CHAPTER-3. Zolmitriptan

Transcription

1 45 CHAPTER-3 Zolmitriptan

2 46 CHAPTER-3 Chapter-3 : Zolmitriptan S. No. Name of the Sub- Title Page No. 3.1 Introduction Experimental Method validation Result& Discussion Conclusion 78

3 47 CHAPTER INTRODUCTION Zolmitriptan compound was considered for the method development and method validation. Chemically it is described as (S)- 4-({3-[2-(dimethylamino)ethyl]-1H-indol-5-yl}methyl)-1,3-oxazolidin-2- one. The empirical formula is C16H21N3O2. The molecular weight of zolmitriptan is The chemical structure of the compound depicted in fig.3.1. Zolmitriptan is a artificial tryptamine unoriginal and show like a white crystalline powder that is freely soluble in water. It is a discriminating serotonin receptor agonist of the 1B and 1D sub types drug, employed for the acute cure of migraine attack with or exclusive of aura and cluster headaches Fig.3.1. Chemical structure of zolmitriptan So many analytical methodologies be cited in analytical journals for the quantitative willpower of Zolmitriptan and its appropriate metabolites in plasma of human and further biological fluids A little number of achiral and chiral HPLC techniques were noticed in favor of the identification and quantification of related substances in the drug of zolmitriptan

4 48 A Significant quantity of analytical HPLC technique has been described for the strength of mind of Zolmitriptan and its related substances in bulk drugs as well as formulations. Numerous HPLC, LC-MS and HPTLC techniques were useful in analytical magazines throughout method enrichment, A few of the methodologies were revealed by LC-MS for serum and blood samples. In this Chapter, we made attention for the improvement of a new stability representative isocratic RP-UPLC methodology for the willpower of assay and process impurities in Zolmitriptan. The impurity details of Zolmitriptan is shown in table.3.1. Forced humiliation studies were conducted to find the stability representative character of the methodology. System suitability, method precision, detection limit (DL), Accuracy, quantification limit (QL) and linearity were in arrangement as stated by ICH procedures Table 3.1: Details of Zolmitriptan process impurities S.No Impurity structure Chemical name Molecular weight Impurity-1 (S)-4-(3-amino benzyl ) oxazolidin-2-one

5 49 Impurity-2 (S)-4-({3-[2-(dimethyl one N-Oxide Impurity-3 (S)-4-({3-[2-(dimethyl amino)ethyl]-1h-indol- 5-yl}methyl)-oxazolidin- amino)ethyl]-1h-indol- 5-yl}methyl)- oxazolidin-2-one 3.2 EXPERIMENTAL Materials Zolmitriptan and its related impurities such as impurity-1, 2 and 3 were received as gift samples with characterized data from M/S Inogent Laboratories private Ltd, Hyderabad, India. Purity was greater than 99% for zolmitriptan and greater than 97% for its related impurities. Acetonitrile, methanol and ammonium dihydrogen phosphate was procured from M/S. Merck. All supplementary aqueous solutions as well as the buffers for the eluent were acquired with high purified water yield in house from Millipore water refinement system.

6 Equipment The employed Acquity UPLC system mainly outfitted of sample manager, solvent manager with PDA detector. The productivity indication was scrutinized and managed employing Empower software. Water bath was employed for degradation experimentation. Photo stability experimentations were performed in chamber of photo stability. Thermal degradation stability experiments were employed in dry hot air oven Chromatographic conditions UPLC system outfitted by a degasser and quaternary pumps along with auto sampler was used to 1µL of the sample onto the BEH C18, 1.7 μm column, 100 mm length,2.1 mm i.d., which was reserved at column high temperature at 40ºC. The isocratic eluent, is a blend of 0.01 M ammonium dihydrogen phosphate adjust ph9.5±0.02 with dilute solution of ammonia and acetonitrile in the combination of 830:170 v/v, it was permit through a 0.22μm membrane filter and finally degassed and sonication for some time and distributed at flow rate of 0.3 ml/min keen on the detector Method development Wave length Selection The UV absorption spectra were generated for Zolmitriptan, impurity-1,2 &3 with the help of PDA detector. Zolmitriptan and its process impurities were initiate to have unreliable absorption of UV maxima over a series of wavelength. Although it was introduced that at on the substance of 225 nm, Zolmitriptan and process impurities

