CHAPTER-2 A VALIDATED STABILITY-NDICATING ANALYTICAL METHOD FOR THE DETERMINATION OF IMPURITIES IN MONTELUKAST SODIUM

Transcription

1 CHAPTER-2 A VALIDATED STABILITY-NDICATING ANALYTICAL METHOD FOR THE DETERMINATION OF IMPURITIES IN MONTELUKAST SODIUM

2 Introduction of Montelukast sodium and survey of analytical methods Fig: 2.1 Chemical structure of Montelukast Sodium S COO - Na + Cl N HO H 3 C CH 3 Montelukast Sodium, the active ingredient in Singular*, is a selective and orally active leukotriene receptor antagonist that inhibits the cysteinyl leukotriene cys LT1 receptor [1]. Montelukast sodium is described chemically as [[[(1R)-1-[3-[(1E)-2-(7-Chloro-2- Quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1- methyethyl)phenyl]propyl]sulfinyl]methyl]cyclopropane acetic acid, mono-sodium salt (Fig: 2.1). Montelukast sodium is a hygroscopic, optically active, white to off-white powder. It is freely soluble in ethanol, methanol, water and practically in soluble in acetonitrile. The empirical formula is C35H35ClNNaO3S. The molecular weight of Montelukast Sodium is [[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-Quinolinyl)ethenyl]phenyl]-3-[2-(1- hydroxy-1-methyethyl)phenyl]propyl]-sulfinyl]methyl]cyclopropane acetic acid, mono-sodium salt. Molecular Formula C35H35ClNNaO3S Molecular Weight

3 The different analytical techniques reported so far for the determination of this drug and its metabolites in biological samples include capillary electrophoresis [2] and spectrophotometry [3]. The determination of Montelukast sodium in plasma by RP-LC [4-6] and in solid dosage forms by RP-LC [7-8] was also reported. Forced degradation or stress studies of drug substance and products play an integral role in the development of pharmaceuticals [9]. The results of degradation studies facilitate the stability-indicating method (SIM) development. As on date, no stability-indicating HPLC method for the quantitative determination of montelukast in montelukast sodium bulk drug was reported in the literature. The current ICH guidelines requires that the analysis of stability samples should be done by using stability-indicating methods (SIAM S) developed and validated after stress testing on drug under variety of conditions, including acid, base hydrolysis, oxidation, photolysis and thermal degradation [10]. Unfortunately, this route for the development of stabilityindicating related substances method was not found in most of the stability-indicating methods reported in literature [11]. The target is to develop a suitable stability-indicating HPLC related substances method for montelukast sodium. In this chapter we describe a stability-indicating LC method for the determination of montelukast sodium and its potential and degradation impurities and also the method validation.

4 Development of a novel stability-indicating analytical method for Montelukast Sodium Materials Reference standard of Montelukast and seven impurities namely, Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F, Imp-G (Fig: 2.2 (a) to Fig: 2.2 (g)) were synthesized and characterized by use of LC-MS, NMR and IR in Aurobindo Pharma Ltd., Hyderabad, India. Montelukast sodium were provided by Chemical Research Division of Aurobindo Pharma Ltd. All reagents used were of analytical reagent grade unless stated otherwise. Milli Q water, HPLC-grade acetonitrile, HPLC-grade orthophosphoric acid (OPA) were purchased from Merck (Darmstadt, Germany) Equipment The LC system was equipped with quaternary gradient pumps with autosampler and auto injector (Alliance Waters 2695, Milliford, MA, USA) controlled with Empower software (Waters). Stability studies were carried out in humidity chamber (Thermo lab Humidity chamber, India) and photo stability studies were carried out in a photo stability chamber (sanyo photo stability chamber. Leicestershire, U.K). Thermal stability studies were performed in a dry air oven ( Merck Pharmaterh, Hyd. India ). The LCMS analysis was performed on waters quatramino TM API system equipped with triple quadrapole check. (Mass Lynax 4.1).

