CHAPTER – 7 DETERMINATION OF RELATED SUBSTANCES...

CHAPTER – 7

DETERMINATION OF RELATED SUBSTANCES OF NICORANDIL IN

TABLET DOSAGE FORM BY USING REVERSE PHASE HIGH PERFORMANCE

LIQUID CHROMATOGRAPHY

CHAPTER – 7

SPP SPTM, SVKM’s NMIMS, Mumbai 156

CHAPTER – 7

Determination of related substances of Nicorandil in tablet dosage form by using reverse phase high performance liquid chromatography

7.0 INTRODUCTION The objective was to develop a method for determination of related substances of

Nicorandil in tablet dosage form. The method was validated as per ICH guidelines Q2

(R1).

7.1 Drug Profile Nicorandil, (2-[(pyridin-3-ylcarbonyl) amino]ethyl nitrate), is a nicotin amide derivative

used as vasodilatory drug used in treatment of angina. The drug act through two

methods, firstly, by activating potassium channels, and secondly by donating nitric oxide

to activate the enzyme guanylate cyclase. This enzyme causes activation of cGMP

leading to both arterial and venous vasodilatation by de -phosphorylation of the myosin

light chain. As it is selective for vascular potassium channels, it has no significant action

on cardiac contractility and conduction. This is a novel drug for treatment of angina

pectoris [24]. Further many studies have suggested that the drug possess similar safety and

efficacy as the other drugs used for angina but efficacy increase after a year on continued

treatment [25, 26]. The drug has also now been evaluated in combination with other drugs

like Lamotrigine [27].

Nicorandil is not official in IP, USP 35 and EP/ BP 2012 General Name : Nicorandil Chemical Structure :

CHAPTER – 7


Chemical Name : 2-[(pyridin-3-ylcarbonyl)amino]ethyl nitrate Molecular Formula : C8H9N3O4 Molecular Weight : 211.175 CAS Number : 65141-46-0 Description : White or slightly yellowish, crystalline powder Solubility : Slightly soluble in water, freely soluble in ethanol (95%), in acetone and chloroform. Drug Category : Antianginal; Potassium Channel Activator/Opener 7.2 LITERATURE SURVEY

The literature revealed that the assay of the drug in pure and dosage forms is not official

in any pharmacopeia and, therefore, requires much more investigation. The estimation of

Nicorandil from biological fluids and/or pharmaceutical formulations has been

conducted using several analytical methods include high-performance thin layer

chromatography [28,29,30] high-performance liquid chromatography [31,32,33,34,35,36,37] and

gas chromatography coupled with mass spectrometry[38]. A review of literature revealed

that there was no related substances method available for detection of known and

unknown impurities by HPLC in tablet dosage form. Only the method provided by drug

supplier for detection of related impurities was available for reference. The details of the

method are given below.

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Table 7.2.1: Related Substances method provided by drug supplier

API Supplier - method

Method HPLC

Column C18

250mm x 4.6mm; 5µ;

Column Temp Not Mentioned

Mobile Phase 70 parts of 0.01M

disodium hydrogen

phosphate and 30 parts

of methanol; adjust pH

to 7.0 with o-

phosphoric acid

Gradient/ Run

time

Three times of the

main peak

Flow Rate 1ml/min

Wavelength 215nm

Inj Vol (µL) 20

CHAPTER – 7

SPP, SPTM, SVKM’s NMIMS, Mumbai 159

7.3 PRESENT WORK AND DISCUSSION 7.3.1 Selection of Chromatographic Method The method available with supplier for Nicorandil was based on reverse phase

chromatographic (RPC) separation. So, all development was conducted using reverse

phase chromatography. Reverse Phase chromatography is a choice because of its ease of

handling and robust nature.

7.3.2 Selection of Stationary Phase

The method available with supplier was based on C18 column. Different reversed phase

columns like C18 and C8 were used as stationary phase to get desired retention. Trials

were conducted by using C8 column because of longer retention time of Nicorandil in

C18 column.

