Chapter 5 STABILITY INDICATING ASSAY METHOD AND IMPURITY PROFILING OF DROTAVERINE HYDROCHLORIDE

Transcription

1 hapter 5 STABILITY INDIATING ASSAY METHD AND IMPURITY PRFILING F DRTAVERINE HYDRHLRIDE 118

2 5. STABILITY INDIATING ASSAY AND IMPURITY PRFILING F DRTAVERINE HYDRHLRIDE (DRT) A simple, sensitive, fast, and accurate RP-HPL stability indicating assay method was described for DRT. The method involves use of simple mobile phase and separation was achieved on octadecyl stationary phase with isocratic mode. For studying the degradation behavior of DRT, extensive stress degradation studies were carried out as per IH guidelines and all degradation products formed in the stress studies were separated with the developed stability indicating assay method. DRT was found to be stable in thermal and solid state photolytic stress conditions and susceptible for degradation in acid, alkaline, neutral, and oxidative and solution state photolysis stress conditions. Total three degradation products were observed with maximum degradation in alkaline and neutral stress conditions. Subsequently, the developed assay method was validated and the results were within the range of acceptance criteria. Finally the applicability of the method was proved when it was applied for the determination of DRT in its pharmaceutical tablet formulations. The simple and sensitive RP-HPL method for the determination of related impurities of DRT was developed. The developed method was selective and could separate all the impurities found in DRT. The method was also validated as per IH guidelines and was found to be reliable from the results of all the validation parameters. The method was then extended for the detection and quantification impurities in tablet formulations of DRT where it was found that the impurities detected in API of DRT were also observed in each tablet formulation of DRT in higher amount (above identification threshold) indicating the possible cause of degradation of DRT. As both the impurities detected in DRT and in its all the formulations were same as the degradation products DP-I and DP II, the same were targeted for their isolation and structural characterization. For this, Prep-HPL method was developed for the isolation and isolated degradation products of DRT, which were further characterized by using spectroscopic techniques like UV, FT-IR, Mass, and NMR spectroscopy. Finally from the results of all the spectroscopic techniques and elemental analysis the structures of all isolated degradation products were proposed. The postulated mechanism for the formation of all the degradation products of DRT from parent drug also helps in knowing the intrinsic stability of DRT. 119

3 5.1. hemicals and materials Analytically pure (98.30 %) Drotaverine Hydrochloride (DRT) Active Pharmaceutical Ingredient was procured from Troikaa Pharmaceutical Ltd., (Ahmedabad, India), with ertificate of Analysis. Methanol, acetonitrile, potassium dihydrogen phosphate, orthophosphoric acid, formic acid, and ammonia used for mobile phase preparation were of HPL grade, Merck, Mumbai, India. Hydrochloric acid, sodium hydroxide, and hydrogen peroxide (30 % w/v) used for stress degradation studies were of analytical reagent grade, DH hemicals, Delhi, India. alibrated micropitte were used for purpose for measurement and transfer. De-ionized water prepared using Milli-Q plus purification system, Millipore (Bradford, USA) was used throughout the study. The description of three tablet formulations of DRT is given in Table 5.1 Sr. No. TABLE 5.1 Detail information about DRT tablet formulations Name of brand and its manufacturer Doverin, Intas A Pharmaceuticals Ltd. Ahmedabad, Gujarat Drotin, Martin and B Harris labs. Ltd. Haridwar, Uttarakhand Beralgan, Aventis Pharma Ltd. Ankleshwar, Gujarat a Average weight of 20 Tablets 5.2. Equipments/ Instruments Label claim (mg) Average Weight (mg) a Batch No. Manufa cturing date Expiry date J003 03/ / Details of Equipments/Instrument are described in section 4.2. TDR / / / /

4 5.3. Identification of Drotaverine Hydrochloride (DRT) The identification of procured sample of DRT was carried out by following methods 1 Melting point determination 2 UV-VIS spectroscopy 3 FT-IR spectroscopy 4 Mass spectroscopy and 5 NMR Spectroscopy ( 1 H and 13 ) Melting point determination Determination of melting point of DRT was carried out using melting point apparatus using open capillary method. TABLE 5.2 omparison of melting point of DRT with reported melting point Drug Reported melting point [1] bserved melting point DRT (º) (º) UV spectroscopy UV spectrum of methanolic solution of DRT (20 µg/ml) was scanned in the range of nm on UV-VIS spectrophotometer. FIGURE 5.1 UV-spectra of methanolic solution of DRT (20 µg/ml) showing λmax at 281 nm 121

5 TABLE 5.3 omparison of reported λmax with obtained λmax of DRT FT-IR Spectroscopy Drug Reported λmax [2] btained λmax DRT 280 nm 281 nm FT-IR spectrum of DRT was recorded in diffused reflectance mode. Theoretical wave numbers responsible for functional groups are compared with observed wave numbers and presented in Table 5.4. Sr. No. FIGURE 5.2 FT-IR spectra of DRT TABLE 5.4 Important frequencies of DRT obtained in FT-IR spectra Functional group Theoretical frequency (cm -1 ) [3-4] 1 Secondary Amines (-NH) Str Aromaticity Methyl (-H 3 ) Str Aromaticity(Benzene overtones) Amine (-N) Str ethoxy bserved frequency (cm -1 ) 122

