CHAPTER - IV. Acharya Nagarjuna University, Guntur 105

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1 CHAPTER - IV Acharya Nagarjuna University, Guntur 105

2 A STABILITY-INDICATING LC METHOD FOR LENALIDOMIDE Lenalidomide, 3-(4-amino-1-oxo-3H-isoindol-2-yl) piperidine-2, 6-dione (fig. 4.1), is a novel oral immunomodulatory drug, with antiangiogenic and antineoplastic properties (Kastritis, E., 2007). It is structurally related to thalidomide but has an improved toxicity profile and more potent immunomodulatory activity (Mitsiades, C.S., 2004). Lenalidomide is currently being used in the treatment of hematological malignancies, particularly multiple myeloma (MM) and a subset of myelodysplastic syndrome (MDS) harboring the deletion 5q chromosome abnormality (Dimopoulos, M. A., 2005; Richardson, P. G., 2006; Weber, D., 2006; List, A., 2006;). 1. Experimental 1.1 Chemicals Lenalidomide and its related impurities (fig. 4.2a, 4.2b) were obtained from Natco Pharma Limited (Kothur, Hyderabad,India). Capsules containing 5, 10, 15 and 25 mg lenalidomide were obtained commercially. HPLC-grade acetonitrile and methanol was from Merck (Darmstadt, Germany). Analytical reagent-grade orthophosphoric acid and Triethylamine were from Merck. High-purity water was prepared by use of a Millipore MilliQ plus water-purification system. 1.2 Chromatography HPLC analysis for method development, forced degradation studies, and method validation was performed with a Waters 2695 binary pump plus auto-sampler and a Waters 2996 photodiode-array detector (PDA). The output signal was monitored and processed using Empower software resident in a Pentium computer (Digital Equipment). Compounds were separated on a Inertsil ODS -3V, (250 x 4.6 mm), 5 µm column with a 50:50 (v/v) mixture of orthophosphoric acid buffer, ph 3.0, and acetonitrile as mobile phase. The buffer was prepared from mix 1mL of orthophosphoric acid to 1000 ml of purified water. Adjust ph to 3.0 with triethylamine. Filter the solution through 0.45µ membrane filter. The injection volume was 15 µl, the mobile phase flow rate was 1.0 ml min -1, the column temperature was 30 C, and the detection wavelength was 240 nm. Acharya Nagarjuna University, Guntur 106

3 1.3 Preparation of solutions Standard solutions A stock solution of lenalidomide (5.0 mg ml -1 ) was prepared by dissolving an appropriate amount in acetonitrile. Working solutions containing (2.5 µg ml -1 ) were prepared from this stock solution for determination of related substances and for assay determination, respectively. A mixed stock solution (2.5 µg ml -1 ) of the impurities (Imp-A and Imp-B) was also prepared in acetonitrile Sample solution Twenty capsules were weighed, powder transferred to a clean, dry volumetric flask. Powder equivalent to 50 mg drug was then transferred to a 100 ml volumetric flask, 60 ml acetonitrile was added, and the flask was attached to a rotary shaker for 15 min to disperse the material completely. The mixture was then sonicated for 15 min and diluted to volume to give a solution containing (500 µg ml -1 ). This solution was centrifuged at 3,000 rpm for 5 min and 10 ml of the supernatant was diluted to 100 ml with acetonitrile, to give a solution containing (50 µg ml -1 ). This solution was filtered through a 0.45 µm pore size Nylon 66 membrane filter. 1.4 Specificity The specificity of a method is its suitability for analysis of a substance in the presence of potential impurities (I. C. H., 2003). Stress testing of a drug substance can help identify likely degradation products, which can, in turn, help establish degradation pathways and the intrinsic stability of the molecule. It can also be used to validate the stability-indicating power of the analytical procedures used. The specificity of the LC method for lenalidomide was determined in the presence of two impurities and degradation products. Forced degradation of lenalidomide was also performed to provide an indication of the stability indicating properties and specificity of the method (Bakshi, M., 2002; Carstensen, J. T., 2000). The stress conditions used for the degradation study included light (conducted as stipulated in ICH Q1B), heat (60 0 C), acid hydrolysis (0.1 M HCl), basic hydrolysis (0.1 M NaOH), aqueous hydrolysis, and oxidation (0.1% H2O2). For studies of the effects of heat and light the study period was 10 days whereas for acidic, basic, and aqueous Acharya Nagarjuna University, Guntur 107

