chapter –chapter –––vvvv -...
TRANSCRIPT
214
CHAPTER CHAPTER CHAPTER CHAPTER ––––VVVV
STABILITY INDICATING
ASSAY AND IMPURITIES METHODS FOR FIXED
DOSE COMBINATION PRODUCT OF
METOPROLOL+ATORVASTATIN+RAMIPRIL
Section (i):Brief account ofMetoprolol,Atorvastatin andRamipril
215
Ramipril(RM)is(1S,5S,7S)-8[(2S)-2-[[(1S)-1-ethoxycarbonyl-3-phenylropyl]amino]-propanoyl]-
8azabicyclo[3.3.0]octane-7-carboxylic acid, potent and specific angiotensin-converting enzyme (ACE) inhibitor
that lower peripheral vascular resistance without affecting heart rate. It is used in treatment of hypertension and
congestive heart failure. The role of this kind of drugs is to inhibit the last step of the biosynthesis of angiotensin
II, a potent vasoconstrictor, and therefore, it causes a general vasodilatation and lowers blood pressure
[1-3]. Ramipril, an angiotensin-converting enzyme inhibitor, is a prodrug which is rapidly hydrolysed after
absorption to the active metabolite Ramiprilate
Ramipril :
N
O
O H
O
H N
O
O
H
H
Atorvastatin calcium (AT) [4], chemically, 1H-pyrrole-1-heptanoic acid, [R-(R*,R*)]-2-(4-
flurophenyl)-β , d-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino) carbonyl]-calcium salt (2:1), is an
antihyperlipoproteinemic drug[5-6], used for treatment of hypercholesterolemia.
Atorvastatin :
NO
NH
F
O
OH OH O
2
Ca+2
216
Metoprololsuccinate(MT),1-[4-(2-methoxyethyl)-phenoxy]-3-[(1-methylethylamino]-
2-propanol is a beta-adrenergic blocking agent, which reduces chest pain and lowers high
blood pressure [7].
Metoprolol :
O NH
CH3
H3COCH3
.
OH
HOOH
O
O
.
2
Metoprolol, Atorvastatin and Ramipril are available as a single or either of two combination dosage forms in
market for effective therapy. As an individual molecule Atorvastatin shows effectiveness in lowering lipid
levels in condition of dyslipidemia and metabolic syndrome. Ramipril as ACE (angiotension converting
enzyme) inhibitor can control hypertension. Metoprololasbeta-adrenergic blocking agent reduces chest pain and
lowers high blood pressure. Combination of Metoprolol SR (sustain release), Ramipril and Atorvastatin will be
beneficial in controlling hypertension, angina associated with dyslipidemia and metabolic syndrome.
All three drug substance monographs are available in United States Pharmacopoeia
(USP), British Pharmacopoeia (BP), Indian Pharmacopeia(IP) and European Pharmacopeia
(EP) where as Metoprolol and Ramipril drug products are official in USP, BP and IP and
Atorvastatin drug product is official in Indian Pharmacopoeia.
Literature survey revealed several analytical methods such as spectrophotometer, spectrofulorimetry, simple
and stability indicating LC, stability indicating TLC, LC-MS methods have been reported for the determination
of Metoprolol, Atorvastatin and Ramipril in single and combination pharmaceutical dosage form and biological
samples [8-29]. Although several methods are reported for assay of individual drugs and
217
combination of two drugs, none of the reported articles described the single method for
estimation of MT+AT+RM from fixed dosage combination product.
Author is also reviewed the literature for impurity methods and few methods have
been reported for estimation of impurities in individual drug substances and drug
products [30-31]. But none of the reported articles described the common method for
estimation of impurities in fixed dosage combination product of MT+AT+RM. Instead of
following three individual methods, author has seen an opportunity to develop a common
analytical method for assay as well as impurities estimation in triple combination
product.This chapter describes development and validation of stability indicating method for assay and
impurities of MT+AT+RM,the method validation performed as per ICH guidelines [32].
Section (ii): Stability Indicating method for Assay of Metoprolol + Atorvastatin + Ramipril by UPLC
This section describes the various aspects related to method development and
validation of stability indicating reversed phase UPLC method for assay of Metoprolol (MT) +
Atorvastatin (AT) + Ramipril (RM) in Capsules.
1. Experimental
1.1. Chemicals
Active pharmaceutical ingredient of MT, AT, RM and its impurities are procured from bulk
manufacturers of Dr. Reddys Ltd. MT+AT+RM Capsules and placebo are procured from formulation Dr.
Reddys Ltd. HPLC grade methanol and acetonitrile are purchased from Merck, Germany. Analytical reagents
sodium lauryl sulfate and ortho phosphoric acid are purchased from Merck, Germany. High pure water is
prepared by using Millipore Milli Q plus purification system.
1.2. Determination of appropriate UV wavelength
The suitable wavelength for the determination of MT, AT and RM in diluent is identified by scanning over
the range 200–400 nm with a double beam UV spectrophotometer.
1.3. Instrumentation and chromatographic conditions
218
The Waters UPLC system with a diode array detector is used for method development, forced degradation
studies and method validation. The output signal is monitored and processed using Empower software. Cintex
digital water bath is used for hydrolysis studies. Photo stability studies are carried out in Sanyo photo stability
chamber. Thermal stability studies are performed in a Cintex dry air oven. The pH of buffer is measured using
Thermo Orion pH meter.
The chromatographic column Zorbax XDB-C18, 4.6 x 50 mm, 1.8 µm is used for developing a method.
The buffer is prepared using 0.0045M of sodium lauryl sulfate with 0.06% ortho phosphoric acid. Buffer and
acetonitrile in the ratio of 50:50 v/v is used as mobile phase. The flow rate of the mobile phase is 1.0 mL/min.
The column is maintained at 55°C and the wavelength of 210 nm for detection of MT, RM and AT. The
injection volume is 2 µL.
1.4. Diluent
Methanol is used as diluent.
1.5. Preparation of standard solution
A standard solution is prepared in methanol containing 420 µg/mL of MT, 80 µg/ mL of
AT and 40 µg/mL of RM. The Specimen overlay chromatograms of diluent and standard are
shown in Fig 5.2.1.
219
Fig 5.2.1 Overlay chromatogram of diluent and 420 µg/ mL of MT, 80 µg/ mL of AT and 40
µg/ mL of RM
1.6. Preparation of Test solution
Open and transfer contents of 10 capsules along with capsule shell (each capsule containing 50 mg MT,
10 mg AT and 5 mg RM) into 250 mL volumetric flask. 150 mL of methanol is added and sonicated for 30
minutes with intermediate shaking (maintain the sonicator temperature between 20°C-25°C), followed by 15
minutes shaking on rotary shaker and then madeup to volume with methanol. A part of solution is centrifuged to
get clear solution. 5 mL of clear centrifuged solution is transferred into 25 mL volumetric flask and made up to
volume with methanol to obtain sample solution concentration of 420 µg/mL, 80 µg/mL and 40 µg/mL of MT,
AT and RM respectively. Placebo samples are prepared in the same way by taking the placebo equivalent its
weight present in a test preparation. The Specimen overlay chromatogram of placebo and test sample is shown in
Fig 5.2.2.
Fig 5.2.2 Overlay chromatogram of placebo and test.
220
1.7. Specificity:
As per ICH guidelines, stability indicating methods are required for evaluating
quality of finished dosage forms. The current ICH guidelines do not talk about
degradation conditions for stress study. The forced degradation conditions, stress agent
concentration and time of stress, are found to be effective based on % degradation.
Preferably not more than 30% of degradation is recommended for active material to make
the right assessment of stability indicating nature of the chromatographic methods. The
optimization of such stress conditions which can yield not more than 30% degradation is
based on experimental conditions. Chromatographic run times are decided for placebo and
samples subjected to force degradation in order to provide an indication of the stability
indicating properties and specificity of the method. The stress conditions employed are
acid, base, neutral, peroxide, heat, humidity and light. After the degradation treatments are
completed, the samples are allowed to equilibrate to room temperature, neutralized with
acid or base (as necessary), and resulting solution is prepared as per test preparation. The
samples are analyzed against a freshly prepared control sample (with no degradation
treatment) and evaluated for peak purity by using photo diode array detector. Specific
conditions are described below:
1.7.1. Placebo (excipients) interference
Placebo Sample solutions are prepared in duplicate by taking the weight of placebo
approximately equivalent to its weight in the test preparation as described in 1.6.