7.3.3 Selection of Wavelength for Analysis

Photo Diode Detector (PDA) was used and Lambda Max was used to select the optimum

wavelength used for analysis. 262 nm was found to be suitable.

Figure: 7.3.3.1 UV spectra of Nicorandil

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7.3.4 Selection and Optimization of Mobile Phase The method given by supplier was used initially for the separation of Nicorandil and its

impurities on C18 column. The isocratic elution led to poor resolution for initial eluting

impurities (Impurity 1 & 2) and broader peak shape for Nicorandil. To overcome this

problem, gradient elution program was tried. It was observed that gradient program

improved resolution between impurities 1 & 2 but led to poor peak shape for Nicorandil.

Various gradient elations were tried to improve peak shape of Nicorandil but the results

were not satisfactory. Further trials were planned by changing stationary phase from C18

to C8. It dramatically improved peak shape for Nicorandil. The peak shapes for

Nicorandil and its related impurities were further improved by addition of acetonitrile

along with methanol. The optimized chromatographic conditions are given below.

Optimized Chromatographic Conditions:

Chromatographic condition for related substances:

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

Module, (UV/PDA), or equivalent.

Analytical Balance, Make –Mettler Toledo, Model-

XS205DU, or equivalent.

Column : Inertsil C8-3, 250 x 4.6 mm, 5µm or equivalent

Flow rate : 1.0 ml/minute

Column temperature : 30°C

Wavelength : 262 nm

Sample temperature : 15°C

Injection volume : 20 l

Run time for sample : 35 minutes

Retention time : About 17 minutes

Diluent : Mixture of Water: Methanol in the ratio 50:50 (v/v), mix

and degas.

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Gradient Program:

Time in minutes Mobile phase A % Mobile phase B % Mobile phase C %

0 90 6 4

5 90 6 4

15 70 20 10

25 70 20 10

26 90 6 4

35 90 6 4

Mobile phase A: Weigh accurately 1.42 gm of Disodium hydrogen phosphate anhydrous

transfer into 1000 ml of water. Adjust the pH 6.4 with ortho phosphoric acid. Filter through

0.45 nylonmembrane filter and sonnicate to degas.

Mobile phase B: Acetonitrile

Mobile phase C: Methanol

7.4 FORCED DEGRADATION STUDIES

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

Nicorandil. They were carried out and chromatographed along with a non-stressed

sample (control).

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

Acid degradation

5 tablets of Nicorandil were weighed into 100 ml amber colour volumetric flask, to this

10 ml of diluent was added and sonnicated for 15minutes. 2 ml of 5N Hydrochloric acid

was further added. The solution was heated on the water bath at 70°C for 3 hours. The

solution was cooled and neutralized with same volume and same strength alkali. The

solution was made up to the volume with diluent. Placebo was weighed equivalent to

tablet and treated similar to sample.

The sample as well as the placebo was injected into HPLC system.

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Base Degradation


10 ml of diluent was added and sonnicated for 15minutes. 2 ml of 0.5N sodium

hydroxide was further added. The solution was heated on the water bath at 70°C for

1hour. The solution was cooled and neutralized with same volume and same strength

alkali. The solution was made up to the volume with diluent. Placebo was weighed

equivalent to tablet and treated as sample.


7.4.2 Oxidative condition: hydrogen peroxide-induced degradation.


10 ml of diluent was added and sonnicated for 15minutes. 1 ml of 50 % Hydrogen

peroxide was further added. The solution was heated on the water bath at 70°C for

45minutes. The solution was cooled and made up to the volume with diluent. Placebo

was weighed equivalent to tablet and treated as sample.


7.4.3 Thermal degradation.


10 ml of diluent was added and sonnicated for 15minutes. The solution was heated on

the water bath at 70°C for 60minutes. The solution was cooled and made up to the

volume with diluent. Placebo was weighed equivalent to tablet and treated as sample.


