-spiked stainless steel 10 cm×10 cm plate as descried.

Fig: 6.4 Chromatogram obtained from (a) assay sample solution

Condition % Assay

Candesartn Irbesartan Telmisartan Valsartan

Sample as such 100.96 100.87 100.11 100

Thermal sample

80°C for 24 h

100.58 95.04 96.29 94.88

Acid sample1N HCL

60°C ½hr

79.29 84.62 88.92 83.21

Base sample 1N

NaOH 60°C ½ hr

0 87.22 87.79 85.53

10 % H2O2 sample

60°C ½ hr

7.49 81.14 85.121 86.55

Chapter VI

210

TELM

ISA

RTA

N -

0.51

3

IRB

ES

AR

TAN

- 0.

663

VA

LSA

RTA

N -

1.46

7

CA

ND

ES

AR

TAN

- 2.

245

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

Fig: 6.5 Chromatogram obtained from Cleaning sample solution

Fig: 6.6 Chromatogram obtained from back ground control samp

Fig: 6.7 Chromatogram obtained from negative swab control sample

Chapter VI

211

Fig: 6.8 Chromatogram obtained unspiked stainless steel sample

Peak purity has been verified for all of the impurities and for both main peaks.

Peak purity shows that impurity peaks as well as main peaks are homogeneous under all

the stress conditions. By the above-mentioned fact we can confirm that the method is a

stability-indicating method. The chromatograms and purity plots of the stressed samples

are shown in Fig: 6.9

Sample As such

Telmisartan Irbesartan

TE

LM

ISA

RT

AN

- 0

.429

PurityAuto Threshold

AU

Degre

es

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes0.410 0.415 0.420 0.425 0.430 0.435 0.440 0.445 0.450 0.455 0.460 0.465 0.470 0.475 0.480 0.485 0.490 0.495 0.500

IRB

ES

AR

TA

N -

0.5

21


AU

Degre

es

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes0.500 0.505 0.510 0.515 0.520 0.525 0.530 0.535 0.540 0.545 0.550 0.555 0.560

Chapter VI

212

Valsartan Candesartan

VA

LS

AR

TA

N -

1.1

33


AU

Degre

es

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes1.100 1.105 1.110 1.115 1.120 1.125 1.130 1.135 1.140 1.145 1.150 1.155 1.160 1.165 1.170

CA

ND

ES

AR

TA

N -

1.9

55


AU

Degre

es

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

0.034

0.036

0.038

0.040

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes1.940 1.942 1.944 1.946 1.948 1.950 1.952 1.954 1.956 1.958 1.960 1.962 1.964 1.966 1.968 1.970 1.972 1.974 1.976 1.978

Acid degraded Sample


TE

LM

ISA

RT

AN

- 0

.443


AU

Degre

es

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

Minutes0.428 0.430 0.432 0.434 0.436 0.438 0.440 0.442 0.444 0.446 0.448 0.450 0.452 0.454 0.456 0.458 0.460 0.462 0.464 0.466 0.468 0.470 0.472 0.474 0.476 0.478

IRB

ES

AR

TA

N -

0.5

27


AU

Degre

es

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes0.510 0.512 0.514 0.516 0.518 0.520 0.522 0.524 0.526 0.528 0.530 0.532 0.534 0.536 0.538 0.540 0.542 0.544 0.546 0.548 0.550 0.552 0.554 0.556 0.558 0.560


VA

LS

AR

TA

N -

1.1

40


AU

Degre

es

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes1.085 1.090 1.095 1.100 1.105 1.110 1.115 1.120 1.125 1.130 1.135 1.140 1.145 1.150 1.155 1.160 1.165 1.170

CA

ND

ES

AR

TA

N -

1.9

55


AU

Degre

es

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

0.034

0.036

0.038

0.040

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes1.936 1.938 1.940 1.942 1.944 1.946 1.948 1.950 1.952 1.954 1.956 1.958 1.960 1.962 1.964 1.966 1.968 1.970 1.972 1.974 1.976 1.978 1.980 1.982

