Development of A Rapid and Sensitive HPLC Assay Method for Lenalidomide Capsules and its Related Substances

A chromatographic method was established for the determination of lenalidomide and related substances in 10 mg and 5 mg capsules using Sunfire C-18(250×4.6 mm ID, 5 μm) HPCL column with 85:15 v/v ratio of mobile phases A (mixture of phosphoric acid buffer and 1-octane sulphonic acid sodium salt) and B(55: 45 v/v ratio of methanol and acetonitrile) at 40C and 210 nm wave length. The degradation studies were performed using 0.1N HCl, 0.1 N NaOH, 1% v/v hydrogen peroxide, humidity, UV at 254 nm, Sun light, and heat to 60C. No significant degradation of lenalidomide was observed. However, the slight degradation was observed in presence of NaOH. The developed HPLC method gave the peaks purity angle was less their threshold angle, indicating it to be suitable for stability studies. It was validated with respect to linearity, accuracy, precision, ruggedness, and robustness.

They are many methods were reported for stability studies of lenalidomide but it's have lack of selectivity by degradation with base or acid and is also applicable to capsules of 5.0 mg and 10.0 mg.With best of my knowledge there is no any validated method for lenalidomide capsules and its related substances.This paper deals with the development of a selective and valid chromatographic assay for quantitative determination of lenalidomide and its related substances from its capsules in view of stability studies.

Experimental
All chemicals are analytical reagent grade otherwise stated here.10 mg and 5.0 mg capsules of lenalidomide, chemical name, 3-(4-Amino-1-oxo 1,3-dihydro-2H-isoindol-2-yl) piperidine-2,6-dione (Figure 1) and its related substances, namely, Imp-B, placebo were received from the Process Research Department of High Potent Active Pharmaceutical Ingredients Operations of Dr. Reddy's Laboratories Limited, Hyderabad, India.HPLC grade acetonitrile, methanol, potassium dihydrogen orthophosphate, and phosphoric acid were purchased from Merck, Schuchardt OHG, Darmstodt Germany for preparation of mobile phase A & B and conditioning of HPLC column.High pure water was prepared with a Millipore Milli Q plus purification system.

Instrumentation
A photodiode array UV detector containing Waters 2489 model LC system was used for chromatographic analysis.The output signals were monitored and processing using advanced waters empowered software on a Pentium computer (Digital Equipment Co).

Preparation of Buffer and Mobile Phase A&B
Weighed and dissolved about 1.36g of potassium dihydrogen orthophosphate in to 1000 mL of milli-Q-water and then the adjusted pH to 2.0 with diluted orthophosphoric acid.Added accurately about 1.0 g of 1-octane sulphonic acid sodium salt (mono basic) to the 1000 mL of buffer and mix well and filter through 0.45µM filter and degassed in a sonicator for 10 min, which was used as a mobile phase-A and methanol and acetonitrile in the ratio of 55:45v/v was used as a mobile phase-B.100:400v/v ratio of buffer and mobile phase-B was used as diluents for the preparation of standard and test samples.

Preparation of Standard and Sample Solutions
A working standard solution of 1.0 ppm was prepared using lenalidomide working standard and make up with above diluent.Taken lenalidomide contain 10 capsules of 5.0 mg dose/5 capsules of 10.0 mg dose as such and then drop in to 200 mL volumetric flask.Added sonicated for about 15 min (Intermediate shaking) after that made up to the mark with diluent.Centrifuge a portion of solution at 3500 rpm for 10 min.

Placebo Preparation
The placebo solution as mentioned under sample preparation was prepared by taking five placebo capsules of 10 mg or ten placebo capsules of 5 mg.

Chromatographic Conditions
Sunfire C-18(250×4.6 mm ID, 5 µm) HPCL column equipped with photo diode array UV detector was used at 40 o C in the present study.At a 1.0 mL/min flow rate of the mobile phases A and B were programmed as following ratios: 85:15v/v of A:B was used at starting and kept constant up to 30 min.After 30 min increase the volume of mobile phase B up to 60 min and kept constant 30:70v/v (A:B) ratio for 70 min were used in it.Then slowly changed the ratio of A: B as 85:15v/v within 1 min.Finally kept constant at 85:15v/v (A:B) up to 85 min.The injection volume was 10 µL and detection was carried out at 210 nm.

Method Validation Summary
System Suitability and System Precision: A standard solution was prepared by using lenalidomide (Lot No.: AH2WS077AA) working standard as per test method and was injected six times into the HPLC system for testing system suitability and precision.

Specificity
The separation of lenolidomide and the specificity of the method was checked by conducting placebo (B.No.: LLC5-036) and using degradants, such as acid (0.1N HCl), base (0.1N NaOH), hydrogen peroxide(1% hydrogen peroxide for 15 min), thermal water for 15 min, humidity, UV light, and sun light degradation at 60 o C.

Precision and Accuracy of the Method
The precision and accuracy of the test method was evaluated by analysing six samples prepared by spiking test preparation (Lenolidomide 5. Mg Capsules) with lenalidomide impurity-B of 0.5% in test solution (250 ppm).Samples were prepared in triplicate at each level by spiking test preparation with 20%, 50%, 100%, 125%, and 150% to the target concentration (0.5%) of lenalidomide impurity-B.The intermediate precision and accuracy was also checked by using different analyst and different instruments in the same laboratory 8 .

Linearity, Limit of Detection and Limit of Quantification of Test Method
Plotted linearity graph for Lenalidomide Impurity-B, at LOQ Level, 20%, 50%, 100%, 125%, and 150% spike levels with 'average µg/mL found' in accuracy section versus 'average µg/mL added'.Limit of detection and limit of quantification was established based on signal to noise ratio.A series of impurity spike solutions having different concentrations of lenalidomide Impurity-B and lenalidomide were injected.Limit of detection for lenalidomide Impurity-B and lenalidomide was established by identifying the concentration, which gives signal to noise ratio of about 3. Limit of quantification for Lenalidomide Impurity-B and Lenalidomide were established by identifying the concentration, which gives signal to noise ratio of about 10.