5 Fig: 2.2 Chemical structures of impurities of Montelukast Sodium O S COOH Cl N HO H 3 C CH 3 Molecular formula C35H36ClN04S Molecular Weight [[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1- hydroxy-1-methylethyl)phenyl]propyl]sulfinyl]methyl] Cyclopropane aceticacid (Imp-A1&A2) Fig: 2.2 (a) Cl O S COOH O N HO H 3 C CH 3 Molecular formula C35H36ClN05S Molecular Weight [[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1- hydroxy-1-ethylethyl)phenyl]propyl]sulfonyl]methyl] Cyclopropane aceticacid (Imp-B) Fig: 2.2 (b) OH Cl N HO H 3 C CH 3 Molecular formula C29H28ClN02 Molecular Weight (2-(3(S)-(3-((1E)-2-(7-Chloro-2-quinolinyl)ethenyl)phenyl)-3-51 hydroxypropyl)-phenyl)-2-propanol] (Imp-C) Fig: 2.2 (c)

6 113 S COOH Cl N O CH 3 Molecular formula C34H32ClN03S Molecular Weight [[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-3-(2- acetylphenyl)-propyl]thio]methyl] cyclopropane aceticacid [Imp-D] Fig: 2.2 (d) S COOCH 3 Cl N HO H 3 C CH 3 Molecular formula C36H38ClN03S Molecular Weight [[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1- hydroxy-1-methylethyl)phenyl]-propyl]thio]methyl] Cyclopropane aceticacid, methylester (Imp-E) Fig: 2.2 (e) S COOH Cl N H 2 C CH 3 Molecular formula C35H34ClN02S Molecular Weight [[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(prop- 1-en-2-yl)phenyl]propyl]thio]methyl] Cyclopropane aceticacid (Imp-F) 52 Fig: 2.2 (f)

7 114 O CH 3 CH 3 Cl N Molecular formula C29H26ClN0 Molecular Weight Chloro-2- ((E)-2-(3-(1,3,4,5-tetrahydro-1,1-dimethylbenzo-[C] oxepin-3-yl)phenyl)-vinyl) quinoline (Imp-G) Fig: 2.2 (g) Preparation of sample solutions The stock solutions of montelukast sodium (1.04 mg/ml) and spiked with 0.50% of Imp-A and 0.15% of Imp-B, Imp-C, Imp-D, Imp- E, Imp-F and Imp-G with respect to the Montelukast analyte concentration. The stock solutions were further diluted with diluent to obtain a standard solution of 0.5 mg/ml (500 µg/ml) for related substances determination. The specification limit of Imp-A 0.50% and 0.15% of Imp-B, Imp-C, Imp-D, Imp-E, Imp-F and Imp-G in montelukast sodium bulk drug sample was 0.15% w/w Generation of stress samples One lot of montelukast sodium drug substance selected for stress testing. From the ICH stability guideline (Q1AR2): Stress testing likely to be carried out on a single batch of material [12]. Different kinds of stress conditions (i.e., acid hydrolysis, base hydrolysis, oxidative stress, heat, humidity, and light) were employed

8 on one lot of montelukast sodium drug substance based on the guidance available from ICH stability guideline (Q1AR2). The details of the stress conditions studies are as follows: Stress study under hydrolytic condition: a) Acid Degradation: drug in 1.0 M HCl solution was kept at 85 C for 15 mins. b) Base Degradation: drug in 5 M NaOH solution was kept at 85 C for 120 mins. c) Oxidative stress: drug in 3.0% H2O2 solution was kept at room temperature for 10 mins. d) Thermal stress: drug was subjected to dry heat at 80 C for 120 hrs. e) Photolytic degradation: drug was subjected to UV at 254 nm (10 K Lux ) for 48 hrs. The photolytic degradation studies was carried out by exposing the Montelukast sodium samples in solid state to light producing on overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200 wh/m2, which took about 10 days period in our photostability chamber Optimization of chromatographic conditions: Forced degradation studies were performed to develop a stability-indicating HPLC method for the quantitative determination

