Method Development and validation for Related substances of Montelukast & Levocetirizine in combination by HPLC

CHAPTER 5 Method Development and validation for Related substances of Montelukast & Levocetirizine in combination by HPLC Introduction Montelukast Formula : C 35 H 35 ClNO 3 S Na CAS Number : 151767-02-1 Molecular Weight : 608.17 Synonyms : Cyclopropaneacetic acid,1-[[[(1r)-1-[3-[(1e)-2-(7-chloro-2- quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy- 1- methylethyl)phenyl]propyl]thio]methyl]-,monosodium salt;singulair (TN);Singulair;sodium 2-[1-[[(1R)-1-[3-[2-(7- chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2- yl)phenyl]propyl]sulfanylmethyl]cyclopropyl]acetate; Melting point : 135.5 C Montelukast is chemically belongs to leukotriene receptor antagonist (LTRA) used for the maintenance treatment of asthma and to relieve symptoms of seasonal allergies.1,2 It is usually administered orally in the form of tablets and oral granules etc. Montelukast is a CysLT1 antagonist; it blocks the action of leukotriene D4 (and secondary ligands LTC4 and LTE4) on the cysteinyl leukotriene receptor CysLT1 in the lungs and bronchial tubes by binding to it. Montelukast is a once-daily leukotriene receptor antagonist, in asthma and allergic rhinitis in both adults and children 3 Page 212 of 305

Cetrizine Dihydrochloride Levo isomer (levocetrizine) Molecular Formula : C 21 H 25 ClN 2 O 3 2HCl CAS No. : 83881-51-0 (Base) 83881-52-1 (Dihydrochloride) 130018-87-0 (Levocetrizine) Molecular Weight : 461.81 Synonyms :(1S.2S)-2-Methylamino-1-phenyl-1-propanol dichloride; (2-(4-((4-Chlorophenyl)phenylmethyl)-1- piperazinyl)ethoxy)acetic acid dichloride; Melting point : 110 to 115 C 215-220 ºC (Levocetrizine) Cetrizine is chemically (±)-[2-[4-[(4-chlorophenyl)phenylmethyl]-1- piperazinyl]ethoxy] acetic acid. It is a second-generation 4 antihistamine, is a major metabolite of hydroxyzine, and a racemic selective H1 receptor inverse agonist used in the treatment of allergies, hay fever, angioedema, and urticaria. The most commonly it is used in reducing the severity of common cold. Levocetirizine (as levocetirizine dihydrochloride) is a third-generation non-sedative antihistamine, developed from Cetirizine. Chemically, levocetirizine is the active enantiomer of cetirizine 5. It is the R-enantiomer of the Cetirizine which is a racemate. Levocetirizine works by blocking histamine receptors. It does not prevent the actual release of histamine from mast cells, but prevents it binding to its receptors. This in turn prevents the release of other allergy chemicals and increased blood supply to the area, and provides relief from the typical symptoms of hay fever Page 213 of 305

Montelukast and Cetirizine/levocetirizine combination therapy Allergic rhinitis is the most common allergic disease worldwide and affects about 18% to 40% of the general population. Combination therapy (Montelukast plus levocetirizine) is a more effective strategy than monotherapy in the treatment of persistent allergic rhinitis 6. Montelukast sodium is a selective and orally active leukotriene receptor antagonist that inhibits the cysteinyl leukotriene (CysLT 1), receptor. Levocetirizine is the R-enantiomer of Cetirizine. Levocetirizine is an orally active, potent, selective and long acting H 1 -histamine receptor antagonist with no anticholinergic activity. Montelukast sodium is alkaline, stable and levocetirizine Dihydrochloride is acid stable, when we prepare a matrix tablet, both the drugs would be in contact and make it unstable during the shelf life of the formulation thus it becomes very important to develop a method to determine the impurities in a combination product and check for its stability during the shelf life. Literature study shows many methods estimation methods for Cetirizine- A.M.Y. Jaber et al described Determination of Cetirizine Dihydrochloride, related impurities and preservatives in oral solution and tablet dosage forms using HPLC 7. Paw B et al published Development and validation of a HPLC method for the determination of Cetirizine in pharmaceutical dosage forms 8. Estimation methods are also available for Montelukast- Ibrahim A. Alsarra, development of a stability-indicating hplc method for the determination of Montelukast in tablets and human plasma and its applications to pharmacokinetic and stability studies 9. R. M. Singh et al Development and Validation of a RP-HPLC Method for Estimation of Montelukast Sodium in Bulk and in Tablet Dosage Form 10. Many articles are available for simultaneous assay determinations of both these drugs Atul S. Rathore et al Development of Validated HPLC and HPTLC Methods for Simultaneous Determination of Levocetirizine Dihydrochloride and Montelukast Sodium in Bulk Drug and Pharmaceutical Dosage Form 11, Arindam Basu et al, Simultaneous RP-HPLC Estimation of Levocetirizine Hydrochloride and Montelukast Sodium in Tablet Dosage Form 12, Laskhmana Rao et al development and validation of a reversed phase hplc method for simultaneous determination of levocetirizine and Page 214 of 305

montelukast sodium in tablet dosage form 13. A few more articles are published for simultaneous determination of Levocetirizine and Montelukast by HPLC 14-17 No article could be traced containing simultaneous determination method for determination of all the impurities of both the drugs. In this chapter a related substances method for determining the impurities in such combination products was developed. For this purpose, the drug substances, standard and impurities were gifted by Dr Reddy s laboratories ltd. The drug product used for this exercise was obtained commercially from the market. The brand called Alerfix from Eris. Alerfix tablets contain levocetirizine hydrochloride 5 mg and Montelukast 10 mg. Page 215 of 305

Montelukast Structure confirmation: The following physicochemical techniques were used to confirm the structure of Montelukast Sodium. These are given below Thermal study UV study FTIR NMR spectrophotometry Mass spectrophotometry 1. Thermal Analysis 1.86 mg of the sample was weighed into an aluminum crucible of 25µL and placed into DSC. The thermogram was recorded from 30ºC to 200ºC which is carried out under nitrogen atmosphere at 50mL/min, at 5ºC /min. The thermogram exhibited two endotherms. The 1 st endotherm was at 54.7 ºC, which may be due to the loss of solvent or water. The second endotherm at 135.5 ºC 2. UV Study The Ultraviolet spectrum was recorded from 200 nm to 400 nm, with API concentration of 0.0007% in methanol. The spectrum showed five λ maxima at 212, 283, 327, 344 and 358 nm. 3. FTIR Study The FTIR of spectrum of Montelukast Sodium was recorded by preparation of pellet with KBr. The assignments are given in the below table. Page 216 of 305

