Lamotrigine, 6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine. novel anticonvulsant drug used in the treatment of epilepsy, bipolar

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1 INTRODUCTION : Lamotrigine, 6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine is novel anticonvulsant drug used in the treatment of epilepsy, bipolar disorder and pain syndromes. In psychiatry, there are several studies reporting the efficacy of lamotrigine in the treatment of bipolar disorder, schizophrenia, cocaine dependence, post traumatic stress disorder and borderline personality disorder in these studies, lamotrigine was seen to be well tolerated, to be an effective mood stabilizer, to be significantly more effective in schizophrenia when given as adjunctive therapy to clozapine. However it requires medical attention when combined with other antipsychotics except for clozapine due to possible iatrogenic worsening of psychiatric symptoms. For epilepsy it is used to treat partial seizures, primary and secondary tonic-clonic seizures, and seizures associated with Lennox-Gastaut syndrome. Lamotrigine also acts as a mood stabilizer. It is the first medication since lithium to be granted approval by the U.S. Food and Drug Administration (FDA) for the maintenance treatment of bipolar type I. Chemically unrelated to other anticonvulsants 42, 38, lamotrigine has relatively few side-effects and does not require blood monitoring in monotherapy. The exact way lamotrigine works is unknown. Some think that it is a Na + (sodium) channel blocker, though it is interesting to note that lamotrigine shares very few sideeffects with other, unrelated anticonvulsants known to inhibit sodium channels, (e.g. Oxcarbazepine), which may suggest that lamotrigine has a

2 31 different mechanism of action. Lamotrigine is inactivated by hepatic glucuronidation. Stability testing is an important part of a process of drug substance development. The purpose of the stability testing is to provide evidence on how the quality of a drug substance or drug products varies with time under influence of a variety of environmental factors such as temperature, humidity, and light and enables recommendations of storage conditions, retest periods, and shelf life to be established. The main aspects of the drug product that play an important role in shelf life determinations are the assay of active drug and degradants generated during the stability studies. During synthesis of drugs, small quantities of impurities are usually carried into the bulk drugs. The impurities in drugs are also formed during the formulation process and long term storage conditions (Kovaleski et al. 2007) 53. Degradation products are the common impurities in the medicines resulting from storage or formulation to different dosage forms. It is required to detect the impurities in the drug substance obtained from batches manufactured during the development process, batches from the commercial process, and stress conditions (ICH Topic Q3 B (R2) 2006). Several methods have been reported for the determination of Lamotrigine in biological samples using high performance liquid chromatography (HPLC) (E. Vidal; D. Londero ;M. A. Saracino;P.Angelis-Stoforidis; M. E. C. Queiroz;M. Contin; E. Forssblad;

3 32 G. L. Lensmeyer; M. Torra; B. Levine; T. May; G. Dumortier; M. A. P. D. Saracino; E. Greiner-Sosanko and K. M. Matar) Lamotrigine can be determined by using UV-Vis spectroscopy (M. Madi and S.J. Rajput) O.Dominguez et.al (2008) reported the determination of Lamotrigine by Stripping Voltammetry 52.In the literature, the analytical techniques for determination of phenyltriazine compound lamotrigine by gas chromatohraphy (M. Watelle) 54.lamotrigine can be determines by different methods i.e in pharmaceutical formulations (B. R. Danielsson; R.Dixon; N. V. Acharya and U. A. Argikar) In literature determination of Lamotrigine by different cases but there was no reported methods for determination of process related impurities in Lamotrigine by using RPLC method.in this work a sensitive, reliable and accurate method has been developed for separation and quantification of six related impurities using RPLC isocratic elution.the developed method was a stability indicating method and established for each impurity accordance to ICH guidelines (ICH Q2(R1)). During the development of an analytical procedure, the HPLC method was developed for the determination of In-House synthesized Lamotrigine and the impurities arising during its manufacturing. In the present study, we described a reverse phase high performance column liquid chromatography method for the separation and quantification of process related impurities of Lamotrigine. The accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) and robustness of the

4 33 method was determined in accordance with ICH guidelines and found to be suitable for quality assurance of Lamotrigine. Cl Cl N N H 2 N N NH 2 Fig: 2.1 structure of Lamotrigine Table 2.1 Lamotrigine Details : Chemical name : (-(2, 3-dichlorophenyl)-1, 2, 4- triazine-3, 5-diamine) Molecular weight (amu) : CAS Registry Number : Chemical formula : C8H7Cl2N5 Therapeutic category : Antiepileptic 2.1 Experimental: Chemicals: Lamotrigine and its impurities (Table 2.1) were synthesized and structure was confirmed with the aid of Mass, NMR and FT-IR spectral data. The Spectra of Mass, NMR and IR of all impurities and Lamotrigine were shown in Fig 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 and Fig 2.9 respectively.