9 and purity evaluation of Montelukast sodium bulk drug substance. The main objective of the chromatographic method was to seperate Montelukast from Imp-A (A1 and A2), Imp-B, Imp-C, Imp-D, Imp-E, Imp-F and Imp-G. Impurities were coeluted using different stationary phases such as C8, phenyl and cyano as well as different mobile phases. During the evaluation of ph study, no effect was observed in elution order and retention times towards acidic side. Elution of impurities required higher ratios of organic modifier, hence 0.1% OPA was chosen as buffer solution to rule out precipitation of aqueous salt buffers with combination of higher organic modifier ratios. During the evaluation of various column chemistries, C18 was observed to give better resolution. Resolution between Montelukast and Imp-D was critical and conditions were optimized. The chromatographic separation was achieved on a Waters 250 x 4.6 mm, Atlantis dc18, 5 µm particles. The gradient LC method employs solution A and solution B as mobile phase. The solution A contains aqueous 0.1% orthophosphoric acid and solution B contains a mixture of water : acetonitrile (5:95 v/v). The flow rate of the mobile phase was 1.5 ml/min and the peak shape of the Montelukast sodium was found to be homogeneous and symmetrical. The HPLC gradient program was set as: time% solution B: 0.01/60, 10/70, 15/90, 20/100, 30/100, 32/60, 40/60 with a post run time of 10 min. The column temperature was thermostated at 20 C and the UV detection was monitored at a wavelength of 225 nm. The injection volume was 20 µl. A mixture of water : methanol (30:70 v/v) was used as a diluent.

10 In the optimized chromatographic conditions Montelukast, Imp-A (A1 and A2), Imp-B, Imp-C, Imp-D, Imp-E, Imp-F and Imp-G were separated with a resolution greater than 2, typical relative retention times were approximately 0.40, 0.45, 0.55, 0.63, 1.04, 1.35, 1.45, 1.59 with respect to Montelukast eluted at Degradation was not observed in Montelukast sodium samples when subjected to forced degradation studies like thermal, photolytic and base hydrolysis. Montelukast sodium was degraded to Imp-A (11.6%) under oxidation (3.0% H2O2/rt/10 mins, Imp-F (0.2%) under acidic conditions (1.0M HCl/85 C/10 mins] and some unknown degradants observed (10%) under photolytic conditions (10K Lux /48 Hours]. Peak purity test results done by using a PDA detector confirmed that the Montelukast peak is homogenous and pure in all the analyzed stress samples. The mass balance of Montelukast Sodium in all stress samples was close to 99.5% (%Assay + %Degradation). It is clearly indicating that the developed HPLC method was found to be specific for Montelukast in presence of its all impurities (Imp-A (A1 and A2), Imp-B, Imp-C, Imp-D, Imp-E, Imp-F and Imp-G) and degradation compounds.

11 Optimized liquid chromatographic conditions Column : Atlantis dc18, 250 x 4.6 mm, 5µ particle size Mobile phase : The solution A contains aqueous 0.1% OPA and Solution B contains a mixture of Water: Acetonitrile (5:95 v/v). Pump mode : Gradient Flow rate : 1.5 ml/min Column oven temperature : 20 C UV detection : 225 nm Injection volume : 20 l Run time : 30 min Retention time : Relative Retention Time (RRT) : Impurity-A (A1 and A2) about 0.40, 0.45, Impurity-B about 0.55 Impurity-C about 0.63 Impurity-D about 1.04 Impurity-E about 1.35 Impurity-F about 1.45 Impurity-G about 1.59

12 Diluent : A mixture of water : methanol (30:70 v/v) Figures: Fig 2.3 to Fig 2.7 is the typical HPLC chromatograms showing the degradation of Montelukast sodium in various stress conditions and also the corresponding peak purity plots. Fig: 2.3 Typical HPLC chromatograms of Acid hydrolysis Blank Chromatogram of Acid hydrolysis (1N HCl) Fig: 2.3 (a)

13 Montelukast Sodium stressed with 1N HCl at 85 C for 10 mins Fig: 2.3 (b) Fig: 2.3 (c) Peak purity plot of Acid hydrolysis Purity Angle Purity Threshold Purity Flag Peak Purity No Pass Fig: 2.3 (c)

14 Fig: 2.4 Typical HPLC chromatograms of Base hydrolysis Blank Chromatogram of Base hydrolysis (5N NaOH) Fig: 2.4 (a) Montelukast Sodium stressed with 5N NaOH at 85 C for 120 mins Fig: 2.4(b)

15 Fig: 2.4 (c) Peak purity plot of Base hydrolysis Purity Angle Purity Threshold Purity Flag Peak Purity No Pass Fig: 2.4 (c)