Chapter VI

213

Base degraded sample


IRB

ES

AR

TA

N -

0.5

96


AU

Degre

es

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

Minutes0.580 0.582 0.584 0.586 0.588 0.590 0.592 0.594 0.596 0.598 0.600 0.602 0.604 0.606 0.608 0.610 0.612 0.614 0.616 0.618 0.620

TE

LM

ISA

RT

AN

- 0

.521


AU

Degre

es

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

Minutes0.505 0.510 0.515 0.520 0.525 0.530 0.535 0.540 0.545 0.550 0.555 0.560 0.565


VA

LS

AR

TA

N -

1.2

15


AU

Degre

es

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

55.00

60.00

65.00

Minutes1.185 1.190 1.195 1.200 1.205 1.210 1.215 1.220 1.225 1.230 1.235 1.240 1.245 1.250

CA

ND

ES

AR

TA

N -

1.9

75


AU

Degre

es

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes1.945 1.950 1.955 1.960 1.965 1.970 1.975 1.980 1.985 1.990 1.995 2.000 2.005 2.010

Peroxide degraded sample


TELM

ISAR

TAN

- 0.4

43


AU

Deg

rees

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes0.415 0.420 0.425 0.430 0.435 0.440 0.445 0.450 0.455 0.460 0.465 0.470 0.475 0.480 0.485 0.490 0.495

IRBE

SART

AN -

0.52

8


AU

Deg

rees

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes0.505 0.510 0.515 0.520 0.525 0.530 0.535 0.540 0.545 0.550 0.555 0.560

Chapter VI

214


VA

LSA

RTA

N -

1.13

8


AU

Deg

rees

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes

1.100 1.105 1.110 1.115 1.120 1.125 1.130 1.135 1.140 1.145 1.150 1.155 1.160 1.165 1.170 1.175

CA

ND

ES

AR

TAN

- 1.

955


AU

Deg

rees

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

0.034

0.036

0.038

0.040

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes1.938 1.940 1.942 1.944 1.946 1.948 1.950 1.952 1.954 1.956 1.958 1.960 1.962 1.964 1.966 1.968 1.970 1.972 1.974 1.976 1.978 1.980 1.982

Thermal degraded sample


TELM

ISAR

TAN

- 0.4

29


AU

Deg

rees

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes0.410 0.415 0.420 0.425 0.430 0.435 0.440 0.445 0.450 0.455 0.460 0.465 0.470 0.475 0.480 0.485 0.490 0.495 0.500

IRB

ES

AR

TAN

- 0.

521


AU

Deg

rees

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes0.500 0.505 0.510 0.515 0.520 0.525 0.530 0.535 0.540 0.545 0.550 0.555 0.560


VA

LSA

RTA

N -

1.13

3


AU

Deg

rees

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes

1.100 1.105 1.110 1.115 1.120 1.125 1.130 1.135 1.140 1.145 1.150 1.155 1.160 1.165 1.170

CAND

ESAR

TAN

- 1.9

55


AU

Degr

ees

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

0.034

0.036

0.038

0.040

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Minutes1.940 1.942 1.944 1.946 1.948 1.950 1.952 1.954 1.956 1.958 1.960 1.962 1.964 1.966 1.968 1.970 1.972 1.974 1.976 1.978

Fig: 6.9 Purity plots for sartans

Precision

The precision of the method was evaluated by performing six independent assays

of formulation sample; cleaning sample and the % RSD was calculated. The %RSD

values for all the four drugs found to be less than 2.

The intermediate precision of the method was investigated by repeating the

precision studies on other days by different analyst on different system using reagents

from different lot. The intermediate precision, expressed as the %RSD was found to be

less than 2. The data obtained suggested that the method exhibited an excellent precision

and intermediate precision. The results are given in Table: 6.5.

Chapter VI

215

Table: 6.5. Results of Precision and Intermediate Precision

Precision Intermediate Precision

Name of the

API

% RSD

(Assay

sample)

% RSD

(Cleaning

Sample)

% RSD

(Assay

sample)

% RSD

(Cleaning

Sample)

Candesartan 0.68 0.73 0.72 0.81

Irbesartan 0.25 0.31 0.42 0.37

Telmisartan 0.21 0.47 0.40 0.41

Valsartan 0.20 0.56 0.38 0.67

Fig: 6.10 Chromatogram obtained from Precision assay sample

TELM

ISA

RTA

N -

0.51

3

IRB

ES

AR

TAN

- 0.