5 34 Chemicals and reagents i.e., Milli-Q-Water, Methanol, Acetonitrile both gradient grade were procured from Sigma-Aldrich Details of Instrumentation: HPLC: The High performance Liquid Chromatography is Waters Alliance 2695 having quaternary pumps including auto injector.this HPLC connected with PDA detector connected with Empower 2 software FT-IR: The FT-IR is Thermo Nicolet 380 connected with Omnic software UV: The UV spectrometer is Shimadzu 2450 with UV probe soft ware, version LC-MS: The LC-MS-MS is waters with Mass lynx soft ware HNMR: The NMR is Varian 400 MHZ with Chem pack soft ware, Version Spectral data of Lamotrigine and impurities (a) Mass spectrum

6 35 (b) IR Spectrum Fig. 2.2a Spectral data of Lamotrigine (a) Mass (b) IR (c) 1 H NMR Spectrum

7 36 (d) UV Spectrum Fig. 2.2b Spectral data of Lamotrigine (c) 1 H NMR and (d) UV a) Mass Spectrum

8 37 b) 1 H NMR Spectrum Fig. 2.3a Spectral data of Lamotrigine Impurity-A (a) Mass and (b) 1 H NMR Spectrum c) IR Spectrum Fig. 2.3b Spectral data of Lamotrigine Impurity-A (c) IR spectrum

9 38 a) Mass Spectrum Fig. 2.4a Spectral data of Lamotrigine Impurity-B (a) Mass b) 1 H NMR Spectrum

10 39 c) IR Spectrum Fig. 2.4b Spectral data of Lamotrigine Impurity-B (b) 1 HNMR (c) IR a)mass Spectrum

11 40 b) NMR Spectrum Fig. 2.5a Spectral data of Lamotrigine Impurity-C (a) Mass (b) 1 H NMR c) IR Spectrum Fig. 2.5b Spectral data of Lamotrigine Impurity-C (c) IR.

12 41 a) Mass Spectrum Fig. 2.6a Spectral data of Lamotrigine Impurity-D.(a) Mass b) NMR Spectrum

13 42 c) IR Spectrum Fig. 2.6b Spectral data of Lamotrigine Impurity-D (b) 1 H NMR (c) IR a) Mass Spectrum

14 43 b) NMR Spectrum Fig. 2.7a Spectral data of Lamotrigine Impurity-E (a) Mass (b) 1 HNMR c) IR spectrum Fig. 2.7b Spectral data of Lamotrigine Impurity-E (c) IR

15 44 a)mass Spectrum Fig. 2.8a Spectral data of Lamotrigine Impurity-F (a) Mass b)nmr Spectrum

16 45 c) IR Spectrum Fig. 2.8b Spectral data of Lamotrigine Impurity-F (b) 1 H NMR (c) IR.

17 Fig. 2.9 Ultra Violet spectra of Lamotrigine Impurities. 46

18 47 Based on the above spectral data the impurities details are as follows. Lamotrigine Impurity-A: N N NH NH 2 Cl O Cl 3-Amino-6-(2,3-dichlorophenyl)1,2,4-triazin-5(4H)-one Mass (M+,257 ), 1 H-NMR (DMSO-d6): δ 7.0 (s, 2H), (m, 2H), (m,1h), 12.5 (s, 1H),IR (cm -1 ): 508(C-Cl), 1492(aromatic), 1559(C=N), 1619(amidic C=O), 2551, 3074, 3415(Amines). Lamotrigine Impurity-B: N N NH 2 NH 2 CN Cl Cl 2-(2, 3-Dichlorobenzyl)-2-(guanidiniimino) Acetonitrile Mass (M+, 256),1 H-NMR (DMSO-d6): δ 7.0 (s, 4H), (m, 2H), (m,1h), IR (cm -1 ): 829, 1179, 1397, 1448, 1547, 1613, 1681, 2206(CN), 3190, (Amines). Lamotrigine Impurity-C: N H N NH O Cl O Cl