6 Mass spectroscopy The MS and MS/MS study of DRT was performed and spectra are shown in Figure 5.3 and 5.4 The (M+1) peak was obtained at m/z which confirms molecular weight of DRT at Figure 5.4 represents daughter ions of DRT at different m/z. The fragmentation pattern of DRT is proposed in Figure 5.5. FIGURE 5.3 Full scan MS spectra of DRT FIGURE 5.4 MS/MS spectra of DRT at molecular peak of

7 DRT m/z 398 N H 3 H 3 H N m/z 370 H H H3 H H H 3 N m/z 354 H H H H H2 m/z 310 N H m/z 326 N H m/z 342 N H H H m/z 190 H H N H m/z 270 N H H H m/z 162 N-H H H H m/z 254 FIGURE 5.5 Proposed fragmentation pattern of DRT from MS/MS spectroscopic studies N NMR spectroscopy The 1 H and 13 NMR spectra of DRT were recorded as described in section 4.2. The 1 H and 13 chemical shifts were reported on the δ scale in ppm, relative to tetra methyl silane (TMS) at δ 0.00 in 1 H NMR and Dl 3 at 77.0 ppm in 13 NMR, respectively. 124

8 FIGURE H NMR spectra of DRT FIGURE NMR Spectra of DRT 125

9 N H 3 H 3 25 TABLE H NMR and 13 NMR spectral assignments for DRT Position 1 H H Position δ (multiplicity, j) δ δ (multiplicity, j) δ (t,7.8) (d,1.8) (t,8.0) (s) (s) (m, 6.9) (s) (m, 6.9) (m) (m) (q, 6.9) (s) (t, 6.9) (m) (dd,1.8) (m) δ= hemical Shift (ppm), j = oupling onstant (Hz) 126

10 5.4 Stability Indicating Assay Method (SIAM) for DRT by RP-HPL Experimental hromatographic conditions Following chromatographic conditions were optimized and were kept constant throughout the analysis. olumn: 18 PURSPHERE STAR Hyber mm i.d., with 5 µm particle size. Mobile phase: Buffer: Acetonitrile (57:43, v/v). Buffer preparation: M potassium dihydrogen orthophosphate; add 0.2 % ammonia and adjust the ph of buffer to 4.0 ± 0.02 with 1 M orthophosphoric acid. Flow rate: 0.8 ml/min; Detection wavelength: 240 nm; Injection volume: 20 µl Preparation of solutions Standard solutions: The standard stock solution 1 mg/ml was prepared by dissolving accurately about 100 mg of DRT with methanol in 100 ml volumetric flask. The aliquots of this stock solution were diluted with diluent (water: acetonitrile, 50:50, v/v) to get concentration of 10 µg/ml. Sample solution for assay of DRT in tablet formulations: DRT tablet powder equivalent to 100 mg DRT for each brand (Table 5.1) was accurately weighed and transferred to a 100 ml volumetric flask with addition of about 80 ml of methanol. The mixture was sonicated for 20 min with shaking, and volume was made up to the mark with methanol. The above solutions were centrifuged in centrifuge tubes at 2500 RPM in the research centrifuge for 15 min and were filtered through 0.45 µm syringe filter. The first 10 ml of the filtrate was rejected and subsequent filtrate was further diluted with diluent to obtain the solution of 10 µg/ml Stress degradation studies [5-8] The stress degradation studies were carried by forcibly degrading DRT under different stress conditions such as hydrolytic, oxidative, dry heat (thermal), photolytic degradation and accelerated stability testing. The stress studies were carried out by preparing DRT solution of 2 mg/ml in respective stressors, and was used for stress studies under optimized conditions as given in Table 5.6. A minimum of four samples were generated for every stressed condition, viz., Initial (zero time) 127

11 sample containing the drug with stressor and the drug solution subjected to stress treatment, the blank solutions stored under normal condition, and the blanks subjected to identical conditions. TABLE 5.6 ptimized stress degradation studies conducted on DRT Stress degradation conditions Acid hydrolysis Alkaline hydrolysis Neutral hydrolysis xidative degradation Thermal degradation Photolytic degradation solution state Photolytic degradation solid state Accelerated stability study Stressor 1 N Hl, reflux at 100 for 24 h 0.1 N NaH, reflux at 100 for 2h water, reflux at 100 for 24 h 3 % 2 reflux at 100 for 4 h drug powder kept in hot air oven at 120 for 48 h aqueous solutions were exposed to direct sunlight for 8 h in total two days drug powder was exposed to direct sunlight for 8 h in total two days drug powder kept in temp. and humidity chamber at 40 º and 75 % RH for 1 month Preparation of forced degraded samples After exposure of DRT to all above stress degradation conditions, the stress study samples were prepared for RP-HPL analysis. The hydrolytic and solution state photolytic samples were suitably diluted with diluent to get concentration 10 µg/ml. Acidic and alkaline hydrolytic stressed samples were appropriately neutralized with equimolar concentrations of NaH and Hl prior to injecting on HPL. The methanolic stock solutions of thermal and accelerated stability stress study samples were prepared with concentration of 2 mg/ml and were suitably diluted with diluent to get concentration 10 µg/ml. All the above samples were analyzed on optimized RP-HPL method as described in section along with their respective initial samples and blanks as described above. All the samples were allowed to run till the 2.5 times of the retention time of DRT. The response of DRT obtained in every stress conditions were compared with the responses of respective initial samples and the degradation of DRT was reported in terms of % degradation Method validation [9-10] The optimized stability indicating assay method for DRT was validated for following parameters. 128