4 hydrolysis and oxidation it was 48 h. The purity of peaks obtained from stressed samples of lenalidomide was checked by use of the PDA. The purity angle is within the purity threshold limit obtained in all stressed samples and demonstrates the analyte peak homogeneity. For all stressed samples of lenalidomide the chromatographic run time was extended to 300 min to check for late-eluting degradation products. Assay of stressed samples was performed by comparison with reference standards and the mass balance (% assay + % impurities + % degradation products) was calculated. Assay was also calculated for bulk samples and drug product by spiking with two impurities at the specification level (i.e. 0.15% of analyte concentration, which was (500µg ml -1 ). 1.5 Method validation Precision The precision of the related-substance method was checked by twofold analysis of (500 µg ml -1 ) lenalidomide spiked with 0.15% of each of the two impurities. The RSD (%) of peak area was calculated for each impurity. To evaluate the intermediate precision (ruggedness) of the method, analysis was performed by a different analyst using a different column and a different instrument in the same laboratory. The precision of the assay was evaluated by performing two independent assays of a test sample of lenalidomide and comparison with a reference standard. The RSD (%) of the two results was calculated Limits of detection (LOD) and quantification (LOQ) LOD and LOQ for the two impurities were estimated as the amounts for which the signal-to-noise ratios were 3:1 and 10:1, respectively, by injecting a series of dilute solutions of known concentration (I. C. H., 1995). Precision was also determined at the LOQ level by analysis of two individual preparations of the two impurities and calculating the RSD (%) of the peak area for each impurity Linearity To test the linearity of the method, solutions at two concentrations from 25 to 150% of the analyte concentration (0.051, 0.633, 1.266, 1.898, 2.531, and µg ml -1 ) were prepared from the stock solution. Least-squares linear regression analysis Acharya Nagarjuna University, Guntur 108

5 was performed on peak area and concentration data. Solutions for testing linearity for the related substances were prepared by diluting the impurity stock solution to two different concentrations from the LOQ to 150% of the permitted maximum level of the impurity (Imp-A: 0.050, 0.629, 1.257, 1.886, and and Imp-B: 0.052, 0.651, 1.302, 1.953, and µg ml -1 ). The correlation coefficients, slopes, and y-intercepts of the calibration plots are reported Accuracy The accuracy of the method for lenalidomide was evaluated in triplicate at three concentrations, 25, 50 and 75µg ml -1, both for bulk sample and drug product, and recovery was calculated. For the impurities, recovery was determined in triplicate for 50, 100 and 150% of the analyte concentration (500 µg ml -1 ), and recovery of the impurities was calculated. (The bulk sample did not contain Imp-A. It contained 0.01% Imp-B) Robustness To determine the robustness of the method the experimental conditions were deliberately changed and the resolution of lenalidomide and the two impurities was evaluated. The mobile phase flow rate was 1.0 ml / minute, to study the effect of flow rate on resolution it was changed to 0.8 and 1.2 ml / minute. The effect of ph was studied at ph 2.9 and 3.1 (instead of ph 3.0). The effect of column temperature was studied at 28 and 32 0 C (instead of 30 0 C). In all these experiments the mobile phase components were not changed Stability in solution and in the mobile phase The stability of lenalidomide in solution was determined by leaving test solutions of the sample and reference standard in tightly capped volumetric flasks at room temperature for 48 h during which they were assayed at 6 h intervals. Stability in the mobile phase was determined by analysis of freshly prepared sample solutions at 6 h intervals for 48 h and comparing the results with those obtained from freshly prepared reference standard solutions. The mobile phase was prepared at the beginning of the study period and not changed during the experiment. The RSD (%) of the results was calculated for both the mobile phase and solution-stability experiments. The stability of lenalidomide and its impurities in solution in the related Acharya Nagarjuna University, Guntur 109