7 51 were carried into being to have superlative imaginable absorption of UV. Consequently, 225 nm was preferred for the experiment and quantification of Zolmitriptan and process impurities. The UV spectra of related impurities are publicized in figure Fig.3.2 :Zolmitriptan UV spectrum: Fig. 3.3: spectrum of Impurity-1 Fig. 3.4: spectrum of Impurity-2

8 52 Fig. 3.5: spectrum of Impurity Selection of mobile phase and stationary phase Zolmitriptan processes impurities such as impurity-1, 2 and 3 were having different functional groups. These impurities, illustrate dissimilar affinities with eluents and stationary phase. Unusual stationary phase(column) with dissimilar selectivity offers superior division for method establishment. Specifically, two constraint are preferred to get essential resolutions and symmetrical peaks and separations for zolmitriptan and process impurities i.e., Selection of the stationary phase and eluent Selection of Mobile phase The processes impurities such as Impurity-1, 2 and 3 be co-eluted with combination of eluents. Zolmitriptan is triptan derivative and related substances of Zolmitriptan are having extensive series of schisms and the partition of impurities primarily influenced by the type of stationary phase (column). An isocratic eluent of buffer is a mixture of 0.01M Ammonium dihydrogen phosphate in milli Q water, adjusted to ph 9.5 with diluted solution of ammonia and acetonitrile was taken for initial method development conditions for the partition

9 53 of Zolmitriptan and its processes impurities. Mobile phase was degasified and filtered with the help of 0.22 microns millipore filter paper Selection of stationary phase Various stationary phases were made sure for the division of Zolmitriptan such as Waters Acquity UPLC BEH C8, C18, Phenyl, BEH Hilic and HSS T3 using by mobile phase specified. The experimentation was started using Acquity HSS T3 100mm X 2.1 mm with 1.7 microns particle size. Experiment-1: The whole test niceties are as mentioned below Column : Acquity HSS T3 100mm, 2.1 mm. with 1.7 microns particle size. Eluent Sample Preparation : : Mix Acetonitrile and buffer in the relative amount of 25:75(v/v). 0.5 mg/ml solution in diluent Wavelength : 225 nm Flow rate : 0.3 ml/ min Temperature of column : 25 C Diluent : Mobile phase Mode of Elution : Isocratic Elution time : 12 min

10 54 Fig.3.6: Zolmitriptan impurities blend solution Observation: In this case, impurity-1 and Impurity-2 are co eluting with each other and Impurity-3 peak shape also found to be very broad. Further, it is found that zolmitriptan peak shape was not symmetrical. Hence Acquity UPLC HSS T3 column is found to not suitable for the separation of Impurity-1&Impurity-2. Experiment-2: For the better resolution and peak shape, 2 nd experiment was performed with the following circumstances: Column : Acquity BEH Hilic 100mm x 2.1 mm with 1.7µm. Mobile Phase : Acetonitrile and aq. Buffer in the proportion of 75:25(v/v). Sample conc. : 5 mg in 10 ml of diluent Detection : 225 nm Eluent Flow : 0.3 ml/ min Column temperature : 25 C Diluent Elution mode Runtime : : : Mobile phase Isocratic mode 12 min

11 55 Fig. 3.7: Impurities blend solution Observation: Here Impurity-1 and Impurity-2 are found to be co eluting along with each other. Hence Acquity UPLC BEH Hilic column is not appropriate suitable for the separation of Impurity-1&Impurity- 2. The chromatogram is shown in fig.3.7. Experiment-3: Again, 3 rd trial was performed for better resolution and separation of impurities with the following revised parameters: Column : Acquity UPLC BEH Phenyl 100x 2.1 mm 1.7µm. Eluent : Acetonitrile and Aq. buffer and in the proportion of 75:25 v/v Sample conc. : 0.5 mg/ml solution in diluent Detection : 225 nm Flow : 0.3 ml/ min Column temperature : 25C Diluent Elution mode Runtime : : : Mobile phase Isocratic 12 min