Table 5.1 FTIR assignments for Montelukast Sodium Wave number (cm -1 ) Assignment Mode of vibration 3392 O-H Stretching 3058 Aromatic -C-H Stretching 2975, 2928 Aliphatic -C-H Stretching 1637, 1607, 1594, 1497 -C=C Stretching 1563, 1408 -C=O Stretching 1440, 1341 Aliphatic -C-H Bending 1144 -C=O Stretching 1068 -C-Cl Stretching 963, 837, 761 -C-H Bending 4. NMR Study The 1 H and 13 C NMR (Fig 6&7) data of Montelukast Sodium were recorded In DMSO-d 6 at 400 MHz and 100MHz respectively on 400MHz spectrometer. The chemical shift values are reported on 3 scale in ppm with respect to TMS (δ 0.00ppm) and DMSO-d 6 (δ 39.5ppm) as internal standard respectively. The exchangeable proton was observed from M exchange spectrum The NMR assignment are given in the Table below. NMR assignments of Montelukast Sodium. Page 217 of 305

Table 5.2 NMR assignments for Montelukast Sodium Position 1 1 H δ (ppm) J (Hz) 2 13 C 2 - - - 156.8 3 1H 7.95 d,8.2 120.3 4 1H 8.40 d,8.2 136.5 5 1H 8.00 d,8.8 129.7 6 1H 7.58 dd,1.8,8.8 125.7 7 - - - 125.6 8 1H 8.03 d,1.8 127.2 9 - - - 148.0 10 1H - - 134.3 11 1H 7.89 d,16.8 135.1 12 1H 7.50 d,16.8 128.3 13 - - - 136.0 14 1H 7.73 s 126.6 15 - - - 144.1 16 1H 7.04-7.63-128.9 17 1H 7.04-7.63-131.0 18 1H 7.04-7.63-126.3 19 1H 4.02 D,7.2 49.4 20 Ha 2.12 m 39.0 Hb 2.21 m - 21 Ha 2.75 dd,4.0,12.6 32 Hb 3.06 m - 22 - - - 146.8 23 1H 7.04-7.63-126.6 24 1H 7.04-7.63-125.1 25 1H 7.04-7.63-128.4 26 1H 7.04-7.63-125.3 27 - - - 139.9 28 - - - 71.6 28 OH* 5.15 br - 29 3H 1.44 s 31.7 29 3H 1.44 s 31.6 30 Ha 2.00 d,14.6 43.7 Hb 2.13 d,14.6-31 - - - 18.0 32 Ha 0.16-0.28 m 12.4 Hb 0.34-0.45 m - 33 Ha 0.16-0.28 m 12.0 Hb 2.54 m - 34 Ha 2.69 d,12.6 39.9 Hb d,12.6-35 - - - 176.0 Page 218 of 305

5. Mass spectral study The ESI mass spectrum of Montelukast sodium was studied on 400Q trap LCMSMS system. The sample is introduced through HPLC system by bypassing the column. The ESI +ve mass spectrum of Montelukast sodium displayed the protonated molecular ion at m/z =586 which corresponds to the molecular formula C 35 H 36 ClNO 3 S. The possible fragmentation pattern is shown below. Figure 5.1- Mass fragmentation pattern for Montelukast Sodium m/z=568 m/z=440 m/z=442 Page 219 of 305

Impurity Details of Montelukast Impurity 1 Chemical Name methyl] : 1-[[[(1R)-1-[3-[2-(7-Chloro-quinolinyl) ethenyl] phenyl-3- [2- (1- hydroxyl-1-methylethyl) phenyl] propyl]thio] cyclopropane acetic acid Molecular Formula : C 35 H 38 ClNO 3 S Molecular Weight : 588.20 Molecular Structure : Impurity-2 Chemical Name : 1-[[[(1R)-1-[3-[(1E)-2-(7-Chloro-quinolinyl) Ethenyl] phenyl- 3- [2- (1- (1-methyl) ethenyl)] phenyl] propyl]thio] methyl] cyclopropane acetic acid [Or] 1-[[[(1R)-1-[3-[(1E)-2-(7-Chloro-quinolin-2-yl) Ethenyl] phenyl-3- [2- (1-methylenyl)phenyl] propyl]sulfanyl] methyl] cycloprpyl]acetic acid Molecular Formula : C 35 H 34 ClNO 2 S Molecular Weight : 568.17 Molecular Structure : Page 220 of 305

Impurity 3 Chemical Name : 1-[[[(1R)-3-(2-acetylphenyl)-1-[3-[(E)-2-(7-Chloroquin-2-yl) Ethenyl] phenyl]propyl]sulfanyl] methyl] cyclopropyl] acetic acid [Or] 2-[1-(3-(2- acetylphenyl)-1-{3-[(e)-2-(7-chloro-2-quinolyl)-1- Ethyl] phenyl] propyl]sulfanyl] methyl] cycloprpyl]acetic acid Molecular Formula : C 34 H 32 ClNO 3 S Molecular Weight : 570.14 Molecular Structure : Impurity 5: Chemical Name : 2-[2-[3-(S)-[3-[2-[7-Chloro-2-quinolinyl] Ethyl] phenyl]-3- hydroxy propyl]phenyl]-2-propanol Molecular Formula : C 29 H 28 ClNO 2 Molecular Weight : 457.99 Molecular Structure : Page 221 of 305

Impurity-6 Chemical Name :2-(1-{(1r)-1-{3- [(E)-2-(7-chloro-quinolyl)1-1ethrnyl]phrnyl}- 3- [2-(1-hydroxy-1-methyl ethyl ) phenyl]prpyl sufinyl methyl}cycloprpyl] acetic acid. [OR] 1-[[[(1-3-[(E)-2-(7-Chloroquinolin-2-yl)Ethenyl] phenyl]-3-[2- (1- hydroxy-1-methylethyl)phenyl]propyl]sulfanyl] methyl] cyclopropyl] acetic acid Molecular Formula : C 35 H 36 ClNO 4 S Molecular Weight : 602.18 Molecular Structure : Page 222 of 305

Cetirizine Structure confirmation: The following physicochemical techniques were used to confirm the structure of Cetirizine Dihydrochloride. These are given below Thermal study UV study FTIR NMR spectrophotometry Mass spectrophotometry 1. Thermal Analysis 3.12 mg of the sample was weighed into an aluminum crucible of 25µL and placed into the DSC. The thermogram was recorded from 30ºC to 300ºC which is carried out under nitrogen atmosphere at 50mL/min, at 10ºC /min. The thermogram exhibited endotherm at 205 and 214 ºC followed by decomposition. This was confirmed by melting point apparatus which showed melting between 200 and 210 ºC. 2. UV Study The Ultraviolet spectrum was recorded from 200 nm to 400 nm, with API concentration of 0.0007% in methanol. The spectrum showed two λmax at 204 and 231 nm. 3. FTIR Study The FTIR of spectrum of Cetirizine Dihydrochloride was recorded by preparation of pellet with KBr. The assignments are given in the below table. Page 223 of 305

