Stability Indicating HPTLC Method for Bedaquiline
Fumarate
M. C. Damle1*, O. A. Shinde2
1Head of Department, Pharmaceutical Quality Assurance,
AISSMS College of Pharmacy, Pune.
2Department of Pharmaceutical Quality Assurance, AISSMS
College of Pharmacy, Pune.
*Corresponding Author E-mail:
damle_mc@aissmscop.com;
omkarshinde31197@gmail.com
ABSTRACT:
Bedaquiline fumarate is an anti-TB treatment that was
approved in 2012 and has a novel mechanism of action for fighting multidrug
resistance (MDR). Bedaquiline fumarate, a diarylquinoline having significant
bactericidal and sterilising activity, has been licenced by the US Food and
Drug Administration,for the treatment of multidrug-resistant tuberculosis
(MDR-TB).A simple, sensitive and stability indicating High Performance Thin
Layer Chromatography method has been developed and validated for estimation of
Bedaquiline fumarate as per ICH guidelines. Method was developed with silica
gel 60 F254TLC plates using Acetonitrile: Ethyl
acetate (7:3v/v) as mobile phase with densitometric detection at 229 nm. This
system was found to give compact bands for Bedaquiline fumarate at (Rf 0.53
±0.01). The linearity was observed in the range of200-1000 ng/band. Bedaquiline
was exposed to different stress degradation conditions like hydrolysis at
different pH, oxidation, photolytic and thermal degradation. The proposed
method can used for routine quantitative determination of Bedaquiline.
KEYWORDS: Bedaquiline fumarate, Stability Indicating method, HPTLC, Validation,
Multidrug-Resistant Tuberculosis.
1. INTRODUCTION:
Bedaquiline Fumarate (BQF) is chemically (1R,2S)-1-(6-bromo-2-methoxyquinolin-3-yl)-4-(dimethylamino)-2-naphthalen-1-yl-1-phenylbutan-2-ol;
(E)-but-2-enedioic acid. It is white odourless
powder having molecular weight 671.5 g/mol with chemical formula C36H35BrN2O61-4.
A thorough literature survey revealed that few methods
are available for the determination of Bedaquiline fumarate in bulk and
pharmaceutical dosage forms5-7.
It includes high-performance liquid chromatography
(HPLC), Liquid Chromatography with
tandem mass spectrometry
(LC-MS-MS),UV spectrophotometric method for determination as a single
drug or simultaneous estimation of combined drug in bulk and formulation but
only one stability indicating HPLC method was available8-15.
Most of the reported methods are based on hyphenated
techniques, so overall cost of the analysis using these techniques is more as
compared to High Performance Thin Layer Chromatography.
Since there were no articles found, related to HPTLC
method, the main objective of this work was to develop and validate Stability
Indicating HPTLC method for Bedaquiline Fumarate. The present HPTLC method was
validated by following the ICH guidelines16, 17. Few papers were also referred for additional details of
method development, validation by HPTLC technique18-27.
2. EXPERIMENTAL:
2.1 Material and Methods:
All chemicals and reagent used were of HPLC grade and
purchased from Merck Chemicals, India.
2.2 Chromatography:
TLC plates were pre-washed with methanol. Activation
was done in oven at 105ºC. The plates were allowed to cool at room temperature.
The chromatographic estimations were performed using these conditions;
Stationary phase:
Aluminium plates Pre-coated with silica gel 60 F254,
Mobile phase:
Acetonitrile: Ethyl Acetate (7:3v/v),
Chamber saturation period: 20 min,
Detection wavelength: 229nm,
Slit dimensions:
3.00×0.30mm spotting parameters used were,
Band width: 6mm, Space between two bands: 10mm.
2.3 Instrumentation:
A CAMAG HPTLC system equipped with Linomat 5
applicator (semi-automatic applicator), a 100
CAMAG syringe and an integrated software
winCATS version 1.4.3 was used for the analysis. HPTLC was performed on
pre-coated aluminium backed HPTLC plates of silica gel 60 F254 (Merck, India). UV- spectral analysis was performed
on UV-visible spectrophotometer JASCO (Model- V730). Photo-stability study was
performed using Photo-stability chamber (Newtronic, Model- IC DAC version 1.2).
