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RP-HPLC as an
analytical method for
the determination of
bendamustine and its
related impurities in
bulk drug
6.1 Introduction
Bendamustine hydrochloride (BMH) chemically known as 4-{5-[bis-(2-chloroethyl) amino]-
1-methyl- 1Hbenzimidazol-2-yl} butanoic acid, is an active nitrogen mustard [1]. The IUPAC
names of its impurities were impurity-A 4-6-[2-chloro ethyl)-2-hydroxy ethyl amino)3 methyl-
benzimidazolyl-2)-butyric acid (HP-1), impurity-B 4-(-5-[(4-5-(bis(2-hydroxyethyl)amino)-1-
methyl-1H-benzo[d]imidazol-2-yl)butanoic acid and impurity-C isopropyl 4-(5-(bis(2-
chloroethyl)amino-1-methyl-1-H-benzol[d]imidazol-2yl)butanoate. It was used for the treatment
of patients with chronic lymphocytic leukemia [2]. It contains mechlorethamine group and
benzimidazole heterocyclic ring with a butyric acid substituent. Mechlorethamine and its
derivatives form electrophilic alkyl groups. These groups form covalent bonds with electron-rich
nucleophilic moieties, resulting in interstrand DNA cross links. The bi functional covalent
linkage can lead to cell death via several pathways [3]. It was active against both quiescent and
dividing cells [4-7]. It was approved by the US food and drug administration (FDA) for chronic
lymphocytic leukaemia in march 2008 [8, 9].The chemical structure of bendamustine shown in
the Figure-6.1.
Figure-6.1: Bendamustine hydrochloride
Its empirical molecular formula was C16H21Cl2N3O2•HC1 and the molecular weight 394.7.
It was first synthesized in 1963 by Ozegowski and Krebs in East Germany (the former German
Democratic Republic). Until 1990 it was available only in East Germany. The German
investigators found that it was useful for treating chronic lymphocytic leukemia, Hodgkin’s
disease, non-Hodgkin’s lymphoma, multiple myeloma and lung cancer.
Bendamustine was marketed under the trade name Ribomustin by Astellas Pharma. It was
used alone or in combination with other anti cancer agents for indolent non-hodgkin's
lymphoma, multiple myeloma, and chronic lymphocytic leukemia. SymBio Pharmaceuticals Ltd
holds exclusive rights to develop and market bendamustine in Japan and in selected Asia Pacific
countries.
In March 2008,Cephalon received approval from the United States Food and Drug
Administration to market bendamustine in the US, where it sold under the trade name treanda for
the treatment of chronic lymphocytic leukemia[10].
In October 2008, the FDA granted further approval to market treanda for the treatment of
indolent B-cell non-Hodgkin's lymphoma that has progressed during or within six months of
treatment with rituximab or a rituximab-containing regimen [11]. The pH of the reconstituted
solution was 2.5 - 3.5. Each 25-mg vial contains 25 mg of bendamustine hydrochloride and 42.5
mg of mannitol. Each 100-mg vial contains 100 mg of bendamustine hydrochloride and 170 mg
of mannitol. It was supplied as a sterile non-pyrogenic white to off-white lyophilized powder in a
single use vial.
Figure-6.2: Bendamustine hydrochloride vial 100mg
Bendamustine is water soluble microcrystalline powder with amphoteric properties. It acts
as an alkylating agent causing intra-strand and inter-strand cross-links between DNA bases.
After intravenous infusion, it is extensively metabolised in the liver by cytochrome p450. More
than 95% of the drug is bound to protein. Only free bendamustine is active. Its elimination is
biphasic with a half-life of 6–10 minutes and a terminal half-life of approximately 30 minutes. It
is eliminated primarily through the kidneys.
Figure-6.3: Clinical mechanism of bendamustine hydrochloride with DNA
In the available literature, many analytical procedures have been reported for the
quantitative determination of bendamustine in pure form as well as in pharmaceutical dosage
formulation by different analytical techniques like high performance liquid chromatography [13-
16], and spectrophotometric methods [17-18]. Annapurna et.al proposed a stability-indicating
liquid chromatographic method for the determination of bendamustine hydrochloride in
parenterals. Reversed phase chromatography was performed using C18 (250 mm × 4.6 mm, 5 µm
particle size) column with acetonitrile: tetra butyl ammonium hydrogen sulphate (80:20, V/V) as
mobile phase at a flow rate of 0.8 mL/min. with UV detection at 233nm [14]. Pavani priya et.al
developed stability indicating RP-HPLC method for the determination of bendamustine in
parenterals by using Agilent Zorbax poroshell C18 RP column. The mobile phase of water and
acetonitrile (with 0.01% TFA), mixed in the ratio of 50:50 (pH 6.9-7.2) was used in this study
[15]. A simple, rapid and sensitive spectrophotometric method was developed for the
determination of bendamustine hydrochloride in phosphate buffer (pH 9.0) by Mathrusri
Annapurna et.al. [18]. Some HPLC methods were also reported in literature for the determination
of bendamustine [19-31].