12 1. System suitability The system suitability test was performed to ensure that the complete testing system was suitable for the intended application and it was performed by injecting five replicate injections of standard preparation (10 µg/ml). The parameters measured were retention time, peak area, theoretical plates, and asymmetry of DRT. 2. Linearity and range For establishment of linearity of DRT by proposed method, the calibration curve was obtained at seven levels in the concentration range of 2-25 µg/ml for DRT. The solutions (20 µl) were analyzed in triplicate as described in section Peak area and concentrations were subjected to least square regression analysis to calculate calibration equation and correlation coefficient. 3. Specificity Specificity is ability of an analytical method to measure the analyte free from interference due to blanks (diluent and mobile phase) and degradation products formed in forced degradation studies and was performed as described in section Precision A. Method precision For repeatability study, six sample sets were prepared by individually weighing DRT in different volumetric flasks to get concentration of 1.0 mg/ml and were further diluted with diluent individually to get concentration of 10 µg/ml. All the samples were analyzed as described in section The response obtained from each sample was extrapolated to find out the mean assay value with RSD. B. Intermediate precision The intermediate precision study was performed at three different levels i.e. intraday, interday, and different analysts precision. For intraday and interday precision studies, the procedure described in repeatability study, was repeated three times at the interval of three hours on same day and on different consecutive days respectively. For intermediate precision by different analyst study, the whole method precision experiment was performed by different analyst. The results of intermediate precision studies were reported as mean assay of DRT and RSD of assay results obtained in each intermediate precision studies. 129

13 5. Accuracy Accuracy of stability indicating assay method for DRT was performed by recovery studies. Most widely used synthetic mixture of tablets excipients (i. e. lactose, starch, magnesium stearate and talc) were prepared (placebo) in the ratio of their permitted concentration in formulation of tablets. Known amounts of DRT corresponding to % of the label claim (40 mg) were added to placebo mixtures at three different levels in triplicate. For level I, II and III approximately 32, 40 and 48 mg of DRT (which correspond to 80, 100 and 120 % of the label claim) was weighed and mixed with constant weight of placebo in 100 ml volumetric flask, about 80 ml methanol was added and the flasks were sonicated for 15 min and volumes were made upto the mark with methanol. All the solutions were filtered through whatman filter paper 41. From the above filtrate, 0.1 ml from each flask were taken and diluted to 10 ml with diluent and the resulting solutions were analyzed as described in section Robustness Deliberate changes in the following parameters which affects % assay of DRT and system suitability parameters were studied. i. hange in % organic phase of mobile phase by ± 5.0 % ii. hange in ph of buffer of mobile phase by ± 0.05 of set ph iii. hange in the flow rate of the mobile phase by ± 10 % of the original flow rate. 7. Solution stability The solution stability was also carried out to check the stability of both the solutions (standard and sample) till 48 h when stored at ambient temperature in laboratory. It was performed by doing the analysis of both the solutions at 0, 12, 24, and at 48 h intervals and comparing the results with the freshly prepared standard solutions analyzed simultaneously Method application to pharmaceutical formulations of DRT The stability indicating assay method was used for the quantification of DRT in three different brands of pharmaceutical tablet dosage forms of DRT. The description of tablets formulations of DRT is given in Table

14 The sample solutions of various marketed tablet formulations of DRT were prepared (as described in section ) and analyzed as described in section The percentage assay of DRT was calculated from responses of the standard solution with the same concentration as that of samples Results and discussion The presence of isoquinoline ring with two diethoxy groups makes the drug liable to loss of ethyl groups leading to formation of alcoholic products. The resonating double bond present around methylidine makes the drug to susceptible for the attack of nucleophilic agents which leading to hydroxylation which may lead to formation keto structure Method development and optimization[11] Non-polar stationary phase was tried for bringing the retention of the drug as the drug non-polar in nature. Several modifications in the mobile phase composition were tried in order to bring about proper good peak shape of drug and selectivity between the degradation products. The modifications included the changing type and strength of buffer used for bringing ionization of drug, ph of buffer, type, and ratio of the organic modifier, and flow rate. From the different mobile phases tried mobile phase consisting mixture of phosphate buffer and acetonitrile (57: 43, v/v) was found to be satisfactory when separation was carried on 18 stationary phase. Ammonia (0.2 %) was added in the buffer preparation as peak reagent and the ph of the buffer was set to 4.0 ± 0.02, which resulted in good peak with acceptable asymmetry and theoretical plates for DRT as shown in system suitability parameters (Table 5.8). The RP-HPL chromatogram obtained from developed RP-HPL method for DRT is shown in Figure

15 FIGURE 5.8 RP-HPL chromatogram of DRT (10 µg/ml) for stability indicating assay method Stress degradation behavior of DRT From the results of stress degradation studies of DRT it was observed that three major degradation products (designated as DP I, DP II and DP III) were seen in acid, alkaline, neutral and oxidative hydrolysis (Figure 5.9 a, b, c, d), however the % of degradation observed in these hydrolytic conditions were different i.e. 9.9, 26.74, and % respectively (Table 5.8). Two degradation products i.e. DP I and DP II were observed in solution state photolytic degradation with 3.98 % degradation of DRT. The results of forced degradation study shows that DP I, DP II and DP III are hydrolytic degradation products of DRT formed due to neutral hydrolysis and their % of formation was enhanced in presence of alkaline condition. Amongst all the major degradation products of DRT, DP III was formed only under refluxed condition as it was not seen in photolytic degradation. From the Table 5.8, it can been concluded that, DRT is stable in dry heat/thermal and solid state photolytic stress studies as there was no change in peak area of stress samples compared to initial peak area of DRT. DRT is however susceptible for hydrolysis in all the hydrolytic conditions with order of degradation as alkaline neutral oxidative acidic solution state photolytic degradation. The mass balance was calculated, from the responses obtained DRT and all the degradation 132