16 Fig: 2.5 Typical HPLC chromatograms of Peroxide degradation Blank Chromatogram of Peroxide Degradation ( 3% H2O2) Fig: 2.5 (a) Montelukast Sodium stressed with 3% H2O2 at room temperature (intial) Fig: 2.5 (b)

17 Fig: 2.5 (c) Peak purity plot of Peroxide Degradation Purity Angle Purity Threshold Purity Flag Peak Purity No Pass Fig: 2.5 (c)

18 Fig: 2.6 Typical HPLC chromatograms of Photolytic degradation Blank Fig: 2.6 (a) Montelukast Sodium stressed with 10K Lux for 48 hours Fig: 2.6 (b)

19 Fig: 2.6 (c) Peak purity of Photolytic Degradation Purity Angle Purity Threshold Purity Flag Peak Purity No Pass Fig: 2.6 (c)

20 Fig: 2.7 Typical HPLC chromatograms of Thermal Degradation Blank Fig: 2.7 (a) Montelukast Sodium stressed at 80 C for 120 mins Fig: 2.7 (b)

21 Fig: 2.7 (c) Peak purity plot of Thermal Degradation Purity Angle Purity Threshold Purity Flag Peak Purity No Pass Fig: 2.7 (c)

22 Validation of Analytical method and its results: The developed and HPLC method was taken up to method validation. The analytical method validation was carried out in accordance with ICH guideline [13] System suitability : A mixture of Montelukast sodium reference standard, Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F and Imp-G injections were injected into HPLC system and good resolution was obtained between impurities and Montelukast Sodium. The system suitability results are tabulated (Table: 2.1). Typical Blank, Montelukast Sodium Sample and SST Chromatograms (Fig: 2.8). Table: 2.1 System Suitability results Compound (n=3) No. of theoretical plates (N) USP Tailing factor (T) USP Resolution (Rs) Imp-A Imp-A Imp-B Imp-C Montelukast Imp-D Imp-E Imp-F Imp-G n = Number of determinations

23 Fig: 2.8 Typical Blank, Montelukast Sodium Sample and SST Chromatograms Blank Fig: 2.8 (a) Montelukast Sodium Sample Fig: 2.8 (b)

24 Montelukast Sodium sample spiked with impurities Fig: 2.8 (c) Precision: The precision of an analytical process expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under prescribed conditions. Precision may be considered at three levels: System precision, Method precision and Intermediate Precision. Assay method precision study was evaluated by carrying out six independent assays of Montelukast Sodium test sample against qualified Montelukast sodium reference standard and RSD of six consecutive assays was 0.6% (Table: 2.2 to Table: 2.4). The results showed insignificant variation observed in response. Which indicated that the assay method was repeatable with RSD s below 0.4%.

25 Table: 2.2 System Precision results of the Assay method Injection ID Montelukast Area Mean SD % RSD % Confidence Interval ± Table: 2.3 Method Precision results of the Assay method Sample ID Assay (% w/w) Mean 99.2 SD 0.15 % RSD % Confidence Interval ± 0.2

26 Table: 2.4 Intermediate Precision results of the Assay method Sample ID Assay (% w/w) Mean 99.2 SD 0.21 % RSD % Confidence Interval ± 0.2 The precision of the related substance method was checked by injecting six individual preparations of Montelukast (1.04 mg/ml) spiked with 0.50% of Imp-A and 0.15% of Imp-B, Imp-C, Imp-D, Imp- E, Imp-F and Imp-G with respect to the Montelukast analyte concentration. The % RSD of the area percentage of each impurity (imp-a, -B, -C, -D, -E, -F and -G) for six consecutive determinations was respectively as below (Table: 2.5 to Table: 2.7 ]. The results showed insignificant variation in measured response. Which demonstrated that the related substances method was repeatable with RSD s below 1.3%.