663

VA

LSA

RTA

N -

1.46

7

CA

ND

ES

AR

TAN

- 2.

245

AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

Fig: 6.11 Chromatogram obtained from Cleaning assay sample

Chapter VI

216

Limit of Detection and Quantification

The limit of detection and limit of quantification were determined by means

signal to noise ratio method. Low concentrations of solutions were prepared using the

working standards of drugs and injected into the system. The signal to noise ratios of the

peaks was checked. The concentration of solution was lowered till the signal to noise

ratio is between 2 and 3 for limit of detection (LOD) and signal to noise ration ratio is

between 9.5-10.4 for limit of quantification (LOQ). The solution was in six replicates at

LOQ concentration level and injected into the system. The % RSD of peak areas was

calculated to prove method repeatability at LOQ.

The LOD for Candesartan, Irbesartan, Telmisartan and Valsartan found to be 0.20

µg/ml, 0.15µg/ml, 0.30µg/ml and 0.15µg/ml respectively. The LOQ values found to be as

0.60 µg/ml, 0.40 µg/ml, 0.85µg/ml and 0.50 µg/ml for candesartan, irbesartan,

telmisartan and valsartan respectively. % RSD at LOQ found to be 0.43, 0.74, 0.63 and

0.54 for Candesartan, Irbesartan, Telmisartan and Valsartan respectively.

Linearity

Linearity was demonstrated by injecting impurities at limit of quantification level,

25%, 50%, 100%, 150%, 200% and 300% with respect to the specification level of assay

and cleaning method. Plotted the calibration curve by taking concentration on X-axis and

peak area on Y-axis, calculated the correlation coefficient and % y-intercept (Table: 6.6).

Table: 6.6 Results of Linear regression analysis of sartans

Impurity name/Statisticalparameter

Correlationcoefficient

Slope Intercept Bias at100%

Candesartan 0.9994 6320 517.8 0.8892

Irbesartan 0.9992 9225 174.4 0.4919

Telmisartan 1.0000 21725 881.4 1.2570

Valsartan 0.9986 7849 651 2.9548

Chapter VI

217

Accuracy

The accuracy of the method was studied by recovery studies. The sample solution

was prepared at six different concentration levels i.e. 25%, 50%, 100%, 150%, 200% &

300%, specified amounts of standard had been added to these solutions and assay of these

solutions was performed. The added amounts were calculated in terms of recovery, which

were found to be between 98 – 102% (Table: 6.7).

Table: 6.7 Results of recovery studies of assay samples

Level Candesartan Irbesartan Telmisartan Valsartan

25% 100.97 99.56 100.58 100.48

50% 99.76 98.12 100.39 100.72

100% 99.99 101.05 100.71 101.20

150% 101.42 101.69 99.03 98.95

200% 99.11 98.40 98.09 99.57

300% 100.82 98.85 99.96 99.15

%RSD 0.86 1.47 1.03 0.92

Chapter VI

218

TELM

ISA

RTA

N -

0.43

8

IRB

ES

AR

TAN

- 0.

525

VA

LSA

RTA

N -

1.12

9

CA

ND

ES

AR

TAN

- 1.

961

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

Fig: 6.12 Chromatogram obtained from 50 % accuracy sample

TELM

ISAR

TAN

- 0.43

8

IRBE

SART

AN -

0.525

VALS

ARTA

N - 1

.129

CAND

ESAR

TAN

- 1.96

1

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00


TELM

ISAR

TAN

- 0.43

8

IRBE

SART

AN -

0.525

VALS

ARTA

N - 1

.129

CAND

ESAR

TAN

- 1.96

1

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00


Chapter VI

219

TELM

ISAR

TAN

- 0.43

8

IRBE

SART

AN - 0

.525

VALS

ARTA

N - 1

.129

CAND

ESAR

TAN

- 1.96

1

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00


TELM

ISAR

TAN

- 0.4

38

IRBE

SART

AN -

0.52

5

VALS

ARTA

N - 1

.129

CAND

ESAR

TAN

- 1.9

61

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00


TELM

ISAR

TAN

- 0.4

38

IRBE

SART

AN -

0.52

5

VALS

ARTA

N - 1

.129

CAND

ESAR

TAN

- 1.9

61

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00


The cleaning sample recovery studies were performed by spiking the cleaning

sample with known concentrations of standard. The added amount was calculated in

terms of recovery, the assay was performed in six replicates, %RSD was calculated

Chapter VI

220

which was found to be less than 2 for all the four drugs, that proves method accuracy

(Table: 6.8).