Ruggedness of Test Method
System to system/analyst to analyst/column to column variability study was conducted on different HPLC system, different column, and different analyst under similar conditions at different times for testing of ruggedness of the method.Six samples prepared by spiking test preparation (Lenalidomide 5.0mg Capsules) with Lenalidomide Impurity-B of 0.5%, on test solution (250ppm) were analyzed as per test method.A study to establish stability of lenalidomide standard and test preparation on bench top was conducted over period of two days.Lenalidomide test preparation with target concentration spiking is injected at initial, 1 day and 2 days.

Robustness
The experimental conditions were purposely altered 1,9 for the determination of robustness of method as follows: A study was conducted to determine the effect of variation in organic phase composition in mobile phase (Mobile phase-B).Standard solution prepared as per the test method was injected into the HPLC system using two mobile phases.The system suitability parameters were evaluated within the limits of mobile phase-B having 90% and 110% (Acetonitrile) of method organic phase.A flow rates was changed 0.8 mL/min and 1.2 mL/min and were evaluated the system suitability parameters at column temperature changed from 30 o C to 45 o C. The pH of standard and sample solutions was changed from 2.2 to 1.8 to fixing the pH of the system.A study was conducted to determine the effect of variation in Initial Gradient composition in mobile phase in the range of +/-5%.

Optimization of Chromatographic Conditions
The optimisation of chromatographic conditions were evaluated by studying various HPLC columns with various mobile phases containing phosphate, sulphate, acetate buffers at pH 7-10 by using various organic solvents such as acetonitile, methanol, and ethanol and finally conclude that the optimum condition for the separation of lenalidomide and Imp-B from capsules, as described above under section chromatographic conditions.Under these evaluated conditions, lenalidomide and Imp-B were well separated from each other and other impurities to be present.

Results of System Suitability and Precision
The system suitability and precision were conducted as described above under system suitability and precision.The system suitability parameters were evaluated from standard chromatograms and found to be within the limits.Calculated the % RSD (0.8%) from six replicate injections for lenalidomide peak area and was found to be within the limits (The acceptable % of relative standard deviation of peak areas of lenalidomide should not be more than 10%), the area ratio between two peaks is within the limit.

Results of Specificity and Degradation
The specificity and degradation of lenolidomide and Imp-B in capsules was conducted using placebo standard and stressed condition such as acid, base hydrolysis, hydrogen peroxide, UV light, and heat as described in experimental section and the results shown in Figure 2. The placebo standard peak does not interfere with the retention peaks of lenolidomide and its impurity-B in capsules.The results found that under degradation condition the lenalidomide peak is homogeneous and the purity angle of lenalidomide was less than its threshold angle (Table 1) was indicates that method was more specificity and stability for the separation of lenalidomide in capsules.However, slight degradation of lenalidomide was observed in presence of 0.

Precision and Accuracy of Test Method
The precision and accuracy of the method was tested by using lenalidomide impurity-B and the results of accuracy were shown in Table 2.The reproducibility of lenalidomide impurity-B determination and its % RSD, 2.7% indicates the method was more precise.The mean recovery of Imp-B was in the range 93.6% to 99.4% in Table 2 indicates the more accuracy of the developed method.

Linearity of the Method
The linearity of the method was checked using Imp-B and the results shown in Figure 3.The correlation coefficient, 0.999 and slope of the curve, 0.978 were indicates the linearity of the test method.

Limit of Detection and Quantification
Limit of detection and quantification were calculated from signal to noise ratio of lenalidomide and Imp-B chramatograms.From the results the detection limits, 3.61 and 3.82 for lenalidomide and Imp-B respectively.The quantification limits, 10.36 and 9.52 for lenalidomide and Imp-B respectively, which were more precise and accuracy.

Ruggedness of the Method and Solution Stability
The ruggedness of the method was checked using Imp-B by means of analyst to analyst/system to system/column to column variability and bench top stability of standard preparations.The % RSD of lenalidomide Impurity-B from the six sample preparations was less than 2.7% and more than 1.3% obtained from both the systems, analysts and columns variation.The difference in % of known individual related compounds and % total impurities from initial to 48 hours is within the limits.Lenalidomide standard was injected at initial, 1 day and 2 days and the difference of the standard over period of one day was found to be stable.Confirms the stand and test solution were stable during this assay of 2 days period.Comparison of both the results obtained on two different HPLC systems, different column and different analysts shows that the related substances test method is rugged for system to system/analyst to analyst/column to column variability.

Robustness of the Method
The robustness of the method was checked by deliberately varied chromatographic conditions, such as mobile phase-B composition, initial gradient organic composition, flow rate, column temperature, and pH of the buffer no significant change in assay value was observed, which were confirms the robustness of the method.
Detector response, Area

Conclusion
The developed HPLC test method for the determination lenalidomide and related substances in capsules is more precise, accurate, and selective with good system suitability, ruggedness (analyst to analyst, system to system and column to column variability, bench top/refrigerator stability), and robustness (mobile phase composition variation, variation in initial gradient, flow rate variation, temperature variation) by satisfactory results.This method can be successfully used for determination lenalidomide and its related substances in capsules.

Figure 1 .
Figure 1.Chemical structures of lenalidomide and its impurity.

Table 1 .
1 N NaOH at 60 o C. Summary of the degradation results.
Figure 2. Typical purity and HPLC chromatograms of lenalidomide capsules and various stressed samples.