19 48 6-(2, 3-Dichlorophenyl)-2, 3, 4, 5-tetrahydro-1, 2, 4-triazin3, 5-dione Mass (M+, 258), 1 H-NMR (DMSO-d6): δ 7.42 (m, 1H), 7.7 (m, 2H ), 12.2 (S,1H), 12.6 (S,1H),IR (cm -1 ): 782, 1329, 1380, 1604(amidic C=O),1687,3276(Aromatic), 3464(Amides),. Lamotrigine Impurity-D: COOH Cl Cl 2, 3-dichlorobenzoic acid Mass (M+,190),1H-NMR (DMSO-d6):7.6 (t, 1 H), 7.7 (dd, 1H), 7.8 (dd,1h), 13.7, (S, 1H) &IR (cm -1 ): 829, 1179, 1397, 1448, 1547, 1613, 1681(Acid C=O), 2206, 3190 (Aromatic CH), 3350 (Acid OH). Lamotrigine Impurity-E: N N N H N O Cl Cl NH Cl 2 Cl N-[5-Amino-6-(2,3-dichlorophenyl)1,2,4-triazine-3yl]-2,3- dichlorobenzamide Mass (M+, 426),1H-NMR (DMSO-d6): δ 7.26 (d, 1H), (m, 5H), (m, 2H), 11 (bs, 1H)&IR (cm -1 ): 740(C-Cl), 1412, 1681(Amide C=O), 1738, 2821, 3021(Aromatic -CH), 3132(amides), 3231(Amines). Lamotrigine Impurity-F:

20 49 Cl Cl CN O 2-(2,3-dichlorophenyl)-2-oxoacetonitrile Mass (M+, 200), 1 H-NMR (DMSO-d6): δ 7.7 (m, 1H (b)), 8.1 (m, 1H (a)).8.2 (m, 1H(c))&IR (cm -1 ): 585, 950, 1260, 1305, 1418, 1683(-C=O), 2638(- CN), Method development: Wave length Selection: The UV absorption spectra of Lamotrigine and its impurities were established with help of UV spectrophotometer and found that 210 nm (Fig 2.2(b)) was the maximum absorbance for all impurities (Fig 2.9) and Lamotrigine. Hence, 210 nm is selected for assay and purity methods Selection of Suitable stationary phase and Mobile phase: The separation of impurities from main component and from adjacent components (impurities) with proper resolution depends on the stationary phase selected for the analysis. The selection of stationary phase is important to achieve the proper separation, because Lamotrigine and its impurities are having different polarities and different functional groups Selection of Mobile phase:

21 50 As Lamotrigine is high polar in nature reverse phase chromatography is suitable. Lamotrigine belongs benzodiazepine class and all impurities are different possesses different polarities, to resolve all impurities from Lamotrigine is largely depending on the stationary phase (HPLC column) and mobile phase. A gradient mobile phase consisting of Mobile phase-a (0.05% Trifluoroaceticacid in water) and Mobile phase-b (0.05% Trifluoroaceticacid in Acetonitrile) was found suitable for the separation. The mobile phases was degassed and filtered through 0.45 microns Millipore filter, prior to its use for chromatographic separation Selection of stationary phase: The different stationary phases such as Luna C18, YMC C8, Zorbax SB phenyl and Symmetry C18, 75 x 4.6 mm, 5µ were examined to achieve chromatographic separation. The experimentation was started using Ace C18, 4.6 mm X 150mm, 5.0 µm. Trail-1: The typical chromatographic conditions considered are as follows Mobile phase-a : 0.05% trifluoroaceticacid in water Mobile phase -B : 0.05% trifluoroaceticacid in Acetonitrile Wavelength : 210 nm

22 51 Column : Ace C18, 150 mm X 4.6 mm X 5.0 µ. Sample preparation : 10 milligram in 50 ml of methanol Flow rate : 1.0 ml min -1 Column oven : 25 o C Diluent : Methanol Elution : Gradient Run time : 20 minutes Gradient program : Time(min) % Mobile phase-a % Mobile phase-b Observation: As Ace C18 is suitable for separating acidic, basic and neutral molecules I had chosen this column as the first priority. But by using Ace C18 column Impurity-B and Impurity-C are co eluting together and the peak shapes of Imp-B, imp-c are not symmetrical shape (Fig 2.10). Changing of Mobile phase to change selectivity and obtain good separation while a common practice, can be tedious and time consuming. Instead of this can change column to obtain good separation and selectivity. So I had proceeded for next column while the experimental conditions are remains same.