Though large numbers of assay methods are available in literature for bendamustine, only
very few of them are standard, sensitive and selective. In view of the importance of
bendamustine in drug formulation in the treatment of chronic lymphocytic leukemia, a more
simple, sensitive, selective and robust method is needed for its validation in bulk drug
formulations. All the reported methods were used for the determination of bendamustine in bulk
and formulations but no method was reported for the determination of bendamustine and its
related impurities. We are now reporting a simple sensitive and selective RP-HPLC method for
the validation of bendamustine and its related impurities which is robust and rugged method.
6.2 Experimental:
6.2.1 Chemicals, reagents and samples:
The standard, samples of bendamustine and known related impurities such as impurity-A,
impurity-B and impurity–C were received from Bio-Leo Labs India (P) Ltd., Hyderabad. HPLC
grade methanol, acetonitrile and trifluroacetic acid were purchased from Merck, Mumbai, India.
High purity water was prepared by using Millipore Milli-Q plus water purification system. The
purity of all samples and impurities used in this study was greater than 99%.
6.2.2 Instrumentation
For initial method development studies Waters prominence HPLC system was employed.
This was equipped with a quaternary UFLC LC-20AD pump, DGU-20A5 degasser, SPD-M20A
diode array detector,SIL-20AC auto sampler,CTO-20AC column oven and CBM-20A
communications bus module. Agilent 1200 series with high pressure liquid chromatographic
instrument provided with Auto sampler and VWD UV detector with thermostatted column
compartment connected with EZ Chrom software was employed for the validation of the drug
and its related impurities. The analysis was carried out on Zorbax SB-C18, 4.6 mm x 25 cm
column with 5µm particle size.
6.2.3 Standard and sample solutions:
6.2.3.1. Preparation of standard solution:
20.0 mg of bendamustine hydrochloride standard were accurately weighed and transferred
into a 20 ml Volumetric flask dissolved and diluted to the Volume with methanol. 2.0 ml of this
solution was further diluted to 100 ml with methanol. The resultant solution was filtered through
0.45 micron filter.
6.2.3.2 Sample preparation:
25.0 mg of bendamustine hydrochloride sample were weighed into a 25 ml Volumetric
flask dissolved in methanol and diluted to the Volume with methanol. The resultant solution was
filtered through 0.45 micron filter.
6.3 Evaluation of system suitability:
The system suitability was evaluated by injecting a known Volume of sample containing a
known amount of bendamustine into chromatograph and calculated the number of theoretical
plates and asymmetry of the chromatogram. The number of theoretical plates was found to be
greater than 3000 and the tailing factor of bendamustine was calculated and found to be more
than 2.0 which showed that the selected column is suitable for the analysis.
6.4 Results and discussion:
6.4.1 Method development and optimization:
The method development was initiated with the solubility study of bendamustine. Based on
the solubility studies, methanol was chosen as solvent for the preparation of sample solutions.
Bendamustine is polar in nature due to the presence of one –COOH group. Hence, non polar
silica based C18 was selected for developing reverse phase high performance liquid
chromatogram. From the molecular structure, it was observed that, there is chromophore group
present in bendamustine. Hence there is possibility for its UV–Visible detection. The UV
experiment was performed on bendamustine, which showed maximum absorbance at 230nm.
To arrive at the optimal chromatographic conditions suitable for the validation of
bendamustine and its related impurities, various trail chromatograms were recorded with
different conditions.
Trail-1
The first trail method was performed by using isocratic mode and mobile phase 0.1%
trifluroacetic acid in water and acetonitrile in the ratio 90:10v/v as the mobile phase. The
injection Volume was 10µL.
Column : Zorbax SB-C18,(4.6 mm x 25 cm) 5µm particle size
Pump mode : Isocratic
Flow rate : 1.0 ml/min
Detection wavelength : UV , 230 nm
Injection Volume : 10µL
Column temperature : 300C
Run time : 60 min
Mobile phase : 0.1% Trifluroacetic acid in water: acetonitrile (90:10v/v)
Diluent : Methanol
In this trail there was no elution of impurity peaks up to a run time of 60 minutes and asymmetry
of bendamustine peak was more than 2.0
Trail-2
To over the limitations of the above trail method, the experiment was repeated by changing
the mode of mobile phase, composition and isocratic mode to gradient mode, keeping the
remaining conditions constant. The mobile at present is a gradient mixture of mobile phase-A
(80:20v/v) and mobile phase-B (60:40v/v).
