Chapter 6 Determination of 1,2-Dibromoethane as a genotoxic impurity in Escitalopram oxalate drug substance by GC. 91Introduction: Escitalopram oxalate (Brand name: Lexapro or Cipralex) is used as an antidepressant belongs to the class of selective serotonin reuptake inhibitor (SSRI) It is used in the treatment of major depressive disorder for adults and children’ s aged minimum of 12 years. Itis also used in the treatment of generalised anxiety disorder. It can be administered orally by means of tablets or oral solution, Escitalopram is 8: somer of the antidepressant drug Citalopram. Escitalopram known chemically as (S)-1-[3-(Dimethylamino)propyl]|-1-(4- fluorophenyl)-1,3-dihydroisobenzofuran-S-carbonitrileThe molecular weight is 414.40 while molecular formula is C20H21FN20 + C2H20+ 1,2-Dibromoethane is a colorless organo bromine compound also known as Ethylene dibromide, It is used as the pesticide for various crops and can be used in pest control a: fumigant for beetles and termites. It is also used in the synthesis of Escitalopram oxalate 1,2-Dibromoethane proved as a genotoxic carcinogen in rodents, It also shown effect on behavior of young rat whose male parents are exposed to 1,2-Dibromoethane earlier. IARC classified 1,2-Dibromoethane as Group 2A (Probably carcinogen to humans) Fig 6.1: Escitalopram oxalate B Br" SO r Fig 6.2: 1,2-Dibromocthane 2Review of literatm Literature survey shows that there are number of methods available for the determination of 2-Dibromoethane by gas chromatography using mass detector, Sonu Sundd et al. (Sonu Sunda et al, 2004) reported a LC-electrospray ionisation MS method for the determination of escitalopram in human plasma and its application in bioequivalence study. S. Sharma et al, (S. Sharma et al, 2010) developed zero order spectrophotometric method for estimation of Escitalopram oxalate in tablet formulations. Tapobana et al. (Yapobana et al, 2011) reported RPHPLC method for the estimation of Escitalopram in bulk and in dosage forms. T. Vetrichelvan et (T. Vettichelvan et al, 2010) reported Colorimetric method for the estimation of Escitalopram oxalate in tablet dosage form. H. Zheng (H. Zheng, 2008) reported method for determination of plasma Escitalopram with LCMS. Y. Shete et al. (Y. Shete et al, 2009) reported spectrophotometric estimation of Escitalopram oxalate in pharmaceutical dosage forms. Elham (Elham, 2009) reported micelle enhanced fluorimetric and TLC densitometric methods for the determination of (+ its S-enantiomer Escitalopram, Dhaneshwar (Dhaneshwar, 2008) reported HPLC with UV ) citalopram and detection and LCMS method for evaluation of stress degradation behavior of escitalopram oxalate, C. Author (C. Author, 2011) reported LC method for estimation of Escitalopram oxalate in tablet dosage forms. Soliman (Soliman, 2011) reported Enantiomeric TLC assay of Escitalopram in presence of in-process impurities. BG Chaudhari et al. (Chaudhari et al., 2010) developed spectrophotometric method for determination of Escitalopram oxalate from tablet formulations. C. Tananaki (C. Tananaki, 2005) reported method for the determination of 1, 2- Dibromoethane, 1, 2-Dichloroethane and naphthalene residue in honey by GCMS using purge and trap desorption extraction. JR King et al.( JR King et al, 1980) reported method of electron capture GC method for determination of residues of 1, 2-dibromoethane in fumigated grapefiuit, William Newsome (William Newsome, 1977) reported method for the determination of 1, 2-Dibromoethane in food crops by using gas chromatography with lable in the current literature for electron capture detector. There is no method @ determination of 1, 2-Dibromoethaneas a genotoxic impurity in Escitalopram oxalate drug substance, 93Development and optimizat 1,2-Dibromoethane is a colorless liquid boils at 131°C It is having molecular weight of 187.88 with specific