27 Table: 2.5 System Precision results of the Related substance method Injection ID Montelukast Area Mean SD 0.3 % RSD % Confidence Interval ± 136

28 Table: 2.6 Method Precision results of Related Substance method Preparation Imp-A (Sum of Imp-A1& Imp-B Imp-C Imp-D Imp-A Mean %RSD SD % Confidence level ±0.004 ±0.001 ±0.002 ±0.001 Preparation Imp-E Imp-F Imp-G Mean %RSD SD % Confidence level ±0.002 ±0.002 ±0.002

29 Table: 2.7 Intermediate Precision results of Related Substance method Preparation Imp-A (Sum of Imp-A1& Imp-B Imp-C Imp-D Imp-A Mean %RSD SD % Confidence level ±0.001 ±0.000 ±0.001 ±0.001 Preparation Imp-E Imp-F Imp-G Mean %RSD SD % Confidence level ±0.001 ±0.001 ± Limit of Detection (LOD) and Limit of Quantification () The detection limit of an individual analytical procedure is the lowest amount of analyte is a sample, which can be detected but not necessarily quantitated as an exact value (Table: 2.8).

30 The quantitation limit () of an analytical procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. The quantitative limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products (Table: 2.9). Table: 2.8 LOD values of the Montelukast Sodium and its impurities Preparation Imp-A Imp-B Imp-C Montelukast Mean SD %RSD Con. (µg/ml) Con. (%w/w) Preparation Imp-D Imp-E Imp-F Imp-G Mean SD %RSD Con. (µg/ml) Con. (%w/w)

31 Table: 2.9 values of the Montelukast Sodium and its impurities Preparation Imp-A Imp-B Imp-C Montelukast Mean SD %RSD Con. (µg/ml) Con. (%w/w) Preparation Imp-D Imp-E Imp-F Imp-G Mean %RSD SD Con. (µg/ml) Con. (%w/w) Linearity: The linearity of an analytical procedure is its ability to obtain test results, which are directly proportional to the concentration of analyte in the test sample. The linearity of the assay method was developed by injecting test sample at 80%, 90%, 100%, 110% and 120% of Montelukast sodium assay concentration (i.e.100 µg/ml).

32 Average Area Each solution injected twice (n=2) into HPLC and the average area at each concentration calculated (Table: 2.10). Calibration curve drawn by plotting average area on the Y-axis and concentration on the X-axis (Fig: 2.9). Table: 2.10 Linearity results of the Assay method % Concentration Average area Slope Intercept % Y - Intercept -1 Residual Sum of Squares 2742 Correlation Coefficient Linearity Plot (Concentration Vs Response) Conc.(µg/mL) Fig: 2.9 Linearity Plot for Assay method

33 Linearity of the Related Substance method: Linearity experiment were carried by preparing the Montelukast sodium sample solutions containing Imp- A, Imp- B, Imp- C, Imp- D, Imp- E, Imp- F and Imp- G from to 150% (i.e. 25%, 50%, 150%) with respect to their specifications limit (0.15%). Calibration curve was drawn by ploting average value of the impurities. (Imp- A, Imp- B, Imp- C, Imp- D, Imp- E, Imp- F and Imp- G on the y-axis and concentrations on the X-axis (Fig: 2.10 to Fig: 2.18).

34 Area Linearity results of the Related Substance method Table: 2.11 Linearity results of the Imp-A Imp-A Concentration (µg/ml) Area Statistical Analysis Slope Intercept Residual Sum of Squares Correlation Coefficient Response factor* 1.01 Linearity Plot (Concentration Vs Area) Con. (µg/ml) Fig: 2.10 Linearity plot for Imp-A

35 Area Table: 2.12 Linearity results of the Imp-B Imp-B Concentration (µg/ml) Area Statistical Analysis Slope Intercept Residual Sum of Squares Correlation Coefficient Response factor Linearity Plot (Concentration Vs Area) Con. (µg/ml) Fig: 2.11 Linearity plot for Imp-B

36 Area Table: 2.13 Linearity results of the Imp-C Imp-C Concentration (µg/ml) Area Statistical Analysis Slope Intercept Residual Sum of Squares Correlation Coefficient Response factor Linearity Plot (Concentration Vs Area) Con. (µg/ml) Fig: 2.12 Linearity plot for Imp-C

37 Area Table: 2.14 Linearity results of the Montelukast sodium Montelukast sodium Concentration (µg/ml) Area Statistical Analysis Slope Intercept Residual Sum of Squares Correlation Coefficient Response factor Linearity Plot (Concentration Vs Area) Con. (µg/ml) Fig: 2.13 Linearity plot for Montelukast sodium

