2.1 2,3 Dichloro Benzoyl Cyanide (2,3 DCBC) and survey of. manufactured commonly for the bulk drug industry, few references were

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1 . Introduction.,3 Dichloro Benzoyl Cyanide (,3 DCBC) and survey of analytical methods,3-dcbc substance, is the although advanced intermediate,3-dcbc is a of lamotrigine well-known bulk drug chemical manufactured commonly for the bulk drug industry, few references were found in the literature regarding separation and detection of its related isomers and impurities -. The,3-DCBC has five regio isomers. In the present case we have reported a simple and effective analytical method for the development and validation of,3-dcbc and its regio isomers. This method is also capable to detect all regio isomer analog impurities. Fig...F: The Chemical structure of,3-dcbc Name Structure,3-DCBC Chemical name Molecular weight/ Molecular formula,3-dichloro benzoyl cyanide. C8H3ClNO. Material and Experimental.. Materials Test samples and reference standards of,3-dcbc and its regio isomers were taken from R&D, Dr. Reddys laboratories, Bollaram, CTO-

2 3, Hyderabad, Andhra Pradesh. Merck gradient grade methanol high pure gradient grade, Analytical grade (AR) Glacial acetic acid (Merck, Germany) and Analytical grade Ammonium acetate (Fluka) were used. Water purified (in-house) by a Millipore system (Milford, USA) was used for making the solutions... Equipment HPLC systems utilized for method validation and development were Waters binary system, with a 996 DAD (Waters Pvt. Limited, USA) and Agilent (Wilmington, Delaware, USA) system equipped with DAD and VWD. The output signal was monitored and processed using Waters (Milford, MA, USA) Empower software. Fig...F: Chemical structures of,3 DCBC and its isomers Name Structure Chemical names,dcbc, Dichloro benzoyl cyanide,dcbc, Dichloro benzoyl cyanide,6dcbc,6 Dichloro benzoyl cyanide Molecular wt./molecular formula. C8H3Cl NO

3 3,DCBC 3, Dichloro benzoyl cyanide 3,DCBC 3, Dichloro benzoyl cyanide.. Preparation of test solutions... Sample /Standard preparation.mg/ml concentration stock solution of,3-dcbc,,-dcbc,,dcbc,,6-dcbc, 3,-DCB and 3,-DCBC were individually prepared in methanol. All standard stock solutions were further diluted with methanol to achieve the standard dilution solutions of.mg/ml concentration for related substances estimation. The specification limit of each,-dcbc,,-dcbc,,6-dcbc, 3,-DCB, 3,-DCBC in,3-dcbc key starting material (KSM) sample was.% w/w (as per ICH controlling limit for any known impurity)..3 HPLC Method optimization and development To develop a stability indicating LC method for the quantitative determination of,3 DCBC Key starting material, all isomers blend solution was used for HPLC method development.

4 .3. Wavelength selection All isomers and,3-dcbc solution spectrums were collected using water PDA system. All the positional isomers and,3-dcbc were contains UV absorption maximum at about nm. Hence the detection was selected for method development was chosen at nm. Weight percentage of each isomer present in the sample was calculated by area normalization method because of all the regio isomers were having same response to that of,3-dcbc and the responses of all regio isomers are as per pharmacopoeial guidelines i.e. between.8 and.. Hence all isomer responses fall under the same range (.98-.). The LC procedure was developed to resolve the possible regio isomers and process impurities in,3-dcbc..3. Column selection Four different C-8 (L column-as per USP: Inertsil ODS-3V, Zorbax C-8, Waters symmetry shield RP-8 and Kromasil C-8 with cm length,.6mm Id, micron particle size) and four different C-8 Columns (Inertsil C-8, Zorbax C-8, Waters symmetry shield RP-8 and Waters symmetry C-8 with cm length,.6mm Id, micron film coated) were used for method development. The column stationary phase was found to be great influence on the retention time, peak shape, USP tailing factor and USP resolution. When inertsil ODS-3V cm,.6mm id, micron column was used in initial experiment with volatile buffer (.mm ammonium acetate (AA), ph adjusted to. with dilute acetic acid (ml

