Chapter VI Development and validation of an UPLC method...
Transcript of 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
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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
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Candesartan
Chemical Formula : C24H20N6O3
CAS number : 139481-59-7
Molecular weight : 440.45
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
Molecular weight : 428.53
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
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Telmisartan
Chemical Formula: C33H30N4O2
CAS number : 144701-48-4
Molecular weight : 514.617
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
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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-
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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.
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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
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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.
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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
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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.
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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
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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.
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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:
Design-Expert® SoftwareFactor Coding: ActualRs1
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
A: Column temperature
C: Flow rate
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Factor A (column temperature) kept constant:
Design-Expert® SoftwareFactor Coding: ActualRs1
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)
Design-Expert® SoftwareFactor Coding: ActualRs1
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
A: Column temperature
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
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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;
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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
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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
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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
PurityAuto Threshold
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
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Valsartan Candesartan
VA
LS
AR
TA
N -
1.1
33
PurityAuto Threshold
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
PurityAuto Threshold
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
Telmisartan Irbesartan
TE
LM
ISA
RT
AN
- 0
.443
PurityAuto Threshold
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
PurityAuto Threshold
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
Valsartan Candesartan
VA
LS
AR
TA
N -
1.1
40
PurityAuto Threshold
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
PurityAuto Threshold
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
Telmisartan Irbesartan
IRB
ES
AR
TA
N -
0.5
96
PurityAuto Threshold
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
PurityAuto Threshold
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
Valsartan Candesartan
VA
LS
AR
TA
N -
1.2
15
PurityAuto Threshold
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
PurityAuto Threshold
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
Telmisartan Irbesartan
TELM
ISAR
TAN
- 0.4
43
PurityAuto Threshold
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
PurityAuto Threshold
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
Valsartan Candesartan
VA
LSA
RTA
N -
1.13
8
PurityAuto Threshold
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
PurityAuto Threshold
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
Telmisartan Irbesartan
TELM
ISAR
TAN
- 0.4
29
PurityAuto Threshold
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
PurityAuto Threshold
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
Valsartan Candesartan
VA
LSA
RTA
N -
1.13
3
PurityAuto Threshold
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
PurityAuto Threshold
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
Fig: 6.13 Chromatogram obtained from 75 % 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
Fig: 6.14 Chromatogram obtained from 100 % accuracy sample
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
Fig: 6.15 Chromatogram obtained from 150 % accuracy sample
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
Fig: 6.16 Chromatogram obtained from 200 % accuracy sample
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
Fig: 6.17 Chromatogram obtained from 300 % accuracy sample
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
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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