16 products obtained after stress studies. a a b 133

17 c d 134

18 e FIGURE 5.9 RP-HPL chromatograms of DRT after a. Acid b. alkaline c. Neutral d. xidative and e. Solution state photolytic treatment (I, II, and III are the major Degradation Products (DPs) of DRT) 135

19 Stress degradation condition Method validation 1. System suitability TABLE 5.7 Results from stress degradation study of DRT Initial peak area The system suitability parameters were evaluated for the developed method by calculating the RSD values of retention time, peak area, asymmetry, and theoretical plates of five standard replicates (Table 5.8). The values are within the acceptable range indicating that the system is suitable for the intended analysis. Total Peak area after stress Appr. degradation observed (%) Acid hydrolysis Alkaline hydrolysis Neutral hydrolysis Rt. (min) of major DPs and peak purity 9.61 (0.998), (0.997), (0.998) (0.995), (0.996), (0.998), 9.94 (0.996), (0.998), (0.999), (0.997), (0.995), TABLE 5.8 System suitability parameters of developed method for DRT Parameters bservation RSD Rt (min) Peak area Theoretical plates Asymmetry a Mean of five replicates % Mass balance achieved xidative hydrolysis (0.996), 87.6 Photolytic solution 9.97 (0.996), state (0.994), 96.0 Thermal/Dry Heat NSD - - Photolytic solid NSD state - Accelerated NSD stability - NSD = No Significant Degradation

20 2. Linearity and range The correlation coefficient values obtained in linearity study for developed RP-HPL method (Figure 5.10) which confirms the good linearity of the method over the range studied (Table 5.9). TABLE 5.9 Linearity of DRT by developed RP-HPL method Linearity Level onc. of DRT ( µg/ml) Response observed (AU) a RSD I II III IV V VI a Mean of three replicates FIGURE 5.10 alibration curve of developed RP-HPL method for DRT 3. Specificity The specificity was evaluated from the forced degradation studies as described in section where Figure 5.9 a, b, c, d and e shows, DRT peak well separated from all the degradation products formed during the different stress conditions with sufficient resolution ( 2). The peak 137

21 purity for DRT and all the degradation products of DRT were more than indicating purity of each separated peak and absence of interference due to other co eluting peaks. Thus specificity study ensures the selectivity of the developed analytical method which is able to separate and quantify DRT in presence of different degradation products. 4. Precision The results (Table 5.10) of all the precision studies (Repeatability, intraday, interday and different analysts), shows that the mean assay values and RSD values are within the acceptance criteria ( %, 2 respectively) which proves the good precision of developed method. TABLE 5.10 Precision study for DRT by SIAM Precision study bservation Mean Assay a RSD Repeatability Intraday b Interday c Different analyst d a n= 6; b Mean value of initial, 3 h, 6 h interval observations; c Mean value of day I and day II observations; d Mean value of analyst I and analyst II observations 6. Accuracy The recovery for DRT was between 98.2 and % with RSD of 1.0 % (Table 5.11), indicates that the developed method was accurate for the determination of DRT in pharmaceutical formulations. TABLE 5.11 Accuracy study of DRT by SIAM Level % Recovery Mean Recovery RSD 99.0 I 98.2 (80 % wrt to L) II (100 % wrt L) III 99.0 (120 % wrt L) 99.0 Mean

22 7. Robustness The results of robustness studies are summarized in Table In any condition assay value of sample is not deviating more than 2.0 % indicating that the method is robust in nature. TABLE 5.12 Robustness study of DRT by SIAM Robustness condition bservation System suitability % % difference RSD a Rt T A Assay in assay b - 5% Acetonitrile (Buffer: Acetonitrile 59:41 v/v) % Acetonitrile (Buffer: Acetonitrile 54:46 v/v) hanged ph of buffer of mobile phase hanged ph of buffer of mobile phase % hange in flow rate ml/min % hange in flow rate ml/min a from five values of standard area; b % difference compared from the method precision result; T= Theoretical plates; A = Asymmetry 8. Solution stability From the results of the solution stability study (Table 5.13), it was found that the % difference of impurities is more than 2 % when compared with initial showing standard and sample solution stability of DRT is upto 24 hr. Interval TABLE 5.13 Solution stability study of DRT by SIAM bservation % Assay % Difference STD* Sample* STD Sample Initial h h h * Result are from duplicate injection of same solution Method application 139

23 Figure 5.11 a, b and c represents the chromatograms of DRT in three different tablet brands. The assay results obtained by the applied stability indicating assay method were found to be satisfactory, accurate, and precise for estimation of DRT without interference of excipients, as indicated by the good recovery and acceptable standard deviation (SD) values (Table 5.15). (a) (b) 140

24 (c) FIGURE 5.11 Representative RP-HPL chromatograms of DRT (10 µg/ml) in Brand A (a); Brand B (b) and Brand (c) TABLE 5.14 Summary of results for DRT assay in marketed tablet dosage forms Formulation Amount of drug recovered a (mg) ± SD b % Assay ± SD b A 39.5 ± ± 0.51 B 39.2 ± ± ± ± 0.40 a Label claim = 40 mg; b n = onclusion The developed stability indicating RP-HPL method for the determination of DRT was found to be simple, selective, sensitive, and economical. The results from the stress degradation studies shows that DRT is susceptible for degradation in all the hydrolytic degradation conditions and maximum degradation was observed in alkaline and neutral stress degradation conditions. The developed stability indicating assay method was reliable as the results from all the validation parameters produced were satisfactory. The applicability of the method was proved when it was applied for the estimation of DRT in pharmaceutical tablets formulations of DRT. 141