5 in ml of MQ water): Solvent-A:- buffer : methanol :::; Solvent-B:methanol: water :: 8: v/v and. ml flow rate with µl of injection volume. The 3, DCBC was highly retained and the USP resolution between 3,-DCBC and,6-dcbc is.. When the experiments with other C-8 and C-8 columns with cm length,.6 mm id, micron size obtained similar kind of results. It could be due to ability of these compounds to from strong hydrogen bonds with residual silinols of above mentioned C8 and C8 columns. To reduce the RT and separation of all regio isomeric peaks different type particles and stationary phase mechanism were required. The experiment showed that the best results were obtained with Cosmosil MS-II cm length,.6mm, m particle size. It provided with good peak shape and selectivity, with this column,3 DCBC and its five regio isomers as well as other process impurities were separated well (USP resolution between,3 DCBC and other isomeric peaks was more than.), USP tailing was about.. Hence this Cosmosil HPLC column was chosen for further method development..3.3 Effect of organic solvent When acetonitrile was used as a solvent in the mobile phase (buffer: acetonitrile: : v/v) the resolution between,6-dcbc and 3, DCBC was not satisfactory. To improve the resolution between regio isomeric pairs the solvents ratio has been changed i.e reduced acetonitrile content and introduced small portion of methanol (buffer: acetonitrile: methanol:: :3: v/v/v). The resolution between above mentioned pair was poor.

6 After changing different compositions with isocratic mobile phase not reached best resolutions have not been achieved between isomeric pairs, it has been chosen to introduce gradient composition of the mobile phase A & B (as mentioned in the experimental section) were found to be appropriate for the separation of all process impurities and its regio isomers..3. Effect of ph When the phosphate buffer ph adjusted to 3. with phosphoric acid (ml in ml) the retentions were observed very early (before mins). The C8 column used in LC has only -3% of surface of silica particles bonded with C8 phase and remaining will be in the form free silinols. These are acidic at moderate ph values. The cause of early peak in RPLC is the column (stationary) contains highly active silinol groups and isomers were also acidic nature. The problem was solved by using mobile phase at higher ph (-6) reduces ion exchange interaction operating at ph. protanates silinols groups at the silica particles and there by makes the silinol layer available for interacting with the higher acidic analyte. At ph. satisfactory results were obtained. Tailing factors of all isomers were close to. and the resolution between any pair of peaks was found grater than 3.. Hence ph. mobile phase was used for further method development.

7 .3. Optimization of gradient elution A gradient elution programme mode system was designed as described below (Table..T-T3 & Fig...F-F). Change in the composition of gradient programme and the investigational conditions were chosen within the best region forecasted by the gradient mode. Due to the late eluting of isomers the buffer concentration was chosen according to separation of all isomers.,3-dcbc, the related regio isomers and all impurities were resolved with base to base separation. Table..T: Gradient experiment- Time Solution-A Solution-B 3 3 Fig..F: Typical spiked chromatogram Table..T: Gradient experiment- Time Solution-A 7 7 Solution-B 3 3

8 Fig..F3: Typical spiked chromatogram Table..T3: Gradient experiment-3 (Flow-.ml/min) Time. 6 6 Solution-A Solution-B 3 3 Fig..F: Typical spiked chromatogram Based on the above experimental data, the resolution among all isomers has found to be very good. This method has been further taken into validated.

9 .3. Finalized HPLC conditions for the estimation of regio isomer impurities by,3 DCBC Column : COSMOSIL MS-II cm x.6mm, m Wavelength : nm Flow :. ml/min Load : l Diluent : Methanol Oven temperature : 3 C Sample concentration :. mg/ml Buffer preparation:.mm ammonium acetate, ph.. Solution-A: Buffer: methanol: : v/v Solution-B: Methanol: water: 9: v/v Retention time of,3-dcbc was observed around 3. mins. Table..T: Gradient programme Time. 6 6 Solution-A Solution-B 3 3. Peak purity The peak homogeneity examination was executed for the,3 DCBC and its isomeric peaks by using DAD and confirmed that the homogeneity of the all peaks were well proved.. Analytical method validation and its results The optimized and developed related substances by HPLC method was taken up for complete method validation. As per ICH and USP guidelines the method validation has been initiated 6-8.