23 52 Fig 2.10 Typical Chromatogram obtained from trail-1 Trail-2: The typical chromatographic conditions considered as follows Column : Zorbax phenyl 50 x 4.6 mm 5µm. Mobile Phase-A : 0.05% trifluoroaceticacid in water Mobile phase-b : 0.05% trifluoroaceticacid in Acetonitrile. Wavelength 210 nm Flow : 1.0 ml min -1 Column temperature : 25 o C Diluent : Methanol Gradient program : Time(min) % Mobile phase-a % Mobile phase-b

24 53 Fig 2.11 Typical Chromatogram obtained from trail-2 Observation: Zorbax phenyl column is basic column. The column dimensions for the column are as follows Pore size: 80 A, Surface area: 180 m 2 /g, Temperature limit: 80 C, ph range : and Carbon load:5.5% As though the column has wide ph range and temperature limit is very high the impurities B and C are co eluting.(due to low carbon load) (Fig 2.11). So to improve the resolution and as well as to shorten run time I had proceed for the next column which C8. Trail-3: The experiment details are as follows. Mobile Phase-A Mobile Phase-B : 0.05% trifluoroaceticacid in water. : 0.05% trifluoroaceticacid in Acetonitrile.

25 54 Column Wavelength : YMC Pack pro C8 150mm x 4.6 mm, 5µm. : 210 nm Flow rate : 1.0 ml min -1 Column oven : 25 C Diluent : Methanol Gradient program : Time(min) %Mobile Phase-A % Mobile Phase-B Fig 2.12 Typical Chromatogram obtained from trail-3 Observation: YMC pack pro C8 column is lower hydrophobic in nature. As all phases in pro family are ideal for the analysis of organic molecules, especially for basic molecules, the ultra pure silica and very low residual silanol activity means that these columns do not require ion pair reagents. Due to shorten the run time of the analysis I used this column but here also the resolution is not proper So I had proceed for the next column which is C18 (Fig 2.12).

26 55 Trail-4: By using symmetry C18 column Lamotrigine and its impurities are well separated (Fig 2.13). The experiment details are as follows. Mobile Phase-A : 0.05% trifluoroaceticacid in water. Mobile Phase-B : 0.05% trifluoroaceticacid in Acetonitrile. Column : Symmetry C18, 75mm X 4.6 mm, 3.5 µm. Wavelength : 210 nm Flow : 1.0 ml.min -1 Column : 25 o C Diluent : Methanol Gradient Program : Time(min) % Mobile phase-a % Mobile phase-b Fig 2.13 Typical Chromatogram obtained for trail-4

27 56 Observation: C18 column is commonly known as traditional reverse phase matrix since C18 has High degree of hydrophobicity and it interacts with widest range of compounds. C18 is more hydrophobic than C8 because of longer carbon chain. The column dimensions are % of carbon content: 19.1%, particle shape: spherical, pore size is 100 A and surface area is 335, in which the column has wide ph range from 2-8. Because of selectivity, column to column and batch to batch reproducibility this column is more suitable for Lamotrigine which has shorter run time, better resolution and good peak shapes. The proper chromatographic separations were achieved on Symmetry C18, 75mm x 4.6mm, 3.5 micron column and all the impurities are found to be symmetrical and well resolved from main component. Conclusion: Since all compounds are base line separated in a reasonable time symmetry C18 column is the best column for Lamotrigine. 2.3 Specificity: Thermal degradation: About 1.0 gm of Lamotrigine sample is taken and kept under thermal condition i.e., at 105 C for 48 hours, sample collected after 48 hours and analyzed.