25 The two of the three degradation products of DRT (DP I and DP II) were found in all the hydrolytic and in solution state photolytic stress degradation conditions where DP III was obtained only in refluxed conditions of stress. Hence it was concluded that the alkaline and neutral conditions are required for the formation of DP I and DP II however DP III is formed only in harsher conditions i.e. refluxing DRT at higher temperatures. It is further required to do L-MS/MS study for characterization of degradation products and elucidation of degradation pathway of DRT. 142

26 5.5. Related Impurities Method for Drotaverine Hydrochloride by RP-HPL Experimental hromatographic conditions The optimized method for stability indicating assay of DRT as described in section 5.4. was applied for detection and quantification of related impurities in DRT with same chromatographic conditions as described in section Preparation of solutions Diluted standard preparation: The diluted standard solution of DRT with concentration of 0.5 µg/ml was prepared from the standard stock preparation of DRT as described in section Preparation of sample solution: The standard stock solution described in section was further diluted with diluent to get the concentration of 100 µg/ml. Sample solution for related impurities of DRT in tablet formulations: The same procedure as described in section for assay of DRT from marketed formulations was followed for related impurities detection with only change in sample concentration 100 µg/ml of DRT Method validation The optimized related impurities method for DRT was validated for following validation parameters. 1. System suitability The system suitability test was performed by injecting five replicate injections of diluted standard preparation of DRT. The parameters measured were retention time, peak area, theoretical plates, and asymmetry of DRT. 2. Linearity and Range The linearity was determined over the range of LQ to 200 % of the specification limit. (LQ is the reporting threshold as specified by IH guidelines (i. e %). The sample solutions for linearity of DRT were prepared by making the dilution given in Table 5.1. Samples at each 143

27 linearity level were analyzed in triplicate as described in section and the response was measured in the form of area under the curve of DRT. Linearity level TABLE 5.15 Linearity study of DRT (Unknown impurity) Volume (ml) taken from standard stock a Diluted with diluent (ml) onc. of the solution (µg/ml) I (LQ) (0.05 %) 0.05 II III IV V VI a Stock solution: 0.1 mg/ml of DRT 3. LD and LQ LD, LQ, and Precision at LQ for DRT by related impurities method was determined as described in section Specificity Specificity is ability of an analytical method to measure the analyte free from interference due to blanks (diluent and mobile phase) and degradation products formed in forced degradation studies and was performed as described in section Precision A. Method Precision For repeatability study, the six sample sets of DRT were prepared having concentration of 100 µg/ml and were analyzed as described in section The responses of impurities detected in each sample sets were measured and % of individual and total impurities in each sample set was calculated. The mean of total impurities in six samples sets was found with RSD. B. Intermediate Precision The intermediate precision study was performed at three different levels i.e. intraday, interday, and different analysts precision. 144

28 For intraday and interday precision studies, the samples were prepared and analyzed as described in repeatability studies, three times at the interval of three hours on same day and on different consecutive days, respectively. For intermediate precision by different analyst study, the whole method precision experiment was performed by different analyst. 6. Accuracy The accuracy of the method for unknown impurity was studied with respect to recovery of DRT. The accuracy of unknown impurity with respect to DRT was determined over the range of LQ to 200 % of the specification limit of impurity (LQ being 0.05 µg/ml to 1.0 µg/ml) at IV levels. The procedure as described in accuracy study of section was followed in accuracy study for unknown impurity. The placebo mixture was prepared and standard stock solution of DRT (0.1 mg/ml), was spiked (0.05, 0.25, 0.5, and 1.0 ml) at different levels in 100 ml volumetric flasks in triplicate containing constant weights of placebo, followed by addition of 80 ml of diluent and the solution was then was sonicated for about 10 min and volume was made upto the mark with diluent. All the solutions were filtered through whatman filter paper 41 and the resulting solutions were analyzed as described in section Robustness The robustness was studied by making the deliberate changes in chromatographic parameters and procedure as described in section Solution Stability The solution stability was also carried out to check the stability of both the solutions (diluted standard solution and sample solution) till 48 h when stored at ambient temperature in laboratory. It was performed by doing the analysis of both the solutions at 0, 12, 24, and at 48 h and comparing the results with the freshly prepared diluted standard solutions analyzed simultaneously as described in section

29 Method application to pharmaceutical tablet formulations of DRT The developed and validated related impurities method was successfully applied for the estimation of impurities in three different brands of tablet formulations of DRT. The description of tablet formulations of DRT is given in Table 5.1. The sample solutions for related impurities of DRT in different tablet brands (prepared as described in section ) were analyzed as described in section The impurities detected above 0.05 % were taken in consideration and the % of each individual impurity and total impurities were calculated Results and discussion Development and optimization of related impurities method[11] The method of related impurities of DRT was developed with the aim to detect and quantify all the impurity at low concentration of reporting threshold as specified by IH guidelines (0.05 %). With keeping sensitivity and selectivity in mind the optimized conditions of stability indicating assay method of DRT was applied for the detection and quantification of impurities of DRT. Figure 5.12 shows good peak shape of DRT at 100 µg/ml concentration. The system suitability parameters are mentioned in Table At this concentration of DRT, two additional peaks were also seen at Rt 9.9 (designated as IMP I) and at 18.2 min (designated as IMP II) respectively (Figure 5.12). The peak area of IMP II was more (0.48 %) as compared to peak area IMP I (0.12 %). Both the detected impurities were found to be well separated from each other and from parent drug with good resolution ( 2). 146

