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ORIENTAL JOURNAL OF CHEMISTRY
www.orientjchem.org
An International Open Access, Peer Reviewed Research Journal
ISSN: 0970-020 X
CODEN: OJCHEG
2019, Vol. 35, No.(1):
Pg. 140-149
This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC- BY).
Published by Oriental Scientific Publishing Company © 2018
Development and Validation of Stability-Indicating RP-HPLC
Method for the Estimation of Lenalidomide and its Impurities
in Oral Solid Dosage Form
SOMANA SIVA PRASAD1, G.V. KRISHNA MOHAN*2 and A. NAGA BABU1
1,2Department of Chemistry, Koneru Lakshmaiah Education Foundation,
Vaddeswaram, Guntur District, Andhra Pradesh-522502, India.
*Corresponding author E-mail: drkrishnamohangv@kluniversity.in
http://dx.doi.org/10.13005/ojc/350115
Received: November 24, 2018; Accepted: January 12, 2018)
ABSTRACT
In this study, a novel, simple and precise RP-HPLC method has been developed for the
quantitative analysis of Lenalidomide (LLM) in pharmaceutical formulations using analytical quality by
design approach. An X-bridge-C18 column (150 mm × 4.6 mm × 3.5 µ) with mobile phases containing
a Potassium dihydrogen orthophosphate anhydrous buffer and methanol in the ratio of (90:10 v/v)
and (35:65 v/v) are used for the estimation of LLM and its degradation products. The flow rate of
0.8 mL/min is maintained and all degradation studies are performed at 210 nm using photodiode array
(PDA) detector. Method Validation is carried out according to International Council for Harmonisation
(ICH) guidelines and the parameters namely; precision, accuracy, specificity, stability, robustness,
linearity, limit of quantitation (LOQ) and limit of detection (LOD) are evaluated. The present developed
RP-HPLC method shows the purity angle of peaks is less than their threshold angle, signifying that
it to be suitable for stability studies. Hence, the developed method can be used for the successful
separation of LLM and its impurities in the pharmaceutical dosage formulations.
Keywords: RP-HPLC; Lenalidomide, Degradation products, ICH guidelines, Method validation.
INTRODUCTION
Lenalidomide (Revlimid®) is an immune
modulatory drug with an extraordinary medical activity
in the treatment of Myelodysplastic Syndrome (group
of cancers) patients1-3. It is a structural analogue of
thalidomide but has an enhanced side effect profile
and high immunomodulatory activity than its native
composite thalidomide4,5. Lenalidomide (LLM) is
chemically designated as 3-(4-amino-1-oxo 1,3-
dihydro-2-H-isoindole-2-yl) piperidine-2,6-dione with
the molecular formula and molar mass of C13H13N3O3
and 259.261 g/mol respectively. In 2006, LLM has
granted approval by US-FDA for the treatment
of patients with multiple myeloma, transfusion-
dependent anaemia and myelodysplastic syndromes
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in combination with dexamethasone 6. Further, it has
been employed to clinical trials for the treatment of
advanced cancers such as blood cancers: Hodgkin’s
lymphoma and non-Hodgkin’s lymphoma, bone
marrow cancer: Chronic lymphocytic leukaemia and
solid tumour cancers: Carcinoma and Pancreas7,8.
Basically, LLM increases the functional capacity of
the T-cells and inhibits the angiogenesis in vitro in
human systems9. The recommended dose of LLM
is 10.0 mg for daily but it can be abridged to 5.0 mg
all other days if neutropenia or thrombocytopenia
occurs. Hence, there is an increasing demand for
appropriate analytical technologies to ensure the
quality of LLM formulations.
In the recent past, different analytical
techniques have been developed with various
detectors in the pharmaceutical analysis10. Few
analytical methods are reported in the literature
for the estimation of LLM and it impurities by using
HPLC, HPLC assay and LC-MS methods1,11-13. These
methods are associated with some major drawbacks
such as lack of selectivity and the methods doesn’t
deal with the forced degradation studies. Moreover,
some of the related substance methods are found
highly pH sensitive due to the buffer solution which
is used in the mobile phase.
Hence, to overcome this problem, attempted
to validate and develop a simple, precise, sensitive
and stability indicating RP- HPLC method for
determination of Lenalidomide related substance
with the inclusion of another lenalidomi
de Impurity-III by using Qbd approach to overcome
this risk-sensitive to pH of buffer solution. The method
is comprehensively validated as per the guidelines of
International Conference on Harmonisation (ICH)14-16.