221
1.7.2. Effect of acid hydrolysis
Ten capsules contents are transferred (each capsule containing 50 mg MT, 10 mg AT and 5 mg RM)
into 100 mL round bottom flask, treated with 20 mL of 0.1N HCl for 30 minutes at 60°C. The sample is allowed
to equilibrate to room temperature, neutralized with base and resulting solution is prepared as per test procedure
to obtain final concentration of 420 µg/mL, 80 µg/mL and 40 µg/mL of MT, AT and RM respectively.
1.7.3. Effect of base hydrolysis
Ten capsules contents are transferred (each capsule containing 50 mg MT, 10 mg AT and 5 mg RM)
into 100 mL round bottom flask, treated with 20 mL of 0.1N NaOH for 30 minutes at 60°C. The sample is
allowed to equilibrate to room temperature, neutralized with acid and resulting solution is prepared as per test
procedure to obtain final concentration of 420 µg/mL, 80 µg/mL and 40 µg/mL of MT, AT and RM respectively.
1.7.4. Effect of neutral hydrolysis
Ten capsules contents are transferred (each capsule containing 50 mg MT, 10 mg AT and 5 mg RM)
into 100 mL round bottom flask, treated with 20 mL of water for 1 hour at 60°C. The sample is allowed to
equilibrate to room temperature and resulting solution is prepared as per test procedure to obtain final
concentration of 420 µg/mL, 80 µg/mL and 40 µg/mL of MT, AT and RM respectively.
1.7.5. Effect of oxidation
Ten capsules contents are transferred (each capsule containing 50 mg MT, 10 mg AT and 5 mg RM)
into 100 mL round bottom flask, treated with 10 mL of 3% H2O2 for 30 minutes at 60°C. The sample is allowed
to equilibrate to room temperature and resulting solution is prepared as per test procedure to obtain final
concentration of 420 µg/mL, 80 µg/mL and 40 µg/mL of MT, AT and RM respectively.
1.7.6. Effect of humidity and heat
To evaluate the effect of humidity and heat, capsules contents are distributed over a petri dish and exposed
to 25ºC/90% RH (Relative Humidity) for 7 days. A similar sample is kept in an oven at 105ºC for 15 hours.
Then, the samples are prepared in methanol as described in test preparation.
1.7.7. Effect of UV and visible light
222
To study the photochemical stability of the drug product the capsule contents is exposed to 1200 K Lux of
visible light and 200 W h/ m2 of UV light by using photo stability chamber. After exposure the samples are
prepared in methanol as described in test preparation.
1.8. Method validation
1.8.1. Precision
Precision (intra-day precision) of the assay method is evaluated by carrying out six independent assays of
test sample of MT+AT+RM capsules against qualified reference standard. The % of RSD of six assays obtained
is calculated. The intermediate precision (inter-day precision) of the method is also evaluated using two different
UPLC systems and different UPLC columns in different days in the same laboratory.
1.8.2. Linearity
Linearity test for assay is established using six different concentration levels in the range of about 105-
840 µg/mL for MT (corresponding to 25% to 200% of assay concentration), 20-160 µg/mL for AT
(corresponding to 25% to 200% of assay concentration) and 10-80 µg/mL for RM (corresponding to 25 to 200%
of assay concentration). The peak area versus concentration data is performed by least-square regression
analysis.
1.8.3. Accuracy
A study of recovery of MT, AT and RM from spiked placebo is conducted. Samples are prepared by
mixing placebo with MT, AT and RM equivalent to 20%, 50%, 80%, 100% and 160% of the assay of highest
test concentration. Sample solutions are prepared in triplicate for each spike level as described in the test
preparation. The % recovery is calculated.
1.8.4. Robustness
To determine the robustness of the developed method, experimental conditions are purposely altered
one after the other to estimate their effect. Five replicate injections of standard solution is injected under each
parameter change. The effect of flow rate, column temperature and organic phase composition (acetonitrile) in
mobile phase is studied by verifying tailing factor and %RSD for peak areas of replicate injections of MT, AT
and RM standard. The effect of flow rate is studied at 0.8 mL/min and 1.2 mL/min. Column temperatures of
223
50ºC and 60ºC and organic phase compositions (acetonitrile) in mobile phase at + 10% along with the actual
method conditions of 1.0 mL/min, 55ºC and 100% organic phase composition is studied.
1.8.5. Solution stability and mobile phase stability
The solution stability of test sample and reference standard is established by allowing solutions on bench
top at controlled room temperature at 24 and 48 hours interval. The solutions are stored in volumetric flasks by
tightly capping. The assay is determined for both test and reference standard solutions by using freshly prepared
reference standard at each interval.
The mobile phase stability is also established by determining the assay of freshly prepared sample
solutions against freshly prepared reference standard solutions at 24 hour interval for 48 hours. Mobile phase
prepared is kept constant during the study period. The tailing factor and % RSD of peak area of standard for
replicate injections is evaluate for the study period.
2. Results and discussion
2.1. Determination of suitable wavelength
The UV spectrum of MT, AT and RM recorded in the range 200-400 nm is illustrated in Fig 5.2.3. The
spectrum indicates that 210 nm gives a good sensitivity for the assay of MT, AT and RM.
Fig 5.2.3 The overlay UV Spectra of MT, AT and RM
224
2.2. Optimization of chromatographic conditions
Assay method plays a major role in dosage form, to quantify the amount of analyte. The main target of
the chromatographic method is to get the separation of all potential impurities of MT, AT and RM without
interfering with the main analyte peaks in single chromatographic condition.
Method optimization is initiated by taking 0.045M sodium lauryl sulfate and 0.06% ortho phosphoric
acid in Milli Q water as buffer; buffer and acetonitrile in the ratio 50:50 at 1.0 mL/min flow at ambient
temperature on C18 column; it is observed that impurity E and impurity D peaks of RM are merged with MT
and AT peaks. To get effective separation of peaks, the buffer and acetonitrile ratio is modified to 55:45 at
ambient temperature and impurity E and impurity D are well separated from MT and AT peaks but RM peak
symmetry is not satisfactory i.e. peak tailing observed more than 2.0. In order to achieve peak symmetry, column
temperature is increased from ambient to 55°C, it is observed that all three analyte peaks AT, RM and MT are
well separated from impurity peaks, but analysis time is too large i.e. 45 minutes
Hence it is decided to further reduce the runtime for the analysis to increase the system efficiency and
cost effectiveness. The same experiments are conducted on UPLC system using Zorbax Eclipse plus-C8, 2.1 mm
x 50 cm, 1.8 µm with mobile phase flow rate of 0.6 mL/min. The runtime is reduced up to 10 minutes but MT
peak symmetry is found to be not satisfactory. i.e. tailing more than 2.0, hence analytical column , flow rate and
column temperature are modified. Analytical column is replaced to Zorbax XDB-C18, 4.6 mm x 50 cm, 1.8 µm,
flow rate is increased to 1.0 mL/min and column temperature increased to 55°C. The adequate separation is
achieved with following chromatographic condition; Isocratic
program using buffer as 0.045M sodium lauryl sulfate and 0.06% ortho phosphoric acid in Milli Q water; buffer
and acetonitrile in the ratio of 50:50. The flow rate of the mobile phase is 1.0 mL/min. The column is maintained
at 55°C and the wavelength of detection is 210 nm. The injection volume is 2 µL. The typical retention times of
MT, AT and RM are 1.324 min, 2.148 min and 2.684 min respectively are achieved. This method is capable to
separate all impurities from its analyte peak within 6 minutes.