10 .. System suitability test The standard solution was used as the system suitability solution. The system suitability solution (SST) was determined from the principal peak of standard solution and established Theoretical plates (N) and USP Tailing (T) (Table..T). Table..T: System suitability solution data Name Tailing factor (T),3 DCBC theoretical RSD in % plates (N) (n=) n = Number of determinations... Limit of detection (LOD) and Limit of quantification (LOQ) LOD and LOQ were estimated for,-dcbc,,-dcbc,,6-dcbc, 3,-DCBC and 3,-DCBC based on the signal to noise ratio methodology...3 Limit of detection Prepared a series of dilutions of,-dcbc,,-dcbc,,6-dcbc, 3,-DCBC and 3,-DCBC in different concentrations and injected them into the chromatographic system until to get the signal to noise ratio about or 3 (Table..T6).

11 Table..T6: Limit of detection Impurity Name S/N Ratio LOD in µg/ml.,-dcbc.7.9.,-dcbc ,-DCBC..9.,6-DCBC ,-DCBC Limit of quantification Prepared a series of dilutions of,-dcbc,,-dcbc,,6-dcbc, 3,-DCBC and 3,-DCBC region isomeric impurities in different concentrations, until to get the signal to noise ratio : (9. to.) (Table..T7). Table..T7: LOQ values of regio isomers Impurity name S/N Ratio LOQ in µg/ml.,-dcbc..3.,-dcbc ,-DCBC ,6-DCBC..6. 3,-DCBC Precision at limit of quantification level The LOQ precision of the regio isomer impurities related substance by HPLC method was verified by loading six different preparations of,dcbc (mg/ml) spiked with LOQ level of,-dcbc,,-dcbc,,6-

12 DCBC, 3,-DCBC and 3,-DCBC w.r.t the,3-dcbc concentration. Calculated standard deviation for the area % of,-dcbc;,-dcbc;,6-dcbc; 3,-DCBC and 3,-DCBC for six (n=6) successive points as follows (Table..T8). The above data shows that no major difference in calculated responses which reveals that the developed HPLC method was reproducible and repeatable at quantification level with less than. % RSD (Table..T8). Table..T8: LOQ precision Impurity name Pre- Pre- Pre-3 Pre- Pre- Pre-6 %RSD,-DCBC ,-DCBC ,-DCBC ,6-DCBC ,-DCBC LOQ accuracy LOQ recovery experiments have been conducted to find out recovery of the developed analytical related substances method for the estimation of residuals organic impurities present in the,3-dcbc test sample at limit of quantification level. The accuracy studies conducted for,-

13 DCBC,,-DCBC,,6-DCBC, 3,-DCBC and 3,-DCBC were passed out by loading each solution thrice (n=3) at quantification level (w.r.t mg/ml). The % recoveries of,-dcbc,,-dcbc,,6-dcbc, 3,-DCBC and 3,-DCBC were established (Table..T9). The regio isomer impurities by HPLC method shows that the average LOQ accuracy values were between 9.% and 6.%. The achieved total recoveries were usually dispersed about the mean with consistent percent RSD values and got low % bias (<.). Table..T9: Accuracy at LOQ Name of the isomer Recovery in %,- DCBC 3.3,- DCBC ,- DCBC 6.,6- DCBC 8. 3,- DCBC 9. Fig..F: % Recovery graph % Recovery graph % Recovery 8 6,- DCBC,- DCBC 3,- DCBC Impurity,6- DCBC 3,- DCBC

14 ..7 Precision Method repeatability/intermediate precision The method precision was established, and prepared all regio isomer impurities were spiked with test sample and prepared six individual preparations and loaded (n=6) into a HPLC, the spiked sample solution of,3-dcbc with respect to the test.mg/ml by two different analysts using two different make or same brand of HPLC systems on day by day. Percent RSD values were between.7 and 8.8, which shows that achieved very good method precision results (..T). Table..T: Intermediate precision of method Impurity Pre- Pre- Pre-3 Pre- Pre- Pre-6 name %RSD,-DCBC ,-DCBC ,-DCBC ,6-DCBC ,-DCBC Linearity of response Linearity was checked for all regio isomers at eight different concentration levels covering the range LOQ % w.r.t their target level (normally as recommended by ICH limits i.e.%). The curve was derived by plotting average area counts of the regio isomer impurities