28 Photo degradation: About 1 gm of sample is taken and kept in UV chamber i.e., at 254 nm for 48 hours, sample collected after 48 hours and sample analyzed Acid hydrolysis: Dissolved 10 mg of sample in 50 ml of 5N HCl solution, kept for 48 hours at 60 C with continuous stirring and analysed after 48 hours Base hydrolysis: Dissolved 10 mg of sample in 50 ml of 5N NaOH solution, kept for 48 hours at 60 C with continuous stirring and analyzed after 48 hours Peroxide hydrolysis: Dissolved 10 mg of sample in 50 ml of 3% Peroxide solution, kept for 48 hours at 60 C with continuous stirring and analyzed after 1 hour Water hydrolysis: Dissolved 10 mg of sample in 50 ml of water, kept for 48 hours at 70 C with continuous stirring and injected after 48 hours Results and discussion: Lamotrigine samples are stable in Thermal, Photo degradation and water hydrolysis. Lamotrigine was degraded under oxidative hydrolysis (peroxide), acid hydrolysis and base hydrolysis. All samples are analyzed and found that degradation products are well separated from known impurities and Lamotrigine. Peak purity were established with PDA detector and proved that Lamotrigine is peak is pure and homogeneous in all the above conditions. The studies are summarized in Table 2.2.

29 58 Table 2.2 Lamotrigine Degradation data : Stressed condition Time (h) % Purity Potency (%) Thermal degradation Photo degradation Peroxide hydrolysis Acid hydrolysis Base hydrolysis Water hydrolysis Optimized method: Based on the above study, the below mentioned HPLC parameters are finalized for related substances by HPLC method for Lamotrigine. The typical chromatogram of Blank and Lamotrigine sample + spiked are shown in the below figure Mobile Phase-A Mobile Phase-B : 0.05% trifluoroaceticacid in water. : 0.05% trifluoroaceticacid in Acetonitrile. Column : Symmetry C18, 75 mm X 4.6 mm, 3.5 µ. Wavelength : 210 nm Flow : 1.0 mlmin -1 Column temperature Diluent : 25 o C : Methanol

30 59 Gradient program : Time(min) % Mobile phase-a % Mobile phase-b Fig 2.14 A typical chromatogram of Lamotrigine and impurities

31 Method validation: Related substances by HPLC: System suitability: a) Preparation of Impurities stock solution: Transfer 10 mg of each impurities i.e A,B,C,D,E and F was transferred into a 50 ml volumetric flasks individually and dilute with diluent to make up to the mark with diluent. b) Preparation of Sample solution: Transfer 10 mg of Lamotrigine was transferred into a 50 ml volumetric flask and dilute with diluent to make up to the mark with diluent. c) Preparation of sample + all impurities spiked: Spiked 0.5% of each impurity-a, B, C, D, E and F to sample solution with respect to the test concentration. Injected all above solutions once and determined the system suitability parameters i.e. the resolution between adjacent peaks, Tailing factor and tangent for each impurity. Results and discussion: Under optimized chromatographic conditions, Lamotrigine eluting at 1.64, Impurity-A eluting about 2.57, Impurity-B eluting about 3.43, Impurity-C eluting at 4.82, Impurity-D eluting about at 6.82, Impurity-E eluting about at 7.45 and Impurity-F eluting about at min, respectively. The system suitability results are given in Table 2.3.

32 Table 2.3 System suitability results S. No Name Retention time Relative retention time Resolution, Rs Theoretical plates, N Tailing factor, T Lamotrigine Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F

33 Relative response factor (RRF): a) Preparation of RRF solutions (0.10, 0.2 and 0.3% solution): Transferred 10, 20 and 30 µl of each impurity stock and sample solution into 5 ml of diluent containing 10 ml volumetric flask and diluted to volume with diluent. Prepared the solutions three times individually. Analyzed all above sample preparations each once and determined the Relative response factor for each impurity. Slope regression line of impurity RRFimpurity = Slope regression line of Lamotrigine d) Results and discussion: RRF s are determined for Lamotrigine and its impurities with injecting the dilution solution of impurities and Lamotrigine i.e. 0.1% to 0.3% and RRF s are tabulated in Table 2.4. Table 2.4 Relative response factor for all impurities Impurity Retention Relative Relative response time retention time factor(rrf) Impurity-A Impurity-B Impurity-C Impurity-D Impurity-E Impurity-F

34 Limit of detection(lod) and limit of quantification(loq): Preparation of LOD/LOQ solutions: Transfer 5, 7.5, 10, 12.5,15 and 20µL of each impurity stock solution into 5 ml diluent containing 10 ml volumetric flasks individually and diluted to volume with diluent. i.e., 0.05%, 0.075%, 0.10%, 0.125%, 0.15% and 0.2% respectively. Injected each above solution once and determined the Limit of detection and limit of quantification for each impurity. Results and discussion: Limit of detection of all impurities namely Imp- A, Imp-B, Imp-C, Imp-D, Imp-E and Imp-F were determined to be 0.016% and the Limit of quantification values for all impurities were determined to be 0.048%. The results are summarized in the following given Table 2.5.