2.3. Method validation
2.3.1. Precision
225
Method repeatability (intra-day precision) is evaluated by assaying six samples,
prepared as described in the test preparation. The mean % assay values of MT, AT and RM
are found to be 101.9, 102.1 and 101.4, the %RSD for assay values of MT, AT and RM are
found to be 0.4, 0.3 and 0.8 respectively. These values are within the acceptable limit of
between 97.0%-103.0% and %RSD not more than 2.0. The intermediate precision (inter day
precision) is performed by assaying six samples on different UPLC systems and different
UPLC columns in different days as described in the sample preparation. The mean % assay
values of MT, AT and RM are found to be 101.1, 101.6 and 101.3 respectively, % RSD for
assay values of MT, AT and RM are found to 0.5. The precision and intermediate precision
results are summarized in Table 5.2.1.
Table 5.2.1 Precision and Intermediate precision results
Sample No.
% Assay
Intra-day precision Intra-day precision
MT AT RM MT AT RM
1 101.9 102.4 102.2 100.8 102.0 101.5
2 101.5 101.7 100.4 101.1 102.3 100.7
3 102.0 102.0 101.7 100.9 100.9 101.5
4 101.3 102.3 102.3 101.5 101.5 100.9
5 102.3 102.1 100.4 101.9 101.1 102.1
6 102.3 102.3 101.5 100.5 101.8 101.2
Mean 101.9 102.1 101.4 101.1 101.6 101.3
%RSD 0.4 0.3 0.8 0.5 0.5 0.5
226
2.3.2. LOQ and LOD
The LOQ and LOD are determined based on signal-to-noise ratios at responses of 10
and 3 times the background noise, respectively. The LOQ is found to be 0.21 µg/mL with a
resultant %RSD of 0.5 (n = 6) for MT, 0.15 µg/mL with a resultant % RSD of 0.6 (n=6) for
AT and 0.32 µg/mL with a resultant % RSD of 0.4 (n=6) for RM. The LOD is found to be
0.064 µg/mL for MT, 0.045 µg/mL
for AT and 0.097 µg/mL for RM.
2.3.3. Linearity
A linear calibration plot for assay of MT, AT and RM is obtained over the calibration
range of 105-840 µg/ mL of MT (corresponding to 25% to 200% of assay concentration), 20-
160 µg/mL of AT (corresponding to 25% to 200% of assay concentration) and 10-80 µg/mL
of RM (corresponding to 25% to 200% of assay concentration) and the correlation co-efficient
is found to be 1.000 for MT, AT and RM. The graphical plots shown in Fig 5.2.4 to 5.2.6
indicates that a good correlation exists between the peak area and concentration of the analyte.
Fig 5.2.4 Linearity of detector response graph for MT
y = 2033.264x + 5139.971R² = 1.000
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
0 100 200 300 400 500 600 700 800 900
Are
a
Concentration in ppm
Linearity of Detector Response of Metoprolol
227
Fig 5.2.5 Linearity of detector response graph for AT
Fig 5.2.6 Linearity of detector response graph for RM
2.3.4. Accuracy
The percentage recovery of MT, AT and RM from drug product is found to be 101.0 to 102.1 for MT,
100.2 to 102.5 for AT and 100.6 to 102.0 for RM which indicates the high accuracy of the method. The results
are shown in Table 5.2.2.
Table 5.2.2 Recovery results of MT, AT and RM from capsules
Spike level
(%)
Average
‘µg/mL’ added
Average
‘µg/mL’ found
MT AT RM MT AT RM
20 99.960 20.565 10.283 100.923 20.894 10.342
50 246.954 52.036 26.018 251.319 52.123 26.547
80 392.084 80.700 40.350 400.184 82.691 40.901
100 495.629 102.731 51.366 506.206 105.162 51.862
160 783.014 162.870 81.435 798.414 166.806 82.250
y = 7928.515x + 5806.824R² = 1.000
0
200000
400000
600000
800000
1000000
1200000
1400000
0 20 40 60 80 100 120 140 160
Are
a
Concentration in ppm
Linearity of Detector Response of Atorvastatin
y = 4045.254x + 339.373R² = 1.000
0
50000
100000
150000
200000
250000
300000
350000
0 10 20 30 40 50 60 70 80 90
Are
a
Concentration in ppm
Linearity of Detector Response of Ramipril
228
Spike level
(%)
Mean % recovery % RSD
MT AT RM MT AT RM
20 101.0 101.6 100.6 0.4 0.5 0.9
50 101.8 100.2 102.0 0.8 0.4 0.2
80 102.1 102.5 101.4 0.5 0.1 0.5
100 102.1 102.4 101.0 0.3 0.1 0.2
160 102.0 102.4 101.0 0.6 0.3 0.4
2.3.5. Robustness
In all the deliberately varied chromatographic conditions studied (flow rate, column
temperature and ratio of acetonitrile in mobile phase), the tailing factor and the % RSD for the
MT, AT and RM peak areas for five replicate injections of standard is found to be within the
acceptable limits of not more than 2 for tailing factor and not more than 2 for %RSD, the
results are summarized in Table 5.2.3
Table 5.2.3 Results of Robustness study
Parameter
Observed value
Variation Tailing factor
% RSD for five
injections of standard
MT AT RM MT AT RM
Flow rate
0.8 mL/min 1.6 1.2 1.2 0.3 0.1 0.3
1.0 mL/min 1.5 1.1 1.1 0.2 0.1 0.4
1.2 mL/min 1.4 1.1 1.1 0.1 0.3 0.2
Column temperature
50ºC 1.4 1.2 1.1 0.3 0.4 0.2
55ºC 1.5 1.1 1.1 0.2 0.1 0.4
60ºC 1.4 1.1 1.0 0.1 0.2 0.2
Mobile phase
composition(Acetonitrile)
90% 1.6 1.2 1.1 0.2 0.4 0.1
100% 1.5 1.1 1.1 0.2 0.1 0.4
110% 1.3 1.0 1.1 0.1 0.5 0.4
229
2.3.6. Solution stability and mobile phase stability
The difference in % assay of test and standard preparations upon storage on bench top
is found to be less than 1.0 up to 48 hours. Mobile phase stability experiments showed that
tailing factor and % RSD are less than 1.6 and 0.7 respectively up to 48 hours. The solution
stability and mobile phase stability experimental data confirmed that sample solutions and
mobile phase used during assay determination are stable up to 48 hours.
2.3.7. Results of specificity studies
Placebo and stressed samples solutions are injected into the UPLC system with
photodiode array detector as per the described chromatographic conditions. Chromatograms
of placebo solutions have shown no peaks at the retention time of MT, AT and RM peaks.
This indicates that the excipients used in the formulation do not interfere in estimation of MT,
AT and RM in capsules.
All degradant peaks are well resolved from MT, AT and RM peaks in the chromatograms of all stressed
samples. The chromatograms of the stressed samples are evaluated for peak purity for MT, AT and RM peaks
using Waters Empower Networking software. For all forced degradation samples, the purity angle (the weighted
average of all spectral contrast angles calculated by comparing all spectra in the integrated peak against the peak
apex spectrum) is found to be less than threshold angle (the sum of the purity noise angle and solvent angle, the
purity noise angles across the integrated peak) for MT, AT and RM peaks (Table 5.2.4). This indicates that there
is no interference from degradants in quantification of MT, AT and RM in capsules. Thus, this method is
considered "Stability indicating”. The typical chromatogram and purity plots of all stressed samples are shown in
Fig 5.2.7 to 5.2.14.