The molecular structure of the LLM is presented in
the Figure 1.
MATERIALS AND METHODS
Instruments and chemical reagents
Waters High-performance liquid
chromatography system (HPLC) with Photodiode
Array (PDA) detector was used throughout the
analysis. This can be used for in-vitro diagnostic
testing to analyze many compounds, including
diagnostic indicators and therapeutically monitored
compounds. The minimum of five pure standards
were employed to calibration of the instrument
and the concentration range has been covered the
entire range of typical specimens, quality-control
samples and atypical specimens. Waters Empower
Networking Software was employed for obtaining the
chromatographic data and the electronic analytical
balance (Thermo Fisher Scientific, Hyderabad,
India) was used for weighing purposes. Standard
Lenalidomide (purity, ≥99.9%) and all impurities
were acquired from Dr. Reddy’s Laboratories Ltd.,
Hyderabad, India. Anhydrous potassium dihydrogen
orthophosphate and orthophosphoric acid were used
for the preparation of buffer solutions and methanol
and acetonitrile were HPLC and analytical grade
chemicals purchased from Merck KGaA (Darmstadt,
Germany). High purified Millipore water from MilliQ
water system was used.
Chromatographic conditions
Waters HPLC instrument with PDA detector
was used for the method development and validation
of the samples at 210 nm. A stationary phase was
developed with the C18 column (4.6 mm internal
diameter, 150 mm length and 3.5µ particle size) to
separate the impurities of LLM. Two mobile phases A
and B were used for the rapid separation of impurities.
The temperature of the column was kept at 27oC
and gradient flow mode was sustained throughout
the analysis. The 10.0 µL volume of the sample was
injected into the system per run by maintaining the
flow rate of mobile phase 0.8 mL/min. The diluents
were prepared by mixing the Buffer and methanol
solutions in the ratio 90:10 and 35:65 for extraction
of LLM and its impurities from formulation matrix.
Preparation of solutions
Mobile phase- A & B
Buffer and methanol solutions were mixed
in the ratio of 90:10 v/v and 35:65 v/v to prepare the
mobile phase-A and mobile phase-B respectively. The
concentration of buffer solution in mobile phase-A
Fig.1. Lenalidomide structure
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is more than the concentration of buffer solution in
mobile phase-B to separate all the impurities and
the concentration of the organic solution in mobile
phase-B is high than mobile phase-A to elude all
impurities.
Standard LLM solution
Accurately weighed about 20 mg of LLM
was transferred into a 100 mL volumetric flask and
70 mL of diluents was added to the flask. Then, the
volumetric flask was sonicated until the complete
digestion of the drug and make up the volume with
diluent. The prepared stock solution of 5.0 mL was
taken into a 50 mL volumetric flask and makeup to
the volume with the same diluent.
Preparation of sample solution
About 25 mg of Lenalidomide tablet powder
was taken into a 50 mL volumetric flask, and then
35 mL of diluents was added, sonicated for 15 min
at 4000 RPM and makeup with the prepared diluent.
Centrifuged and filtered the sample and filtrate were
used to inject into the HPLC system.
Preparation of impurity stock solution
1.0 mg of each impurity-I, impurity-II and
impurity-III standards were accurately weighed and
transferred to a 20 mL volumetric flask. Then the
each impurity was dissolved with diluent and the
solution was makeup to the mark with diluent.
Preparation of spiked sample
Accurately weighed 25 mg of LLM tablet
powder was transferred into the 50 mL volumetric
flask and 35 ml of the diluent was added to the
flask. Then, 1.0 mL of stock impurity solution was
added. The resulting solution was sonicated until the
complete drug digestion, make up the volume with
diluent and mix well. Centrifuge the above solution
for 5 more minutes at 4000 RPM.
Specificity
The LLM and its impurities were injected
into the optimized system to demonstrate the
specificity of the developed method in the formulation
of LLM (ICH 2005). The effectual separation of
known impurities and degradants of LLM peak by
forced degradation studies of LLM tablets were
conducted at different stress conditions like acid (0.5
N HCl), base (0.2 N NaOH), peroxide (30% H2O2),
water (80oC on water bath), thermal (80oC in hot
air oven), humidity (90% RH in humidity chamber)
and photolytic stress (photostability chamber at
200 watt-hour/square meter).