12 56 Fig. 3.8: Zolmitriptan impurities blend solution chromatogram Observation: Here also observed that Impurity-1 and Impurity-2 are co eluting with each other and impurity-3 found to elute very close to impurity-1&2. Therefore Acquity BEH Phenyl column is not suitable for the separation of Impurity-1& 2. Typical chromatogram as revealed in fig.3.8. Experiment-4: Experimentation- 4 was proficient by captivating in view of the above concern and alter the following parameters were recognized for better division and resolution. Column Mobile Phase Sample conc. Detection Eluent Flow Column temperature Diluent Mode of Elution Runtime : Acquity BEH C8 100mm x2.1 mm 1.7µm. : Phosphate buffer plus Acetonitrile in the percentage of 85:15 v/v. : 0.5 mg/ml in diluent : 225 nm : 0.3 ml/ min : 25oC : Mobile phase : Isocratic : 12 min

13 57 Fig.3.9: Zolmitriptan impurities blend solution chromatogram Observation: From fig.3.9, it is found that Impurity-1, 2 and 3 were well divided, but Impurity-3 peak shape is not resolved completely. So the method further needed to refine for improvement of the Impurity-3 peak shape. Hence BEH C18 column is optional to get symmetrical peak nature for the Zolmitriptan and impurities by varying the column with the same constraints. Experiment-5: To conclude, experiment- 5 was proficient by attractive into consideration of the above, the subsequent refined constraint reputable for better and high resolution and severance of impurities. Column : Acquity UPLC BEH C18, 100 mm X 2.1 mm X 1.7 micron. Buffer preparation : 0.01M Ammonium dihydrogen phoshate in water ph adjusted to 9.5 with dil. ammonia solution. Mobile phase Sample preparation : : Buffer :Acetonitrile in the percentage of 83:17 v/v. 0.5 mg/ml in diluent Detection : 225 nm Flow : 0.3 ml/min

14 IMP IMP IMP AU ZOLMITRIPTAN Column temperature : 40 C Diluent Elution Runtime : : : Mobile phase Isocratic 12 min Minutes Fig. 3.10: Typical chromatogram of Zolmitriptan impurities blend solution Observation: From the chromatogram shown in fig.3.10, it is found to that all impurities are fine divided and found symmetrical peak nature with good resolution. Hence Acquity BEH C18 stationary phase is appropriate for the division of Zolmitriptan and its impurities. Conclusion Based on the above all experimentation on fixed phase, it was originated to facilitate Impurity-1, 2, 3 and Zolmitriptan compounds were well separated from each other in Acquity BEH C18, 100mm x 2.1 mm 1.7 microns column and same was applying for organization of specificity and method of validation Specificity The specificity of the UPLC methodology for Zolmitriptan was conceded out in the occurrence of its related impurities namely

15 59 impurity-1,2 and 3. Stability representative nature and specificity of the anticipated technique was conducted with the aid of forced degradation studies for Zolmitriptan bulk drug. Standard degradation studies were undertaken for stress circumstance of UV light at 254 nm, thermal at 105 C, acid hydrolysis in 0.1N HCl at 60 C, hydrolysis of base conducted in 0.5N NaOH at 60 C, oxidation at 0.01% H2O2 and hydrolysis of water conducted at 60 C to examine the capability of the developed LC method to divide Zolmitriptan from its humiliation products. The degradation study period for thermal was 7 days and UV light studies were 48 hours, 12 hours for the acid, base, water hydrolysis and oxidation studies. The PDA detector was used to evaluate the Peak purity of harassed samples of Zolmitriptan. Quantification assay investigation was executed for stress samples in opposition to qualified Zolmitriptan reference standard. The assay of Zolmitriptan also calculated in the presence of all the three impurities in test requirement level (i.e., 0.15%) 3.3 Method validation Precision The precision for assay method was estimated for zolmitriptan test sample in opposition to a eligible primary reference standard. Six independent assays were conducted and calculated the % RSD for replicate assay determination. Six different measures of Zolmitriptan (0.5 mg/ml) added with 0.15% level of related impurities namely impurity-1, 2 and 3 with admiration to zolmitriptan analyte concentration were considered for