7.4.4 Photolytic degradation.

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

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

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

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

For photo stability testing 5 tablet of Nicorandil was transferred to each of 100 ml clear

volumetric flask, flask covered with aluminium foil and amber coloured flask. To each

flask, 10 ml of diluent was added and sonnicated for15minutes. The flasks were kept

under UV and white light for 1.2 million lux hours in photo stability chamber. After

study the sample was cooled and diluted up to the mark with diluent. Placebo was treated

CHAPTER – 7


similarly.


7.4.5 Observations in forced degradation studies.

It was observed that overall Nicorandil is a highly degradable molecule. It degraded in

all conditions.

The quickest degradation was observed under basic conditions. It was very difficult to

control the reaction. The solution had turned brown in a short while. However the colour

disappeared and become colourless once more when the degraded sample was

neutralized with acid. This suggested that the reaction with base was reversible with

addition of acid. The main degradants were observed at RRT of about 0.28 and 0.37.

Under acidic conditions the degradation took some time to be achieved and the reaction

was easier to control. The main degradants were observed at RRTs of about 0.28, 0.37

and 1.06.

Under oxidative conditions the main degradant observed was at RRT of about 0.28.

Under thermal conditions the main degradant was again observed at RRT of about 0.28

suggesting that it is the primary degradant for Nicorandil molecule since this was

obtained in all stress conditions.

Under photolytic conditions, apparently, degradation was observed. However it was

noted that the control sample had also degraded equally. This indicated that the

degradation is due to the inherent instability of the sample solution over a period of about

seven days (time taken for completion of exposure to stipulated light energy) and not due

to photolytic effect.

For identification of major degradants an LC-MS compatible method was developed.

The method details and finding are given in the next sections.

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Table: 7.4.1 Samples injected under different stress conditions

Condition % Degradation

Acidic 10.49%

Basic 7.16%

Oxidation 4.61%

Thermal 9.27%

Photolytic Not significant with respect to control

The chromatograms are given below in the following figures.

Figure-7.4.1: Chromatogram of sample in Acid.

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Figure-7.4.2: Chromatogram of sample in Base.

Figure-7.4.3: Chromatogram of sample in Peroxide.

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7.5 Identification of the major degradants by LC-MS

Nicorandil was susceptible under all stress conditions. Total three degradants were

generated under stress degradation. Out of these three degradants, two degradants eluted

at same retention time as that of impurities 1 & 2 while third degradant was eluted after

the peak of Nicorandil. All the three major degradants were identified with the help of

LC-MS

The phosphate buffer which was used in HPLC system was not compatible with MS

detectors. Hence phosphate buffer was replaced with Ammonium acetate buffer. Rest of

the method parameters were kept same.

The optimized conditions for LC-MS

Column : Inertsil C8-3, 250 x 4.6 mm, 5µm or equivalent

Flow rate : 1.0 ml/minute

Column temperature : 30°C

Wavelength : 262 nm

Sample temperature : 15°C

Injection volume : 20 l

Run time for sample : 35 minutes

Retention time : About 17 minutes

Diluent : Mixture of Water: Methanol in the ratio 50:50 (v/v), mix

and degas.

Gradient Program:

Time in minutes Mobile phase A % Mobile phase B % Mobile phase C %

0 90 6 4

5 90 6 4

15 70 20 10

25 70 20 10

26 90 6 4

35 90 6 4

Mobile phase A: Pipette 2ml glacial acetic acid in 1000ml water. Adjust the pH 6.4 with

ammonia solution. Filter through 0.45 Nylonmembrane filter and sonnicate to degas.

CHAPTER – 7


Mobile phase B: Acetonitrile

Mobile phase C: Methanol

Optimized conditions of mass spectrometer

Interface :ESI

Interface Temperature :350 C

DL Temperature :300 C

Nebulizing Gas Flow :3.00 L/min

Heat Block :400 C

Drying Gas :On

Drying Gas Flow :15.00 L/min

CHARACTERIZATION OF DEGRADATION PRODUCTS BY LC-MS

The LC-MS spectra for Nicorandil and its degradants (impurities 1, 2 & 3) were recorded

by using positive mode of electrospray ionization (ESI). The output of mass spectrometer

was validated by injecting standard drug solution of Nicorandil. The obtained m/z value

212 was found to be exactly matching with molecular weight of Nicorandil (M+H).