Table 5.3 FTIR assignments for Cetirizine Dihydrochloride Wave number (cm -1 ) Assignment Mode of vibration 3461 -N-H, O-H Stretching 2981,2949 Aliphatic -C-H Stretching 2629, 2358 N-H + Stretching 1743 Acid C=O Aromatic -C=C Stretching 1601 Aromatic -C=C Stretching 1496, 1382, 1357 Aliphatic -C-H Bending 1319 -C-N Stretching 1135 Ether C-O Stretching 1092 Aromatic C-Cl Stretching 805, 757, 699 Aromatic C-H Stretching 4. NMR study The 1 H and 13 C NMR (Fig 6&7) data of Cetirizine Dihydrochloride were recorded In DMSO-d 6 at 400 MHz and 100MHz respectively on 400MHz spectrometer. The chemical shift values are reported on 3 scale in ppm with respect to TMS (δ 0.00ppm) and CD 3 COOD (δ 39.5ppm) as internal standard respectively. The exchangeable proton was observed from M exchange spectrum The NMR assignment are given in the Table below. NMR assignments of Cetirizine Dihydrochloride Page 224 of 305

Table 5.4 NMR assignments for Cetirizine Dihydrochloride Position 1 1 H δ (ppm) J (Hz) 2 13 C 2&6 Ha 2H 4.20 m 50.02 Hb 2H 4.06 2&6 Ha 2H 3.96 m 50.02 Hb 2H 3.69 7 1H 5.72 s 76.34 8 - - - 133.52 9 1H 7.48 m 130.77 10 1H 7.95 m 131.51 11 - - 136.65 12 1H 7.95 m 131.51 13 1H 7.48 m 130.77 14 - - 134.49 15 1H 7.48 m 130.77 16 1H 7.95 m 129.68 17 1H 7.42 m 130.77 18 1H 7.95 m 129.68 19 1H 7.48 m 130.77 20 2H 3.59 m 57.44 21 2H 4.06 m 65.90 22 2H 4.24 s 68.35 23 - - - 178.11 5. Mass spectral study The ESI mass spectrum of Cetirizine Dihydrochloride was studied on 400Q trap LCMSMS system. The sample is introduced through HPLC system by bypassing the column. The chemical ionization was performed by using isobutene gas to enhance ionization. The CI mass spectrum showed base peak at m/z =389. The possible mass fragmentation is shown below. Page 225 of 305

Figure 5.2- Mass Fragmentation pattern of Cetirizine dihydrochloride C 21 H 25 ClN 2 O 3 C 19 H 22 ClN 2 Exact Mass 388 Exact Mass 313 C 18 H 20 ClN 2 C 13 H 10 Cl + Exact Mass 299 Exact Mass 201 Page 226 of 305

Impurities of Cetirizine 1. Impurity A Chemical Name: 1-[(4-Chlorophenyl) phenylmethyl] pipeazine Molecular Formula: C 17 H 19 ClN Molecular Weight: 286.80 Chemical Structure: 1. Impurity B Chemical Name: acid 2-[4-[(4-Chlorophenyl) phenyl methyl] pipeazin-1-yl] acetic Molecular Formula: C 19 H 21 N 2 O 2 Cl Molecular Weight: 344.84 Chemical Structure: Page 227 of 305

2. Impurity C Chemical Name: ethoxy] 2-[2-[4-[(4-Chlorophenyl) phenylmethyl] pipeazin-1-yl] acetic acid Molecular Formula: C 21 H 25 ClN 2 O 3 Molecular Weight: 388.89 Chemical Structure: 3. Impurity D Chemical Name: Bis-[(4-Chlorophenyl) phenyl methyl] pipeazine Molecular Formula: C 30 H 28 N 2 Cl 2 Molecular Weight: 487.46 Chemical Structure: Page 228 of 305

4. Impurity E Chemical Name: ethoxy] 2-[2-[2-[4-[(4-Chlorophenyl) phenylmethyl] pipeazin-1-yl] acetic acid Molecular Formula: C 23 H 23 N 2 O 4 Cl Molecular Weight: 432.94 Chemical Structure: 5. Impurity F Chemical Name: 2-[2-[4-(diphenylmethyl) pipeazin-1-yl] ethoxy]acetic acid Molecular Formula: C 21 H 26 N 2 O 3 Molecular Weight: 354.44 Chemical Structure: Page 229 of 305

Method Development By HPLC Objective: To develop an analytical method for determination of related substances in a combination drug product i.e. tablets containing Montelukast and Cetirizine. Scope: This method can be used for routine analysis in Quality control laboratories. This method will also be checked for its applicability during the Stability studies for determination of related substance and also degradation products in a combination product of Montelukast and Cetirizine. Chemicals and reagents: All the solvents used i.e. Acetonitrile, Methanol, Water were of HPLC grade. The Selection of Mobile phase: Mobile phase was selected on the basis of chemical properties of Cetirizine and Montelukast. Cetirizine and its impurity showed different polarity as Imp A,B,C,E were eluting faster and Imp D was eluting slower, whereas for Montelukast and its impurities, elution is dependent on high ratio of organic modifiers, thus 0.1% of OPA buffer was selected. The selection of buffers was made by taking into account, the solubility of the buffers in the organic phase. In order to ensure that difference in readings of different ph meters does not affect the method performance, buffers were avoided initially. Organic modifiers used in the beginning was a combination Acetonitrile and Water in ratio 95:5, but in this combination, Cetirizine and all its impurity except Imp D eluted at around 2 minutes, hence instead of Water, Methanol was used to provide optimum polarity so that the Cetirizine and its impurity retentions time increase. This change however increased the retention of Montelukast also but it turned out to be beneficial as it provided sufficient space for Cetirizine and its impurity to elute. Selection of Column: Column study was done initially using Inertsil ODS 3V, 250X4.6mm, 5µm, but as the organic modifier combination was changed from Acetonitrile: Methanol: 90:10% v/v to 90:15:Acetonitrile: Methanol, Impurity 2 and Impurity 5 of Montelukast resolution decreased significantly, hence column study was done on 5 different column of almost same chemical property, Page 230 of 305

1. Inertsil C8, 250X4.6 mm, 5µm 2. Waters symmetry shield RP18, 250X4.6mm, 5µm 3. Xterra RP-18, 250X4.6mm, 5µm 4. Unison US-C18, 250X4.6mm, 5µm 5. YMC pack ODS, 250X4.6mm, 5µm Among these set of columns Waters symmetry shield RP-18, 250X4.6mm, 5µm showed enhanced resolution and peak shape to its High carbon load of 17%, whereas for Inertsil ODS 3V is 15%, Xterra RP-18 is 15%, Unison US-C18 is 15% and YMC ODS-AQ is 14%. Selection of Diluent: On the basis of solubility of both the compounds diluents was selected to be 70:30 :: Methanol : Water. Selection of wavelength: Absorption maxima of Cetirizine is around 243 and 229 and that of Montelukast is 266 and 283 wavelength was selected to be 225nm since 10 nm below this the response of Montelukast and its impurities were significantly reduced along with the irregular baseline due to proximity of cut off wavelength of Acetonitrile, 10 nm above this response of Cetirizine Imp F was significantly reduced and 15 nm above this wavelength, the peak responses of all the impurities decreased drastically. Experiment 1: Buffer: Pipetted out 1mL of 85%v/v Orthophosphoric Acid in to a 1000mL volumetric flask, 500mL water added, shaken for 10min, subjected to ultrasonication, cooled to room temperature and made up to the volume with water Mobile Phase A: Buffer Mobile Phase B: Acetonitrile::Water :: 95:5% v/v Diluent: Methanol: Water :: 70:30% v/v Page 231 of 305