6 substance method was determined by leaving spiked sample solution in a tightly capped volumetric flask at room temperature for 48 h and measuring the amounts of the two impurities every 6 h. The stability of lenalidomide and its impurities in the mobile phase was also determined by analysis of a solution of the two impurities every 6 h for 48 h. The mobile phase was not changed during the study period. 2. Results and discussion 2.1 Method development and optimization The main objective of the chromatographic method was to separate critical closely eluting impurities Imp-A and Imp-B and to elute lenalidomide as a symmetrical peak. When Inertsil C8 and Waters Symmetry C18 columns (both 250 x 4.6 mm, containing 5 µm particles) were used, lenalidomide was much too strongly retained (approx. 50 min) when the mobile phase flow rate was 1.0 ml min -1. Use of the 250 x 4.6 mm, 5 µm particle, Zorbax CN column reduced the retention of lenalidomide and its impurities. The resolution between Imp-A and Imp-B was 1.1. The tailing factor for the lenalidomide peak was very high (3.3) when orthophosphoric acid buffer ph was adjusted with triethylamine. To check the effect on resolution of the impurities, peak shape for lenalidomide and the impurities. Ammonium buffer (20 mm ammonium acetate adjusted to ph 7.2 with aqueous ammonia solution) was evaluated (buffer acetonitrile, 50:50, v/v) but this resulted in early elution of lenalidomide (retention time approx. 2.5 min) and co-elution of Imp- A and Imp-B. It was found that use of buffer prepared by adjusting the ph of 0.1% orthophosphoric acid to 3.0 with triethylamine (again buffer acetonitrile, 50:50, v/v) enabled separation of Imp-A and Imp-B and elution of lenalidomide as a symmetrical peak (fig. 4.3). Detailed results from the method-development trials are listed in table 4.1. At a column temperature of 30 0 C the peak shape of lenalidomide was symmetrical. When acetonitrile was used as blank there was no interference with the peaks of the two impurities or lenalidomide. Interference from the excipients (Anhydrous Lactose, empty hard gelatin capsule) was also checked by injection of solutions of these materials. No interference was observed (fig. 4.4). Under optimized conditions lenalidomide and the two impurities were well separated with resolution greater than two; typical retention times were approximately 8.7, 21.5 and 42.0 min Acharya Nagarjuna University, Guntur 110

7 for lenalidomide, Imp-B and Imp-A, respectively. Results from determination of system suitability are given in table 4.2. The method was found to be specific for lenalidomide and its impurities (table 4.3). Analysis was performed on different batches of bulk drug (n = 3) and on pharmaceutical dosage forms (n = 3). The results were given in table 4.4. Results from stability studies performed in accordance with ICH Q1A (R2) recommendations are given in tables 4.5 and Validation of the Method Precision RSD (%) in the study of the precision of the assay of lenalidomide was within 0.1%. RSD (%) of peak area for the two impurities in the study of the precision of the assay of the related substances was within 1.5%. These results confirmed the method was highly precise. RSD (%) for lenalidomide in the study of intermediate precision was 0.3%; for the two impurities RSD (%) of peak area was well within 2.0%. These results confirmed the ruggedness of the method (table 4.7) Limits of Detection and Quantification The limits of detection for Imp-A and Imp-B were, respectively, , and % of the concentration of lenalidomide, i.e µg ml -1, for a 15µL injection. The limits of quantification for Imp-A and Imp- B were, respectively, , and % of the concentration of the analyte, i.e µg ml -1, respectively, for a 15µL injection. Precision at the LOQ concentration for the two impurities was <2% Linearity The calibration plot for assay of lenalidomide was linear over the calibration range tested ( µg ml -1 ), and the correlation coefficient was > These results show there was an excellent correlation between the peak area and analyte concentration. The slope and y intercept of the calibration plot were and , respectively. Calibration plots for Imp-A (0.050 to µg ml -1 ) and Imp-B (052 to µg ml -1 ) were linear over the ranges tested. The correlation coefficients were > When linearity for the related substances was checked over the same concentration range for three consecutive days RSD (%) for the slopes and y intercepts of the Acharya Nagarjuna University, Guntur 111

8 calibration plots were 0.5 and 0.3, respectively. These results show there was an excellent correlation between the peak area and concentration for the two impurities Accuracy Recovery of lenalidomide from bulk drug samples ranged from to % (table 4.8) and that from pharmaceutical dosage forms ranged from to % (table 4.9). Recovery of the two impurities from bulk drug samples ranged from to 102.4% Robustness When mobile phase flow rate and ph, and column temperature were deliberately varied resolution between closely eluting impurities, i.e. between Imp-A and Imp-B, was greater than 2.0, illustrating the robustness of the method (table 4.10) Stability in Standard Solution and in the Mobile Phase RSD (%) for assay of lenalidomide during standard solution stability was within 1%. changes in the amounts of the two impurities were observed during solution stability, but sample in mobile phase are not stable at room temperature (25 C ± 2 C) and refrigerator condition (2 C 8 C). The results from standard solution stability experiments confirmed that for up to 48 h during assay and determination of related substances. Stability in the mobile phase was not with in the limit at room temperature and refrigerator and solutions were prepared freshly at the time of analysis Results from Forced Degradation Studies Degradation was not observed when lenalidomide was subjected to light and heat. Degradation was not observed when the drug substance was subjected to acidic, basic and to oxidative conditions (fig. 4.5). Lenalidomide is highly sensitive to aqueous hydrolysis and was degraded into Imp-B by basic for 48 h (fig. 4.6). This was confirmed by co-injection with Imp-B standard. Peak-purity test results from the PDA detector confirmed the lenalidomide peak obtained from all the stress samples analyzed was homogeneous and pure. Peak purity results from the PDA detector for the peaks produced by degradation of lenalidomide confirmed that all these peaks were homogeneous and pure for all the stress samples analyzed (fig. 4.7a- 4.7g). For Acharya Nagarjuna University, Guntur 112