229
Table 5.2.4 Specificity study results
Stress
conditions
Metoprolol Atorvastatin Ramipril
%
degradation
Purity
Angle
Purity
Threshold
%
degradation
Purity
Angle
Purity
Threshold
%
degradation
Purity
Angle
Purity
Threshold
Treated with 0.1 N HCI
solution for 30 minutes at
60 °C
Nil 0.052 2.258 1.2 0.068 6.127 2.7 0.373 45.662
Treated with 0.1 N NaOH
solution for 30 min at 60 °C
0.1 0.053 2.279 0.7 0.068 5.516 2.6 0.349 39.835
Treated with 3% H2O2
solution for 30 minutes at
60 °C
0.3 0.058 3.798 2.2 0.063 10.178 4.8 0.408 90.000
Exposed UV light (200 W h
m -2
)
0.1 0.053 2.446 0.6 0.055 5.834 0.3 0.389 43.127
Exposed to Heat for 15 hour
at 105 °C
0.4 0.123 2.095 32.3 0.096 6.503 33.5 0.922 78.380
Exposed to Visible light(
1,200 K lux hours)
0.2 0.053 2.235 0.9 0.068 5.963 1.5 0.364 41.429
Treated with purified water
for 1 hour at 60°C
0.1 0.052 2.268 0.9 0.058 5.937 0.7 0.377 43.397
Exposed to humidity at 25
°C, 90% RH for 7 days
0.0 0.051 2.299 1.2 0.050 5.613 1.4 0.358 41.430
239
Fig 5.2.7 Chromatogram and purity plot of acid stress sample
240
Fig 5.2.8 Chromatogram and purity plot of base stress sample
241
Fig 5.2.9 Chromatogram and purity plot of H2O2 stress sample
242
Fig 5.2.10 Chromatogram and purity plot of water stress sample
243
Fig 5.2.11 Chromatogram and purity plot of visible light stress sample
244
Fig 5.2.12 Chromatogram and purity plot of UV light stress sample
245
Fig 5.2.13 Chromatogram and purity plot of heat stress sample
246
Fig 5.2.14 Chromatogram and purity plot of humidity stress sample
3. Conclusion
247
A Stability-indicating UPLC analytical method has been developed for the determination of MT, AT
and RM in its fixed dose capsule dosage forms. The results of stress testing undertaken reveal that the method is
selective and stability-indicating. The proposed method is simple, accurate, precise, specific and has the ability
to separate the drug from degradation products. The method is suitable for the routine analysis of MT, AT and
RM in either bulk powder or in pharmaceutical dosage forms. The UPLC procedure can be applied to the
analysis of samples obtained during accelerated stability experiments to predict the expiry period of bulk drug
and pharmaceutical dosage forms.
Section (iii): Stability Indicating HPLC method for determination of impurities in
Metoprolol + Atorvastatin + Ramipril capsules
This section describes the various aspects related to method development and validation of stability
indicating HPLC methods (Method A related to MT impurities and Method B related to AT & RM impurities)
for determination of impurities in Metoprolol (MT) + Atorvastatin (AT) + Ramipril (RM) capsules.
1. Experimental
1.1. Chemicals
Active Pharmaceutical ingredient samples of MT, AT and RM as well as impurities are received from
bulk manufacturer of Dr. Reddy’s Laboratories Limited, Hyderabad, India. Finished product capsules received
from Formulation R&D, Dr. Reddy’s Laboratories Ltd. HPLC grade acetonitrile and methanol are purchased
from Merck, Germany. Analytical reagents monobasic potassium phosphate, disodium hydrogen phosphate,
orthophosphoric acid and sodium hydroxide are purchased from Merck, Germany. High pure water is prepared
by using Millipore Milli Q plus purification system. Chemical names and structures of actives and impurities are
mentioned in Table 5.3.1 to 5.3.3
248
Table 5.3.1 Chemical name and structure of MT and its three impurities
Name Structure IUPAC Name
Metoprolol
(RS)-1-(Isopropylamino)-3-[4-
(2-
methoxyethyl)phenoxy]propan-
2-ol
Impurity A
(±)1-(ethylamino)-3-[4-(2-
methoxyethyl) phenoxy]-
propan-2-ol
Impurity C
(±) 4-[2-hydroxy-3-(1-
methylethyl)aminopropoxy]ben
zaldehyde
Diol
(±)1,2-Hydroxy-3-[4-(2-
methoxyethyl) phenoxy]-
propane
Table 5.3.2 Chemical name and structure of AT and its two impurities
249
Name Structure IUPAC Name
Atrvastatin
NO
N H
F
O
O H O H O
2
C a + 2
1H-pyrrole-1-heptanoic acid,
[R-(R*,R*)]-2-(4-flurophenyl)-
β , d-dihydroxy-5-(1-
methylethyl)-3-phenyl-4-
[(phenylamino) carbonyl]-
calcium salt
Desfluoro
impurity
(3R,5R)-3,5-dihydroxy-7-[5-(1-
methylethyl)-2,3-diphenyl-4-
(phenylcarbamoyl)-1H-pyrrol-
1-yl]heptanoic acid
Lactone impurity
N
N H
O
O O
F
O H
(2R trans)-5-(4-fluoro phenyl)-
2-(1-methyl ethyl)-N,4-
diphenyl-1 [2-(tetrahydro-4-
hydroxy-6-oxo-2H-pyran-2-
yl)ethyl]-1H-pyrrole-3-
carboxamide
Table 5.3.3 Chemical name and structure of RM and its three impurities
Name Structure IUPAC Name
250
Ramipril
(1S,5S,7S)-8 [(2S)-2-[[(1S)-1-
ethoxycarbonyl-3-phenyl
propyl]amino]-
propanoyl]-
8azabicyclo[3.3.0]octane-7-
carboxylic acid
Impurity E
HN
N
O HCOOH
H
H
HH
O
HOCH3
(2S,3aS,6aS)-1-[(S)-2-[[(S)-1-
carboxy-3-phenylpropyl]-
amino]propanoyl]octahydrocycl
openta[b]pyrrole-2-carboxylic
acid (ramipril diacid)
Impurity N
ethyl (2S)-2-[(3S,5aS,8aS,9aS)-
3-methyl-1,4-dioxodecahydro-
2H-cyclopenta[4,5]pyrrolo[1,2-
a]pyrazin-2-yl]-4-
phenylbutanoate (ramipril
diketopiperazine)
Impurity D
N
HH
O
OC H 3
H 3 C
N
O
O
H
H
H
(2R,3aR,6aR)-1-[(S)-2-[[(S)-1-
(ethoxycarbonyl)-3-
phenylpropyl]amino]propanoyl]
octahydrocyclopenta[b]pyrrole-
2-carboxylic acid ((S,S-R,R,R)
isomer of ramipril
1.2. Determination of appropriate UV wavelength
The suitable wavelength for the determination of MT, AT, RM and its impurities is identified by taking the
overlay spectra from 200–400 nm for all impurities, MT, AT and RM from PDA detector.
251
1.3. Instrumentation and chromatographic conditions
The Waters HPLC system with a diode array detector is used for method development and forced
degradation studies. The output signal is monitored and processed using Empower software. Cintex digital water
bath is used for hydrolysis studies. Sanyo Photostability chamber is used for photo stability studies. Thermal
stability studies are performed in a Cintex, dry air oven. The pH of the solutions is measured by a Orion pH
meter.
In method A, the chromatographic column Vydac-C8 150 x 4.6 mm, 5µm is used for method
development and validation. Gradient method is developed with different ratios of buffer, water and acetonitrile.
Mobile Phase A contains 0.01M monobasic potassium phosphate buffer pH adjusted to 3.0 using
orthophosphoric acid solution. Mobile phase B contains water and acetonitrile in the ratio of 10:90 (v/v). The
flow rate of the mobile phase is 1.0 mL/min. The column temperature is maintained at 40°C and the wavelength
of 223 nm is used for detection of impurities of MT. The injection volume is 20 µL. Gradient program as follow:
Time in minutes %A %B
0 90 10
40 50 50
50 50 50
51 90 10
60 90 10
In method B, the chromatographic column Xterra RP-18, 250 x 4.6 mm, 5 µm is used for method
development and method validation. The buffer solution is prepared by using Sodium perchlorate and disodium
hydrogen phosphate in water, pH adjusted to 5.2 with dilute phosphoric acid. Gradient method is developed
with different ratios of buffer, acetonitrile and methanol. Mobile phase A consists of buffer and acetonitrile in
the ratio of 80:20 (v/v). Mobile phase B consists of buffer, acetonitrile and methanol in the ratio of 20:70:10
(v/v). The flow rate of the mobile phase is 0.8 mL/min. The column temperature is maintained at 60°C and the
wavelength of 210 nm is used for detection of both compound impurities. The injection volume is 20 µL.
