Stability
Indicating RP-HPLC Method Development and Validation for the Simultaneous
Estimation of Tezacaftor and Ivacaftor in Bulk and Pharmaceutical Dosage Form
G. Indira Priyadarshini, V.
Mounika, G. Anjani, B. Sowmya
Department
of Pharmaceutical Analysis, Hindu College of Pharmacy, Amaravathi Road,
Guntur-522002, A. P. India
*Corresponding Author E-mail: darshinipharma@gmail.com
ABSTRACT:
A simple, precise, accurate,
sensitive, reliable and cost effective Stability indicating RP-HPLC method was
developed and validated for the estimation of Tezacaftor and Ivacaftor in Bulk
and pharmaceutical dosage form. Chromatographic separation was done by using a Phenomenex C18 column (4.6×250mm,
5µm). Mobile phase containing 0.2% TEA (pH 3.5), methanol and acetonitrile in
the ratio of 40:50:10v/v was pumped through column at a flow rate of 1ml/min in
isocratic mode. Temperature was maintained at Ambient. Optimized wavelength for
Tezacaftor and Ivacaftor was 259 nm. Retention time of Tezacaftor and Ivacaftor
were found to be 4.916 and 2.891 min respectively. The linearity was
established over the concentration ranges of 20–100μg/ml and
30–150μg/ml with correlation coefficient (R2) 0.999 for both
Tezacaftor and Ivacaftor. % RSD for Intra-day Precision for Tezacaftor and
Ivacaftor were 0.7 and 0.6 respectively. % RSD for Intermediate Precision for
Tezacaftor and Ivacaftor were 0.8 and 0.9 respectively. Mean % recovery was
found to be 99.36% and 99.27% respectively. S/N ratio values of LOD, LOQ for
Tezacaftor were 2.98 and 10.02, for Ivacaftor were 3.04 and 9.96 respectively.
Percentage assay of Tezacaftor and Ivacaftor was found to be 99.69 and 100.21
respectively. Tezacaftor and Ivacaftor were subjected to stress conditions like
Acidic, Alkaline, Oxidation, Thermal and Photo degradation and results showed
that Tezacaftor was more sensitive towards alkaline degradation and Ivacaftor
was more sensitive towards photo degradation. The % degradation results were
within the limits. Hence the developed method can be successfully
employed for the routine analysis of Tezacaftor and Ivacaftor in bulk and
pharmaceutical dosage forms.
KEYWORDS: Tezacaftor,
Ivacaftor, RP-HPLC, Validation and Stability indicating.
INTRODUCTION:
Tezacaftor
is chemically 1-(2,2-difluoro-2H-1,3-benzodioxol-5-yl)-N-{1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl}cyclopropane-1-carboxamide.
Its molecular formula is C26H27F3 N2O6
and its molecular weight is 520.505 g/mol. Tezacaftor exerts its effect by acting as a corrector of the
CFTR protein. Ivacaftor is chemically
N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide.
Its molecular formula is C24H28N2O3 and
its molecular weight is 392.490 g/mol.
Fig 1: Structure of Tezacaftor
Fig 2: Structure of
Ivacaftor
Ivacaftor
exerts its effect by acting as a potentiator of the CFTR protein.
Tezacaftor
and Ivacaftor were introduced into the market in combined dosage form (SYMDEKO)
for the treatment of cystic fibrosis. The combined effect of Tezacaftor and
Ivacaftor is increased quantity and function of CFTR at the cell surface,
resulting in increases in chloride transport, airway surface liquid height, and
ciliary beat frequency.
The literature review reveals
that few analytical methods have been reported for the individual analysis of
Ivacaftor and simultaneous estimation of Lumacaftor and Ivacaftor in bulk,
pharmaceutical dosage forms and in biological samples. They are UV
Spectrophotometric, HPLC and LC-MS/MS methods. [32-39] Few
analytical methods are reported for the Tezacaftor and Ivacaftor in bulk and
pharmaceutical formulations. They are UV Spectrophotometric, HPLC and UPLC
methods. [41- 44] But still there is a need for the development of
more sensitive and cost effective analytical method for simultaneous estimation
of Tezacaftor and Ivacaftor. Hence an attempt has been made to develop a
simple, precise, accurate, sensitive, reliable and cost effective stability
indicating RP-HPLC method for the simultaneous estimation of Tezacaftor and
Ivacaftor in bulk and pharmaceutical dosage form.