9 all the stress conditions investigated no degradation product peaks were observed after 30 min in chromatograms acquired for the extended runtime of 300 min. The mass balance for the stressed samples was close to 99.6%. Assay of lenalidomide was unaffected by the presence of the two impurities / degradation products, confirming the stability-indicating power of the method. 3. Conclusions The isocratic RP-LC method developed for quantitative analysis of lenalidomide and related substances in both bulk drug and pharmaceutical dosage forms is precise, accurate, and specific. Satisfactory results were obtained from validation of the method. The method is stability indicating and can be used for routine analysis of production samples and to check the stability of samples of lenalidomide. Acharya Nagarjuna University, Guntur 113

10 Fig 4.1: Lenalidomide: 3-(7-Amino-3-oxo-1H-isoindol-2-yl) piperidine-2, 6-dione Fig 4.2a: Impurity-A: Methyl 2-bromomethyl-3-nitrobenzoate Fig 4.2b: Impurity-B: 3-(4-nitro-1-oxoisondolin-2-yl)-piperidine-2, 6 dione Acharya Nagarjuna University, Guntur 114

11 Fig 4.3: Typical chromatogram obtained under the optimum conditions from LLM spiked with impurities at the 0.15% level Acharya Nagarjuna University, Guntur 115

12 Fig 4.4: Typical chromatogram obtained from blank Acharya Nagarjuna University, Guntur 116

13 Fig 4.5: Typical chromatogram obtained from oxidative conditions stressed LLM samples Acharya Nagarjuna University, Guntur 117

14 Fig 4.6: Typical chromatogram obtained from aqueous hydrolysis stressed LLM samples Acharya Nagarjuna University, Guntur 118

15 Fig 4.7a: Control sample preparation Fig 4.7b: Treatment of sample with 0.1N HCl Acharya Nagarjuna University, Guntur 119

16 Fig. 4.7c: Treatment of sample with 0.025N NaOH Fig. 4.7d: Treatment of sample with 10 % H2O2 Acharya Nagarjuna University, Guntur 120

17 Fig. 4.7e: Treatment of sample with Purified Water Fig. 4.7f: Sensitivity to heat at 105 C Acharya Nagarjuna University, Guntur 121

18 Fig. 4.7g: Sensitivity to UV radiation at 254 nm Figure Peak purity results from the PDA detector for the peaks produced by degradation of lenalidomide for all the stress samples Acharya Nagarjuna University, Guntur 122

19 Trial no. HPLC conditions Remarks 1 Flow: 1.0 ml / minute Column: Inertsil C8, 250 x 4.6 mm, 5 µm particles. Mobile phase: 50:50 (v/v) buffer acetonitrile: buffer Prepared by adjusting 20mM sodium dihydrogen phosphate monohydrate to ph 3.0 by addition of trifluoroacetic acid 2 Flow: 1.0 ml / minute Column: Zorbax CN (250 mm 4.6) mm, 5 µm particles Mobile phase: 50:50 (v/v) buffer acetonitrile; buffer prepared by adjusting 20 mm sodium dihydrogen phosphate monohydrate to ph 3.0 by addition of phosphoric acid 3 Flow: 1.0 ml / minute Column: Zorbax CN (250 mm 4.6) mm, 5 µm particles Mobile phase: 50:50 (v/v) buffer acetonitrile; buffer prepared by adjusting 20 mm ammonium acetate to ph Flow: 1.0 ml min) Column: Zorbax CN (250 mm 4.6) mm, 5 µm particles Mobile phase: 50:50 (v/v) buffer acetonitrile; buffer prepared by adjusting 20 mm sodium dihydrogen phosphate monohydrate to ph 3.0 by addition of trifluoroacetic acid The retention times of lenalidomide and Imp- A were approximately 50alidomide and 120 min approximately 50 and 120 min The tailing factor of lenalidomide was 3.3; RS between Imp-B and Imp-3 was 1.1; RS between Imp-A andimp-4 as 1.3 The retention time of lenalidomide was approximately 2.5 min; Imp Imp-3, and Imp-4 co eluted by addition of aqueous ammonia solution. The tailing factor of lenalidomide was 1.1. RS between Imp-A and Imp-B was 2.2; RS between Imp-3 and LLM was 2. Table 4.1: Results from different method development trials Acharya Nagarjuna University, Guntur 123