Gradient programme is as follow:
Time (Minutes) Mobile Phase A (%) Mobile Phase B (%)
0.0 100 0.0
15.0 95.0 5.0
25.0 85.0 15.0
30.0 75.0 25.0
35.0 70.0 30.0
45.0 70.0 30.0
252
65.0 65.0 35.0
75.0 55.0 45.0
90.0 60.0 40.0
95.0 60.0 40.0
100.0 70.0 30.0
120.0 100.0 0.0
1.4. Diluent
Methanol.
1.5. Preparation of standard solution
MT standard solution (Method A): A standard stock solution is prepared by dissolving appropriate
amount of MT in diluent (600 µg/mL). Stock solution is further diluted with diluent to obtain a standard solution
of 6 µg/mL of MT. The typical chromatograms of diluent and standard are shown in Fig 5.3.1 & 5.3.2
Fig 5.3.1 Typical chromatogram of diluent of method A
Fig 5.3.2 Typical chromatogram of MT standard
AT and RM standard solution (Method B) : A standard stock solution of AT and RM is prepared in
diluent by dissolving appropriate quantity of AT and RM (600 µg/mL of AT and 300 µg/mL of RM). Stock
253
solution is further diluted with diluent to obtain a standard solution of 3µg/mL and 1.5 µg/mL respectively. The
typical chromatograms of diluent and standard are shown in Fig 5.3.3 & 5.3.4
Fig 5.3.3 Typical chromatogram of diluent of method B
Fig 5.3.4 Typical chromatogram of AT+RM standard
1.6. Preparation of Test solution
MT+AT+RM (50+10+5 mg) capsules contain MT in the form of pellets and AT+RM in
the form of powder. Open contents of capsules, separate MT pellets and AT+RM powder
using mesh. Pellets equivalent to 100 mg of MT is dissolved in diluent with aid of sonication
for 15 min to give a solution containing 2 mg/mL of MT. Part quantity of solution is
centrifuged and used for analysis. Placebo sample is prepared in the same way by taking the
placebo equivalent its weight present in a test preparation (Method A).
254
Blend powder equivalent to 100 mg of AT & 50 mg of RM is dissolved in diluent with
aid of sonication for 20 min to give a solution containing 1 mg/mL of AT and 0.5 mg/mL of
RM. Part quantity of solution is centrifuged and used for analysis. Placebo sample is prepared
in the same way by taking the placebo equivalent to its weight present in a test preparation
(Method B). The typical chromatograms of placebo and sample are shown in Fig 5.3.5 to
5.3.8
Fig 5.3.5 Typical chromatogram of Placebo (Method A)
Fig 5.3.6 Typical chromatogram of sample (Method A)
255
Fig 5.3.7 Typical chromatogram of Placebo (Method B)
Fig 5.3.8 Typical chromatogram of AT+RM sample (Method B)
1.7. Impurity stock preparations
Impurity stock solutions are prepared by accurately transferring about 20 mg each of MT impurities and
20 mg each of AT & RM impurities into two separate 100 mL volumetric flasks. 25 mL of diluent is added to
each of the above flasks. The impurities are dissolved with aid of sonication, both the stocks are made up to
volume with diluent and mixed to obtain stock solutions of 200 µg/mL each of MT impurities and 200 µg/mL
each of AT & RM impurities.
1.8. Specificity
As per ICH guideline, requires development and validation of stability indicating methods for all
pharmaceutical dosage forms. The current ICH guidelines do not describe degradation conditions for stress
study. The forced degradation conditions, stress agent concentration and time of stress, are found to effect the %
256
degradation. Preferably between 20% to 30% is recommended for active materials to make the right assessment
of stability indicating nature of the chromatographic methods. The optimization of such stress conditions which
can yield desired % degradation is based on experimental study. Chromatographic runs of placebo solution and
samples subjected to force degradation are performed in order to provide an indication of the stability indicating
properties and specificity of the method. The stress conditions employed are acid, base, neutral, peroxide, heat,
humidity and light. After the degradation treatments are completed, the samples are allowed to equilibrate to
room temperature, neutralized with acid or base (as necessary), and diluted with diluent to 2000 µg/mL of MT
(Method A); 1000 µg/mL of AT and 500 µg/mL of RM (Method B). Peak purity test is carried out for the MT,
AT and RM peaks by using PDA detector in stress samples. Specific conditions are described below:
1.8.1. Placebo (excipients) interference
Samples are prepared in duplicate by taking the weight of placebo approximately
equivalent to its weight in the test sample for both methods as described in the Test
preparation.
1.8.2. Effect of acid, base and neutral hydrolysis
Method A: Transferred MT pellets from capsules equivalent to 100 mg of MT into three individual 100
mL round bottom flasks, treated with 10 mL of 0.1N HCl, 0.1N NaOH and purified water for 3 hours at 60°C.
The samples are allowed to equilibrate to room temperature, neutralized acid and base samples as appropriate
and resulting solutions are prepared as per test procedure to obtain final concentration of 2.0 mg/mL of MT .
Method B: Transferred AT+RM contents from capsules equivalent to 100 mg of AT and 50 mg of RM
into three individual 100 mL round bottom flask, treated with 20 mL of 0.1N HCl, 0.1N NaOH and purified
water for 30 min at 60°C. The samples are allowed to equilibrate to room temperature, neutralized acid and base
samples as appropriate and resulting solutions are prepared as per test procedure to obtain final concentration of
1.0 mg/mL and 0.5 mg/mL of AT and RM respectively.
1.8.3. Effect of oxidation
257
Method A: Transferred MT pellets from capsules equivalent to 100 mg of MT into 100 mL round
bottom flask, treated with 10 mL of 3%H2O2 for 3 hours at 60ºC. The sample is allowed to room temperature and
resulting solution is prepared as per test procedure to obtain final concentration of 2.0 mg/mL of MT.
Method B: Transferred AT+RM contents from capsules equivalent to 100 mg AT and 50 mg of RM
into 100 mL round bottom flask, treated with 10 mL of 3%H2O2 for 30 minutes at 60ºC . The sample is allowed
to room temperature and resulting solution is prepared as per test procedure to obtain final concentration of 1.0
mg/mL and 0.5 mg/mL of AT and RM respectively.
1.8.4. Effect of humidity and heat
To evaluate the effect of moisture and heat, capsule contents (Pellets and powder) are distributed over a
petri dish and exposed to 25ºC/90% RH (Relative Humidity) for about 7 days. Similarly sample is kept in an
oven at 105ºC for 7 days for Method A and 105ºC for 24 hours for Method B. The stressed samples are used for
analysis by preparing the solution as described in the test preparation.
1.8.5. Effect of UV and visible light
To study the photochemical stability of the drug product capsule contents (Pellets and powder) are exposed
to 1200 K Lux of visible light and 200 W h/ m2 of UV light by using photo stability chamber. The stressed
samples are used for analysis by preparing the solution as described in the test preparation.
1.9. Method validation
1.9.1. Relative retention times and relative response factors
Relative retention times (RRT) and Relative response factors (RRF) are established for all the known
impurities of MT against MT in method A and all the known impurities of AT against AT and all known
impurities of RM against RM in method B .
RRFs are established as the ratio of slope of impurities and slope of respective active moiety peaks.
Slope value obtained with linearity calibration plots. Established RRT and RRF values of MT, AT and RM
impurities are shown in Table 5.3.4 and 5.3.5
Table 5.3.4 RRT and RRF values of MT impurities (Method A)
S.No. Name RRT RRF
1 Impurity A 0.92 0.96
2 Impurity C 0.56 0.81
258
3 Diol impurity 1.22 0.74
Table 5.3.5 RRT and RRF values of AT and RM impurities (Method B)
S.No. Name RRT RRF
1 Impurity E (Ramipril ) 0.18 0.76
2 Impurity N (Ramipril) 0.96 1.06
3 Impurity D (Ramipril) 1.87 0.99
4 Atorvastatin Desfluoro 1.67 0.98
5 Atorvastatin Lactone 3.00 0.96
1.9.2. Precision
The precision of the method is verified by analyzing six individual test preparations spiked with MT
impurities at 0.3% in method A and AT + RM samples spiked with 0.3% of AT & RM impurities in method B.