Table: 6.8 Results of recovery studies of cleaning samples

Level Candesartan Irbesartan Telmisartan Valsartan

25% 99.02 101.82 101.47 99.71

50% 99.42 100.82 101.44 99.20

100% 99.38 99.05 100.62 98.53

150% 99.21 99.90 100.77 98.95

200% 97.64 101.64 100.81 99.7

300% 97.17 98.84 100.84 98.41

%RSD 0.99 1.28 0.36 0.56

Application of the method to assay dissolution samples

The accurate determination of sartans in complex media such as dissolution

requires selective and sensitive analytical methodologies. The dissolution samples of all

four sartans were prepared according to current USP dissolution methods. The samples

were injected into the system and assay of the solutions was performed. The results are

shown in Table: 6.9.

Table: 6.9 Application of method to dissolutions studies

Dissolution media Valsartan Irbesartan Telmisartan Candesartan

0.1N HCl 100.98 99.73 99.58 NA

pH 4.5 Acetate

Buffer

103.32 95.44 98.02 99.70

pH 6.8 Phospate

buffer

102.13 100.56 100.33 NA

pH 7.5 phosphate

buffer

NA NA 98.81 NA

Chapter VI

221

Conclusion

A simple, accurate, precise LC method is developed for the determination of

Candesartan, Irbesartan, Telmisartan and Valsartan. This method has also been validated

as per ICH guidelines. Forced degradation studies are carried out by stressing at variety

of conditions. All the degradant peaks are well separated from the principle peaks. The

method is validated with respect to and found to be precise. The accuracy is carried out

on 7 levels from LOQ to 300% of the specification limit and the recoveries of all the

peaks are within acceptable limits. The linearity is carried out on 7 levels from LOQ to

300% of the specification limit. The correlation coefficient is found to be more than

0.998 for all the 8 peaks. Limit of detection (LOD) and Limit of quantification (LOQ)

results demonstrated the extremely high sensitivity of the method. The method is found to

be specific, precise, linear and accurate in the range of its intended application. The QbD

based method optimization helped in generating a design space and operating space with

knowledge of all method performance characteristics and limitations and successful

method robustness within the operating space. A single method can be used for

determination these drugs in cleaning swabs and suitable for assay of active

pharmaceutical ingredient for these four drugs. This method can be applied to dissolution

testing of all the four drugs.

On the basis of this study, it appears that the use of UPLC for the quantification of

API residues in cleaning validation samples in product formulation area is practical. The

time reducing and solvent saving characteristics of UPLC method are very advantageous,

compared to the most widely used conventional HPLC technique. The concept of

applying a generic method for several API residues for a product line is feasible and

practical if the structure and properties of compounds to be determined are similar.

Chapter VI

222

References

1. Israili Z.H., J Hum Hypertens 14 supp1, 73-86, 2000.

2. Hemandez-Hemandez R., Sosa-Canache B., Velasco M., Armas-Hermanadez

M.J., Armas-Padilla M.C., Cammarata R., J. Hum Hypetens 16 supp 1, 93-99,

2002.

3. Tronvik E., Stovner L.J., Helde G., Sand T., Bovim G. JAMA 1 (289 Pt 1): 65–9,

2003.

4. Cernes R., Mashavi M., Zimlichman R.,Vasc Health Risk Manag 7:749–59, 2011.

5. Gales B.J, Bailey E.K, Reed A.N, Gales M.A., Ann Pharmacother 44 (2), 360–6,

2010.

6. Klinkenberg R., Streel B., Ceccato A., J Pharm Biomed Anal. 32(2), 345-52,

2003.