Following figure depicts LC-MS spectra of Nicorandil.

Figure 7.5.1: LC-MS spectra of Nicorandil in ESI positive mode

The mass spectra of impurity 1, 2 and 3 were depicted in following figures

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A

B

C Figure 7.5.2: LC-MS spectra of impurity 1 (A), 2 (B) and 3(C) recorded in positive mode of electrospray ionization From the results of LC-MS and route of synthesis, obtained from US Patent 4200640 and Baker data, structure of the impurities were assigned for impurity 1, 2 and 3. The structures are depicted in figure 7.5.3.

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Impurity 1 Impurity 2

Impurity 3 Figure 7.5.3: Assigned structure of impurities. Impurity 1 This is a primary degradation product from Nicorandil, which is generated after the

aqueous hydrolysis of amide linkage of Nicorandil. Nicotinic acid, is itself used as a drug

and thus its toxicity is well established. ORAL (LD50): Acute: 7000 mg/kg [Rat]

Impurity 2

This is a primary degradation product from Nicorandil, which is generated after the

elimination of –NO2 from the Nicorandil main moiety.

Impurity 3

This is not considered as a degradation product as this molecule can only be formed from

the esterification of Nicotinic acid (impurity 1) with methanol. When studied with

alternate diluents, (Acetonitrile), the peak due to impurity 3 was not observed. Thus it

was concluded that the impurity 3 is not a degradation product of Nicorandil

CHAPTER – 7


With Methanol in Diluent (Acid Degradation)

With Acetonitrile in Diluent (Acid Degradation). No peak was observed. Figure 7.5.4: Acid degradation using methanol and acetonitrile as diluent respectively.

CHAPTER – 7


Figure 7.5.5: Nicorandil Route of Synthesis

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7.6 EXPERIMENTAL WORK 7.6.1 Instrumentation

Equipment Make Model

HPLC Waters 2695Alliance Separation

Module, (PDA/UV

Detector) 2996/2487

Column AKZO

NOBEL

Inertsil C8-3,

250 x 4.6 mm, 5µm

pH meter Thermo

Electron

Corp.

Orion-4star 1117000

Analytical

Balance

Mettler

Toledo

XS205DU

Micro Balance Mettler

Toledo

UMX-2

Ultrasonnicator Spectralab -

Photostability

Chamber

Thermolab 400litr

Water Bath Spectralab

7.6.2 Chemicals and Reagents

Name Grade Manufacturer

Disodium Hydrogen phosphate HPLC grade Merck

Acetonitrile HPLC Gradient grade Rankem Methanol HPLC Gradient grade Merck

Ortho Phosphoric acid GR Merck

Sodium Hydroxide GR Merck Hydrochloric acid GR Merck Hydrogen peroxide GR Merck Water HPLC milli-Q In-house

CHAPTER – 7


7.6.3 Working Standard

Working Standard:

Standard Lot .No. Potency (as is) %

Nicorandil 6712008002 99.4

Test Sample:

Batch. No. Label claim

NCT/20/23 20mg

Placebo:

Batch. No.

NCT/23P

7.6.4 Solution Preparation

Preparation of Standard solution:

Accurately weigh and transfer about 42.0 mg of Nicorandil standard and transfer to a 250 ml

amber coloured volumetric flask. Add 100 ml of diluent, sonnicate to dissolve and dilute to

volume with diluent. Dilute 3 ml of this solution to 250 ml with diluent.

Preparation of Sample solution:

Weigh 5 tablets and transfer these tablets into 100ml amber coloured volumetric flask, add

70ml of diluent and sonnicate it for 15minutes. Allow it to cool at room temperature. Make

up the volume up to the mark with diluent. Filter the sample solution through 0.45 Nylon

membrane filter.