%RSD is calculated for each impurity. The intermediate precision (Inter-day precision) of the method A and B is
evaluated using two different HPLC systems and different HPLC columns in different days in the same
laboratory.
1.9.3 Limits of Detection (LOD) and Quantification (LOQ)
The LOD and LOQ for impurities of MT, AT and RM are estimated at a signal-to-noise ratio of about
3:1 and 10:1 respectively by injecting a series of diluted solutions with known concentration. Precision study is
carried at the LOQ level by injecting six individual preparations of all impurities. % RSD is calculated for each
impurity.
1.9.4. Linearity
Linearity solutions for the impurities are prepared by diluting impurity stock solution to get the
solutions of impurities having different concentrations. The solutions are prepared at different concentration
levels from LOQ to 200% of specification limit for all impurities. The peak area versus concentration data is
treated by least-squares linear regression analysis.
1.9.5. Accuracy
Recovery experiments are conducted to determine accuracy of method for the quantification of all
known impurities. The study is carried out in triplicate at LOQ, 25%, 50%, 100%, 150% and 200% of
specification limit. The % recovery is calculated for MT, AT and RM impurities.
259
1.9.6. Robustness
To determine the robustness of the method, experimental conditions are deliberately altered one after
other to establish for their effect. The relative retention times for all impurities and trailing factor for main
analyte peaks are verified. The effect of flow rate is studied at ± 0.2 mL/ min of actual flow rate. The effect of
column temperature is studied at ± 5 ºC. The effect of organic solvent present in mobile phase is studied by
varying acetonitrile percentage from -10% to +10% while the other mobile phase components are held constant.
The effect of pH of the buffer is studied at ± 0.2 pH.
1.9.7. Solution stability and mobile phase stability
The solution stability of MT, AT and RM standards and test preparation spiked with impurities is
established by allowing solutions on bench top at controlled room temperature for 24 hours. The solutions are
stored in volumetric flasks by tightly capping. The amounts of MT, AT and RM and its impurities in the above
solutions are measured. The stability of mobile phase is also determined by analyzing freshly prepared solution
of MT, AT and RM and its impurities at 24 hours interval for 48 hrs using same lot of mobile phase.
2. Results and discussion
2.1. Determination of suitable wavelength
Based on the UV spectra (Fig 5.3.9 & 5.3.10) MT and its impurities are having absorption maxima at
224 nm which is selected for quantification all the impurities of MT for method A. AT and its impurities are
having absorption maxima at 246 nm, where as RM is having absorption maxima at 210 nm. To get the optimum
absorbance for both AT, RM and its impurities, 210nm is selected for quantification all the impurities of AT and
RM in method B.
Fig 5.3.9 The UV spectra of MT and its impurities
260
Fig 5.3.10 The UV spectra of AT, RM and its impurities
2.2. Optimization of chromatographic conditions
The drug product consists three active moieties. Since polarity of components are different and
developing a single chromatographic method is not viable option. MT present as pellets and AT and RM is
present as blend powder filled in a capsules. Hence it is decided to separate metformin pellets and blend powder
and develop a two individual methods for determination of impurities in MT in one method and impurities in AT
and RM in another method.
Method A for determination of Metoprolol impurities in drug product
261
MT Impurity-A, C and Diol impurity are the potential impurities in MT. The main target of the
chromatographic method is to get the separation of all potential impurities along with AT and RM, since sample
preparation involves separation of MT pellets from AT+RM blend powder. The impurities and degradants are
estimated at specific wavelength of 223 nm. Initially separation is tried by using official methods of USP and
EP.
In EP Method (Acetate buffer) unknown impurity is merging with Diol impurity and Atorvastatin and
Ramipril peaks are eluted at longer time point i.e. after 80 minutes. In case of USP method (Sodium dodecyl
sulfate buffer) Diol impurity and few unknown impurities are not properly separated from MT peak and AT and
RM peaks are eluted at 25 minutes. USP method is selected for further optimization and tried various ratios of
USP mobile phases along with different flow rates but separation between MT, Diol impurity and unknown
impurities are is not up to the mark. Sodium dodecyl sulfate buffer is replaced with phosphate buffer and verified
separation. Better separation is observed in phosphate buffer but separation between unknown impurities needs
further improvement.
Based on above experiments isocratic elution is replaced with gradient elution mode and tried with
different pH range buffers in mobile phase with various ratios of organic modifiers like acetonitrile and methanol
using different gradient programs. Finally separation is achieved in following chromatographic conditions: RP
Vydac-C8 150 x 4.6 mm, 5 µm particle size column operated at 40°C with gradient elution at 1.0 mL/min using a
mobile phase buffer as 0.01M monobasic potassium phosphate pH adjusted to 3.0 using orthophosphoric acid
solution; detection wavelength at 223 nm; injection volume is 20 µL. The mobile phase A consists of pH 3.0
buffer; mobile phase B consists of water and acetonitrile 10:90 v/v. The LC gradient program is set as time
(min)/% mobile phase B: 0.01/10, 40/50, 50/50, 51/10 and 60/30. All the impurities are well separated and no
chromatographic interference observed due to the blank (diluent) or other excipients (placebo) at the retention
time of MT and impurities peaks. The relative retention time and relative response factors are evaluated for
impurities. The developed LC method is found to be specific for MT and their impurities.
Method B for determination of Ramipril and Atorvastatin impurities in drug product
Desmethoxy, Lactone impurities of AT, Impurity-E, N and D of RM are the potential impurities and
main target of the chromatographic method is to get the separation of all potential impurities in single
262
chromatographic condition. The impurities and degradants pertaining to individual active moiety are estimated at
specific wavelength of 210 nm.
Initially separation is tried by using RM BP method, but AT, AT Desfluoro and RM impurity D are
merging with each other. Also separation is not adequate between Lactone and few unknown impurities .The
separation of all impurities are tried using different mobile phases containing acetate and phosphate buffers like
ammonium acetate and Di sodium hydrogen phosphate along with various ratios of organic modifiers like
acetonitrile and methanol. Separation is not achieved in any of the above mentioned buffers and impurity peak
shapes are not symmetric.
Based on above results, experiments are performed by modifying BP method mobile phase by using
Hypersil BDS C18, 250 x 4.6 mm, 5 µm column with pH 5.0 sodium perchlorate and disodium hydrogen
phosphate buffer. Mobile phase A consists of buffer and acetonitrile in the ratio of 90:10 v/v. Mobile phase B
consists of buffer, acetonitrile and methanol in the ratio of 10:80:10 v/v/v. All the known impurity peaks are well
separated but Lactone is merging with one unknown impurity. Also desfluoro impurity is eluting very close to
AT. Experiments are conducted with different gradient programmes by using different mobile phase mixtures.
Separation of all the impurities from main actives is obtained but more number of gradient peaks are observed at
the retention times of unknown impurities.
Further tried with different columns by using different pH of the mobile phases and the separation
achieved with good baseline on reversed phase XTerra RP18, 250 x 4.6 mm, 5 µm column operated at 60°C
with gradient elution at 0.8 mL/min, detection wavelength 210 nm and injection volume 20 µL using a mobile
phase buffer as pH 5.2 phosphate buffer. The buffer prepared by dissolving 3.0 g of Sodium perchlorate and 1.0
g of Disodium hydrogen phosphate in 1000 mL of water, adjusted the pH to 5.2 with dilute phosphoric acid; The
mobile phase A consists of pH 5.2 buffer and Acetonitrile (80:20 v/v); mobile phase B consists of pH 5.2
buffer, acetonitrile and methanol (20:70:10 v/v/v). The LC gradient program is set as time (min)/% mobile
phase B: 0.01/0, 15/5, 25/15, 30/25, 35/30, 65/35, 75/45, 90/40, 95/40, 100/30 and 120/30. All AT, RM and
its known and unknown impurities are well separated with a resolution greater than 2. No chromatographic
interference due to the blank (diluent) and other excipients (placebo) at the retention time of AT, RM and all
impurities are observed.