30 FIGURE 5.12 RP-HPL chromatogram of DRT (100 µg/ml) for related impurities method showing IMP I at Rt 9.99 and IMP II at mins respectively Method validation [12-13] 1. System suitability The system suitability was evaluated by calculating the RSD values of retention time, peak area, asymmetry, and theoretical plates of five standard replicates. The experimental results (Table 5.16) showed that the values are within the acceptable range indicating that the system is suitable for the intended analysis. TABLE 5.16 System suitability parameters for DRT diluted standard preparation (Unknown impurity) Parameters bservation a RSD Rt (min) Peak area Theoretical plates Asymmetry a Mean of five replicates 147

31 2. Linearity and range The correlation coefficient value obtained in related impurities method (Figure 5.13) confirms the good linearity of the method over the range studied. FIGURE 5.13 Plot of linearity curve for DRT by the developed method 3. LD and LQ From the triplicate results of linearity study, SD and slope value was found to be and respectively which is further used to calculate LD and LQ values. LD value was found to be 0.02 µg/ml and LQ was 0.05 µg/ml. The RSD value of theoretically calculated LQ preparation was found to be 5.5 with mean area Specificity The developed RP-HPL method for determination of impurities of DRT was specific as proved from the results of the stress degradation studies. The peak purity for all the observed degradation products of DRT were more than indicating purity of each separated peak and absence of interference due to other co eluting peaks. 5. Precision 148

32 The results of all the precision studies (Table 5.17) obtained in related impurities method (repeatability, intraday, interday and different analyst), shows that RSD values are within the acceptance criteria which proves the good precision of developed method. TABLE 5.17 Precision study DRT by related impurity method (Unknown impurity) Presicion study Mean of total Impurities (%) a RSD Repeatability a Intraday b Interday c Different analyst d a n= 6 b Mean value of initial, 3 hrs, 6 hrs interval observations; c Mean value of day I and day II observations; d Mean value of analyst I and analyst II observations 6. Accuracy The mean recovery at LQ level is % with 3.2 % RSD which is within the acceptance criteria. Similarly, the recovery range at level II, III, and IV between to % which also is within the acceptance criteria (Table 5.18). TABLE 5.18 Accuracy study of DRT by related impurity method (Unknown impurity) Level % Recovery Mean Recovery RSD I LQ (0.05 %) II (50 % wrt to specification limit) III (100 % wrt to specification limit) IV (200 % wrt to specification limit) Mean

33 7. Robustness The results of robustness studies are summarized in Table The assay value of sample is not deviating more than 2.0 % indicating that the method is robust in nature. TABLE 5.19 Robustness study of DRT by related impurities method Robustness condition System suitability bservation % Total impurities Absolute difference % RSD a Rt T A - 5% Acetonitrile (Buffer: Acetonitrile 59:41 v/v) % Acetonitrile (Buffer: Acetonitrile 55 : 45 v/v) hanged ph of buffer of mobile phase hanged ph of buffer of mobile phase % hange in flow rate ml/min % hange in flow rate ml/min a from five values of standard area; b % difference compared from the method precision result; T= Theoretical plates; A = Asymmetry 8. Solution stability There was no significant difference in the % assay of diluted standard preparation from initial and also the not much deviation in % of total impurities found in samples indicates that standard and samples solutions are stable at ambient temperature for 24 h (Table 5.20) TABLE 5.20 Solution stability study of DRT by related impurity method bservation Interval Absolute difference % Assay of STD* Total Impurities (%)* STD Sample Initial h h h * Result are from duplicate injection of same solution 150

34 Method application The proposed method of related impurities was applied for the determination of impurities in tablet formulations of DRT in three different brands. The impurities detected in DRT at Rt 9.9 min and 18.2 min as described in section were also found in all the tablet formulations of DRT in different amount. The results from impurity analysis of DRT tablet formulations are summarized in Table TABLE 5.21 Summary of results for related impurities for DRT in marketed tablet dosage forms % of impurities Found a Formulation IMP I IMP II Total impurities a (%) A B a Mean value of three determinations From Table 5.21, it was observed that IMP I and IMP II found in all the three formulations above the identification threshold specified by IH guidelines, thus it is very much essential to characterize these two impurities found in DRT formulations onclusion The developed related impurities method for DRT is simple, involves use of simple mobile phase with isocratic elution and easy extraction procedure. The method could detect, separate, and quantify all the found impurities in DRT API with sufficient resolution. The reliability of the method was proved form the acceptable results of all the validation parameters. It was observed that both the impurities (IMP I and IMP II) detected in DRT and its tablets formulation are degradation impurities of DRT since their Rt. was same as degradation products (DP I and DP II) of stress studies of DRT and also gave same PDA spectra in the range of nm. Further, it is necessary to elucidate the structures of both the Degradation Products (DPs) of DRT as they are quantified above the identification threshold as prescribed by IH guidelines. 151