Preparation of Placebo solution:

Weigh accurately placebo equivalent to 5 tablets (450 mg) and transfer into 100 ml amber

coloured volumetric flask, add 70ml of diluent and sonnicate it for 15minutes. Allow it to cool

at room temperature. Make up the volume up to the mark with diluent. Filter the sample

solution through 0.45 Nylon membrane filter.

CHAPTER – 7


Evaluation of System suitability:

Inject the standard solution six times. The relative standard deviation of six replicate injections

should not be more than 5.0%. The USP tailing factor for Nicorandil peak should not be more

than 2.0. The USP plates should not be less than 50000.

Procedure:

Inject equal volumes of Blank (diluent), Standard solution (6 replicate), placebo solution and

sample solution.

Calculation Formula:

AT WS 3 100 P % Impurity = -------- x --------- x ------ x --------------------- x -------- AS 250 250 Wt. of 5 Tablets LC

Where,

AT = Area count of Impurity in the sample solution.

AS = Area count of peak due to Nicorandil in standard preparation.

WS = Weight of Nicorandil standard in mg.

LC = Label claim of Nicorandil per tablet in mg.

P = Potency of Nicorandil working standard on as is basis.

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7.7 VALIDATION OF THE DEVELOPED METHOD 7.7.1 Validation parameters and acceptance criteria The Table 7.7.1.1 summarizes the validation acceptance criteria along with the obtained results.

Table 7.7.1.1: Validation Summary

Sr.No. Parameters Acceptance criteria Result obtained

1.0 System suitability

% RSD for Standard solution.

USP Tailing Factor

USP Plates

NMT 5.0 %

NMT 2.0

NLT 50000

1.28

1.14

84820

2.0

2.1

Specificity

Identification

Results should be

comparable with respect to

the retention time and

relative retention time.

Complies

2.2

Interference

No interference from blank

and placebo to main

component and impurities

Complies

2.3

Peak purity

Purity angle should be less

than purity threshold.

Standard peak should be

pure.

Complies

2.4

Forced degradation

The peak due to major

impurities should be pure

as shown on the PDA.

Complies

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Table 7.7.1.1: Validation Summary (continued) Sr. No. Parameters Acceptance criteria Result obtained

3.0 Limit of Detection

% RSD for LOD:

between 10% and 33%

% RSD Concentration

(%)

11.70 0.005

4.0 Limit of Quantitation % RSD for LOQ: NMT

10% 1.71 0.015

5.0 Linearity

Response should be

Linear Response is linear

Correlation coefficient

should not be less than

0.99.

1.0000

Y- Intercept should be

within

± 10.0% of the

corresponding Y-co-

ordinate of the working

level.

Complies

6.0 Accuracy (Recovery)

At LOQ Level mean

recovery should be in the

range 75.0 % to 125.0 %.

% Mean Recovery

97.8

Mean recovery should be

in the range of 80.0%-

120.0%.

100.5

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7.0

7.1

7.2

System Precision

System suitability

% RSD for Standard

solution.

Should be fulfilled

NMT 5.0 %

Complies

1.28

Method Precision

RSD for % Single max

Impurity content.

NMT 10.0%.

RRT-0.27 RRT-0.97

1.66 5.11

8.0 Intermediate Precision

(Ruggedness)

System suitability

% RSD for Standard

solution.

RSD for % Single max

Impurity content

RSD for pooled result

( Analyst-I and II )

Should be fulfilled

NMT 5.0 %

NMT 10.0%.

NMT 10.0%.

Complies

RRT-0.27 RRT-0.97

1.58 8.21

2.86 8.46

9.0 Stability in analytical

solution

The RSD of Initial and

after x hours result NMT

10.0%.

If the peak amount is

below 0.2% the initial

and final should not be

more than 0.05% apart.