Standard and sample solutions were applied to the plates, as 6 mm bands,
distance between tracks 10mm, under a stream of nitrogen.
2.4 Selection of detection wavelength:
Stock solution of Bedaquiline was prepared in 10ml of
methanol and UV spectrum was taken and it was found that BQF showed maximum
absorbance at 229nm.
Fig 3: UV Absorption Spectrum of Bedaquiline in Methanol (20 μg/ml)
2.5 Preparation of standard stock solution:
Weighed accurately 10mg Bedaquiline, transferred into
separate 10ml volumetric flask, about 3ml of methanol was added and shaken well
until dissolved. Then volume was made up with methanol up to the mark 10ml and
mixed (1000μg/ml). It was appropriately diluted to get working solution of
Bedaquiline (BQF) 500μg/ml.
2.6 Chromatographic Separation:
Plates were then developed, at room temperature, with
Acetonitrile: Ethyl acetate (7:3v/v) as mobile phase, in a 10cm×10cm CAMAG
twin-trough chamber previously saturated for 20 min. The development distance
was 80 mm. After development the plates were removed from the chamber, dried in
air and densitometric scanning was performed at 229nm with a CAMAG TLC
Scanner-III with winCATS software at a slit width of 3.00×0.30mm and scanning
speed of 20mm/s.
The retention factor of BQF were found to be
0.53±0.01.
Fig2: Representative densitogram of standard solution
of BQF (200 ng/band, Rf= 0.53 ± 0.01)
2.7 Forced degradation studies:
The degradation conditions were as per ICH guidelines
Q1A (R2). The strength of reagent and the time of exposure were optimised to
obtained 10-30% degradation16,17. The optimised conditions are as follows:
2.7.1 Acid degradation:
1ml of the BQF working standard solution (500μg/ml)
was mixed with 1mL of 0.5 N HCl and volume was made up to 10ml with methanol.
After 24hours of storage at room temperature, the resultant solution of a
concentration of 50μg/ml was applied to a TLC and developed using
optimised mobile phase.
2.7.2 Base degradation:
To 1ml of 0.5 N NaOH added 1ml working standard
solution of BQF (500 μg/ml) and the volume was made up with methanol to
10ml. The resultant solution was kept for 4 hrs. And applied on TLC plate. The
plate was developed and scanned at 229 nm.
2.7.3 Neutral degradation:
1 ml of the BQF working standard solution (500
μg/ml) was mixed with 1 mL of water and volume made up to 10 ml with
methanol. After 24 hours of room temperature storage. The resultant solution a
concentration of 50 μg/ml was applied to a TLC plate to develop a
chromatogram.
2.7.4 Thermal degradation:
The thermal degradation was carried out by placing
(BQF)solid state in an oven at 80°C for 6 hours. A sample was taken from oven,
cooled to room temperature, weighed and diluted in methanol to provide a final
concentration of 50μg/ml of Bedaquiline fumarate, which was then applied
to HPTLC and evaluated under optimum chromatographic conditions.
2.7.5 Photolytic degradation:
It was carried out using photo stability chamber as
per ICH Q1B Guidelines.
UV degradation:
Photolytic degradation was carried out by exposing
solid state powdered sample ofBedaquiline fumarate to UV energy not less than
200 watt hours/ square meter. Sample was weighed, dissolved in methanol to get
concentration of 50μg/ml of Bedaquiline fumarate. The resulting solution
was applied to TLC and analysed under optimized chromatographic conditions.
Fluorescence degradation:
Photolytic degradation was carried outby exposing
solid state powdered sample ofBedaquiline fumarate to cool white Fluorescence
illumination not less than 1.2 million lux hours. Sample was weighed, dissolved
in methanol to get concentration of 50μg/ml of Bedaquiline fumarate. The
resulting solution was applied to TLC and analysed under optimized
chromatographic conditions.