20 Compound No. of theoretical USP USP tailing plates, N (USP Resolution factor tangent method) Lenalidomide Imp-B Imp-A Table 4.2: Results from system-suitability test S No. Sample name Average Retention time (minutes) % Interference (Respective to Lenalidomide and Impurities) 1 Diluent , NO 2 Placebo NO 3 Lenalidom ide NA 4 Impurity A NA 5 Impurity B NA Table 4.3: Results from specific and selectivity Acharya Nagarjuna University, Guntur 124

21 Sample Imp-A Imp-B Purity by Assay by HPLC HPLC Bulk drug; batch no ND Bulk drug; batch no ND Bulk drug; batch no ND Drug product; batch no ND Drug product; batch no ND Drug product; batch no. CDGH0022 ND Table 4.4: Results (%) from batch analysis Duration Related substance by HPLC Imp-A Imp-B Total impurities Assay by HPLC Bulk drug batch no Initial ND ND days ND days ND days ND Drug product batch no Initial ND ND days ND days ND days ND Remarks Table 4.5: Results (%) from accelerated stability study (storage conditions 40 ± 2 0 C and RH 75 ± 5%) Acharya Nagarjuna University, Guntur 125

22 Related Duration Assay substance by Total by HPLC impurities HPLC Imp-A Imp-B Bulk drug batch no Initial ND ND days ND days ND days ND Drug product batch no Initial ND ND days ND days ND days ND Remarks Table 4.6: Results (%) from long-term stability study (storage conditions 25 ± 2 0 C and RH 60 ± 5%) Acharya Nagarjuna University, Guntur 126

23 Variation Different system Waters 2695 Alliance system Agilent 1100 series VWD system RSD (%) for RSD (%) for related assay substances Different column Different analyst Column Column Analyst Analyst Table 4.7: Results from determination of intermediate precision Added (µg) Recovered (µg) Recovery (%) RSD (%) Table 4.8: Results from study of accuracy for bulk drug substance Added (µg) Recove red (µg) Recove ry (%) RSD (%) Table 4.9: Results from study of accuracy for drug product Acharya Nagarjuna University, Guntur 127

24 Condition Temperature (±5 C of optimum temperature) Flow rate (±20% of the optimum flow rate) ph (±0.1 unit of set ph) Resolution Resolution Variation (RS) between (RS) between lenalidomide Imp-B and and Imp-B Imp-A ml / min ml / min Table 4.10: Results from study of robustness Acharya Nagarjuna University, Guntur 128

25 BIBLIOGRAPHY: Bakshi, M., Singh, S., J. Pharm. Biomed. Anal., 28, 6, 2002, Carstensen, J. T., Rhodes, C. T., (ed) (2000) Drug stability principles and practices, 3rd edn., Marcel Dekker, NY Dimopoulos, M. A., Spencer, A., Attal, M., Blood., 106, 6, 2005, International Conference on Harmonization (2003) Stability testing of new drug substances and products Q1A (R2). International Conference on Harmonization., IFPMA, Geneva International Conference on Harmonization (1995) ICH guidelines on validation of analytical procedures., text and methodology Q2 (R1): FDA. Federal Register 60,11260 Kastritis, E., Dimopoulos, M. A., Expert. Opin. Pharmacother., 8, 4, 2007, List, A., Dewald, G., Bennett, J., N. Engl. J. Med., 355, 14, 2006, Mitsiades, C.S., Mitsiades, N., Curr. Opin. Investig. Drugs., 5, 6, 2004, Rajkumar, S. V., Hayman, S. R., Lacy M. Q., Blood., 106, 13, 2005, Richardson, P. G., Blood, E., Mitsiades, C. S., Blood., 108, 10, 2006, Weber, D., Wang, M., Chen, C., Blood., 108, 6, 2006, Acharya Nagarjuna University, Guntur 129