38 Area Table: 2.15 Linearity results of the Imp-D Imp-D Concentration (µg/ml) Area Statistical Analysis Slope Intercept Residual Sum of Squares Correlation Coefficient Response factor Linearity Plot (Concentration Vs Area) Con. (µg/ml) Fig: 2.14 Linearity plot for Imp-D

39 Area Table: 2.16 Linearity results of the Imp-E Imp-E Concentration (µg/ml) Area Statistical Analysis Slope Intercept Residual Sum of Squares Correlation Coefficient Response factor Linearity Plot (Concentration Vs Area) Conc(µg/ml) Fig: 2.15 Linearity plot for Imp-E

40 Area Table: 2.17 Linearity results of the Imp-F Imp-F Concentration (µg/ml) Area Statistical Analysis Slope Intercept Residual Sum of Squares Correlation Coefficient Response factor Linearity Plot (Concentration Vs Area) Conc(µg/ml) Fig: 2.16 Linearity plot for Imp-F

41 Area Table: 2.18 Linearity results of the Imp-G Imp-G Concentration (µg/ml) Area Statistical Analysis Slope Intercept Residual Sum of Squares Correlation Coefficient Response factor Linearity Plot (Concentration Vs Area) Conc(µg/ml) Fig: 2.17 Linearity plot for Imp-G

42 Accuracy/Recovery The accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a conventional true value or an accepted reference value and the value found. Accuracy of the assay method Accuracy of the assay method was developed by injecting three different preparations of test sample at 80%, 100%, 120% of analyte concentration (i.e.100 µg/ml). Each solution was injected twice (n=2) into HPLC and the mean peak area of Montelukast sodium peak was calculated. Assay (%w/w) of test solution was determined against three injections (n=3) of qualified Montelukast sodium standard (Table: 2.19). The method was showed consistent and high absolute recoveries at all three concentration (80%, 100%, 120% ) levels with mean absolute recovery ranging from 99.3 % to 99.5%. The obtained absolute recoveries were normally distributed around the mean with uniform RSD values. The method was found to be accurate with low% bias (< 1.0).

43 Table: 2.19 Accuracy results of the Assay method S.NO Concentration (%) Mean recovery (%)(n=3) %RSD Accuracy of the related substances method established at 50% 100% and 150% of the impurities specification limit (0.15%). Accuracy at 50% impurity specification level: Test solution prepared in triplicate (n=3) with impurities (Imp-A, B, C, D, E, F and G) at 0.25% (Imp-A] and 0.1% (Imp- B, C, D, E, F, G) level w.r.s analyte concentration (i.e 1.04 mg/m l). Each solution was injected thrice into HPLC system (Table: 2.20).

44 Table: 2.20 Accuracy at 50% specification level S.NO Impurity name Mean recovery(%) SD %RSD 1 Imp-A1&Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F Imp-G Accuracy at 100% impurity specification level: Test solution prepared in triplicate (n=3) with impurities (Imp-A, B, C, D, E, F and G) at 0.5% (Imp-A] and 0.15% (Imp- B, C, D, E, F, G) level w.r.s analyte concentration (i.e 1.04 mg/m l). Each solution was injected thrice into HPLC (Table: 2.21). Table: 2.21 Accuracy at 100% specification level S.NO Impurity name Mean recovery(%) SD %RSD 1 Imp-A1&Imp- A Imp-B Imp-C Imp-D Imp-E Imp-F Imp-G

45 Accuracy at 150% impurity specification level: Test solution prepared in triplicate (n=3) with impurities (Imp-A, B, C, D, E, F and G) at 0.75% of (Imp-A] and 0.25% of (Imp- B, C, D, E, F, G) level w.r.s analyte concentration (i.e 1.04 mg/ml). Each solution was injected thrice into HPLC system (Table: 2.22). Table: 2.22 Accuracy at 150% specification level S.NO Impurity name Mean recovery(%) SD %RSD 1 Imp- A1&Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F Imp-G The related substance method was showed consistent and high accurate recoveries of all six impurities at all three different concentrations (50, 100, 150%) levels in drug substance Solution state stability: The solution state stability of Montelukast sodium in diluent in the assay method was carried out by leaving both the test solutions of sample and reference standard in tightly capped volumetric flasks kept at room temperature for two days. The same sample solutions were assayed for every one hour interval up to the study period. The %RSD of assay of Montelukast during solution stability experiments was with in 1.0%.