16 60 the evaluation of precision of the related substance method and the RSD percentage of area for impurity-1, 2 and 3 were deliberate for six replicate injections Quantification limit (QL) and Detection Limit (DL) The quantification and detection limit were measured by using signal-to-noise ratio of analyte peak 10:1 and 3:1, correspondingly, by injecting a sequence of various concentrations of solutions with known impurities. Precision experimentation was also executed at the LOQ level by establishing different arrangements of all impurities for six times, scheming the % RSD of the area of each impurity Linearity Test solutions to evaluate linearity for the assay attitude were arranged from Zolmitriptan stock solutions at dissimilar concentration range from 50% to 150 % of analyte deliberation i.e, 50, 75, 100, 125 and 150 respectively. The concentration against peak area statistics was derived by least-squares linear degeneration analysis. Test solutions to evaluate linearity for the purity methodology were arranged by reduced stock solutions to the necessary concentration. The test solutions be organized at dissimilar concentration intensity beginning 0.05% to 0.30 % Accuracy Accuracy for the assay technique was examined at three different levels and each concentration was conceded in triplicate, i.e. 50 %, 100% and 150 % with admiration to analyte test concentration. The

17 61 recovery percentage was measured from the Y-intercept and slope of the calibration arc taken in the linearity study. The accuracy of the related substances technique was examined at six concentration levels and each concentration was executed in triplicate, i.e. at 0.05 %, 0.10%, 0.15%, 0.2 %, 0.25 % and 0.3% of the zolmitriptan analyte concentration (0.5 mg/ml). % Recoveries of the interrelated impurities was considered by the Y-intercept and slope of the calibration arc Robustness The robustness of the made-up LC technique was appraised by intentionally changing investigational situations and the resolution between Zolmitriptan, impurity-1, 2 and 3 was credentials. The flow rate for the eluent was 0.3 ml/min. The consequence of flow rate of eluent on the resolution was deliberate at different flow rates of 0.25 and 0.35 ml/min respectively, instead of 0.3 ml/min. The consequence of the temperature on resolution was research at 35 C and 40 C as an alternative of 45 C. The consequence of ph on the eluent was deliberate by changeable ph by 0.1 to units, although other eluent composition was detained constant Solution stability The stability of solution studies of Zolmitriptan assay methodology was achieved by preparing the analyte sample solution and reference standard in diluents and leaving together in closely capped in suitable flasks at ambient situation for 48 hours. The similar analyte

18 62 solutions were performed for assay for each 6 hours gap up to the achievement of the study era. Stability of solution of Zolmitriptan and its suitable impurities in the concerned substance methodology was achieved by parting spiked analyte solutions in strongly capped flask at ambient temperature up to 48 hours. comfortable of impurities namely impurity-1, 2 and 3 were resolute for every 6 hours gap up to the completion of the study period Eluent (Mobile phase) stability The steadiness of mobile phase for assay methodology was executed by assaying the newly arranged analyte solutions versus freshly organized reference standard solution for 6 hours gap up to the end of the period i.e.48 hours. Eluent arranged was reserved invariable throughout the study period. The % RSD for the assay of Zolmitriptan was deliberated throughout eluent and solution steadiness experimentation. The eluent steadiness of related substances method was accomplished for 48 hours by injecting the recently primed analyte sample solutions for each 6 hrs gap. Stuffing of impurities namely impurity-1, 2 and 3 were determined in the analyte sample. 3.4 Results and discussion Specificity During the forced degradation study, Zolmitriptan sample was shown stable at Photo degradation, Thermal, water hydrolysis and Acid hydrolysis. Zolmitriptan was despoiled into Impurity-2 in

19 63 oxidation condition and unknown peaks were observed in base hydrolysis. All degraded samples are analyzed and initiated that humiliation peaks are alienated from known impurities and Zolmitriptan. The results from Peak purity assessment inveterate that the Zolmitriptan peak is pure and homogenous in all the pressure samples. There was no change in assay of zolmitriptan in the occurrence of related substances (impurity-1,2 and 3) and its humiliation products authenticates the stability demonstrating strength of the methodology. The review of forced humiliation studies is specified in the table Peak purity study were conventional through PDA detector and demonstrate that Zolmitriptan is peak is uncontaminated in all circumstances. The degradation studies results are précis in Table 3.2. Table 3.2: Zolmitriptan Degradation studies result Stressed condition Time (hr) % Purity Peak Purity Thermal humiliation Pass Photo humiliation Pass Peroxide hydrolysis Pass Acid hydrolysis Pass Base hydrolysis Pass Water hydrolysis Pass