Chromatographic Conditions: Flow rate 1.5 ml/min Wavelength 225 nm Sample temperature Ambient Column temperature 25 C Column Inertsil ODS 3V, 250X4.6 mm, 5µm Gradient Program Time %A %B 0.01 40 60 10 30 70 15 10 90 20 0 100 30 0 100 32 40 60 40 40 60 Figure 5.3 Chromatogram for experiment No 1showing Montelukast and related impurities Page 232 of 305

Figure 5.4 Chromatogram for experiment No 1showing Cetirizine and related impurities Observation: Montelukast and all its Impurities eluted within 25 minutes. Cetirizine and all its impurity were eluted within 2 minutes. Way forward: Introduction of methanol in mobile phase B with replacement of water with methanol should help in retention Cetirizine and its impurity. It should also extend the retention time of Montelukast and its impurities. This change would also ensure proper separation of all impurities. Experiment 2: Buffer: Pipetted out 1mL of 85%v/v Orthophosphoric Acid in to a 1000mL volumetric flask, 500mL water added, shaken for 10min, subjected to ultrasonication, cooled to room temperature and made up to the volume with water Mobile Phase A: Buffer Mobile Phase B: Acetonitrile::Water :: 90:10% v/v Diluent: Methanol: Water :: 70:30% v/v Page 233 of 305

Chromatographic Condition: Flow rate 1.5 ml/min Wavelength 225 nm Sample temperature Ambient Column temperature 25 C Column Inertsil ODS 3V, 250X4.6 mm, 5µm Gradient Program Time %A %B 0.01 70 30 10 70 30 15 65 35 20 50 50 30 30 40 40 10 90 50 0 100 60 0 100 62 70 30 70 70 30 Page 234 of 305

Figure 5.5 Chromatogram for experiment No 2 with all the peaks Observation: This change has achieved the required results up to a certain extent. Montelukast and all its Impurities were well separated. The decrease in Acetonitrile content in mobile phase B impacted on Montelukast and its impurities which seem to be relatively nonpolar and the runtime was extended upto 45 min. Cetirizine and all its impurities eluted within 20 minutes but with very less resolution. Way forward: Slight increment in the percentage of methanol and corresponding reduction in the percentage of Acetonitrile should improve the resolution of Cetirizine and its impurities. Page 235 of 305

Experiment 3: Buffer: Pipetted out 1mL of 85%v/v Orthophosphoric Acid in to a 1000mL volumetric flask, 500mL water added, shaken for 10min, subjected to ultrasonication, cooled to room temperature and made up to the volume with water Mobile Phase A: Buffer Mobile Phase B: Acetonitrile:: Methanol :: 85:15% v/v Diluent: Methanol: Water :: 70:30% v/v Chromatographic Conditions: Flow rate 1.5 ml/min Wavelength 225 nm Sample temperature Ambient Column temperature 25 C Column Inertsil ODS 3V, 250X4.6 mm, 5µm Gradient Program Time %A %B 0.01 70 30 10 70 30 15 65 35 20 50 50 30 60 40 40 10 90 50 0 100 55 0 100 58 70 30 65 70 30 Page 236 of 305

Figure 5.6 Chromatogram for experiment No 3 showing all peaks Observation: Changing the percentage of Acetonitrile and Methanol in mobile phase B worked as expected. The resolution was improved for Cetirizine and all its impurity whereas in case of Montelukast resolution between Imp 2 and Imp 5 was reduced. This indicates that it is necessary to keep the percentage of Acetonitrile to 90% for optimum separation of Montelukast and its impurity and 15% of methanol for improved separation of Cetirizine and its impurity. Way forward: Increasing the percentage of Acetonitrile to 90% and maintaining the same percentage of Methanol should workout logically to separate all the impurities. Page 237 of 305

Experiment 4: Buffer: Pipetted out 1mL of 85%v/v Orthophosphoric Acid in to a 1000mL volumetric flask, 500mL water added, shaken for 10min, subjected to ultrasonication, cooled to room temperature and made up to the volume with water Mobile Phase A: Buffer Mobile Phase B: Acetonitrile:: Methanol :: 90:15% v/v Diluent: Methanol: Water :: 70:30% v/v Chromatographic Conditions: Flow rate 1.5 ml/min Wavelength 225 nm Sample temperature Ambient Column temperature 25 C Column Inertsil ODS 3V, 250X4.6 mm, 5µm Gradient Program Time %A %B 0.01 70 30 10 70 30 15 65 35 20 50 50 30 60 40 40 10 90 50 0 100 55 0 100 58 70 30 65 70 30 Page 238 of 305

Figure 5.7 Chromatogram for experiment No 4 showing all peaks Observation: Changing the percentage of Acetonitrile and Methanol in mobile phase B didn t work as expected, resolution was not improved between Imp 2 and Imp 5 of Montelukast. This shows that the impurity 2 and impurity 5 peaks are not organic phase sensitive. Way forward: Need to study the impact of change in stationary phase parameters thus perform column study on equivalent columns keeping all the other chromatographic conditions same as that of previous Experiment. Page 239 of 305

Experiment 5: Buffer: Pipetted out 1mL of 85%v/v Orthophosphoric Acid in to a 1000mL volumetric flask, 500mL water added, shaken for 10min, subjected to ultrasonication, cooled to room temperature and made up to the volume with water Mobile Phase A: Buffer Mobile Phase B: Acetonitrile:: Methanol :: 90:15% v/v Diluent: Methanol: Water :: 70:30% v/v Chromatographic Conditions: Flow rate 1.5 ml/min Wavelength 225 nm Sample temperature Ambient Column temperature 25 C Column Inertsil C8, 250X4.6 mm, 5µm Gradient Program Time %A %B 0.01 70 30 10 70 30 15 65 35 20 50 50 30 60 40 40 10 90 50 0 100 55 0 100 58 70 30 Page 240 of 305

Figure 5.8 Chromatogram for experiment No 5 showing all peaks Observation: The separation between the impurities did not improve significantly. Needs to improve more in order to finalize the method Way forward: Need to check for similar columns for solutions Experiment 6: Buffer: Pipetted out 1mL of 85%v/v Orthophosphoric Acid in to a 1000mL volumetric flask, 500mL water added, shaken for 10min, subjected to ultrasonication, cooled to room temperature and made up to the volume with water Mobile Phase A: Buffer Mobile Phase B: Acetonitrile:: Methanol :: 90:15% v/v Diluent: Methanol: Water :: 70:30% v/v Page 241 of 305