46 The solution state stability of Montelukast sodium related substance method was carried out by leaving sample solution in tightly capped volumetric flask at room temperature for two days. Content of impurities (Imp A, B, C, D, E, F and G) were checked for every six hours internal up to the study period. No significant change was observed in the content of all six impurities in drug solution stability experiments up to the study period. Hence Montelukast sodium sample solutions are stable for atleast 48 hours in the developed method. In assay method the standard and test solution injected at each 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h. (Table: 2.23).

47 Table: 2.23 Solution stability results of the Assay method S.No Time in Hours Assay (% w/w) 1 initial % RSD 0.42 In related substances method the stability of Montelukast sodium sample in diluent was established for 15 hr by injecting test solution for every one hour interval up to the study period. The impurity profiles obtained at different interval were very consistent and matched with initial value.

48 Robustness The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication and of its reliability during normal usage. To determine the robustness of the developed analytical method experimental conditions were purposely altered and the resolution between Montelukast and its Imp-D was evaluated. In each of the deliberately altered chromatographic condition (flow rate 1.3 ml/min and 1.7ml/min, acetonitrile 58% and 62% in the mobile phase, column temperature 15 C and 25 C) the resolution between Imp-B, Imp-C and Imp-D, Imp-E and Imp-F was greater than 2.0, to illustrating the robustness of the method Mass balance The mass balance is a process of adding both the assay value and the levels of degradation products to see how closely these add up to 100% of the initial value, with due consideration of the margin of analytical error. Its establishment hence is a regulatory requirement. The mass balance is very closely linked to the development of stability- indicating assay method as it acts as an approach to establish its validity. The stressed studies samples of Montelukast sodium bulk drug were assayed against the qualified reference standard and the results of mass balance obtained were very close to 99.8%. The results of mass balance obtained in each condition is presented below (Table: 2.24).

49 Table: 2.24 Mass balance of the Assay method Degradation Mechanism Acid Base Peroxide Thermal Photolytic Degradation Condition 1M HCl / 85 C/15 min. 5M NaOH / 85 C /120min 30% H 2O 2/ RT /10 min 80 C/120 Hours 10K Lux/48 Hours % Assay of active substance Mass balance (% Assay+ % impurities+ % degradants) Remarks Degraded to Imp- F and some unknown degradants observed No degradation observed Degraded to Imp- A and some unknown degradants observed No degradation observed Some unknown degradants observed 2.3 Analysis of Montelukast sodium drug substance stability samples One manufacturing lot of Montelukast drug substance was placed on stability study in chambers maintained at ICH set conditions [12]. The analysis of stability samples were carried up to 24 months period using the above optimized method. The stability data results obtained are presented in Table: 2.25 and Table: The developed HPLC method performed satisfactorily for the quantitative evaluation of stability samples.

50 Table: 2.25 Accelerated stability data ( Storage conditions 40 C/75%RH) Batch No: PS(781)194 Packing & storage conditions: Each sample packed in a polyethylene bag in a triple laminated bag and kept in a HDPE drum Stability study duration: 6 months Temperature %Relative humidity 40 C/75%RH Tests Specifications Initial 1M 2M 3M 6M Description A white to off-white, amorphous powder A white powder A white powder A white powder A white powder A white powder Water content (%w/w, KF) NMT 0.5 Identification IR spectrum should concordant with that of standard Assay (By HPLC, %w/w, on anhydrous basis) NLT 98.0 and NMT Complies Complies Complies Complies Complies 99.0 Related substances details on next page.

51 Related Substances (%w/w) LOD (%w/w) Related Substances (By HPLC, %w/w) INITIAL 1M 2M 3M 6M Imp-A1 ND ND ND ND ND Imp-A Imp-B ND ND ND ND ND Imp-C Imp-D Imp-E ND ND Imp-F Imp-G Highest unknown Total unknown Total RS ND: Not detected NA: Not available

52 Table: 2.26 Long-Term stability data ( Storage conditions 25 C/60%RH) Batch No: PS(781)19 Packing & storage conditions: Each sample packed in a polyethylene bag in a triple laminated bag and kept in a HDPE drum Stability study duration: 12 months Temperature %Relative humidity 25 C/60%RH Tests Specifications Initial 1M 2M 3M 6M 9M 12M 24M Description A white to off-white, amorphous powder A white powder A white powder A white powder A white powder A white powder A white powder A white powder A white powder Water content (%w/w, KF) NMT 0.5 Identification IR spectrum should concordant with that of standard Assay (By HPLC, %w/w, on anhydrous basis) NLT 98.0 and NMT Complies Complies Complies Complies Complies Complies Complies Complies 99.9 Related substances details on next page.