RESULTS AND DISCUSSION
Optimization of chromatographic conditions
Several mobile phase compositions and
different stationary phases were investigated in the
preliminary studies to get the best resolution between
LLM and its impurities. All analytes have the different
retention behaviours and hence it is a challenging
development to separate all analytes in the shorter
method without interfering Placebo components
and degradation impurities. Based on the Design
of Experiments (DOE), the final chromatographic
conditions were optimized which gives a powerful
suite for a statistical methodology and the obtained
findings are presented in Table 1 and Table 2. The
DOE was performed using fractional design by in
view of the pH of the buffer in mobile phase-A, flow
rate, the percentage of methanol in mobile phases-
A and B and resolution between the close eluting
impurities (Impurity-I&II) as responses.
Table 1: CMP, CQA, and QTMP of LLM related substance stability-indicating analysis method
CMP Range of each parameters used for DOE QTMP (Quality Target CQA(Critical
(Critical Method Method Profile) Quality Attribute)
Parameter) Low As such High Targeted QTMP
A) pH of the buffer in mobile phase -A 3.4 3.6 3.8 Resolution b/w Resolution b/w
B) % Methanol in mobile phase-B 60 65 70 Impurity-I&II Impurity-I&II
C) % Methanol in mobile phase-A 5 10 15 not less than-1.5
D) pH of the buffer in mobile phase -B 3.4 3.6 3.8
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Table 2. Design of Experiments runs – LLM related substance method
Std Run Canter pH of %Methanol %Methanol pH of Resolution b/w
Order Order Point M.P-A in M.P-B in M.P-A M.P-B Impurity-I&II
3 1 1 3.4 70 5 3.8 2.2
2 2 1 3.8 60 5 3.8 4.2
9 3 0 3.6 65 10 3.6 3.8
1 4 1 3.4 60 5 3.4 2.1
6 5 1 3.8 60 15 3.4 3.5
5 6 1 3.4 60 15 3.8 2.5
7 7 1 3.4 70 15 3.4 1.8
8 8 1 3.8 70 15 3.8 2.1
4 9 1 3.8 70 5 3.4 3.9
The main effect chart for the resolution
between LLM Impurity-I&II is presented in the
Fig. 2 and further, the interaction plot for the
resolution between LLM Impurity-I&II is presented in
the Fig. 3. Minitab software was used for evaluating
the effects of factors on resolutions and to generate
the Pareto chart with three-dimensional plots. pH of
the buffer solution in mobile phase-A and percentage
of methanol in mobile phase-B plays a major role in
the separation of impurities. Further, the acquired
data was employed for setting the upper and lower
boundaries for all variables. Moreover, the design
space was demonstrated and experimentally
proposed values were nearer to the suggested
parameters. Interactive effects were carried out
from various overlay graphs plotted between two
parameters at a time using visual inspections and
modelled data. The parameters of the developed
Table 3: Optimized HPLC method conditions
Column X-Bridge-C18 150 x 4.6 mm, 3.5µm
Flow rate 0.8 mL /min
Column oven temperature 27 °C
Wave length 210 nm
Injection Volume 10 µL
Run time 65 minutes
Time (min) % Mobile Phase-A % Mobile Phase-B
0 100 0
5 100 0
35 60 40
Gradient program 45 50 50
58 50 50
58.1 100 0
65 100 0
and validated HPLC method are presented in
Table 3. The Pareto chart for standardized effects
on the resolution between LLM Impurity-I&II is
presented in the Fig. 4 and the contour plot for the
resolution between Acetyl and Diacryloyl impurities
are presented in the Figure 5.
Based on the recovery and shape of
the peak, the diluents were finalized, and test
concentrations and injection volumes were optimized
to contain greater reporting threshold than the limit of
quantification (LOQ). The gradient was optimized to
get the best resolution among main analyte and all
impurities. Fig. 6. presents the typical chromatogram
of standard solution, Fig. 7. Presents the typical
chromatogram of impurity spiked chromatogram
and furthermore, the Fig. 8. presents the typical
chromatogram of spiked test chromatogram.
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Fig. 2. The main effect chart for the resolution between LLM Impurity-I&II
Fig. 3. The interaction plot for the resolution between LLM Impurity-I&II
Fig. 4. The Pareto chart for standardized effects on the resolution between LLM Impurity-I&II
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Fig. 5. The contour plot for the resolution between Acetyl and Diacryloyl impurities
Fig. 6. Typical chromatogram of standard solution
Fig. 7. Typical chromatogram of impurity Spiked
chromatogram
System suitability
The standard level of the LLM test solution
was introduced into HPLC system and found that
system suitability parameters are within the limits.
The percentage of relative standard deviation (RSD)
was calculated for peak areas and USP plate count.