35 5.6. Isolation and characterization of Degradation Products (DPs) of DRT Experimental hromatographic conditions For the isolation of DPs of DRT, method was developed and optimized on analytical and then transferred to Prep-HPL. hromatographic conditions (analytical HPL) olumn: 18 column (Inertsil DS 3V, 250 x 4.6 mm id 5µm). Mobile phase: Water (ph of water adjusted to 3.60 ± 0.02 with formic acid after addition of 0.2 % ammonia as peak reagent): Acetonitrile (55:45, v/v). Flow rate: 0.8 ml/min; Detection wavelength: 240 nm; Injection volume: 20 µl Solution preparation The solution for sample loading on Prep-HPL was prepared by dissolving 1 g of DRT in 100 ml distilled water (1 % solution). The prepared solution was then subjected to the stress degradation as described in section Since major degradation products were found in neutral conditions of degradation, neutral degradation was considered as a source for the generation of degradtion products of DRT. The prepared aqueous solution of DRT was then stressed by refluxing at 100 for h to generate all the degradation products in maximum ammount Isolation of DPs of DRT by Prep-HPL[14-16] The Prep-HPL method developed as described in section was scaled up for column (Semi-Prep-HPL column), Flow rate (6.0 ml/min), and injection volume (1.0 ml) but was modified for gradient programming for reducing run time on Prep-HPL system. Table 5.22 depicted gradient program used for Prep-HPL method in which solution A and B represents mobile phase and acetonitrile respectively. 152

36 Table 5.22 Gradient programming of mobile phase for Prep-HPL method Time (min) Solution A Solution B The neutral degraded sample solution of DRT prepared as described in section was loaded on Prep-HPL and eluents containing targeted DPs were collected and concentrated by evaporating acetonitrile portion of eluents at room temperature under high vacuum on rota evaporator. The concentrated aqueous layers were further dehydrated with solid sodium sulphate (approximately 1 g) and further extracted with chloroform (50 ml each time) thrice for each DP of DRT. The collected combined chloroform layers were then evaporated individually in rota evaporator to get solid masses of DPs of DRT. Before characterization of isolated DPs using different spectroscopic techniques, chromatographic purity of each DP was checked using developed RP-HPL method as described in section haracterization of isolated DPs by spectroscopic techniques The isolated and purified DPs (designated as DP I and DP II) were further analyzed by different spectroscopic techniques like UV, FT-IR, Mass and NMR spectroscopy for characterization and structural elucidation UV Spectroscopy The standard solution (20 µg/ml) of DP I and DP II were prepared individually in methanol and used to do analysis in UV-VIS region from nm to determine their ƛ max FT-IR spectroscopy The FT-IR spectroscopic analysis was performed by diffused reflectance technique. The FT-IR spectra of DP I and DP II were recorded in the range of wave number cm -1 and compared with spectra of DRT recorded as described in section Mass spectroscopy The MS and MS/MS experiments were performed on a Varian MS system as described in section 4.2. The analysis was performed in positive ionization mode with Electrospray interface. 153

37 The mass to charge (m/z) ratio was recorded in the range of m/z. The parameters for capillary and Rf voltage were 80 V, with nebulizer gas as air at a pressure of 35 psi and curtain gas as nitrogen at a pressure of 10 psi NMR spectroscopy The 1 H and 13 NMR studies on DP I and DP II were carried out on instrument as described in section Elemental Analysis The elemental analysis was carried out to determine the amounts of carbon, hydrogen and nitrogen in isolated DPs on HN- element analyzer as described in section Results and discussion Method development and optimization The isolation and purification of all the DPs of DRT, Prep-HPL method was developed on analytical HPL. Method development was initiated using neutral degraded sample of DRT where all targeted degradation products (DP I, DP II, and DP III) were formed in sufficient quantity (Figure 5.9 c). Satisfactory separation between all the DPs of DRT was achieved when mobile phase described in section was used where DP I, DP II and DP III eluted at Rt.7.8, 25.8 and 30.8 mins, respectively (Figure 5.14) which were well separated from DRT peak (Rt 5.66). Sufficient resolution was obtained between DRT and all the DPs which would be helpful for isolation of DPs in pure form. 154

38 FIGURE 5.14 RP-HPL chromatogram (10 µg/ml) of neutral treated DRT showing well resolved DPs of DRT Isolation and purification of DPs The eluent fractions were collected containing DP I, DP II and DP III and processed as described in section to get solid mass of DP I (yield 70 mg) and DP II (yield 50 mg). However DP III was not obtained in sufficient quantity for further characterization. DP I and DP II showed melting point , and respectively hromatographic purity of isolated DP-I and DP-II Before carrying out the spectroscopic experiments, the purity of isolated DPs was carried out as described in section The chromatographic purity of isolated DP I and DP II was found to be 95.4 and 99.1% respectively. Figure 5.15 represents the RP-HPL chromatograms of isolated DP I (a) and DP II (b) showing the chromatographic purity. 155

39 a b FIGURE 5.15 RP-HPL chromatogram of isolated DPs of DRT (a) DP I and (b) DP II showing chromatographic purity 156

40 Spectroscopic characterization of isolated DPs UV spectroscopic analysis The results from UV spectroscopic analysis of both the DPs of DRT are depicted in Table The UV spectra of both the DPs (Figure 5.16 a, and b) suggests the probable presence of parent chemical moiety in structure of both the DPs as both have very near UV absorption maxima as that was observed for DRT (281nm). TABLE 5.23 Results from the UV spectral analysis of DPs of DRT ompound bserved absorption maxima (ƛ max) DP I 250, 285, 360, DP II 228, 283, 328 a b FIGURE 5.16 UV spectra of (20 µg/ml) DP I (a) and DP II (b) in methanol 157

