DETERMINATION OF RELATED SUBSTANCES OF NICORANDIL IN TABLET DOSAGE FORM BY USING REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
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 : SPP SPTM, SVKM s NMIMS, Mumbai 156
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. SPP SPTM, SVKM s NMIMS, Mumbai 157
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 Three times of the time main peak Flow Rate 1ml/min Wavelength 215nm Inj Vol (µl) 20 SPP SPTM, SVKM s NMIMS, Mumbai 158
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 SPP, SPTM, SVKM s NMIMS, Mumbai 159
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. SPP, SPTM, SVKM s NMIMS, Mumbai 160
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 nylon membrane 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. SPP, SPTM, SVKM s NMIMS, Mumbai 161
Base 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 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. The sample as well as the placebo was injected into HPLC system. 7.4.2 Oxidative condition: hydrogen peroxide-induced 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. 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. The sample as well as the placebo was injected into HPLC system. 7.4.3 Thermal 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. 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. The sample as well as the placebo was injected into HPLC system. 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 SPP, SPTM, SVKM s NMIMS, Mumbai 162
similarly. The sample as well as the placebo was injected into HPLC system. 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. SPP, SPTM, SVKM s NMIMS, Mumbai 163
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. SPP, SPTM, SVKM s NMIMS, Mumbai 164
Figure-7.4.2: Chromatogram of sample in Base. Figure-7.4.3: Chromatogram of sample in Peroxide. SPP, SPTM, SVKM s NMIMS, Mumbai 165
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 Nylon membrane filter and sonnicate to degas. SPP, SPTM, SVKM s NMIMS, Mumbai 166
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 SPP, SPTM, SVKM s NMIMS, Mumbai 167
A B Figure 7.5.2: LC-MS spectra of impurity 1 (A), 2 (B) and 3(C) recorded in positive mode of electrospray ionization C 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. SPP, SPTM, SVKM s NMIMS, Mumbai 168
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 SPP, SPTM, SVKM s NMIMS, Mumbai 169
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. SPP, SPTM, SVKM s NMIMS, Mumbai 170
Figure 7.5.5: Nicorandil Route of Synthesis SPP, SPTM, SVKM s NMIMS, Mumbai 171
7.6 EXPERIMENTAL WORK 7.6.1 Instrumentation Equipment Make Model HPLC Waters 2695Alliance Separation Module, (PDA/UV Detector) 2996/2487 Column ph meter Analytical Balance Micro Balance AKZO NOBEL Thermo Electron Corp. Mettler Toledo Mettler Toledo Inertsil C8-3, 250 x 4.6 mm, 5µm Orion-4star 1117000 XS205DU UMX-2 Ultrasonnicator Spectralab - Photostability Chamber Water Bath Thermolab Spectralab 400litr 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 SPP, SPTM, SVKM s NMIMS, Mumbai 172
7.6.3 Working Standard Working Standard: Standard Lot.No. Potency (as is) % Nicorandil 6712008002 99.4 Test Sample: Batch. No. NCT/20/23 Label claim 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. SPP, SPTM, SVKM s NMIMS, Mumbai 173
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. SPP, SPTM, SVKM s NMIMS, Mumbai 174
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 2.0 Specificity 2.1 Identification Results should be comparable with respect to the retention time and relative retention time. 2.2 Interference No interference from blank and placebo to main component and impurities 1.28 1.14 84820 Complies Complies 2.3 Peak purity 2.4 Forced degradation Purity angle should be less than purity threshold. Standard peak should be pure. The peak due to major impurities should be pure as shown on the PDA. Complies Complies SPP, SPTM, SVKM s NMIMS, Mumbai 175
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 5.0 Linearity 6.0 Accuracy (Recovery) % RSD for LOQ: NMT 10% Response should be Linear Correlation coefficient should not be less than 0.99. Y- Intercept should be within ± 10.0% of the corresponding Y-coordinate of the working level. At LOQ Level mean recovery should be in the range 75.0 % to 125.0 %. Mean recovery should be in the range of 80.0%- 120.0%. 1.71 0.015 Response is linear 1.0000 Complies % Mean Recovery 97.8 100.5 SPP, SPTM, SVKM s NMIMS, Mumbai 176