35 Table 2.5 Limit of detection and Limit of quantification data for all impurities S.No Conc. (%) Correlation coefficient STEYX SLOPE LOD LOQ Imp-A area Imp-B area Imp-C area Imp-D area Imp-E area Imp-F area

36 Precision and accuracy at limit of quantification level: Precision at LOQ level: Preparation of authentic solution: Prepared six times the solution in diluent containing all impurities at their limit of quantification level. Injected each preparation once, determined the % RSD of six preparations for each impurity. Accuracy at LOQ level: Prepared three times the sample solution containing all impurities at their limit of quantification level. Preparation of sample solution: Prepared three times the sample in diluent having concentration 0.2mg.mL -1. Injected each preparation once and determined the % Recovery for each impurity at Limit of quantification level. Results and discussions: The repeatability at the LOQ level is less than 5.2 % only and the recovery more than 98.6% and less than 101.2%. The results are summarized in the Table 2.6 below.

37 66 Table 2.6 Precision and Accuracy at Limit of quantification level S. No Impurity % RSD (n=6) % Recovery (n=3) 1 Impurity-A Impurity-B Impurity-C Impurity-D Impurity-E Impurity-F Linearity: Preparation of Linearity solutions: Transfer 5, 7.5, 10, 12.5,15 20, 25 and 30 µl of each impurity stock solution into 5 ml diluent containing 10 ml volumetric flasks individually and diluted to volume with diluent. i.e., 0.05%, 0.075%, 0.10%, 0.125%, 0.15%, 0.2% 0.25% and 0.3% respectively prepare the solutions three times. Results and discussion: Linearity determined at 0.05%, 0.075%, 0.10%, 0.125%, 0.15%, 0.20%, 0.25% and 0.30%. The correlation coefficient (r 2 ) is more than The above results reveal that method is linear from LOQ level (0.05%) to 0.30%. The results and graphs are summarized in the following Tables 2.7, 2.8, 2.9, 2.10, 2.11 and 2.12 for Imp-A, Imp-B, Imp-C, Imp-D, Imp-E and Imp-F respectively.

38 67 Table 2.7 Linearity data of Lamotrigine Impurity-A Level (%) Average area of Imp-A (n=3) r Y-Intercept Slope Table 2.8 Linearity data of Lamotrigine Impurity-B Level (%) Average area of Imp-B (n=3) r Y-Intercept Slope

39 68 Table 2.9 Linearity data of Lamotrigine Impurity-C Level (%) Average area of Imp-C (n=3) r Y-Intercept Table 2.10 Linearity data of Lamotrigine Impurity-D Level (%) Average area of Imp-D (n=3) r Y-Intercept Slope 394.2

40 69 Table 2.11 Linearity data of Lamotrigine Impurity-E Level (%) Average area of Imp-E (n=3) r Y-Intercept Slope Table 2.12 Linearity data of Lamotrigine Impurity-F Level (%) Average area of Imp-F (n=3) r Y-Intercept Slope 394.2

41 Accuracy: Preparation of accuracy solutions: Lamotrigine sample was spiked three times at each level 0.05%, 0.075%, 0.10%, 0.125%, 0.15%, 0.20% and at 0.30% of each impurity to the sample solution with respect to the test concentration. Injected each above preparation once and determined the Recovery for each impurity at each level.

42 Table 2.13 Accuracy data for all impurities ( % Recovery) Concentration (%) 0.05% 0.075% 0.10% 0.125% 0.150% 0.200% 0.250% 0.300% Imp-A (%) Imp-B (%) Imp-C (%) Imp-D (%) Imp-E (%) Imp-F (%) Results and discussion: The percentage recovery of impurities in Lamotrigine samples varied from 91.1 to 104.4%. The results are shown in the below table 2.12.