Sample is stable for 6 hours

at 15°C

CHAPTER – 7



10.0 Robustness

Change in Flow rate

(± 0.1 ml/min)

Change in wavelength

(± 5 nm)

Change in Buffer pH

(± 0.2)

Column oven temperature

(± 5°C)

There should be no

significant change in

system suitability

parameters.

There should be no


system suitability

parameters.

There should be no


system suitability

parameters.

There should be no


system suitability

parameters.

No significant change




11.0 Filter compatibility % Difference for impurity

content of Centrifugate and

filtered should be 0.05.

Complies

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

made on the system. The data obtained is summarized in Table. 7.6.2.1 The data demonstrate

that the Tailing factor is not more than 2.0, USP plates are less than 50000 and % RSD for

Nicorandil in Standard solution is less than 5.0%.Thus the system was suitable.

Table 7.6.2.1: System suitability

Standard Solution

USP Tailing 1.14

USP Plates 84820

Area

39475

38633

40109

39083

39114

39525

Mean 39323

SD 501.81

%RSD 1.28

7.7.3 Specificity:

The Specificity study included Identification of the main peak, Interference study and

Peak Purity.

Blank (diluent), Placebo solution, Standard solution and sample solution were injected. The

data obtained is summarized in Table 7.7.3.1.

Table 7.6.3.1: Specificity (Identification and Interference)

Component Retention

time (min)

RRT Purity

Nicorandil 18.018 1.00 Yes

impurity-1 4.947 0.27 Yes

impurity-2 6.375 0.35 Yes

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The data demonstrate that there is no interference in blank, placebo, Nicorandil and unknown

impurities peaks. All peaks are well resolved.

Chromatograms of Blank (diluent), placebo, Standard solution and Sample solution are given

in following figures.

Figure-7.6.3.1: Chromatogram of Blank

Figure-7.6.3.2: Chromatogram of Placebo

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Figure-7.6.3.3: Chromatogram of Standard

Figure-7.6.3.4: Chromatogram of Sample

Forced degradation:

Forced degradation was conducted under different stress conditions to ascertain that no

major degradants co-elute with the main peak or each other.

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7.7.4 Determination of Limit of Detection (LOD) and Limit of Quantitation (LOQ):

For determining LOD and LOQ, A series of dilutions with decreasing concentrations were

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

plotted and SLOPE of the line was calculated. Also the STEYX of this line (Correction for

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

The prediction linearity data obtained are summarized in Table 7.6.4.1.

Table 7.6.4.1: Prediction Linearity

Sr.No.

Nicorandil

Concentration

(ppm) Area

1 0.512 9695

2 0.409 7656

3 0.307 5491

4 0.205 4058

5 0.102 2340

CORREL 0.9972

STEYX 250.07

SLOPE 17890.09

PREDICTED LOD (ppm) 0.05

PREDICTED LOQ (ppm) 0.14

PREDICTED LOD (%) 0.005

PREDICTED LOQ (%) 0.014

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

and calculated % RSD are summarized in Table 7.6.4.2 and 7.6.4.3. A percentage RSD of

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

LOD. Both the criteria had been fulfilled.

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Table 7.6.4.2: Precision for LOD.

Sr.No. Nicorandil

Conc. (ppm) 0.05

Conc. w.r.t sample (%) 0.005

995

1138

1115

927

824

1008

Mean 1001

SD 117.18

% RSD 11.70

Table 7.6.4.3: Precision for LOQ.

Sr.No. Nicorandil

Conc. (ppm) 0.15

Conc. w.r.t sample (%) 0.015

2918

2939

2911

2973

2918

3042

Mean 2950

SD 50.36

% RSD 1.71

7.7.5 Linearity and Range:

The Linearity of response was determined by preparing different concentrations of standard

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

in Table 7.6.5.1 demonstrates that the linearity had been achieved.