The relative retention time and Relative response factors (RRF) are evaluated for impurities. The
developed LC method is found to be specific for AT and RM and their impurities.
263
2.3. Method validation
2.3.1. Precision
The % RSD of replicate test preparations spiked with impurities in both method A and
B ( Intra-day and inter-day precision study) is found to be less than 3.4 in method A and 3.1
in method B, conforming good precision of both methods. All values are well within the
acceptance criteria i.e. % RSD not more than 15.0 %. The data is presented in Table 5.3.6 to
5.3.9.
Table 5.3.6 Results of precision of test method for MT impurities (Method A)
TEST Impurity A
(%)
Impurity C
(%)
Diol Impurity
(%)
1 0.288 0.289 0.284
2 0.301 0.314 0.290
3 0.301 0.315 0.289
4 0.299 0.311 0.284
5 0.299 0.319 0.285
6 0.298 0.311 0.283
Mean 0.298 0.310 0.286
%RSD 1.6 3.4 1.0
Table 5.3.7 Results of precision of test method for AT & RM impurities (Method B)
TEST AT Desfluro
impurity
(%)
AT Lactine
(%)
RM –
Impurity N
(%)
RM –
Impurity E
(%)
RM – Impurity
D
(%)
1 0.538 1.252 0.269 3.694 1.674
2 0.529 1.244 0.276 3.722 1.669
3 0.534 1.235 0.273 3.756 1.655
4 0.530 1.227 0.273 3.687 1.650
5 0.536 1.243 0.280 3.777 1.662
264
6 0.533 1.228 0.270 3.699 1.674
Mean 0.533 1.238 0.274 3.723 1.664
%RSD 0.6 0.8 1.4 1.0 0.6
Table 5.3.8 Results of intermediate precision of test method for MT impurities
Table 5.3.9 Results of intermediate precision of test method for AT & RM impurities
(Method B)
TEST AT Desfluro
impurity
(%)
AT Lactone
(%)
RM –
Impurity N
(%)
RM –
Impurity E
(%)
RM –
Impurity D
(%)
1 0.448 0.834 0.287 3.219 1.443
2 0.443 0.911 0.303 3.3.6 1.384
3 0.429 0.870 0.302 3.249 1.366
4 0.433 0.796 0.305 3.271 1.410
5 0.462 0.802 0.276 3.309 1.541
6 0.461 0.807 0.276 3.438 1.563
Mean 0.446 0.837 0.289 3.299 1.451
%RSD 3.1 5.5 6.3 2.3 2.3
TEST Impurity A
(%)
Impurity C
(%)
Diol Impurity
(%)
1 0.319 0.297 0.349
2 0.315 0.297 0.336
3 0.316 0.296 0.352
4 0.317 0.290 0.348
5 0.314 0.294 0.345
6 0.321 0.299 0.360
Mean 0.317 0.296 0.348
%RSD 0.8 1.1 2.3
2.3.2. LOQ and LOD
The determined limit of detection
for MT, AT and RM impurities are reported
Table 5.3.10 LOD, LOQ and precision at LOQ for MT, AT and RM impurities
Name of the Impurity
Metoprolol
impurities
Impurity A
Impurity C
Diol impurity
Atorvastatin
impurities
Desfluoro impurity
Lactone
Ramipril
impurities
RM - Impurity
RM – Impurity
RM – Impurity
2.3.3. Linearity
A linear calibration plot for
to 200% specification limit of all impurities and
The results are shown in Fig
area and concentration of the analyte
260
The determined limit of detection (LOD), limit of quantification (LOQ) and precision at LOQ values
impurities are reported in Table 5.3.10.
Table 5.3.10 LOD, LOQ and precision at LOQ for MT, AT and RM impurities
Name of the Impurity
LOD
Concentration in
‘%’
S/N
ratio
Concentration
in ‘%’
A 0.0079 2.961 0.0239
0.0084 3.122 0.0255
Diol impurity 0.0087 2.945 0.0262
Desfluoro impurity 0.002 3.4 0.008
0.003 3.4 0.010
Impurity N 0.016 3.0 0.050
Impurity E 0.011 3.3 0.033
Impurity D 0.010 3.1 0.030
A linear calibration plot for MT, AT and RM impurities is obtained over the calibration range
to 200% specification limit of all impurities and the correlation co-efficient is found to be greater than 0.995.
5.3.11 and 5.3.13 indicates that an excellent correlation exists
area and concentration of the analyte for all the impurities.
and precision at LOQ values
Table 5.3.10 LOD, LOQ and precision at LOQ for MT, AT and RM impurities
LOQ
Concentration
S/N
ratio
%
RSD
LOQ
10.350 2.2
9.889 1.6
9.563 3.1
10.4 1.9
10.4 3.8
9.5 1.4
10.5 3.0
9.7 2.6
is obtained over the calibration range from LOQ
efficient is found to be greater than 0.995.
indicates that an excellent correlation exists between the peak
Fig 5.3.11 Linearity graphs of MT impurities
Fig 4.3.12 Linearity graphs of
261
Fig 5.3.11 Linearity graphs of MT impurities
Linearity graphs of AT impurities
Fig 5.3.13 Linearity graphs of
2.3.4. Accuracy
The percentage recoveries of all
85.0 to 115.0. The % recovery values for
2.3.5. Robustness
To determine the robustness of the developed method, experimental conditions
and the elution pattern, separation
impurities, RM and its impurities
conditions (flow rate, column temperature
all analytes are adequately resolved and elution orders remained unchanged.
all deliberately varied conditions along with original conditions are
262
Linearity graphs of RM and its impurities
The percentage recoveries of all known impurities in MT, AT+RM samples are
recovery values for MT, AT, RM impurities are presented in Table
To determine the robustness of the developed method, experimental conditions
elution pattern, separation between MT and its impurities in method A; separation
impurities, RM and its impurities in method B are recorded. In all the deliberate varied chromatographic
column temperature, pH of the buffer in mobile phase and composition of organic solvent),
re adequately resolved and elution orders remained unchanged. RRT of all the known impurities for
all deliberately varied conditions along with original conditions are summarized in Table
samples are found to be between
Table 5.3.11 and 5.3.12.
To determine the robustness of the developed method, experimental conditions are deliberately altered
in method A; separation between AT and its
In all the deliberate varied chromatographic
and composition of organic solvent),
all the known impurities for
able 5.3.13 and 5.3.14.
263
2.3.6. Solution stability and mobile phase stability
The stability of diluted standard solutions is estimated against freshly prepared standard and found that
solutions are stable up to 2 days, as the difference in assay is found to be less than 1.0% up to 1 day. The results
from test solution stability confirmed that the solution is stable for 24 hours on bench top for impurities
quantification analysis. The results are summarized in Table 5.3.15. The variability in the estimation of MT, AT
and RM impurities is within ± 0.04% during mobile phase stability experiments. The results from mobile phase
stability experiments confirmed that mobile phases are stable up to 48 hours for impurities quantification
analysis. The results are summarized in Table 5.3.16.