MATERIALS AND METHODS:
Chemicals and reagents:
Working standards of
Tezacaftor and Ivacaftor were provided as gift samples by Pharma Train
(Hyderabad, India). SYMDEKO® is supplied as co-packaged tablets (Tezacaftor-100mg/Ivacaftor-150mg
and Ivacaftor-150mg) manufactured by Vertex Pharmaceuticals Incorporated.
Water (HPLC grade) - LiChrosolv (Merck), Acetonitrile (HPLC grade) -
Molychem, Methanol (HPLC grade) - LiChrosolv (Merck), Tri ethyl amine - Finer
Chemicals Ltd, Formic acid - Fisher Scientific, HCl - Fisher Scientific, NaOH -
Fisher Scientific, 30% H2O2 - Fisher Scientific.
HPLC - Waters Alliance 2695
separation module with UV detector and Empower 2 Software
UV-VIS spectrophotometer
(LABINDIA UV 3000+), Weighing machine (Afcoset ER-200A), Ultra
Sonicator (ELMA), pH Analyser (Adwa – AD 1020), Hot air oven (NSW INDIA)
Selection of wavelength:
UV spectrum of 10µg/ml
Tezacaftor and 10µg/ml Ivacaftor in Methanol were recorded by scanning in the
range of 200nm to 400nm against blank separately. Then the suitable wavelength
for the detection of Tezacaftor and Ivacaftor was selected as 259nm by
overlapping the spectrum of both drugs. At this wavelength both the drugs
showed good absorbance.
Chromatographic condition:
The chromatographic separation
of Tezacaftor and Ivacaftor was performed by using a Phenomenex C18 column (4.6´250mm, 5μm) at an ambient temperature in
isocratic mode. The mobile
phase was composed of 0.2% TEA buffer (pH 3.5), methanol and ACN in the ratio
of 40:50:10v/v. The flow rate was 1ml/min and the injection volume was 20µl.
Detection of wavelength was carried out at 259nm. The retention time of
Tezacaftor and Ivacaftor was found to be 4.916 and 2.891 minutes respectively.
Fig 3: Chromatogram of
Tezacaftor and Ivacaftor standard
Preparation of 0.2 % TEA
buffer:
Pipetted 1ml of TEA solution
into 500ml of volumetric flask and volume was made up to the mark with HPLC
water. The pH was adjusted to 3.5 with Formic Acid. Final solution was
sonicated for 10 minutes and filtered through 0.45µ Membrane filter.
Preparation of mobile phase:
Accurately measured 400ml
(40%) of 0.2% TEA buffer, 500ml (50%) of methanol and 100ml (10%) of
Acetonitrile. They were mixed and degassed in an ultrasonicator for 10 minutes
and then filtered through 0.45µ filter under vacuum filtration.
Diluent Preparation:
Methanol was used as diluent.
Preparation of standard
solution:
Accurately weighed and
transferred 20mg of Tezacaftor and 30mg of Ivacaftor working standards
into a 100ml clean dry volumetric flask, added 3/4th volume of
diluent and sonicated to dissolve it completely and volume was made up to
the mark with diluent. (Stock solution - 200µg/ml of Tezacaftor and 300µg/ml of
Ivacaftor)
Further pipetted 3 ml of the
above stock solution into a 10ml volumetric flask and diluted up to the mark
with diluent. (60µg/ml of Tezacaftor and 90µg/ml of Ivacaftor)
Preparation of sample
solution:
20 tablets were weighed and
powdered. Then the powder weight equivalent to 20mg of Tezacaftor and
30mg of Ivacaftor was transferred into a 100ml clean dry volumetric flask and
3/4th volume of diluent was added . The solution was sonicated for
15 mins and volume was made up to the mark with diluent. The solution was then
filtered through a 0.45µ injection filter. From the filtered solution 3ml was
pipetted into a 10ml volumetric flask and diluted up to the mark with
diluent. (60µg/ml of Tezacaftor and 90µg/ml of Ivacaftor)
RESULTS AND DISCUSSION:
Method validation:
The developed method was
validated with respect to system suitability, specificity, linearity, accuracy,
precision, limit of detection, limit of quantification and robustness in
accordance with the ICH Q2 (R1) guidelines.