53 Related Substances Imp-A1 Imp-A2 (%w/w) LOD (%w/w) Related Substances (By HPLC, %w/w) Initial 3M 6M 9M 12M 24M ND ND ND ND ND Imp-B Imp-C Imp-D Imp-E Imp-F Imp-G ND ND ND ND ND ND Highest unknown Total unknown - - ND ND ND ND ND ND - - NA NA NA NA NA NA Total RS ND: Not detected NA: Not available

54 Summary and Conclusions Validated stability-indicating HPLC method was developed for Montelukast sodium after subjecting the samples to stress testing under ICH recommendes conditions. The RPLC method developed for quantitative and related substance determination of Montelukast sodium is rapid precise, accurate, linear and selective. The method was completely validated and showing the satisfactory data for all the method validation parameters tested. The developed method was found to be specific to the drug, as the peaks of the degradation products did not interfere with the degradation peak. Thus the proposed method can be employed for assessing the stability of Montelukast sodium bulk drug samples.

55 100 Table: 2.27 Summary of Analytical method validation data Test Parameter Related Substances method Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F Imp-G Assay method Precision (RSD) LOD (µg/ml) (µg/ml) Linearity (corre coefficient) N/A N/A Accuracy (%) Robustness Resolution b/w Montelukast Imp-D>2 Resolution b/w Montelukast Imp-D>2 Resolution b/w Montelukast Imp-D>2 Resolution b/w Montelukast Imp-D>2 Resolution b/w Montelukast Imp-D>2 Resolution b/w Montelukast Imp-D>2 Resolution b/w Montelukast Imp-D>2 Resolution b/w Montelukast Imp-D>2 Solution stability Stable up to 15hr Stable up to 15hr Stable up to 15hr Stable up to 15hr Stable up to 15hr Stable up to 15hr Stable up to 15hr Stable up to 15hr Mobile phase stability Stable up to 15hr Stable up to 15hr Stable up to 15hr Stable up to 15hr Stable up to 15hr Stable up to 15hr Stable up to 15hr Stable up to 15hr

56 References: 1. Physicians Desk Reference 63 rd Edition., 2009, pp Shakalisava, Y.; Regan, F.; J. Sep Sci., 2008, 31, Alsarra, I.; Khalil, N. Y.; Sultan, M.; AL-Ashban, R.; Belal, F.; Pharmazie., 2005, 60, Radhakrishna, T.; Narasaraju, A.; Ramakrishna, M.; Satyanarayana, A.; J. Pharm. Biomed. Anal., 2003, 31, Sripalakit, P.; Kongthong, B.; Saraphanchotiuritthaya, A.; J.Chromatogr., B Analyt Technol Biomed Life Sci. 2008, 869, Ochiai, H.; Uchiyama, N.; Takano, T.; Hara, K.; Kamei, T.; J. Chromatogr., B 1998, 713, Smith, G. A.; Rawls, C. M.; Kunka, R. L.; Pharm Res., 2004, 21, AL-Rawithi, S.; AL-Gazlan, S.; AL-Ahmadi, W.; Alshowaier, I. A.; Yusuf, A.; Raines, D. A.; J. Chromatogr., B: Biomed Sci., 2001, 754, Reynolds, D. W.; Facchine, K. L.; Mullaney, J. F.; Alsante, K. M.; Hatajik, T. D.; Motto, M. G.; February 2002, Stability Testing of New Drug Substances and Products (Q1AR2). ICH Harmonised Tripartite Guideline. 11. Bakshi M.; Singh S.; J.Pharm. Biomed. Anal. 2002, 28, Steven, W. Baertschi Pharmaceutical Stress Testing Predicting Drug Degradation.

57 Validation of Analytical Procedures: Methodology Q2B ICH Guidelines.