20 IMP IMP IMP AU ZOLMITRIPTAN Fig. 3.11: Reference Chromatogram of Diluent Minutes Fig 3.12: System suitability solution Fig 3.13: Analyte Chromatogram

21 65 Fig 3.14: Chromatogram of Acid hydrolysis Fig 3.15: Reference chromatogram of Base hydrolysis Fig 3.16: Reference chromatogram of Oxidation sample

22 66 Fig 3.17: Reference chromatogram of Thermal humiliation Fig 3.18: Reference chromatogram of Photo Degradation Optimized method From on the over study, the subsequent chromatographic methodology was preferred for the division and quantification of Zolmitriptan and its related substances. The chromatography stationary phase (column) is BEH C18,100 mm, length x 2.1 mm with 1.7 µm particles. The eluent was prepared by amalgamation buffer and acetonitrile (83: 17; v/v) and the buffer consists of 0.01M ammonium hydrogen phosphate ph attuned to 9.5 with ammonia. The eluent flow reserved at 0.3 ml/min, column temperature preserve at 40 C, the nature of the peak of zolmitriptan

23 67 was established to be regular. In chromatographic optimized circumstances of Zolmitriptan, impurity-1, 2 and 3 were alienated with resolution exceeding 2, representative RT were about 5.06, 1.48, 1.19and 1.79 min, respectively (Fig3.12). The outcome of system suitability solution is mentioned in Table-3.03 and the developed RP- UPLC technique was originate to be unambiguous for Zolmitriptan and its associated impurities specifically- impurity-1, 2, and 3 (Table-3.01) Method validation results for purity System suitability In the system suitability analyte solution was inserted into UPLC for related substance method and system suitability constraints like tailing factor, resolution and number of theoretical plates were estimated (Table-3.3). Table 3.3: System suitability results S. No Name R t(min) RRT Resolution, Theoretical Tailing Rs plates, N factor, T 01 Impurity Impurity Impurity Zolmitriptan

24 Limit of detection and quantification The detection limit for Impurity -1, 2 and 3 were established to be %, %, and % correspondingly. The Quantification Limit for Impurity -1, 2 and 3 were established to be 0.04 %, % and 0.02 % correspondingly. The final outcomes are recapitulate in the underneath table. Table 3.4: Results of Detection Limit and quantification Limit Conc. Impurity -1 Impurity -2 Impurity -3 (%) LOD (%) LOQ Precision and accuracy at quantification limit level The impurities percentage RSD at the LOQ level for Impurity-1, 2 and 3 were 1.2%, 1.5% and 5.0 %. The percentage recovery at LOQ level for Impurity-1, 2 and 3 were 99.8, 99.9 and 99.5 correspondingly. The outcome are précis in the beneath table. Table 3.5: Accuracy at quantification level and Precision results S. No Name of the Impurity % RSD (n=6) % Recovery (n=3) 1 Impurity Impurity Impurity Linearity The Linear calibration plots for the processed impurities described as Imp-1, 2 and 3 were found over the calibration vary experienced, i.e. The achieve concentration levels 0.05% to 0.3%. The correlation

25 69 coefficient be beyond The outcome illustrate that an outstanding relationship present between the concentration and the peak area of imp-1, 2 and 3. The linearity computation were prepared right through Microsoft XL data computations. Table 3.6: Impurity-1 Linearity data S. No Concentration Area of Impurity-1 (%) Correlation coefficient Slope Y-Intercept Fig 3.19: Zolmitriptan Impurity-1 Linearity graph

26 70 Table 3.7 Linearity data Impurity-2 S. No Concentration (%) Area of Impurity Correlation coefficient Slope Y-Intercept Fig Zolmitriptan Impurity-2 graph

27 71 Table 3.8 Zolmitriptan Impurity-3 Linearity data S. No Concentration (%) Area of Impurity Correlation coefficient Slope Y-Intercept Fig 3.21: Zolmitriptan Impurity-3 graph Accuracy: The recovery % of all associated impurities in Zolmitriptan analyte is exposed in below Table 3.9