gravity 2.18 (water-1) It is easily miscible with organic solvents like n-Hexane, hence it is used as diluent in the experiment. DB-624 column shows good peak shape for 1,2-Dibromoethane as compared to DB-1 and DB-S5 column. Due to low response of 1,2-Dibromoethane on FID detector injection volume optimized to SuL and split ratio is optimized as 1:5 for better response. Initially diluent selected were methanol, Ethanol and Toluene, but n-Hexane is finalized as the diluent because of response of 1,2- Dibromoethane peak and no interference observed due to n-Hexane. To achieve maximum very high (200 mg/mL) as 1, response on FID detector sample quantity used Dibromoethane shows very less response at such a low level. Chromatographie conditions: Column : DB-624 (30 meter capillary column with inner diameter of 0.53 command film thickness 3.0 wt) Detector :FID Injector temperature 220°C Detector temperature 2 260°C Column oven temperature: 60°C for S min and then raised to 240°C @20°C/min and kept hold for 20 min. Cartier gas : Nitrogen Carrier gas pressure 3 psi (constant pressure) Injection volume 5.0L Split ratio 21:8 Diluent c n-hexane Chemicals and reagents used: 1,2-Dibromoethane was purchased from Aldrich chemicals while n-hexane used is HPLC- grade from Merck chemicals (Mumbai, India) Escitalopram oxalate samples were collected from local market. 94Instrumentation: Gas Chromatograph of Shimadzu with liquid autosampler (GC2010 with AOC 204) with sed. GC solution software was Standard preparation: Accurately weighed about 75 mg of 1, 2-Dibromocthane standard into @ 100 ml. volumetric flask containing 20 mL. of diluent, mix and diluted to volume with diluent. Further diluted 2.0 mL of above solution to 100 mL with diluent, Test solution: ‘Weighed about 1000 mg of sample into a 20 mL. of centrifuge tube, add $ mL. of diluent and cyclomixed for 2 min, Centrifuged the solution and allow to settle, Injected supernatant solution. Experimental results and discussion: System suitability and Specificity: ‘The blank, standard, sample solution and spiked sample solutions was prepared. The indi lual solvent solution and individual impurity solutions were prepared and injected into the GC system. Table 6.1: System suitability results 1,2-Dibromoethane Inj. No. Area 1 36012 2 36258 3 36125 4 37215 5 36958 G 37815 Mean 36731 sD 116.77 % RSD 1.95Table 6.2: System suitability Standard solution Spiked sample solution Name Retention time | Relative retention | Retention time | Relative retention (min) time (min) time n-hexane 3.540 1.00 3.487 1.00 1,2-Dibromoethane 8.981 2.54 8.965 2.57 ‘The solvents used in the reaction i.e. Acetone, Isopropanol, Dichloromethane, N, N- Dimethylformamidedo not show any interference with retention time of any of the peak of interest, Li it of detection (LOD) and limit of quantitation (LOQ): A series of lowest concentration of 1,2-Dibromoethane solutions were prepared by quantitative dilutions of the impurity stock solution and inject into gas chromatography and chromatograms were recorded. From the areas obtained the slope, intercept and residual standard deviation was calculated and predicted the LOD, LOQ values. Six injections of LOQ injection were injected into GC and recorded the chromatograms. The obtained results were presented below, LOD is injected in three replicate which was detected as follows, Table 6.3: LOD results 1,2-Dibromoethane Inj. No. Area 1 1725 2 1626 3 1698 Cone (ppm) 96Table 6.4: LOQ Precision results 1,2-Dibromoethane Inj. No. Area 1 4692 2 4612 3 4631 4 4692 3 4703 6 4780 Mean 4685 sD 59.4845 % RSD 1.27 Cone (ppm) 10 Linearity and range: A series of 1, 2-Dibromoethane solutions were prepared by quantitative dilutions of the stock solution of standard to obtain solutions at LOQ, 50%, 80%, 100%, 120%, 150% and 200% of the standard concentration level. Each solution we injected and peak area was recorded. The slope, intercept, correlation coefficient of the regression line and residual sum of squares were calculated. The values of peak area and corrected concentrations are presented the result table. A graph of corrected concentration (ppm) vs. peak area was plotted. For range, recorded the concentration levels over which the results are linear. 