Table 5.3.11 Recovery results of MT impurities (Method A)
Table 5.3.12
Recovery results of AT & RM impurities (Method B)
Amount
spiked
% Recovery
AT-Desfluoro AT-Lactone RM-Impurity
N RM-Impurity E
RM-Impurity
D
LOQ 101.5±1.8 103.5±3.3 98.9±0.9
102.5±0.7
100.5±1.8
25%
107.3±1.7
109.6±1.4 95.9±0.5 103.0±1.1 98.5±2.7
50%
101.1±0.9
105.9±1.3 103.0±2.2 100.7±3.2 107.5±0.4
100% 99.4±2.4 103.2±3.2
108.3±1.9
105.6±2.2
109.2±2.2
Amount
spiked
% Recovery
MT- Impurity A MT- Impurity C MT+Diol impurity
LOQ 95.6±1.9 102.5±4.2 96.5±3.1
25%
101.5±1.6
104.4±2.4 97.4±3.2
50%
99.5±0.7
104.2±0.8 99.8±1.4
100% 100.3±2.6 104.2±1.8 99.3±2.3
150% 100.9±3.6 104.2±2.2 98.4±3.5
200% 99.2±4.2 104.0±1.5 95.8±0.9
264
150% 93.9±2.2 101.7±1.7
106.6±1.1
101.8±3.6
102.9±1.7
150% 88.4±3.2 94.2±2.5 102.9±0.6
110.5±2.5
100.6±0.9
Table 5.3.13 Results of Robustness study (Method A)
Impurity
Name
RRT’s of the impurities
As per the
method
conditions
Flow rate Column temperature pH of the buffer
0.8
mL/min
1.2
mL/min 35°C 45°C 2.8 3.2
MT-Impurity
A 0.92 0.93 0.91 0.92 0.90 0.91 0.90
MT-Impurity
C 0.56 0.60 0.55 0.58 0.53 0.57 0.56
MT-Diol
impurity 1.22 1.22 1.23 1.22 1.17 1.22 1.19
14
265
Table 5.3.14 Results of Robustness study (Method B)
Table 5.3.15 Results of test solution stability on bench top
Table 5.3.16 Results of mobile phase stability
Impurity Name
RRT’s of the impurities
As per the
method
conditions
Flow rate Column temperature pH of the buffer Acetonitrile composition
0.6
mL/min
1.0
mL/min 55°C 65°C 5.0 5.4
90% 110%
A B A B
AT-Desfluoro 1.67 1.55 1.65 1.65 1.61 1.65 1.74 1.63 1.69 1.80 1.56
AT- Lactone 3.00 2.66 2.91 2.91 2.82 3.11 3.15 2.85 3.15 3.26 2.71
RM - Impurity N 0.96 0.96 0.96 0.96 0.95 0.94 0.95 0.96 0.97 0.94 0.96
RM – Impurity E 0.18 0.19 0.17 0.17 0.17 0.18 0.18 0.16 0.18 0.18 0.17
RM – Impurity D 1.87 1.75 1.80 1.81 1.76 1.84 1.97 1.79 1.85 2.01 1.72
% of impurities
MT-Impurity A MT-Impurity C MT-Diol
impurity
Initial 0.4225 0.4313 0.5386
After 24h 0.3976 0.4125 0.5114
Difference from
Initial 0.025 0.0188 0.0272
% of impurities
AT-
Desfluoro
AT-
Lactone
RM -
Impurity N
RM –
Impurity E
RM –
Impurity D
Initial 0.52 0.52 0.25 3.41 1.63
After 24h 0.52 0.52 0.24 3.41 1.62
Difference
from Initial 0.00
0.02 0.01 0.0 0.0
% of MT impurities
266
MT-
Impurity
A
MT-Impurity C MT-Diol
impurity
Initial 0.306 0.292 0.348
After 48 h 0.316 0.285 0.350
Difference from
Initial 0.01 0.007 0.002
% of AT and RM impurities
AT-
Desflu
oro
AT-
Lactone RM -
Impurity N
RM –
Impurity E
RM – Impurity
D
Initial 0.52 0.52 0.25 3.41 1.63
After 48 h 0.53 0.51 0.23 3.39 1.65
Difference
from Initial 0.01 0.01 0.02 0.02 0.02
267
2.3.7. Results of specificity studies
All the placebo and stressed samples prepared are injected into the HPLC system
with photodiode array detector as per the described chromatographic conditions.
Chromatograms of placebo solutions have shown no peaks at the retention time of MT and AT+RM and its
impurities. This indicates that the excipients used in the formulation do not interfere in estimation of impurities
in MT+AT+RM capsules
Degradation is not observed significantly in light exposure, thermal, acid, base and water hydrolytic.
Degradation observed in oxidative study. All degradant peaks are well resolved from MT in method A and
AT+RM peaks in method B in the chromatograms of all stressed samples. The chromatograms of the stressed
samples are evaluated for peak purity of MT, AT and RM using Empower software. For all forced degradation
samples, purity angle for MT, AT and RM peaks are found to be less than purity threshold. This indicates that
there is no interference from degradants in quantitating the impurities in MT+AT+RM capsules. The %
degradation and peak purity details of MT, AT and RM peaks are summarised in Table 5.3.17. The data
indicates that there is no co elution of any degradants in the MT, AT and RM peaks and no impurity is missing.
Thus, these methods are considered "Stability indicating”. The chromatogram and purity plots of all stressed
samples are shown in Fig 5.3.14 to 5.3.29
268
Table 5.3.17 Summary of forced degradation studies
Stress conditions % degradation Purity Angle Peak Threshold
MT AT RM MT AT RM MT AT RM
Treated with 0.1 N HCI solution 0.11 0.42 0.95 0.212 0.92 0.140 1.008 0.308 0.327
Treated with 0.1 N NaOH solution 0.11 0.29 0.72 0.211 0.06 0.150 1.008 0.292 0.355
Treated with 3% H2O2 10.38 0.65 0.92 0.196 0.106 0.095 1.008 0.305 0.291
Exposed to Heat 0.04 0.28 0.42 0.207 0.657 0.054 1.008 3.850 0.276
Treated with purified water 0.07 0.47 0.85 0.234 0.107 0.150 1.007 0.380 0.355
Exposed to UV light 0.04 0.41 0.25 0.213 0.072 0.138 1.007 0.285 0.282
Exposed visible light 0.05 0.39 0.70 0.208 0.076 0.112 1.007 0.291 0.307
Exposed to humidity at 25 °C, 90% RH 0.03 0.20 0.32 0.203 0.362 0.048 1.015 5.057 0.277
269
Fig 5.3.14 Chromatogram and purity plots of acid stress MT sample.
270
Fig 5.3.15 Chromatogram and purity plots of base stress MT sample
Fig 5.3.16 Chromatogram and purity plot of peroxide stress MT sample
271
Fig 5.3.17 Chromatogram and purity plot of water stress MT sample
Fig 5.3.18 Chromatogram and purity plot of visible light stress MT sample
272
Fig 5.3.19 Chromatogram and purity plot of UV light stress MT sample
Fig 5.3.2 Chromatogram and purity plot of heat stress MT sample
273
Fig 5.3.21 Chromatogram and purity plot of humidity stress MT test
274
Fig 5.3.22 Chromatogram and purity plots of acid stressed AT+RM sample
275
Fig 5.3.23 Chromatogram and purity plots of base stressed AT+RM sample
276
Fig 5.3.24 Chromatogram and purity plots of peroxide stressed AT+RM sample
277
Fig 5.3.25 Chromatogram and purity plots of water stressed AT+RM sample
278
Fig 5.3.26 Chromatogram and purity plots of visible light stressed AT+RM sample
279
Fig 5.3.27 Chromatogram and purity plots of UV light stressed AT+RM sample
280
Fig 5.3.28 Chromatogram and purity plots of heat stressed AT+RM sample
281
Fig 5.3.29 Chromatogram and purity plots of humidity stressed AT+RM sample
282
3. Conclusion
Two RP-LC methods are developed for determination of degradants and impurity in MT+AT+RM capsules.
First method determines the MT impurities and second method determines the RM and AT impurities. Both methods
are precise, accurate, sensitive, specific, robust and rugged. Both stability indicating methods are validated as per
International Conference on Harmonization and capable to determine degradation products and process- related
impurities of MT+AT+RM capsules. The product is subjected to various stress conditions and peak purity is studied
for active moiety and found to be less than the threshold. This demonstrates the stability- indicating power of both
methods and very much useful for checking the quality of product during stability studies. These methods can be
used for determination of impurities in MT+AT+RM in capsule dosage form and their combination of any of these
drugs.
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