The repeated injection of RSD percentage was
observed as 0.2%, where the acceptance criteria
were not more than 10.0%. The obtained results are
presented in Table 4.
Fig. 8. Typical chromatogram of spiked test chromatogram
Specificity
Placebo interference
A study was conducted to establish the
placebo interference. As per the test method, samples
were prepared by taking the placebo, equivalent to
the weight in a portion of test preparation and then
injected into HPLC system. Interference was not
found for the chromatograms of placebo solution,
empty cell solution and impurities solution at the
retention time of LLM and its impurities. The obtained
chromatogram is presented in Figure 9.
Interference from degradation products
The samples were subjected to various
stress circumstances for the efficient separation
of all degradants from forced degradation of LLM.
Separate portions of LLM capsules were exposed
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Table 4: System suitability results table
System suitability parameters Observed Value Acceptance criteria
The relative standard deviation of peak areas of 0.2 Not more than 10.0%
Lenalidomide from three replicate injections of
standard solution
The Tailing factor for Lenalidomide peak from Standard 1.1 Not more than 1.5
The number of theoretical plates for Lenalidomide peak 23503 Not less than 2500
to following stress conditions to induce degradation
and the detailed findings are depicted in Table 5.
a) Acid Degradation- Kept on a water bath at
80°C for 16 h with 0.5N HCl.
b) Base Degradation- Kept on bench top for
15 min with 0.2N NaOH.
c) Peroxide Degradation - Kept on the bench top
in dark for 14 h with 30% H2O2 solution.
d) Water Degradation- Kept on a water bath at
80°C for 16 hours.
e) Thermal Degradation - Kept in a hot-air oven
at 80oC for 7 days.
f) Humidity Degradation - Kept in Humidity
chamber at 90% RH for 7 days at 25°C.
g) Photolytic Degradation - Exposed to 200-watt
h/m2 and 1.2 million lux hours in photostability
chamber for 16 hours.
To find the purity of the main analyte and
impurity peaks, the all stressed samples were
subjected to HPLC system with PDA detector.
Chromatograms of the stressed samples were
evaluated for peak purity of LLM and all impurities
using Waters Empower Networking Software. Impurity
degradant peaks in chromatograms of all stressed
samples and LLM were resolved. The purity angle
was found fewer than the purity threshold for the all
forced degradation samples. This shows that there is
no interference and co-elution from degradants in the
quantification of impurity in the drug product. Hence,
this method is "Stability Indicating" and extremely
specific. The assay and mass balance of degradation
samples are presented in Table 6.
Fig. 9. Typical chromatogram of Alkali stress chromatogram
Table 5: Interference from degradation Products
S. Stress condition % Net degradation (% imp Purity angle Purity threshold Purity flag
No. of stressed sample - % imp (yes/no)
of unstressed sample)
1. Unstressed sample 0.1929 4.205 10..055 No
2. Kept on water bath at 80°C for 16Hrs with 0.5N HCl 12.0141 2.487 8.536 No
3. Kept on bench top for 15min with 0.2N NaOH 3.4424 4.178 10.643 No
4. Kept on bench top in dark for 14 h with 30% H2O2 solution. 11.2247 2.642 8.721 No
5. Kept on water bath at 80°C for 16 hours. 24.0716 1.867 7.354 No
6. Kept in hot-air oven at 80° for 7 days. 0.1879 4.321 11.363 No
7. Kept in Humidity chamber at 90% RH for 7 days at 25°C. 0.1924 4.069 12.375 No
8. Exposed to 200-watt hour/m2 and 1.2 million lux hours in 4.497 12.969 No
Photo stability chamber for 16 h 0.2730
Linearity and range
The linearity was examined between the
series of 0.2 mg/L to 3.4 mg/L for the main analyte
and all impurities. The prepared six dissimilar linearity
solutions were injected into the HPLC system, and
the obtained findings are presented in Table 7.
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Table 6: Mass Balance of degradation sample
S. Stress condition % Degradation % Assay Mass balance
No.
1. Unstressed sample 0.1929 100.00 100.19
2. Kept on water bath at 80°C for 16 h with 0.5N HCl 12.0141 86.44 98.46
3. Kept on bench top for 15 min with 0.2N NaOH 3.4424 95.87 99.31
4. Kept on bench top in dark for 14 h with 30% H2O2 solution. 11.2247 84.33 95.55
5. Kept on water bath at 80°C for 16 hours. 24.0716 79.90 103.98
6. Kept in hot-air oven at 80° for 7 days. 0.1879 100.87 101.05
7. Kept in Humidity chamber at 90% RH for 7 days at 25°C. 0.1924 100.76 100.96
8. Exposed to 200-watt hour/m2 and 1.2 million lux hours in 0.2730 100.00 102.00
Photo stability chamber for 16 hours.