2.7.6 Peroxide induced degradation:
3%w/vH2O2 solution was prepared.
1ml of 3%w/v H2O2 was mixed with 1ml of working standard
solution of Bedaquiline fumarate (500μg/ml) and made up the volume upto
10ml with methanol and kept at room temperature for 4hrs. And the resultant
solution was spotted on TLC plate. Plate was developed and scanned at 229nm.
2.8 Method Validation:
The method was validated as per the ICH guidelines
ICHQ2(R1) in terms of linearity, accuracy and specificity, limit of detection,
limit of quantitation, intra-day and inter-day precision and robustness28-30.
2.8.1 Linearity:
Linearity of proposed method was checked by spotting
4-20μg/mlfrom a solution (50 μg/ml) for Bedaquiline fumarate. Sample
solutions were applied on TLC plate with the help of CAMAG 100 µL syringe,
using Linomat 5 sample applicator. The plate was developed and scanned under
the established chromatographic conditions. Linearity study was repeated six
times. Calibration curve of Bedaquiline fumarate was obtained by plotting
amount spotted vs peak area.
2.8.2Specificity:
Assay:
A blend of commonly used excipients was prepared, BQF
was spiked in this blend. The amount equivalent to average weight of tablet was
taken from blend and diluted with methanol sonicated for 5 minute, filtered
through Whatmaan filter paper and volume made up with methanol to achieve
concentration of 50μg/ml.
2.8.3 Accuracy:
Recovery study
was carried out by performing standard addition method at 50%, 100% and 150%
level. The standard drug solution of BQF was added to preanalysed sample
solution at three levels. The basic concentration of was sample chosen was 400
ng/band of BQF. The peak areas were calculated after development of plate and
the results are shown in Table 2.
2.8.4
Precision:
Precision study was performed as intra day precision
and inter day precision. Intra day precision was performed by analysing 400
ng/band of BQF, as six replicates in same day. For inter day precision, the
procedure was repeated on three consecutive days. The results are shown in
Table 3.
2.8.5 Limit of Detection and Limit of Quantitation:
The LOD and LOQ were calculated using equations,
LOD=3.3 x σ/S; LOQ =10 x σ/S, respectively where σ is the
standard deviation of the y-intercepts or area at lowest concentration and S is
the slope of the calibration curve.
2.8.6 Robustness:
Robustness of developed method was analysed by small
but deliberate changes in mobile phase volume, saturation time and detection
wavelentgh. Mobile phase volume was changed by ± 0.2ml, saturation time was varied by ±2min i.e., 18
min and 22 min. Detection wavelength were varied by ±1nm. One factor at a time
was varied at conc. of 400ng/band for Bedaquiline fumarate to study effect of
all factors on peak area of drug.
3. RESULTS AND DISCUSSION:
3.1 Force degradation study:
In order to evaluate the stability indicating property
of the developed method, forced degradation studies were carried out in
accordance with ICH guidelinesQ1A (R2).
Table 1: Data of forced degradation studies of BQF
|
Sr. No.
|
Parameter
|
Condition
|
% Recovery
|
% Degradation
|
Peak Purity
|
|
r(s,m)
|
r(m, e)
|
|
1
|
Acid
|
0.5 N HCL at Room temperature for 24 hrs.
|
81.07 %
|
18.93 %
|
0.9999
|
0.9994
|
|
2
|
Base
|
0.5 N NaOH at Room temperature for 4 hrs.
|
84.43 %
|
15.57 %
|
0.9997
|
0.9987
|
|
3
|
Neutral
|
1ml water for 24 hrs.
|
89.31 %
|
10.69 %
|
0.9996
|
0.9992
|
|
4
|
Thermal
|
80 0C 6 hrs.
|
83.03 %
|
16.97 %
|
0.9998
|
0.9998
|
|
5
|
UV
|
Not less than 200 Watt hours/square meter
|
97.67 %
|
2.33 %
|
0.9998
|
0.9999
|
|
6
|
Fluorescence
|
Not less than 1.2 million lux hours
|
97.36 %
|
2.64 %
|
0.9996
|
0.9997
|
|
7
|
Oxidation
|
3% w/v H2O2 for 4 hrs.
|
76.02 %
|
23.98 %
|
0.9997
|
0.9995
|
3.2 Linearity and range:
Linearity was determined by plotting amount spotted vs
peak area. Linearity range for Bedaquiline fumarate was found to be 200-1000ng/band. The correlation coefficient was found 0.9974 with
equation of y = 17.456x + 2144.7 of Bedaquiline fumarate. The linearity curve
BQF was shown in Fig.4.