Column : Zorbax SB-C18,(4.6 mm x 25 cm) 5µm particle size
Pump mode : Gradient
Flow rate : 1.0 ml/min
Detection wavelength : UV , 230 nm
Injection Volume : 10µL
Column temperature : 300C
Run time : 60 min
Mobile phase A : 0.1% Trifluroacetic acid in Water: Acetonitrile (80:20v/v)
Mobile phase B : 0.1% Trifluroacetic acid in Water: Acetonitrile (60:40v/v)
Diluent : Methanol
In this trail there was elution of bendamustine and its known impurities but peak shapes of
bendamustine and its impurities were not symmetrical.
Trail -3
To achieve the peak symmetries for bendamustine and its impurities, both the mobile phase
compositions were changed as follows and the experiment was repeated under the optimal
conditions.
Column : Zorbax SB-C18,(4.6 mm x 25 cm) 5µm particle size
Pump mode : Gradient
Flow rate : 1.0 ml/min
Detection wavelength : UV , 230 nm
Injection Volume : 10µL
Column temperature : 300C
Run time : 60 min
Mobile phase A : 0.1% Trifluroacetic acid in Water: Acetonitrile (90:10v/v)
Mobile phase B : 0.1% Trifluroacetic acid in Water: Acetonitrile (50:50v/v)
Diluent : Methanol
In this trail bendamustine peak was eluted at 24 minutes and the impurities-A, B and C of
bendamustine were well separated with main bendamustine peak with retention times of 15, 30
and 38 minutes and all the peaks were obtained with excellent symmetry.
Finally, satisfactory separation with better peak shape was achieved by employing the
optimal conditions of trail-III within a reasonable retention time with gradient mode with a flow
rate of 1.0mL/min at temperature 300C.
6.5 Method validation:
In order to determine bendamustine and its related impurities, the method was validated as
per the ICH guidelines [32-33] individually in terms of system suitability, specificity, precision,
accuracy, linearity, robustness, limit of detection and limit of quantification (LOD and LOQ) and
solution stability.
6.5.1 System suitability test:
The system suitability was studied by injecting diluted blank one injection and diluted
standard six replicate injections. The RSD from six replicate injections of diluted standard
preparation was calculated. The system suitability results are given in Table-6.1
Table-6.1: Results of system suitability
System suitability parameters
Result
Acceptance criteria as per USP
Theoretical plates 778991 > 3000
Tailing factor 1.1 < 2.0
% RSD for six injections 0.9 < 5.0%
.
6.5.2 Specificity of method with related substances:
Specificity is the ability of the method to accurately measure the analyte response in the
presence of all potential sample components. The response of the analyte in test mixtures
containing the analyte and all potential components is compared with the response of a solution
containing only analyte. For specificity determination, solution containing diluent and all related
substances of bendamustine was prepared by mixing in suitable proportions. Then diluent,
standard preparation, sample preparation, sample spiked with impurities were injected into the
chromatograph and the peak homogeneity was verified for bendamustine and its related
substances using EZ Chrom software. The specificity experiment results are given in the Table-
6.2. The specificity chromatograms are shown in Figures-6.4
Table-6.2: Results of specificity experiment
S.No. Name of the impurity/analyte Peak purity Retention time
1 Bendamustine 1.00000 24.109
2 Impurity-A 1.00000 15.306
3 Impurity-B 1.00000 30.505
4 Impurity-C 1.00000 38.638
6.5.3 Precision:
6.5.3.1 System precision (Repeatability):
Six replicate aliquots of sample solution of bendamustine, spiked with impurities, were
injected into RP-HPLC system and the chromatograms were recorded for checking the
performance of the system under the chromatographic conditions on the day tested and
calculated the relative standard deviation. The RSD value obtained was 0.8%, which showed the
good precision of the system. The system precision results are shown in Table-6.3
Table-6.3: System precision of bendamustine hydrochloride
Injection No.