43 Precision solution: Preparation of Sample solution: Prepared the sample solution in diluent having concentration of 0.2mg.mL -1. Precision solution preparation: Spiked six times 0.10% of each impurity stock solution to the sample solution with respect to the test concentration. Injected all above sample preparations and determined the % RSD for each impurity. Results and discussion: The precision of the related substance method was checked by injecting six individual preparations of Lamotrigine spiked with 0.10% of Impurity-A, Impurity-B, Impurity-C, Impurity-D, Impurity-E and Impurity-F. The % R.S.D of the area for each of Impurity-A, Impurity-B, Impurity-C, Impurity-D, Impurity-E and Impurity-F were determined. The results were summarized in the below Table 2.14.

44 73 Table 2.14 Precision data for all impurities. Imp-F Imp-E Imp-D Imp-C Imp-B Imp-A Preparation Average STDEV % RSD

45 Robustness: a) Variation of Flow Rate: Prepared the sample solution and precision solution each once as mentioned in precision. Injected the sample solution and precision solution at flow rates 0.8 ml min -1 and at 1.2 ml min -1 and observed the system suitability parameters, impurities relative retention times and compared with 1.0 ml min -1 results. b) Variation of Temperature: Prepared the sample solution and precision solution each once as mentioned in precision. Injected the sample solution and precision solution at Temperature 20 C and at 30 C and observed the system suitability parameters an impurities relative retention times and compared with 25 Cresults. c) Results and discussion: In all deliberate varied chromatographic conditions i.e., flow rate and temperature the resolution between the critical pairs i.e., the resolution between Lamotrigine and impurity- A was greater than 6.0 shows the robustness of the method. The results are summarized in the Table 2.15

46 Table 2.15 Robustness data ml min -1 ml min -1 ml min -1 Parameter Impurity A RRT Impurity B RRT Impurity C RRT Impurity D RRT Impurity E RRT Impurity F RRT Theoretical plates for Lamotrigine Tailing factor for Lamotrigine Resolution for Lamotrigine 20 C C

47 Solution stability: Preparation of sample solution: Prepared the sample in diluent having concentration of 0.2 mg.ml -1. Injected the sample solution for 0 hrs, 6 hrs, 12hrs, 18 hrs, 24 hrs, 30 hrs, 36 hrs, 42hrs and 48 hrs and performed the impurity content. b) Results and discussion: Impurity-A, Impurity-B, Impurity-C, Impurity-D, Impurity-E and Impurity-F is not increased and other impurities are also not observed during the solution stability and mobile phase stability experiments when performed using the related substance method. The solution stability and mobile phase stability experiment data confirms that the sample solutions and mobile phases used during the related substance determination were stable for at least 48 hours. The results are summarized in the below table 2.16and 2.17for solution stability data and Mobile phase stability data.

48 Table 2.16 solution stability data: Duration Impurity-A (%) Impurity-B (%) Impurity-C (%) Impurity-D (%) Impurity-E (%) Impurity-F (%) Any other impurity (%) Purity (%) SS initial After 6 hrs After 12 hrs After 18 hrs After 24 hrs After 30 hrs After 36 hrs After 42 hrs After 48 hrs

49 Table 2.17 Mobile phase stability data: Duration Impurity-A (%) Impurity-B (%) Impurity-C (%) Impurity-D (%) Impurity-E (%) Impurity-F (%) Any other impurity (%) Purity (%) MS initial After 6 hrs After 12 hrs After 18 hrs After 24 hrs After 30 hrs After 36 hrs After 42 hrs After 48 hrs

50 Batch analysis : Using the above validated method, some Lamotrigine samples were analyzed and the data is furnished in Table Table 2.18 Batch analysis data Lot Number Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F unknown impurity Purity % % 99.98% % 99.95% Conclusion: The simple related substances by HPLC method for quantification of impurities of Lamotrigine is precise, accurate, rapid and specific. The method was fully validated showing satisfactory data for all the method validation parameters tested. The developed method can be used for regular samples and stability samples analysis also.