3.3 Assay of Blend:
Assay of prepared blend was performed and the % drug
content was found to be 101.5%.
Fig 4: Calibration curve of Bedaquiline Fumarate
Fig 5: 3 D Densitogram of Bedaquiline linearity
200ng/band-1000ng/band
3.4. Accuracy:
The % mean recovery was found to be 99.02% for BQF
which indicated that the proposed method is accurate for estimation of drug in
combined tablet dosage form. The percent recovery for Bedaquiline fumarate was
found to be in range as shown in Table 2.
Table 2: Accuracy Studies of BQF:
|
Drug
|
Concentration taken (ng/band)
|
Concentration added (ng/band)
|
% Recovery
|
|
BQF
|
400
|
600
|
98.71%
|
|
400
|
800
|
99.35%
|
|
400
|
1000
|
99.01%
|
*Average
of three determinations
3.5 Precision:
Intra-day and Inter-day precision was performed. The results of precision studies for BQF
are shown in Table 3.
Table 3: Precision:
|
Amount Spotted
|
Condition
|
Mean Area (Average)
|
Standard Deviation (SD)
|
% Relative Standard deviation (%RSD)
|
|
400ng/
band
|
Intra-day
|
9808.2
|
56.76
|
0.57%
|
|
Inter-day
|
9544.6
|
131.29
|
1.37%
|
3.6 Limit of detection (LOD) and limit of quantitation (LOQ):
LOD and LOQ were calculated by two methods and results
were shown in Table 4.
Table 4:Limit of detection (LOD) and limit of
quantitation (LOQ):
|
Method
|
Average Slope
|
S.D
|
LOD (ng/band)
|
LOQ (ng/band)
|
|
By using lowest Concentration
|
5279.35
|
155.30
|
29.37
|
89.00
|
|
By using Y-intercept
|
2144.767
|
28.078
|
5.31
|
16.09
|
3.7 Robustness:
It was observed that there were no marked changes in
the densitograms, which confirmed that the method developed was robust. The
results are shown in Table 5.
Table 5: Robustness Data in Terms of % RSD
|
Parameters
|
|
% RSD
|
|
Mobile Phase Composition
(± 0.2ml)
|
Acetonitrile: Ethyl acetate (7.2:2.8) v/v
|
1.54
|
|
Acetonitrile: Ethyl acetate (6.8:3.2) v/v
|
1.46
|
|
Saturation time
(± 2min)
|
18 min
|
1.51
|
|
22 min
|
0.88
|
|
Wavelength
(± 1nm)
|
228 nm
|
1.61
|
|
230 nm
|
1.37
|
*n = 3
4. CONCLUSION:
This developed HPTLC method is simple, rapid and
stability indicating for routine quantitative analysis of Bedaquiline fumarate
in bulk drug and the tablet dosage form without interference of other
excipient. The developed method was validated as per ICH guidelines. The
results have ascertained methods linearity, precision, accuracy and robustness.
Bedaquiline fumarate was found to be sensitive to oxidative conditions and
considerably stable under UV and Fluorescence degradation. BQF undergoes
degradation under all conditions but there was no any degradant peak in any of
the conditions. The peak purity was within limit Thus, this method can
conveniently used for quantitative analysis of Bedaquiline fumarate on routine
basis.
5. ACKNOWLEDGEMENT:
Authors are thankful to the principal and the
management of the AISSMS College of Pharmacy, Pune, Maharashtra, India for
providing required facilities for research work.
6. CONFLICT OF INTEREST:
The authors declare that there is no conflict of
interest.
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