CHAPTER –CHAPTER –––VVVV - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/33339/10/10...H CH3 H3CO CH3. OH HO OH O O. 2 Metoprolol, Atorvastatin and Ramipril are available

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


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


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


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.


2.3.1. 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


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


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


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


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


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.


1.9.1. 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.


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


2.3.1. 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.


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


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


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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CHAPTER –CHAPTER –––VVVV - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/33339/10/10...H CH3 H3CO CH3. OH HO OH O O. 2 Metoprolol, Atorvastatin and Ramipril are available