15 that are,-dcbc,,-dcbc,,6-dcbc, 3,-DCBC and 3,-DCBC on the Y-axis and concentration level on the X axis, the curve shows that the linear relation ship with a correlation coefficient of more than.999 for each regio isomer impurity (Fig...F6), at all concentration levels, RSD of peak were found to be considerably low and standard deviation was less than.%. Analysis of residuals specified (Table..T) that the residuals were usually dispersed approximately the mean with consistent no variance across all strengths (Fig...F7) suggestive of the uniform nature of information (Table..T to Table..T6). Table..T: Linearity results Level,-DCBC,-DCBC 3,-DCBC,6-DCBC 3,-DCBC area area area area area LOQ NA Correlation coefficient Slope Y-Intercept % Y-intercept

16 Fig..F6,3-DCBC linearity graphs, DCBC linearity graph y = 637.x - 7. R =.999 R = area area y = 78.3x + 8., DCBC linearity graph % Concentration y = 38.76x R =.999 R = area area % Concentration 6 y = 769.6x R = % Concentration % Concentration 3, DCBC linearity graph 8 area,6 DCBC linearity graph 8 y = 68.33x % Concentration 3, DCBC linearity graph

17 Table..T: Residual summary of linearity results of,-dcbc Level Average area y-best fit Residuals (Difference) response achieved LOQ Table..T3: Residual summary of linearity results of,-dcbc Level Average area y-best fit Residuals (Difference) response achieved LOQ

18 Table..T: Residual summary of linearity results of 3,-DCBC Level Average area y-best fit Residuals (Difference) response achieved LOQ Table..T: Residual summary of linearity results of,6-dcbc Level Average area y-best fit Residuals (Difference) response achieved LOQ (8)

19 Table..T6: Residual summary of linearity results of 3,-DCBC Level Average area y-best fit Residuals (Difference) response achieved LOQ Fig..F7:,3- DCBC residual graphs,- DCBC Residual plot graph, DCBC Residual plot graph Residuals Residuals Order of residual Order of residuals 3, DCBC Residual plot graph,6 DBC Residual plot graph Residuals Residuals Order of residuals Order of residuals 6 8

20 3, -DCBC Residual plot graph Residuals Order of residuals..9 Accuracy Recovery of the HPLC method for all regio isomer impurities quantification was verified by spiking pre-analyzed samples with four different concentration levels, i.e. at.7%,.%,.% and.% (w.r.t the analyte concentration) of every regio isomers of,3dcbc and the mixtures were analyzed by proposed method (n=3). The average accuracy showed that the proposed method provides acceptable recovery it was found to be in the range between 9.7% and.9%...9. RS by HPLC accuracy Recovery of the regio isomer estimation by HPLC method established at % to % levels of the regio impurities with respective to the specification limit that is.% as per ICH limits...9. Accuracy at %, 7%, % and % level The spiked,3-dcbc solutions were prepared in thrice with impurities,-dcbc,,-dcbc,,6-dcbc, 3,-DCBC and 3,-DCBC at.7%,.%,.% and.% (%, 7%,% and %) level

21 with respect to,3-dcbc test concentration. Each prepared solution was loaded into HPLC system once. Calculated percentage average recovery for all regio impurities (Fig...F8) in the recovery preparation using the same area of regio impurities working standard authentic solution i.e.% level w.r.t,3dcbc test concentration (Table..T7). Table..T7: Summary of recoveries at %, 7%, % and % level Regio % recovery % recovery %recovery %recovery isomer name At % at 7% at % at %,-DCBC ,-DCBC ,-DCBC ,6-DCBC ,-DCBC Fig...F8: Recovery graph % Recovery graph Series 8 % Recovery Series 6 Series3 3 Series levels The regio impurities quantification by HPLC method shows reliable and higher average accuracy values for each regio isomeric impurity (five

22 isomers) at the entire four level of concentrations such are %, 7%, % and % in,3 DCBC associated average accuracy range between 9.7% and.9%. The above experimental spiking method designated that the achieved or received average recoveries were usually dispersed approximately the mean with consistent percentage RSD through out the four levels of strengths suggestive of uniform environment of the information. Based on the above data it confirms that there was no significant trace level interference of other impurity and the developed HPLC method was found to be accurate with low % of bias values (<.). The above study specified that the regio isomers of related substances by HPLC method was suitable for quantification of isomeric impurities in,3 DCBC... Solution state stability The solution stability of,3 DCBC in the RS by HPLC method was passed out by leaving sample solutions in at ambient temperature on bench top for 8hrs. Calculated the individual isomer impurity content of,-dcbc,,-dcbc,,6-dcbc, 3,-DCBC and 3,-DCBC for every 6 hrs duration of period up to the exercise completion. There are no significant changes observed from initial spiking analysis and after 8 hrs of the analysis in regio isomers content study period. Therefore,3DCBC spiked solutions were stable up to 8 hrs in the above chosen diluent medium developed method (Table..T8).