7. Arayne M.S., Sultana N., Sajid S.S., Ali S.S., J Pharm Sci Technol. 62(5), 353-

61, 2008.

8. Ganesh Akula, Kandikonda Saikrishna, Saikumar Bhupathi, Rasapally Ramesh

Kumar and Santhosh Kumar K., Journal of Pharmaceutical Sciences and

Research 1 (12), 191-196, 2010.

9. Kamalakkannan V., Puratchikody A., Masilamani K., Saraswathy T., Der

Pharmacia Lettre 3(3), 286-296, 2011.

10. Subba Rao D.V., Radhakrishnanand P., Suryanarayana M.V., Himabindu V.,

Chromatographia. 66 (7/8), 499-507, 2007.

11. Naseem A.C., Mohammad B., Enas A., Khalid Ibrahim H.A., Analytical Letters.

42(14), 2232 – 2243, 2009.

12. İncilay Süslü, Nuran Özaltın, Sacide Altınöz, Journal of Applied Electrochemistry

39(9), 1535-1543, 2009.

13. Süslü I., Ozaltin N., Altinöz S., J AOAC Int. 93(2), 562-568, 2010.

14. Kamepally Sujana, International Journal of Pharmaceutical Research and

Development 3(6), 67-73, 2011.

15. Dhanawade P.P., Kane R.N., International Journal of Research in Pharmaceutical

and Biomedical Sciences 3 (3), 1300-1305, 2012.

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16. Kishanta Kumar Pradhan, Uma Shankar Mishra, Subasini Pattnaik, Debananda

Mishra, Ghanshyam Panigrahi, Kanhu Charana Sahu,. International Journal of

Pharmaceutical & Biological Archives 2(4), 1114-1122, 2011.

17. Gupta V.K., Jain R., Agarwal S., Mishra R., Dwivedi A., Anal Biochem. 410(2),

266-71, 2011.

18. Hanaa S. El-Desoky, Mohamed M. Ghoneim and Allia. D. Habazy J. Braz. Chem.

Soc. 22(2), 239-247, 2011.

19. Sujana K., Gowri Sankar D., Bala Souri O., Swathi Rani G., Pharmacie Globale

(IJCP), 1 (4), 1-4,2010.

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Pharm. Sci. 1(4), 493-501,2010.

21. Sahu K., Sahu S., Shaharyar M., Siddiqui A.A., J Chromat Separation Techniq 3,

129, 2012.

22. Sagar Tatane, Journal of Advances in Pharmacy and Healthcare Research 1(1),

23-26, 2011.

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International Journal of ChemTech Research, 3(2), 657-660, 2011.

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Sharadha Ch., International Journal of ChemTech Research 2(2), 1194-1198,

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41, 2008.

Papers published:

1. Simultaneous determination of Omeprazole and Domperidone impurities in active

pharmaceutical ingredients by UPLC. (paper accepted on 04-03-13, Ref. M.S.No: 3200)

(International Journal of Pharma and Bio sciences)

2. Development and Validation of a Stability-Indicating RP-UPLC Method for Assay of 1-

(4-Hydrozinophenyl) methyl-1,2,4-triazole Dihydrochloride and Estimation of its Related

Compounds

(Asian Journal of Research In Chemistry) Asian J. Research Chem. 5(11): Nov., 2012

3. Identification and characterization of oligomersof ampililloc acids impurity in ampicillin

sodium active pharmaceutical ingredient stability studies.

(International Journal Of Chemical and Pharmaceutical Sciences)2012,Sep.,Vol.3 (3)

Papers Communicated:

1. Development and validation of stability indicating method for the determination of

related substances and assay of sparfloxacin by UPLC

(Journal of liquid chromatography and liquid technologies)

2. QBD approach to development and validation of HPLC method for the determination of

related compounds of Nimesulide in drug substance

(International Journal of Chemical and Analytical Science)

Papers under Pipeline:

1. A Quality by Design based approach to stability indicating method for determination of

related substances of Dexlansoprazole by UPLC

2. Simultaneous determination of Amlodipine Besylate and Benazepril HCl impurities in

finished product by UPLC

chapter vi development and validation of an uplc method...

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