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Table 7.6.5.1: Linearity of Nicorandil

Level (%) Concentration

w.r.t sample (%) Area

LOQ 0.015 2928

25 0.251 49484

50 0.501 99000

70 0.702 138102

80 0.802 158904

100 1.002 196019

120 1.203 239186

160 1.604 318596

200 2.004 396481

CORRELATION

COEFFICIENT (r) 1.0000

Y-INTERCEPT -400.807

SLOPE 198238.06

MEDIAN (AREA) 158904

LIMIT OF Y-INTERCEPT ± 10% OF MEDIAN

15890

Figure 7.6.5.1 denotes the linearity plot

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

0.000 0.500 1.000 1.500 2.000 2.500

CONCENTRATION (%)

RE

SP

ON

SE

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7.7.6 Accuracy:

Thirteen samples of dosage in form of spiked placebo with Nicorandil at 4 different levels;

each level in triplicate were prepared. Single placebo preparation (un-spiked), 3x LOQ, 3x

50%, 3x 100% and 3x 200% spiked placebo of the limit concentration were prepared. From

the amount added and the amount found, percentage recovery was calculated. The mean

recovery and % RSD were calculated. The results obtained are summarized in Table 7.6.6.1

demonstrates that the data was well within acceptance criteria.

Table 7.6.6.1: Accuracy for Nicorandil

Level

%

Amount

added

%

Response

Amount

recovered

%

% Recovery

Mean

recovery

%

LOQ

0.015 3037 0.015 100.0

97.8 0.015 3108 0.015 100.0

0.015 2873 0.014 93.3

50 %

0.501 101374 0.503 100.4

100.5

0.501 101023 0.501 100.0

0.501 102233 0.507 101.2

100 %

1.002 202409 1.003 100.1

1.002 203369 1.008 100.6

1.002 203946 1.011 100.9

200 %

2.004 411040 2.038 101.7

2.004 405620 2.011 100.3

2.004 402812 1.997 99.7

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7.7.7 Precision

7.7.7.1 System Precision:

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

made on the system. Please refer to Table 7.6.2.1. All the data were acceptable as per the

system suitability requirements.

7.7.7.2 Method Precision:

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

summarized in Table 7.6.7.2.1. The values are well within acceptable range.

Table 7.6.7.2.1: Method precision

Spl. No. % Unknown impurity % Total

impuritiesRRT-0.27

RRT-0.35

RRT-0.67

RRT-0.97

RRT-1.06

1 0.50 0.21 0.12 0.47 0.05 1.35

2 0.50 0.22 0.13 0.48 0.05 1.38

3 0.49 0.22 0.13 0.50 0.05 1.39

4 0.50 0.22 0.13 0.50 0.05 1.40

5 0.49 0.22 0.14 0.52 0.05 1.42

6 0.48 0.23 0.13 0.54 0.05 1.43

Mean 0.49 0.22 0.13 0.50 0.05 1.40

SD 0.008 0.0063 0.0063 0.0256 0.0000 0.0288

% RSD 1.66 2.87 4.87 5.11 0.00 2.07

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7.7.7.3 Intermediate Precision (Ruggedness):

Same procedure of system precision and method precision is followed by another Analyst on

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

in Table 7.6.7.3.1 (system suitability) and 7.6.7.3.2. All values are well within acceptance

criteria.

Table 7.6.7.3.1: System suitability

Standard Solution

USP Tailing 1.02

USP Plates 94123

Area

38856

38954

38566

38527

38523

38560

Mean 38664

SD 189.75

%RSD 0.49

Table 7.6.7.3.2: Intermediate precision (Ruggedness)

Spl. No. % Unknown impurity % Total

impurities RRT-0.27

RRT-0.35

RRT-0.67

RRT-0.97

RRT-1.06

1 0.51 0.23 0.14 0.40 0.04 1.32

2 0.51 0.23 0.13 0.42 0.05 1.34

3 0.51 0.24 0.14 0.45 0.05 1.39

4 0.52 0.24 0.14 0.47 0.05 1.42

5 0.53 0.24 0.14 0.49 0.05 1.45

6 0.52 0.24 0.14 0.49 0.05 1.44

Mean 0.52 0.24 0.14 0.45 0.05 1.39

SD 0.008 0.0052 0.0041 0.0372 0.0041 0.0535

% RSD 1.58 2.18 2.95 8.21 8.45 3.84

CHAPTER – 7


The pooled data obtained from Analyst-I and Analyst-II is summarized in Table 7.6.7.3.3. The

data demonstrates that the percentage RSD of individually prepared twelve samples are in

acceptable limits.