Table 7.7.1.1: Validation Summary (continued) Sr. No. Parameters Acceptance criteria Result obtained 7.0 7.1 System Precision System suitability % RSD for Standard solution. 7.2 Method Precision RSD for % Single max Impurity content. 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 ) 9.0 Stability in analytical solution Should be fulfilled NMT 5.0 % NMT 10.0%. Should be fulfilled NMT 5.0 % NMT 10.0%. NMT 10.0%. 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. Complies 1.28 RRT-0.27 RRT-0.97 1.66 5.11 Complies RRT-0.27 RRT-0.97 1.58 8.21 2.86 8.46 Sample is stable for 6 hours at 15 C SPP, SPTM, SVKM s NMIMS, Mumbai 177
Table 7.7.1.1: Validation Summary (continued) Sr. No. Parameters Acceptance criteria Result obtained 10.0 Robustness Change in Flow rate (± 0.1 ml/min) There should be no significant change in system suitability parameters. No significant change Change in wavelength (± 5 nm) There should be no significant change in system suitability parameters. No significant change Change in Buffer ph (± 0.2) There should be no significant change in system suitability parameters. No significant change Column oven temperature (± 5 C) There should be no significant change in system suitability parameters. 11.0 Filter compatibility % Difference for impurity content of Centrifugate and filtered should be 0.05. No significant change Complies SPP, SPTM, SVKM s NMIMS, Mumbai 178
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 RRT Purity time (min) Nicorandil 18.018 1.00 Yes impurity-1 4.947 0.27 Yes impurity-2 6.375 0.35 Yes SPP, SPTM, SVKM s NMIMS, Mumbai 179
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 SPP, SPTM, SVKM s NMIMS, Mumbai 180
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. SPP, SPTM, SVKM s NMIMS, Mumbai 181
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 Nicorandil Sr.No. 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. SPP, SPTM, SVKM s NMIMS, Mumbai 182
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. SPP, SPTM, SVKM s NMIMS, Mumbai 183
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 RESPONSE 450000 400000 350000 300000 250000 200000 150000 100000 50000 0 0.000 0.500 1.000 1.500 2.000 2.500 CONCENTRATION (%) SPP, SPTM, SVKM s NMIMS, Mumbai 184
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 50 % 100 % 200 % 0.015 3037 0.015 100.0 0.015 3108 0.015 100.0 0.015 2873 0.014 93.3 0.501 101374 0.503 100.4 0.501 101023 0.501 100.0 0.501 102233 0.507 101.2 1.002 202409 1.003 100.1 1.002 203369 1.008 100.6 1.002 203946 1.011 100.9 2.004 411040 2.038 101.7 2.004 405620 2.011 100.3 2.004 402812 1.997 99.7 97.8 100.5 SPP, SPTM, SVKM s NMIMS, Mumbai 185
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 % Unknown impurity % Total Spl. No. RRT- RRT- RRT- RRT- RRT- impurities 0.27 0.35 0.67 0.97 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 SPP, SPTM, SVKM s NMIMS, Mumbai 186
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) % Unknown impurity % Total Spl. No. RRT- RRT- RRT- RRT- RRT- impurities 0.27 0.35 0.67 0.97 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 SPP, SPTM, SVKM s NMIMS, Mumbai 187
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 I II RRT- 0.27 % Unknown impurity % Total RRT- RRT- RRT- RRT- 0.35 0.67 0.97 1.06 impurities 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 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 RRT-1.06 initial 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 SPP, SPTM, SVKM s NMIMS, Mumbai 188
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 % Retention % RSD % Changes in Tailing USP Single Values Time of Standard RSD parameters Factor Plates max Nicorandil area Impurity Control As per method 18.038 1.14 84820 1.28 0.47 - Flow 0.9 19.027 1.03 94218 3.49 0.43 6.3 (ml/min) 1.1 17.232 1.02 76979 1.55 0.49 2.9 Wavelength 257 18.038 1.17 79805 1.29 0.46 1.5 (nm) 267 18.038 1.13 84535 3.25 0.48 1.5 Temperature 25 C 18.444 1.24 73935 2.09 0.49 2.9 C 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 SPP, SPTM, SVKM s NMIMS, Mumbai 189
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. SPP, SPTM, SVKM s NMIMS, Mumbai 190