Chromatographic Conditions: Flow rate 1.5 ml/min Wavelength 225 nm Sample temperature Ambient Column temperature 25 C Column Unison US-C18, 250X4.6mm, 5µm Gradient Program Time %A %B 0.01 70 30 10 70 30 15 65 35 20 50 50 30 60 40 40 10 90 50 0 100 55 0 100 58 70 30 65 70 30 Page 242 of 305

Figure 5.9 Chromatogram for experiment No 6 showing all peaks Observation: The separation between the impurities did not improve significantly. Needs to improve more in order to finalize the method Way forward: Need to check for similar columns for solutions Experiment 7: Buffer: Pipetted out 1mL of 85%v/v Orthophosphoric Acid in to a 1000mL volumetric flask, 500mL water added, shaken for 10min, subjected to ultrasonication, cooled to room temperature and made up to the volume with water Mobile Phase A: Buffer Mobile Phase B: Acetonitrile:: Methanol :: 90:15% v/v Diluent: Methanol: Water :: 70:30% v/v Page 243 of 305

Chromatographic Conditions: Flow rate 1.5 ml/min Wavelength 225 nm Sample temperature Ambient Column temperature 25 C Column Xterra RP-18, 250X4.6mm, 5µm Gradient Program Time %A %B 0.01 70 30 10 70 30 15 65 35 20 50 50 30 60 40 40 10 90 50 0 100 55 0 100 58 70 30 65 70 30 Page 244 of 305

Figure 5.10 Chromatogram for experiment No 7 showing all peaks Observation: The separation between the impurities did not improve significantly. Needs to improve more in order to finalize the method Way forward: Need to check for similar columns for solutions Experiment 8: Buffer: Pipetted out 1mL of 85%v/v Orthophosphoric Acid in to a 1000mL volumetric flask, 500mL water added, shaken for 10min, subjected to ultrasonication, cooled to room temperature and made up to the volume with water Mobile Phase A: Buffer Mobile Phase B: Acetonitrile:: Methanol :: 90:15% v/v Diluent: Methanol: Water :: 70:30% v/v Page 245 of 305

Chromatographic Conditions: Flow rate 1.5 ml/min Wavelength 225 nm Sample temperature Ambient Column temperature 25 C Column YMC pack ODS, 250X4.6mm, 5µm Gradient Program Time %A %B 0.01 70 30 10 70 30 15 65 35 20 50 50 30 60 40 40 10 90 50 0 100 55 0 100 58 70 30 65 70 30 Page 246 of 305

Figure 5.11 Chromatogram for experiment No 8 showing all peaks Observation: The separation between the impurities did not improve significantly. Needs to improve more in order to finalize the method Way forward: Need to check for similar columns for solutions Experiment 9: Buffer: Pipetted out 1mL of 85%v/v Orthophosphoric Acid in to a 1000mL volumetric flask, 500mL water added, shaken for 10min, subjected to ultrasonication, cooled to room temperature and made up to the volume with water Mobile Phase A: Buffer Mobile Phase B: Acetonitrile:: Methanol :: 90:15% v/v Diluent: Methanol: Water :: 70:30% v/v Page 247 of 305

Chromatographic Conditions: Flow rate 1.5 ml/min Wavelength 225 nm Sample temperature Ambient Column temperature 25 C Column Waters symmetry shield RP18, 250X4.6mm, 5µm Gradient Program Time %A %B 0.01 70 30 10 70 30 15 65 35 20 50 50 30 60 40 40 10 90 50 0 100 55 0 100 58 70 30 65 70 30 Page 248 of 305

Figure 5.12 Chromatogram for experiment No 9 showing all peaks Observation: Significant improvement was observed in the resolution in waters symmetry shield RP18, 250X4.6mm, 5µm Page 249 of 305

Optimized final method: Buffer: 0.1% of 85% Orthophosphoric Acid in water (1ml of 85% OPA in 1000ml Milli-Q water) Mobile Phase A: Buffer Mobile Phase B: Acetonitrile::Methanol :: 90:15 Diluent: Methanol: Water :: 70:30 Sample preparation: Cetirizine 200 ppm, Montelukast: 1000 ppm. Sample preparation was carried out by transferring 20mg of Cetirizine and 100mg of Montelukast in amber colored 100ml volumetric flask, sonicated for 5minutes to dissolve and diluted to volume with diluent. Standard stock Preparations: Standard stock preparation was carried out by adding weighed quantities of 50 mg of Cetirizine and 100 mg Montelukast standards into separate volumetric flasks respectively, 50mL of diluent added in each of the volumetric flasks, subjected to ultra sonication for about 5 mins, cooled and made upto the volume with diluent. Standard Preparation: Cetirizine: 1ppm, Montelukast: 2ppm 2 ml from each of the standard stock solutions were pipetted out into a 100mL volumetric flask, 50mL of diluent added and shaken well and madeup to the volume with the diluent. Chromatographic Conditions: Flow rate 1.5 ml/min Column temperature 25 C Inj Volume 30µL Wavelength 225 nm Sample temperature 10 C Columns Waters symmetry shield RP18, 250X4.6mm, 5µm Page 250 of 305

Gradient Program Time %A %B 0.01 75 25 10 70 30 15 65 35 20 50 50 30 35 65 40 20 80 50 0 100 55 0 100 58 75 25 65 75 25 Table 5.5-Individual limit of impurities considered for method validation Cetirizine Imp Name/No Limit Imp A 0.10% Imp B 0.10% Imp C 0.10% Imp D 0.10% Imp E 0.10% Imp F 0.10% Montelukast Imp 1 0.10% Imp 2 0.15% Imp 3 0.15% Imp 4 0.10% Imp 5 0.10% Page 251 of 305

Figure 5.13 Chromatogram showing all peaks in optimized method Page 252 of 305

Analytical method validation Analytical method validation is a process that demonstrates the suitability of the proposed procedures for the intended purpose. More specifically, it is a matter of establishing documented evidence providing a high degree of assurance with respect to the consistency of the method and results. It evaluates the product against defined specifications. The validation parameters viz., specificity, accuracy, precision, linearity, limit of detection, limit of quantitation, robustness, system suitability have to be evaluated as per the ICH guidelines for all analytical methods developed by HPLC. Validation Characteristics The following validation characteristics were verified as per the ICH guidelines. System suitability Specificity Linearity Accuracy Precision LOD & LOQ System suitability This is an integral part of development of a chromatographic method to verify that the resolution and reproducibility of the system are adequate enough for the analysis to be performed. It is based on the concept that the equipment, electronics, analytical operations and samples constituting an integral system could be evaluated as a whole. Parameters such as plate number (N), asymmetry or tailing factors (A s ), relative retention time (RRT), resolution (R s ) and reproducibility (% R.S.D), retention time were determined. These parameters were determined during the analysis of a "sample" containing the main components and related substances. System suitability terms were determined and compared with the recommended limits (1 A s 2 and R s >1.5). Page 253 of 305