Bendamustine hydrochloride
Retention time Area response
1 24.833 88860
2 24.840 88250
3 24.841 87923
4 24.828 86902
5 24.824 87794
6 24.825 87359
Mean 24.832 87848
% RSD 0.0 0.8
6.5.3.2 Intermediate precision (Ruggedness):
The intermediate precision also called as ruggedness, is the inter-day variation. It is defined
as the degree of reproducibility obtained by following the same procedure as mentioned for
method precision experiment. The ruggedness of the test method was demonstrated by carrying
out precision study in six preparations of sample, a single batch sample by different analysts,
different columns on different days using different instruments and calculated the percentage of
impurities. The RSD for impurities from six spiked sample preparations were found to be 0.7 for
impurity-A, 0.8 for impurity-B and 0.8 for impurity-C showing high ruggedness of the proposed
method. The validated intermediate precision results are given in Table-6.4
Table-6.4: Results of intermediate precision
Injections Impurity- A Impurity -B Impurity -C
1 0.217 0.182 0.175
2 0.217 0.185 0.172
3 0.218 0.186 0.175
4 0.214 0.186 0.175
5 0.217 0.185 0.175
6 0.215 0.184 0.173
Mean 0.216 0.185 0.174
% RSD 0.7 0.8 0.8
6.5.4 Accuracy:
The accuracy of the proposed method was tested by preparing sample solutions with known
quantities of impurities-A, B and C at the level of LOQ, 50%, 100%, 150%, 200% and 300% of
target concentration (i.e., 1.0 % of test concentration).The chromatograms were recorded and the
recovery percentages were evaluated from the peak areas. The results are shown in Table-6.5
Table-6.5: Accuracy results of bendamustine hydrochloride and its impurities
Levels
Mean recovery (%)
Bendamustine Impurity-A Impurity-B Impurity-C RSD (%)
LOQ 101.5 99.7 101.0 99.9 0.9
50% 100.2 101.0 99.7 99.2 0.8
100% 101.5 99.9 101.0 99.7 0.9
150% 99.9 99.7 99.2 99.1 0.4
200% 101.5 99.2 98.8 99.2 1.5
300% 99.8 99.7 102.1 100.5 1.1
6.5.5 Linearity:
The concentration ranges in which bendamustine, its impurity-A, impurity-B and impurity-
C can be determined with good accuracy were evaluated by preparing calibration plots between
the concentration of the analyte and peak areas. Six different aliquots of standard solutions were
injected into the RP-HPLC chromatograph and the chromatograms were recorded. Peak areas of
the resultant chromatogram were plotted against the concentration of the analyte. The
experimental data obtained are shown in Table-6.6 and the resultant linear plots obtained for
these data are given in Figure-6.5.
Table 6.6: Results of linearity experiment of bendamustine and its related impurities
a) Bendamustine b) Impurity-A
Level Amount of
bendamustine(ppm)
Area
Response
1 0.0129 403
2 0.1932 7321
3 0.4895 18611
4 0.7729 30367
5 1.1723 48577
6 1.4814 61200
Correlation coefficient 0.9995
C) Impurity-B D) Impurity-C
The data was subjected to statistical analysis and the results of these analyses are presented in
table 6.6. The values of different statistical analyses like correlation coefficient intercept,
residual sum square and relative standard deviation confirm high precision and accuracy of the
proposed method.
6.5.6 Limit of detection and limit of quantification:
Level Amount of
impurity-A(ppm)
Area
Response
1 0.0195 616
2 0.1461 4482
3 0.2923 9140
4 0.7404 23477
5 1.169 38146
6 1.4807 48508
Correlation coefficient 0.9999
Level Amount of
impurity-C(ppm) Area Response
1 0.0193 1097
2 0.1451 5874
3 0.2902 11670
4 0.7351 27992
5 1.1607 44194
6 1.4702 55895
Correlation coefficient 1.0000
Level Amount of
impurity-B(ppm)
Area
Response
1 0.0196 858
2 0.1467 5410
3 0.2934 11388
4 0.7434 29078
5 1.1737 46234
6 1.4867 59331
Correlation coefficient 0.9999
Limit of quantification and limit of detection were established for bendamustine and its
related impurities based on slope ratio method. The detection of an individual analytical
procedure is the lowest amount of analyte in a sample, which can be detected not necessarily
quantitated as an exact value. The quantification limit of an individual analytical procedure was
the lowest amount of analyte in a sample which can be quantitatively determined with suitable
precision and accuracy. The LOD & LOQ results of bendamustine and its impurities were given
in the Table-6.7.
Table 6.7: LOQ and LOD results of bendamustine and its related impurities
S.No.
Analyte/impurity
LOD concentrations
(ppm)
LOQ concentrations
(ppm)
1. Bendamustine 0.092 0.306
2. Impurity-A 0.072 0.239
3. Impurity-B 0.127 0.383
4. Impurity-C 0.127 0.226
6.6 CONCLUSION
The liquid chromatographic method with gradient elution developed for determination of
bendamustine hydrochloride and its impurities A, B and C in the bulk drug, was fully validated
and proved to be reliable, sensitive, accurate and precise .The method has higher sensitivity
towards the determination of impurities and it is the first time that such method appears in the
literature and can be useful for routine analysis and quality control of bendamustine
hydrochloride in the relevant forms. The method can be used for quality assurance of
bendamustine hydrochloride in bulk drugs and can be extended to validate the pharmaceutical
formulations.
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