51 Assay by HPLC: System suitability: Preparation of standard solution: Prepared standard solution in diluent having the concentration of 0.2 mg.ml -1. Injected standard solution for six times and determined the system suitability parameters from the standard solution i.e., % RSD for six replicate injections, Tailing factor and plate count for Lamotrigine peak were evaluated (Table 2.19). Table 2.19 System suitability data of Lamotrigine assay method S. No. Parameter Observation 1 %RSD Tailing factor Plate count Linearity and Accuracy: Linearity/Accuracy solutions preparation: Prepared three times the sample at each level 25% (0.05 mgml -1 ), 50%(0.1 mgml -1 ), 75%(0.15 mgml -1 ), 100%(0.20 mgml -1 ), 125%(0.25 mgml -1 ), 150% (0.30 mgml -1 ), 200%(0.40 mgml -1 ), 250%(0.50 mg ml -1 ) and at 300%(0.60 mg ml -1 )with respect to the test concentration. Injected all the solutions each preparation once and determined the correlation coefficient, Slope and Y-intercept for Lamotrigine.

52 81 Results and discussion: The linearity and accuracy determined across the range from 0.05 mgml -1, 0.10 mgml -1, 0.15 mgml -1, 0.20 mgml -1, 0.25 mgml -1, 0.30 mgml -1, 0.40 mgml -1, 0.50 mgml -1 and 0.60 mgml -1 for Lamotrigine. Correlation coefficient obtained was greater than and the recoveries are between 99.8 % and 100.0%. The Linearity results are summarized in the Table 2.20 and the Accuracy results are summarized in the Table Table 2.20 Linearity data of Lamotrigine Average wt Average Area of the of Lamotrigine Lamotrigine r Slope Y-Intercept

53 82 Table 2.21 Accuracy data of Lamotrigine S. No Level (%) % Recovery (n=3) Precision: Preparation of standard solution: Prepared the standard solution diluent having the concentration of 0.2 mg.ml -1 Preparation of sample solution: Prepared the sample solution twice in diluent having concentration 0.2 mg.ml -1. Injected above standard solution six times and sample solution each preparation twice and determined the assay content for each preparation.

54 83 c) Results and discussion: The % of assay of Lamotrigine during the assay method precision was within 0.04%. The results are summarized in the following Table Table 2.22 Precision data of Lamotrigine Preparation Lamotrigine Assay (%) Average STDEV 0.04 % RSD 0.04

55 Robustness: a) Variation in flow rate: Preparation of sample solution: Prepared the sample solution in diluent having the concentration of 0.2 mg.ml -1. Injected the above sample solution at flow rates 0.8 ml min -1 and at 1.2 ml min -1 and observed the system suitability parameters and results are compared with 1.0 ml min -1 results. b) Variation in temperature: Preparation of sample solution: Prepared the sample solution in diluent having the concentration of 0.2 mg.ml -1. Injected the above sample solution at Temperature 20 C and at 30 C and observed the system suitability parameters and results compared with the results obtained at 25 C. c) Results and discussion: In all above conditions i.e., the change in temperature and flow there is no significant changes are observed in system suitability results as well as in assay content. The results reveal that method is robust.

56 Solution stability and Mobile phase stability: a) Preparation of sample solution: Prepared the sample solution in diluent having concentration of 0.2mg.mL -1 The solution stability of Lamotrigine in the assay method was carried out by leaving both the test solutions of sample and reference standard in tightly capped volumetric flasks at room temperature for two days. The same sample solutions were performed for 0 hrs, 6 hrs, 12hrs, 18 hrs, 24 hrs, 30 hrs, 36 hrs, 42hrs and 48 hrs. The mobile phase stability was also carried out by performing the freshly prepared sample solutions against freshly prepared was kept constant during the study period. The % RSD for the assay of Lamotrigine was determined during mobile phase and solution stability experiments. b) Results and discussion: During the solution stability and mobile phase stability there is no significant changes observed in the assay content of the sample. So this data reveals that the sample solutions and mobile phases are stable for 48 hours. The results are summarized in the below Table 2.23.

57 86 Table 2.23 Solution stability and Mobile phase stability Duration Solution stability Mobile phase stability % Assay % Assay Initial After 6 hrs After 12 hrs After 18 hrs After 24 hrs After 30 hrs After 36 hrs After 42 hrs After 48 hrs % RSD Conclusion: The assay by HPLC method for Lamotrigine is precise, accurate, rugged and specific. The method was fully validated showing satisfactory data for all the method validation parameters tested. The developed method can be used for regular samples and stability samples analysis also.