41 FT-IR spectroscopy Figure 5.17 shows FT-IR spectra of DP I and DP II obtained in diffused reflectance mode with characteristic frequencies observed reported in Table a b FIGURE 5.17 FT-IR spectra of DP I (a) and DP II (b) 158

42 TABLE 5.24 Important frequencies of DPs obtained in FT-IR spectra ompound Wave number (cm -1 ) DP I 3587, 3093,3014, 2806, 1592, 1139, 1043 DP II 3079, 2994, 2849, 1655, 1590, 1516, 1139,1041, Mass spectroscopy The MS of DP I and DP II exhibited molecular ion at m/z (M+1) 414 and 412 amu respectively (Figure 5.18). Further MS/MS studies on both the DPs was also carried out to study their fragmentation pattern and to predict the structures of both the DPs (Figure 5.19). Table 5.25 depicts the summarized results of mass spectroscopic studies performed. a 159

43 b FIGURE 5.18 Mass spectra of DP I (a) and DP II (b) showing m/z value 414.2, amu respectively a 160

44 b FIGURE 5.19 MS/MS of DP I (a) and DP (II) Table 5.25 shows the summarized results of Mass spectroscopic experiments carried on DP I and DP II ompound DP I DP II TABLE 5.25 Summary of results from the mass spectroscopic analysis bserved parent ions (m+1) and major daughter ions 414, (m+1), 412, 396, 384,370, 367, 357, 354,310, 234, 262, 218, 190, (m+1), 396, 384, 370, 367, 354, 353, 340, 326, 324, 262, , 218, 190, NMR spectroscopy For the further confirmation of proposed structures of DP I, and DP II, NMR spectroscopic experiments were carried out. The NMR experiments could not be done on DP III because of its low recovery. Figure 5.20 (a and b) shows the 1 H NMR of DP I and DP II and Figure (a and b) shows 13 NMR of DP I and DP II. The results from the NMR ( 1 H and 13 ) spectral data for DRT and DP I and DP II are compiled in Table

45 a b FIGURE H NMR spectra of DP I (a) and DP II (b) 162

46 a b FIGURE NMR spectra of DP I (a) and DP II (b) 163

47 TABLE 5.26 NMR ( 1 H and 13 ) spectral assignments for DRT, DP I and DP II Position 1 H δ, (multiplicity, j) δ ( ppm) DRT DP I DP II DRT DP I DP II (t,7.8) 3.94 (t,7.5) 3.91 (t,7.8) (t,8.0) 2.91 (t,7.8) 2.79 (t,7.7) (s) 7.18 (s) 6.73 (s) (s) 6.77 (s) 6.90 (s) , s 6.69, s (dd,1.8) 7.72 (dd,1.5) 7.55 (dd,1.8) (d,1.8) 7.59 (d,1.5) 7.65 (d, 1.5) (s) 6.90 (d,8.4) 6.85 (d,8.7) (m) 4.03 (m) 4.01 (m) (m) 1.40 (m) 1.40 (m) (m) 4.03 (m) 4.01 (m) (m) 1.40 (m) 1.40 (m) (q,6.9) 4.16 (m) 4.18 (m) (t,6.9) 1.49 (m) 1.48 (t,7.1) (m) 4.03 (m) 4.01 (m) (m) 1.40 (m) 1.40 (m) (s) *Refer structures for numbering (Figure 5.27 a. and b.); δ= chemical shift; j= coupling constant in Hz Elemental Analysis of DP I and DP II Table 5.27 depicts the results from the elemental analysis of DP I and DP II, which supports the proposed structures. 164

48 TABLE 5.27 Results from the elemental analysis of DP I and DP II Element estimated (%) DP I DP II arbon Hydrogen Nitrogen Structural elucidation of DPs of DRT Structural elucidation of DP I The spectral data of DP I was compared with that of DRT. As observed in Figure 5.22.a. the mass spectrum of DP I exhibited molecular ion [M+H] + at 414, which is 16 Da more than that of DRT (Figure a). The 1 H NMR spectrum showed all signals corresponding to DRT with small shift in the δ ppm values, additionally a singlet was observed at 4.28 ppm. ompared to DRT, signal obtained due to proton at position 11 was observed at downfield position of 6.69 ppm suggesting substitution at this carbon with electronegative group (Table 5.26). In 13 NMR also, the signal obtained due same numbered carbon at ppm was not observed but downfield signal at ppm near to chemical shift value of hydroxyl group was obtained for DP I supporting 1 H NMR results. This assumption was further supported by the results of FT-IR analysis of DRT and DP I where the characteristic broad peak of hydroxyl group was seen at wave number 3587 cm -1 (Figure 5.24 a), confirming the hydroxylation of the parent drug. The other characteristic frequencies observed at 1042, 1509, 2985, and 3049 at cm -1, due to ethoxy group (---), amine (--N), aliphatic alkanes and aromatic alkanes respectively, obtained for DRT were also observed for DP I with small shift suggesting the retention of parent structure of DRT and only hydroxylation has been taken place in generation of DP I (Table 5.24). Based on these spectral data the molecular formula of DP I was proposed as 24 H 31 N 5 which was finally confirmed from the elemental analysis results (Table 5.27) where the % of carbon, hydrogen and nitrogen practically observed were 69.38, 7.21, and 3.15 % respectively. Finally the structure of DP I was characterized as (6,7-diethoxy-3, 4-dihydroisoquinolin-1-yl) (3, 4-diethoxyphenyl) methanol (Figure 5.22.a.) from the MS/MS study of DP I where the daughter ions obtained were corresponding to the proposed fragmentation pattern of DP I (Figure 5.23) 165