7Table 6.5: Linearity results Level Corrected concentration Area obtained (ppm) LOQ 10.19 4660 50% 36.39 18269 80% 58.23 28931 100% 72.79 37056 120% 87.34 44855 150% 109.18 55951 519.212 -754,263 correlation coefficient 0.9999 Linearity of 1,2-Dibromoethane 420.00, 100.00 20.00 | 0.00 | 40.00 20.00 | 0.09 |} >" ° to000 20000 «30000-40000 50000-60000 98Precision: Repeatability Six sample solutions were prepared by spiking with standard solution at 100% concentration level. The mean and relative standard deviation was calculated. The obtained results are presented as follows, Table 6.6: Precision results Inj. No. 1,2-Dibromoethane content (ppm) 1 73.87 2 7472 3 73.94 4 74.55 5 74.03, 6 73.27 Mean 74.06 sD 0.52 % RSD 0.70 Intermediate precision: ‘The analysis was carried out by different analyst, different day, different instrument and different column. Six sample solutions were prepared by spiking with standard solution at 100% concentration level. The mean and relative standard deviation was calculated. The obtained results are presented as follows, 99Table 6.7: Intermediate results Inj. No. 1,2-Dibromoethane content (ppm) 1 73.98 2 74.21 3 74.25 4 73.68 5 3.96 6 BAL Mean 3.87 sD 0.42 % RSD 0.57 Table 6.8: Cumulative RSD Solvent % RSD in % RSD in % Cumulative RSD Repeatability Intermediate (Twelve precision preparations) 1,2-Dibromoethane 0.70 0.57 0.63 Accuracy: Sample solutions were prepared by spiking at LOQ, 50%, 100% and 150% levels of the standard concentration, Each level was spiked in triplicate and % recovery was calculated The obtained results were represented as follows, Table 6.9: Batch analysis Area in as is sample Sample Area Content (ppm) 1 0 0 2 0 0 3 0 0 Mean 0 100Table 6.10: Recovery results acura Amount added arent Amount found | % Recovery 9.91 0.0 9.92 100.1 Log 991 0.0 9.98 100.7 991 0.0 10.05 1015 38.10 00 38.26 1004 50 38.10 00 37.12 974 38.10 00 38.14 100.1 76.20 0.0 75.96 99.7 100 76.20 0.0 76.18 100.0 76.20 0.0 76.11 99.9 57.15 0.0 58.19 101.8 150 57.15 0.0 58.11 101.7 57.15 00 58.95 103.1 Robustness ‘The Gas chromatographic analysis were carried out for system suitability by altering the chromatographic conditions as follows, 1. Change in carrier gas pressure a, Cartier gas pressure changed to 3.0 psi instead of 3.3 psi b. Carrier gas pressure changed to 3.6 psi instead of 3.3 psi 2. Change in initial column oven temperature a, Initial oven temperature changed to 54°C instead of 60°C b. Initial oven temperature changed to 66°C instead of 60°C 101Table 6.11: Robustness results Conditions % RSD Unaltered 1.95 Flow rate of carrier gas (3.0 psi) 2.48 Flow rate of carrier gas (3.6 psi) 1.96 initial column oven temperature 54°C 215 initial column oven temperature 66° 2.29 Solution stability ‘The solution stability was carried out by injecting the spiked test solution (at the limit concentration level) after every 12 hrs for two days. No significant change was observed which shows that spiked sample solution was stable up to 48 hours (2 days) Table 6.12: Solution stability results Results obtained (ppm) Time interval 1,2-Dibromoethane Initial 75.12 12 hrs 74.56 24 hrs 74.65 36 hrs 7439 48 hrs 74.45 ‘The other impurity “S-Cyanophthalide” is determined using HPLC technique, so it was described in the next chapter. 102CHROMATOGRAMS 50000 ADA 25000 _ o > a — 5 10 15 0 5 30 Fig 6.3: Blank chromatogram THD. 25000- 8.274 / 1,2.Di brommocthase — j 10 15 a) 5 30 Fig 6.4: Standard chromatogram 103Fig 6.6:Spiked sample chromatogram 104Fig 6.7:L0Q Chromatogram 105