Specificity Specificity is the ability of the method to measure the analyte response in presence of its process related impurities. The specificity of the developed HPLC method was performed by injecting blank solution and standard solution spiked with processrelated impurities separately The chromatogram of drug with impurities was compared with the blank chromatogram, to verify the blank interference. No peak was observed at the retention time of Montelukast, Cetirizine and their impurities. Hence the method is specific for the determination of Montelukast, Cetirizine and its combination product. Precision of Test method System precision of the method was evaluated by injecting the standard solution six times and percent relative standard deviation (% R.S.D) for area of Montelukast peak was 2.1% and for Cetirizine peak it was 1.16%. This proves the system precision of the test method. The precision of the method for the determination of impurities related to Montelukast and Cetirizine peaks was studied for repeatability at 100 % level. Repeatability was demonstrated by analyzing the standard solution spiked with impurities for six times. The % R.S.D for peak area of each impurity was calculated. Repeatability for Montelukast, Cetirizine and its impurities were found to be optimum Thus proves that this method is precise. The results are given in Table 5.6. Table 5.6- Precision results for Montelukast impurities Impurity name (Montelukast) RRF of impurities %Imp of SPL-1 %Imp of SPL-2 %Imp of SPL-3 %Imp of SPL-4 %Imp of SPL-5 %Imp of SPL-6 %RSD Impurity 1 0.77 0.116 0.104 0.102 0.106 0.095 0.097 7.13 Impurity 2 1.10 0.166 0.159 0.158 0.150 0.135 0.132 9.14 Impurity 3 0.75 0.169 0.152 0.147 0.144 0.136 0.130 9.16 Impurity 5 1.17 0.099 0.097 0.097 0.095 0.092 0.090 3.45 Impurity 6 0.80 0.110 0.103 0.103 0.101 0.097 0.096 6.95 Page 254 of 305

Table 5.7- Precision results for Cetirizine impurities Impurity name (Cetirizine) RRF of impurities %Imp of SPL-1 %Imp of SPL-2 %Imp of SPL-3 %Imp of SPL-4 %Imp of SPL-5 %Imp of SPL-6 %RSD Impurity A 1.70 0.109 0.113 0.114 0.114 0.113 0.112 1.72 Impurity B 1.26 0.118 0.119 0.116 0.115 0.115 0.117 1.22 Impurity C 0.55 0.088 0.083 0.088 0.087 0.092 0.093 4.01 Impurity D 2.50 0.110 0.115 0.115 0.114 0.111 0.113 1.70 Impurity E 1.20 0.100 0.092 0.090 0.093 0.092 0.092 3.65 Impurity F 0.45 0.094 0.093 0.093 0.094 0.095 0.093 0.91 Page 255 of 305

Linearity Standard solutions at different concentration levels ranging from 50% of the spec level to 300% of the specification limit were prepared and analyzed. In order to demonstrate the linearity of detector response for Montelukast, Cetirizine and their impurities, the linearity plot was drawn taking the concentration on X-axis and the mean peak area on Y-axis. The data were subjected to statistical analysis using a linear-regression model. The regression equations and correlation coefficients (r 2 ) are given in Tables below. Linearity of Montelukast and its Impurities Table 5.8 Linearity table for Montelukast API %Level Conc (ppm) area 50 1 44494 75 1.5 62215 100 2 81877 150 3 123580 200 4 161445 300 6 241958 intercept 3539 Bias at 100% 4.3223 Correlation coefficient 0.9999 Figure 5.14- Linearity graph for Montelukast 300000 Montelukast 250000 200000 150000 100000 y = 39676x + 3539. R² = 0.999 50000 0 0 1 2 3 4 5 6 7 Page 256 of 305

Table 5.9-Linearity table for Montelukast Impurity 1 IMP 1 % Level conc(ppm) area 50 0.5 15693 75 0.75 24388 100 1 33432 150 1.5 50316 200 2 66870 300 3 104907 intercept 397 Bias at 100% 1.1875 Correlation coefficient 0.9996 Figure 5.14- Linearity graph for Montelukast impurity 1 120000 Impurity 1 100000 80000 60000 40000 y = 35428x - 397. R² = 0.999 20000 0 0 0.5 1 1.5 2 2.5 3 3.5 Page 257 of 305

Table 5.10-Linearity table for Montelukast Impurity 2 IMP 2 % Level conc(ppm) area 50 0.75 30909 75 1.125 46554 100 1.5 62363 150 2.25 94701 200 3 127600 300 4.5 191949 intercept 1824 Bias at 100% 2.925 Correlation coefficient 1.0000 Figure 5.15- Linearity graph for Montelukast impurity 2 250000 Impurity 2 200000 150000 100000 y = 43050x - 1824. R² = 1 50000 0 0 1 2 3 4 5 Page 258 of 305

Table 5.11-Linearity table for Montelukast Impurity 3 IMP 3 % Level conc(ppm) area 50 0.75 36571 75 1.125 55638 100 1.5 74477 150 2.25 112545 200 3 149943 300 4.5 224523 intercept 696.3 Bias at 100% 0.9349 Correlation coefficient 1.0000 Figure 5.16- Linearity graph for Montelukast impurity 3 250000 Impurity 3 200000 150000 100000 50000 0 y = 50124x - 696.3 R² = 1 0 1 2 3 4 5 Page 259 of 305

Table 5.12-Linearity table for Montelukast Impurity 5 IMP 5 % Level conc(ppm) area 50 0.5 23344 75 0.75 34977 100 1 46897 150 1.5 72874 200 2 99892 300 3 151532 intercept 651 Bias at 100% 3707 Correlation coefficient 0.9998 Figure 5.17- Linearity graph for Montelukast impurity 5 160000 140000 120000 100000 80000 60000 40000 20000 0 Impurity 5 y = 51630x - 3707. R² = 0.999 0 0.5 1 1.5 2 2.5 3 3.5 Page 260 of 305

Table 5.13-Linearity table for Montelukast Impurity 6 IMP 6 % Level conc(ppm) area 50 0.5 19336 75 0.75 28854 100 1 39267 150 1.5 58960 200 2 78437 300 3 116161 intercept 205.6 Bias at 100% 0.5236 Correlation coefficient 0.9999 Figure 5.18- Linearity graph for Montelukast impurity 6 140000 120000 100000 80000 60000 40000 20000 0 Impurity 6 y = 38832x + 205.6 R² = 0.999 0 0.5 1 1.5 2 2.5 3 3.5 Page 261 of 305

Linearity of Cetirizine and its Impurities Table 5.14-Linearity table for Cetirizine API % Level conc(ppm) area 50 0.5 17649 75 0.75 27055 100 1 35915 150 1.5 54116 200 2 71444 300 3 107398 intercept 63.86 Bias at 100% 0.1778 Correlation coefficient 1.0000 Figure 5.19- Linearity graph for Cetirizine 120000 Cetrizine 100000 80000 60000 40000 y = 35794x + 63.86 R² = 0.999 20000 0 0 0.5 1 1.5 2 2.5 3 3.5 Page 262 of 305