Table 7.6.7.3.3: Pooled data

Analyst % Unknown impurity % Total

impurities RRT-0.27

RRT-0.35

RRT-0.67

RRT-0.97

RRT-1.06

I

0.51 0.23 0.14 0.40 0.04 0.51

0.51 0.23 0.13 0.42 0.05 0.51

0.51 0.24 0.14 0.45 0.05 0.51

0.52 0.24 0.14 0.47 0.05 0.52

0.53 0.24 0.14 0.49 0.05 0.53

0.52 0.24 0.14 0.51 0.05 0.52

II

0.50 0.21 0.12 0.47 0.05 0.50

0.50 0.22 0.13 0.48 0.05 0.50

0.49 0.22 0.13 0.50 0.05 0.49

0.50 0.22 0.13 0.50 0.05 0.50

0.49 0.22 0.14 0.52 0.05 0.49

0.48 0.23 0.13 0.54 0.05 0.48

Mean 0.51 0.23 0.13 0.48 0.05 0.51

SD 0.014 0.010 0.007 0.041 0.003 0.014

% RSD 2.86 4.51 4.98 8.46 5.87 2.86

7.7.8 Stability in Analytical solution:

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

HPLC. The data obtained are summarized in Table 7.6.8.1 demonstrating that the sample

solution is stable for 6 hours.

Table 7.6.8.1 Solution Stability

Condition % single max impurity Difference from

initial RRT-1.06

INITIAL 0.05 0.00

6HRS 0.07 0.02

12HRS 0.14 0.09

18HRS 0.18 0.13

24HRS 0.21 0.16

CHAPTER – 7


7.7.9 Filter Compatibility:

Injection of spiked sample solution filtered through different types of filters

(Centrifuged, Glass, Nylon, PVDF, Nylon + glass) were done on HPLC. All the filters were

found to be suitable.

7.7.10 Robustness:

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

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

solutions. The data obtained are summarized in Table 7.6.10.1. All results are within acceptable

limits.

Table 7.6.10.1 Robustness

System suitability solution

Changes in

parameters Values

Retention

Time of

Nicorandil

Tailing

Factor

USP

Plates

% RSD

Standard

area

%

Single

max

Impurity

%

RSD

Control As per

method 18.038 1.14 84820 1.28 0.47 -

Flow

(ml/min)

0.9 19.027 1.03 94218 3.49 0.43 6.3

1.1 17.232 1.02 76979 1.55 0.49 2.9

Wavelength

(nm)

257 18.038 1.17 79805 1.29 0.46 1.5

267 18.038 1.13 84535 3.25 0.48 1.5

Temperature

°C

25°C 18.444 1.24 73935 2.09 0.49 2.9

35°C 17.618 1.14 76234 1.36 0.53 8.5

Buffer pH 6.2 18.165 0.90 109875 1.72 0.42 7.9

6.6 18.176 0.93 105891 1.88 0.48 1.5

CHAPTER – 7


7.7.11 CONCLUSIONS:

The method has been shown to be specific for Nicorandil Tablets. The method has been shown to be Linear, precise and accurate across the suitable

analytical range and stability indicating. Solution has been shown to be stable for at least 6 hours at 15°c. The method has been shown to be robust towards deliberate minor changes in the

method parameters. Limit of detection and Limit of quantification concentration have been set. The method can be used in quality control laboratory for release of production batches.

CHAPTER – 7 DETERMINATION OF RELATED SUBSTANCES...

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