23 Table..T8: Related substances by HPLC solution stability results %,DCBC. %,DCBC. %3,DCBC. hrs %,6- % 3,DCBC DCBC.. 6hrs.... hrs... 8hrs.. hrs. 3hrs % SMUI %Purity hrs hrs hrs %RSD... Robustness Investigational environments were deliberately changed and the USP resolution between all pair of isomers, USP tailing, and theoretical plates were assessed. In all of the purposely altered LC condition (The flow rate of the mobile phase was. ml/min, it was changed by. units from. to.3 and.7 ml/min. The ph of the mobile phase was.; it was changed by. units from. to.3 and.7. The column oven temperature (T) was 3 C, it was changed by. C units from 3 C to C and 3 C the USP resolution between all pairs was not less than 3., asymmetry was less than. and USP theoretical plate counts was more than plates, illustrating the good robustness of the method (Table..T9 (a)-(f)).

24 Table..T9 (a): Different flow results Parameter Theoretical value value value Flow.7ml/min Flow.3 ml/min Flow. ml/min plates Tailing factor Table..T9 (b): Different column oven temperature results Parameter Theoretical plates Tailing factor value value value C 3 C 3 C Table..T9(c): Different column oven temperature results Parameter value value value ph.3 ph. ph Theoretical plates Tailing factor Table..T9 (d): Flow:.7 ml/min results Impurity Pre- Pre- Pre-3 Pre- Pre- Pre-6 %RSD,-DCBC ,-DCBC ,-DCBC ,6-DCBC ,-DCBC

25 Table..T9 (e): Flow:. ml/min results Impurity name Pre- Pre- Pre-3 Pre- Pre-,-DCBC ,-DCBC ,-DCBC ,6-DCBC ,-DCBC Pre-6 %RSD Table..T9 (f): Flow:.3 ml/min results Impurity name Pre- Pre- Pre-3 Pre- Pre- Pre-6 %RSD,-DCBC ,-DCBC ,-DCBC ,6-DCBC ,-DCBC Summary and Conclusion The simple, rugged and robust gradient RP-LC method has been developed for separation and quantitative determination of,3 DCBC, its regio isomers and its related impurities. The above stated method is selective, precise and accurate. The method was completely validated and showing satisfactory data for all the method validation parameters tested. The developed method is stability-indicating and can be used for

26 assessing the stability of bulk samples of,3-dcbc and also for monitoring the synthetic procedures of,3-dcbc. Table..T:,3-DCBC of analytical method validation data Test Related substances results,-dcbc,-dcbc 3,-DCBC,6-DCBC 3,-DCBC Precision (% RSD) LOD in %.%.7 %.6 % 8.8 % 7.8 % LOQ in % Theoretical plates > & Tailing factor >.9 Theoretical plates > & Tailing factor >.9 Theoretical plates > & Tailing factor >.9 Theoretical plates > & Tailing factor >.9 Intermediate precision (% RSD) Linearity (correlation coefficient) Accuracy.7-.9 (% Recovery) Robustness Solution state stability Mobile phase stability Theoretical plates > & Tailing factor >.9 Stable for 8hrs Stable for 8hrs References. Carrier DJ, Eckers C, Wolff JC, J Pharm Biomed Anal 7(8) Reddy VV, Govardan G, Srinivasulu K, Reddy GM, Himabindu VR, J Chem. (8) Youssef NF, Taha EA, Chem Pharm Bull (Tokyo) (7).

27 . Ashton DS, Ray AD, Valko K, Int JPharm 89(999) 8.. Emami J, Ghassami N, Ahmadi F, J Pharm Biomed Anal. (6) ICH Guide QA: text on validation of analytical procedures: term and Definition, international conference on harmonization, Fed. Reg. (6 FR 6), March ICH Guide QB: validation of analytical procedures: methodology, international conference on harmonization. Fed. Reg. (6 FR 63), 9 May ICH Guide Q3A: impurities in new drug substances, international conference on harmonization. Fed. Reg. (68 FR 69), February 3.