Area Table 5.15-Linearity table for Cetirizine Impurity A IMP A % Level conc(ppm) area 50 0.12 5808 75 0.18 8167 100 0.24 11410 150 0.36 15496 200 0.48 22647 300 0.72 33973 intercept 260.7 Bias at 100% 2.2848 Correlation coefficient 0.9982 Figure 5.20- Linearity graph for Cetirizine Impurity A 40000 35000 30000 25000 20000 15000 10000 5000 0 Impurity A y = 47174x - 260.7 R² = 0.996 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Page 263 of 305

Table 5.16-Linearity table for Cetirizine Impurity B IMP B % Level conc(ppm) area 50 0.12 4214 75 0.18 6533 100 0.24 8569 150 0.36 13219 200 0.48 17332 300 0.72 26251 intercept 132.5 Bias at 100% 1.546 Correlation coefficient 0.9999 Figure 5.21- Linearity graph for Cetirizine Impurity B 30000 Impurity B 25000 20000 15000 10000 5000 0 y = 36626x - 132.5 R² = 0.999 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Page 264 of 305

Area Table 5.17-Linearity table for Cetirizine Impurity C IMP C % Level conc(ppm) area 50 0.12 1295 75 0.18 2038 100 0.24 2720 150 0.36 4294 200 0.48 5668 300 0.72 8700 intercept 196.1 Bias at 100% 7.2096 Correlation coefficient 0.9999 Table 5.22-Linearity table for Cetirizine Impurity C 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Impurity C y = 12330x - 196.1 R² = 0.999 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Page 265 of 305

Table 5.18-Linearity table for Cetirizine Impurity D IMP D % Level conc(ppm) area 50 0.12 9910 75 0.18 13982 100 0.24 17843 150 0.36 26181 200 0.48 34026 300 0.72 49754 intercept 512 Bias at 100% 2.8695 Correlation coefficient 1.0000 Table 5.23-Linearity table for Cetirizine Impurity D 60000 Impurity D 50000 40000 30000 20000 y = 66486x + 2012. R² = 0.999 10000 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Page 266 of 305

Table 5.19-Linearity table for Cetirizine Impurity E IMP E % Level conc(ppm) area 50 0.12 3202 75 0.18 4902 100 0.24 6546 150 0.36 9436 200 0.48 12232 300 0.72 18387 intercept 363.4 Bias at 100% 5.5515 Correlation coefficient 0.9997 Table 5.24-Linearity table for Cetirizine Impurity E 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 Impurity E y = 25012x + 363.4 R² = 0.999 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Page 267 of 305

Area Table 5.20-Linearity table for Cetirizine Impurity F IMP F % Level conc(ppm) area 50 0.12 1199 75 0.18 1510 100 0.24 2083 150 0.36 3470 200 0.48 4220 300 0.72 6370 intercept 64.88 Bias at 100% 3.1147 Correlation coefficient 0.9975 Table 5.25-Linearity table for Cetirizine Impurity F 7000 Impurity F 6000 5000 4000 3000 2000 1000 0 y = 8791.x + 64.88 R² = 0.995 0 0.2 0.4 0.6 0.8 Page 268 of 305

Accuracy of test method Accuracy of the test method was determined by analyzing Motelukast, Cetrizine drug substance spiked with impurities at five different concentration levels of 50 %, 75%, 100 %,150%, 200% and 300 % of each at the specified limit. The mean recoveries of all the impurities were calculated individually and are represented in the below tables individually for Motelukast, Cetrizine and all the impurities Table 5.21- Accuracy results for Montelukast Level Amount Added Amount Found %Recovery 50% 0.9093 1.0090 110.97 75% 1.4397 1.4109 98.00 100% 1.8943 1.8568 98.02 150% 2.8793 2.8026 97.33 200% 3.7886 3.6613 96.64 300% 5.6829 5.4872 96.56 Table 5.22- Accuracy results for Montelukast Impurity 1 Level Amount Added Amount Found %Recovery 50% 0.4733 0.4636 97.95 75% 0.7573 0.7204 95.13 100% 0.9466 0.9876 104.33 150% 1.4200 1.4864 104.68 200% 1.8933 1.9754 104.34 300% 2.8399 3.0990 109.12 Page 269 of 305

Table 5.23- Accuracy results for Montelukast Impurity 2 Level Amount Added Amount Found %Recovery 50% 0.7601 0.7010 92.22 75% 1.2162 1.0558 86.81 100% 1.5202 1.4143 93.03 150% 2.2804 2.1476 94.18 200% 3.0405 2.8937 95.17 300% 4.5607 4.3531 95.45 Table 5.24- Accuracy results for Montelukast Impurity 3 Level Amount Added Amount Found %Recovery 50% 0.8116 0.8294 102.20 75% 1.2985 1.2618 97.17 100% 1.6232 1.6890 104.06 150% 2.4348 2.5523 104.83 200% 3.2463 3.4004 104.75 300% 4.8695 5.0918 104.57 Table 5.25- Accuracy results for Montelukast Impurity 5 Level Amount Added Amount Found %Recovery 50% 0.5163 0.5294 102.54 75% 0.8260 0.7932 96.03 100% 1.0325 1.0635 103.00 150% 1.5488 1.6526 106.70 200% 2.0651 2.2654 109.70 300% 3.0976 3.4365 110.94 Page 270 of 305

Table 5.26- Accuracy results for Montelukast Impurity 6 Level Amount Added Amount Found %Recovery 50% 0.4101 0.4385 106.93 75% 0.6562 0.6544 99.73 100% 0.8202 0.8905 108.57 150% 1.2303 1.3371 108.68 200% 1.6404 1.7788 108.44 300% 2.4606 2.6343 107.06 Table 5.27- Accuracy results for Cetirizine Level Amount Added Amount Found %Recovery 50% 0.4786 0.4912 102.64 75% 0.7577 0.7529 99.36 100% 0.9970 0.9995 100.25 150% 1.5154 1.5060 99.38 200% 1.9940 1.9883 99.71 300% 2.9910 2.9889 99.93 Table 5.28- Accuracy results for Cetirizine Impurity A Level Amount Added Amount Found %Recovery 50% 0.1014 0.0951 93.82 75% 0.1521 0.1664 109.44 100% 0.2027 0.1868 92.14 150% 0.3041 0.3355 110.32 200% 0.4055 0.3707 91.42 300% 0.6082 0.5562 91.45 Page 271 of 305