28 72 Table 3.9: Accuracy in % Recovery: Concentration(%) Imp-1(%) (n=3) Imp-2(%) (n=3) Imp-3(%) (n=3) 0.05% % % % % % Method Precision: In the purity method, method precision was verified by introducing six individual measures of Zolmitriptan impaled by 0.10% of Impurity-1, 2 and 3. The % R.S.D of the area for each of Impurity- 1, Impurity-2 and Impurity-3 were considered. The outcome were recapitulated in the underneath table Table 3.10: Results of method Precision data S. No Preparation Imp-1 area Imp-2 area Imp-3 area Average Standard Deviation %RSD

29 Robustness: The Robustness of the methods was fruitfully demonstrated and in all the intentionally dissimilar chromatographic circumstances (flow rate of eluent, ph of the eluent and temperature ), the resolution between significant pair, i.e., Impurity-2 and 3 was above 2.0, demonstrated the forcefulness of the methodology. The detailed experimentation data is depicted in Table Table.3.11: Results for robustness data Relative retention time ( RRT, in minutes) Parameter Imp -1 Imp 2 Imp 3 Tailing Theoretical Resolution factor(t) plates(n) (Rs) 1. Flow Rate, ml/min (± 0.05 ml/min) Temperature((± 5 C) 35 C C C ph (± 0.1 of the set ph)

30 Solution and Eluent(Mobile phase) stability No considerable difference were identified in the substance of three impurities 1,2&3, and other degradation impurities are also not noticed for the period of the solution and eluent stabilities tests when examined using the related substance methodology. The eluent and solution steadiness trial information authenticate that the analyte solutions and eluent employed throughout the quantitative determination of impurities were stable for as a minimum 48 hours. The outcomes are précis in the beneath table Table 3.12: Results of Solution stability data Duration Imp-1 Imp-2 Imp-3 Any other Purity (%) (%) (%) impurity(%) (%) SS Initial 0.01 ND After 12 hrs 0.01 ND After 24 hrs 0.01 ND After 48 hrs 0.01 ND Table 3.13: Mobile phase stability data Duration Imp-1 Imp-2 Imp-3 Any other Purity (%) (%) (%) imp(%) (%) SS Initial 0.01 ND After 12 hrs 0.01 ND After 24 hrs 0.01 ND After 48 hrs 0.01 ND

31 Method validation results for Assay System suitability The system suitability of analyte sample was injected into UPLC for quantitative estimation and evaluated the plate count, Tailing factor and Relative standard deviation for replicate injections Table 3.14:Zolmitriptan assay System suitability Results S. No. Parameter Observed result 1 %RSD Tailing factor Theoretical plates Linearity The calibration arc for linearity of the assay methodology was acquired in excess of the calibration series tested, i.e μg/ml and correlation coefficient acquired was found exceeding S. No Level (%) Area of Zolmitriptan Correlation coefficient ( r 2 ) Slope Y-Intercept Table 3.15: Zolmitriptan Linearity results

32 76 Fig 3.22: Zolmitriptan Linearity graph Precision The % RSD for precision experimentation was within 0.25% in assay of Zolmitriptan throughout the analysis. Table 3.16: Zolmitriptan precision results Preparation Zolmitriptan Assay(%w/w) Average STDEV %RSD Accuracy The percentage recovery for assay of Zolmitriptan in bulk drug sample was range starting 99.1 to 100.7%.

33 77 Table 3.17: Zolmitriptan Accuracy data Concentration (mg/ml) % Recovery (n=3) Robustness The relative standard deviation for six replicate injection from system suitability solution found below 0.5%. Results mentioned in below table Table 3.18: Robustness data System suitability parameters Parameters and Results Temperature Flow Rate Variation Variation ph Variation As such 0.25 ml/min 0.35 ml/min At 35 C At 45 C ph at 9.4 ph at 9.6 % RSD Stability of solution and Mobile phase There is no change experiential in assay during the solution stability and eluent stability throughout the time period. Results mentioned below in table 3.19.

34 78 Table 3.19 Zolmitriptan Stability of Solution and Mobile phase Interval Solution stability Mobile phase stability % Assay % Assay Initial After 6 hrs After 12 hrs After 24 hrs After 48 hrs Conclusion The simple RP-UPLC isocratic technique developed for quantification of Zolmitriptan and processed impurities is specific, accurate, precise and rapid. The established technique was completely authenticated based on adequate data throughout the method of validation factors tested. The optimized technique can be employed for the analysis of regular production drugs as well as stability samples.