Table 7: Precision of the LLM impurities
S.No % Impurity-I % Impurity-II % Impurity-III
1 0.1749 0.2030 0.2407
2 0.1766 0.2040 0.2383
3 0.1766 0.2058 0.2396
Average 0.1764 0.2043 0.2395
%RSD 0.77 0.69 0.50
Limit of detection (LOD) and limit of quantitation
(LOQ)
In order to examine the LOD and LOQ,
dissimilar concentrations of solutions were prepared by
spiking known amounts of impurities and spiked LLM
in the diluent. The signal-to-noise (S/N) approach was
used to determine the detection limits and quantitation
limits and the average S/N ratio was used for calculating
the all analyses at each concentration level. The
concentration which gives S/N 3 can be readily detected
was reported as the LOD. The slope method was used
for estimation of LOD and LOQ and the equations
used are LOQ=10×σ/S and LOD=3.3×σ/S, where,
S is the calibration curve slope, and σ is the standard
deviation of the response. The observed values are
presented in Table 8.
Table 8: LOQ values of LLM impurities
S.No Spike level Impurity-I Impurity-II Impurity-III
% Recovery Mean % Recovery % Recovery Mean % Recovery % Recovery Mean % Recovery
1 Limit of 94.7 93.9 102.0 102.0 102.6 101.9
2 Quantification 92.4 102.0 102.6
3 level 94.7 102.0 100.4
1
100% level 91.1 92.6 104.0 104.7 104.4 103.6
2 93.0 104.6 103.0
3 93.0 105.6 103.5
1
150% level 98.1 97.9 106.6 106.6 101.9 102.1
2 98.3 106.7 102.7
3 97.2 106.6 101.8
Stability
The stability of the LLM and its impurities
in the spiked sample was examined at room
temperature for 96 hours. All the spiked samples
were kept in the air-tight volumetric flask on
bench-top for observing the stability of the samples
and found that all prepared samples are stable up
to 96 hours.
Accuracy and Precision
Six samples were prepared at 0.2% of
the targeted test solution by spiking the impurities.
The obtained chromatogram is presented in
Fig. 9. Recovery studies were carried out for the
LLM and its impurities, and the values were obtained
between 92.6-106.6 %. The linearity of detector
response was calculated for three impurities along
with LLM and the obtained values were presented
in Table 9. The accuracy was calculated as %bias
(divergence between measured concentrations
and nominal concentrations), and the precision
was calculated within the intra-day (single run) and
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inter-day (different runs). The ensuing percentage of
Relative Standard Deviation (RSD) values of LLM
impurities were observed below 2.0 (n=6). Therefore,
the method is precise and accurate.
Table 9: Linearity of Detector Response of LLM impurity-I
S.No Concentration in mg/L Area Response Slope (m) Intercept (C) Correlation Coefficient (R) Bias at 100% Response
1 0.26 11037 42811.464 24.881 1.0000 0.02
2 0.52 22108
3 1.55 66822
4 2.58 110520
5 3.61 154393
6 5.15 220502
Impurity-II
1 0.25 10717 41432.997 649.190 1.0000 0.62
2 0.50 21272
3 1.51 63698
4 2.51 104810
5 3.51 145877
6 5.02 208580
Impurity-III
1 0.25 18930 74135.872 553.407 1.0000 0.29
2 0.51 37796
3 1.53 114761
4 2.54 189152
5 3.56 264368
6 5.09 377665
CONCLUSION
The RP-HPLC method developed in this
study for the analysis of Lenalidomide (LLM) is
simple, precise, accurate, selective and economical.
The method was found to be robust within the
defined design space. Samples are subjected to
different forced degradation studies and found that
impurity degradant peaks in chromatograms of all
stressed samples and LLM are resolved. The %RSD
for all parameters was found to be within the limit.
The validated method shows the satisfactory data for
all tested parameters. Thus, this developed method
can be used for the purpose of quality control in the
pharmaceutical dosage formulations.
ACKNOWLEDGEMENT
The author thanks the management of
Dr. Reddy’s Laboratories Ltd. for their valuable
support to carry out and publish the work.
Conflicts of interests
The authors declare that there is no conflict
of interests regarding to the publication of the paper.
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