Table 5.29- Accuracy results for Cetirizine Impurity B Level Amount Added Amount Found %Recovery 50% 0.1003 0.0931 92.86 75% 0.1504 0.1443 95.95 100% 0.2005 0.1893 94.40 150% 0.3008 0.2920 97.08 200% 0.4011 0.3828 95.45 300% 0.6016 0.5798 96.38 Table 5.30- Accuracy results for Cetirizine Impurity C Level Amount Added Amount Found %Recovery 50% 0.0721 0.0655 90.87 75% 0.1081 0.1031 95.35 100% 0.1442 0.1376 95.44 150% 0.2163 0.2173 100.49 200% 0.2883 0.2868 99.47 300% 0.4325 0.4402 101.78 Table 5.31- Accuracy results for Cetirizine Impurity D Level Amount Added Amount Found %Recovery 50% 0.0966 0.1103 114.18 75% 0.1449 0.1556 107.38 100% 0.1932 0.1986 102.79 150% 0.2898 0.2914 100.55 200% 0.3864 0.3788 98.03 300% 0.5796 0.5539 95.56 Page 272 of 305

Table 5.32- Accuracy results for Cetirizine Impurity E Level Amount Added Amount Found %Recovery 50% 0.0824 0.0743 90.15 75% 0.1236 0.1137 91.97 100% 0.1648 0.1518 92.09 150% 0.2473 0.2188 88.49 200% 0.3297 0.2837 86.05 300% 0.4945 0.4264 86.23 Table 5.33- Accuracy results for Cetirizine Impurity F Level Amount Added Amount Found %Recovery 50% 0.0711 0.0742 104.35 75% 0.1067 0.0934 87.56 100% 0.1422 0.1288 90.56 150% 0.2133 0.2146 100.60 200% 0.2844 0.2597 91.30 300% 0.4267 0.3939 92.32 Page 273 of 305

Limit of detection (LOD) and limit of quantitation (LOQ) Limit of detection or LOD is the lowest level at which the impurity or API peak can be observed or in other words can be distinguished from that of the system noise. Limit of quantitation or LOQ is the lowest level at which the impurity or API can be quantitatively estimated with an acceptable accuracy. This estimation was performed by means of the slope method. The calculation was carried by means of the following formula. LOD 3. 3 Where LOQ 10 Where S = standard deviation of intercept S = slope of the calibration curve S = standard deviation of intercept S = slope of the calibration curve The high level of sensitivity of the method can be observed by means of low levels of the LOD and LOQ values. Table 5.34- LOD and LOQ of Montelukast impurities Impurity Name LOQ LOD Impurity 1 0.006% 0.002% Impurity 2 0.008% 0.003% Impurity 3 0.008% 0.003% Impurity 5 0.006% 0.002% Impurity 6 0.005% 0.002% Page 274 of 305

Table 5.35- LOD and LOQ of Cetirizine impurities Impurity Name LOQ LOD Impurity A 0.019% 0.005% Impurity B 0.010% 0.003% Impurity C 0.017% 0.006% Impurity D 0.003% 0.001% Impurity E 0.023% 0.008% Impurity F 0.041% 0.015% Figure 5.26- Chromatogram showing LOQ level peaks Page 275 of 305

FORCED DEGRADATION STUDY The forced degradation of a drug product is performed as a part of method development or method validation in order to understand which are the degradation product peaks that are appearing in the chromatogram when the drug product is exposed to extreme conditions. This is essentially to test the capability of the test method to check if the same is able to separate any peak thus formed in any of the degradation conditions. Stability testing of an active substance or finished product provide evidence on how the quality of a drug substance or drug product varies with time influenced by a variety of environmental conditions like temperature, humidity and light etc,. Knowledge from stability studies enables understanding of the longterm effects of the environment on the drugs. Stability testing provides information about degradation mechanisms, potential degradation products, possible degradation path ways of drug as well as interaction between the drug and the excipients in drug product. Forced degradation study was carried out by treating the sample under the following conditions Acid degradation A tablet powder sample containing approx 20 mg of Cetirizine and 100mg of Montelukast was weighed and transferred into 100 ml volumetric flask and 5 ml of 1N HCl was added to it. The solution was warmed on a water bath at 80 C for 2 hr and then neutralized with 5 ml of 1N NaOH. The neutralized solution was made up to the volume with diluent. Alkali degradation A tablet powder sample containing approx 20 mg of Cetirizine and 100mg of Montelukast was weighed and transferred into 100 ml volumetric flask and 5 ml of 1N NaOH was added to it. The solution was warmed on a water bath at 80 C for 2 hr and then neutralized with 5 ml of 1N HCl. The neutralized solution was made up to the volume with diluent. Page 276 of 305

Oxidative degradation A tablet powder sample containing approx 20 mg of Cetirizine and 100mg of Montelukast was weighed and transferred into a 100 ml volumetric flask and 5 ml of 1 % Hydrogen peroxide solution was added to it. The solution was warmed on water bath at 80 C for 1 hr. Then the above mixture was kept aside for few minutes, and the volume was made up with diluent. The above stressed samples were analyzed as per the test procedure using Photodiode Array detector. The results are summarized in below table Table 5.36-Forced degradation Cetirizine Degradation Degradation Condition Net Purity Purity Type degradation angle threshold Acid Base Exposed for 1hrs with 1N HCl at 60 C Exposed for 1hr with 1N NaoH at 60 C 1.8% 0.073 0.269 0.05% 0.068 0.268 Peroxide Exposed for 1hr with 1% H2O2 at 60 C 2.3% 0.072 0.269 Page 277 of 305

Table 5.37-Forced degradation Montelukast Degradation Net Purity Purity Degradation Condition degradation angle threshold Type Acid Exposed for 1 hrs with 1N HCl at 60 C 1.9% 3.481 4.298 Base Exposed for 1hr with 1N NaoH at 60 C 0.51% 2.935 4.164 Peroxide Exposed for 1hr with 1% H2O2 at 60 C 18.0% 2.439 4.050 Chromatograms for forced degradation study. Figure 5.27- Chromatogram for Sample in as such condition Page 278 of 305

Figure 5.28- Chromatogram for Cetrizine degradation in IN HCl Figure 5.29-Chromatogram for Montelukast degradation in IN HCl Page 279 of 305

Figure 5.30-Chromatogram for Montelukast and Cetirizine In 1n HCl Figure 5.31-Chromatogram for Cetirizine In 1N NaOH Page 280 of 305

Figure 5.32-Chromatogram for Montelukast in 1N NaOH Figure 5.33-Chromatogram for Montelukast and Cetirizine in 1N NaOH Page 281 of 305

Figure 5.34-Chromatogram for Cetirizine in 1% H 2 O 2 Figure 5.35-Chromatogram for Montelukast in 1% H 2 O 2 Page 282 of 305

Figure 5.36-Chromatogram for Montelukast and Cetirizine in 1% H 2 O 2 Conclusion: A method for determination of Cetirizine, Montelukast, and their related substances has been successfully developed by HPLC. This method has also been validated as per ICH guidelines. The method has demonstrated the stability indicating capability as it has complied the acceptance criteria of separating all the unknown degradation products arising from various stress studies, namely acid, base and peroxide. The method is found to be specific, precise, linear and accurate in the range of its intended application. This method is suitable for use in routine analysis in any quality control laboratory and if applied will prove to be extremely beneficial for the organization and the end user i.e. the patient. Page 283 of 305

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