System suitability:
System-suitability tests are
an integral part of method development and were used to ensure adequate
performance of the chromatographic system. Retention time (RT), number
of theoretical plates (N), tailing factor (T), and peak asymmetry
(AS), resolution (RS) were evaluated. The results are shown in Table 1.
Table 1: Results of system
suitability parameters
|
Property |
Tezacaftor |
Ivacaftor |
Acceptance Criteria |
|
Retention time (Rt) |
4.921 |
2.891 |
- |
|
Resolution |
6.26 |
- |
NLT 2.0 |
|
Tailing factor (T) |
0.96 |
1.19 |
NMT 2.0 |
|
Theoretical plates (N) |
2556.22 |
3994.84 |
NLT 2000 |
From the above
data it was found that all the system suitability parameters for developed
method were within the limit.
Specificity:
Specificity is the ability to
assess unequivocally the analyte in the presence of components which may be
expected to be present. It was found that there is no interference of any blank
peaks with the drugs of the analysis concern. Hence method was specific.
Linearity:
The linearity of an analytical
method is its ability to elicit test results which are directly proportional to
the concentration of analyte in the sample. Standard solution of Tezacaftor and
Ivacaftor were prepared in such a way that the final concentration of
Tezacaftor and Ivacaftor is in the range of 20-100µg/Ml and 30-150µg/mL
respectively. The peak area was recorded for all the peaks of Tezacaftor and
Ivacaftor. Calibration curves were constructed by plotting peak area vs.
concentration of Tezacaftor and Ivacaftor. Calibration curves for Tezacaftor
and Ivacaftor were plotted and correlation coefficient was calculated. The
results were shown in Table 2 and 3 and calibration curves were shown in Fig 4
and 5
Table 2: Areas of different
concentrations of Tezacaftor and Ivacaftor
|
S. No. |
Tezacaftor |
Ivacaftor |
||
|
Concentration (µg/ml) |
Area |
Concentration (µg/ml) |
Area |
|
|
1 |
20 |
61498 |
30 |
47997 |
|
2 |
40 |
124200 |
60 |
86759 |
|
3 |
60 |
185926 |
90 |
125452 |
|
4 |
80 |
250974 |
120 |
166324 |
|
5 |
100 |
309375 |
150 |
207446 |
Fig 4: Calibration curve of
Tezacaftor
Fig 5: Calibration curve of
Ivacaftor
Table 3: Analytical performance
parameters of Tezacaftor and Ivacaftor
|
Parameters |
Tezacaftor |
Ivacaftor |
|
Slope (m) |
3110 |
1362.8 |
|
Intercept (c) |
173.24 |
3455.6 |
|
Correlation coefficient (R2) |
0.999 |
0.999 |
Correlation
coefficient should be not less than 0.999. The correlation co-efficient
of Tezacaftor and Ivacaftor were found to be 0.999 and 0.999 which
were in the acceptance limit. Hence the proposed method was linear.
Precision:
The precision of an analytical
method is the closeness of a series of individual analyte measurements applied
repeatedly to multiple aliquots of the same sample.
Intraday Precision:
Solution containing 60
μg/ml of Tezacaftor and 90 μg/ml of Ivacaftor were analyzed six times
on the same day and %RSD was calculated. The results were shown in
Table 4.
Table 4: Results of Intra-day
Precision for Tezacaftor and Ivacaftor
|
Injection |
Area of Tezacaftor |
Area of Ivacaftor |
|
Injection-1 |
173994 |
123455 |
|
Injection-2 |
171378 |
123264 |
|
Injection-3 |
173439 |
122336 |
|
Injection-4 |
174594 |
121236 |
|
Injection-5 |
172609 |
123071 |
|
Injection-6 |
173150 |
122527 |
|
Average |
173194.0 |
122648.2 |
|
Standard Deviation |
1122.7 |
814.3 |
|
%RSD |
0.6 |
0.7 |
The % RSD should be not more
than 2. The % RSD for the Tezacaftor and Ivacaftor were found to be 0.6 and 0.7
respectively, which were within the limit. Hence method was precise.
Intermediate Precision
(Ruggedness):
Solution containing 60
μg/ml of Tezacaftor and 90 μg/ml of Ivacaftor were analyzed six times
on different day and %RSD was calculated. The results were shown in
Table 5.
Table 5: Results of
Intermediate precision for Tezacaftor and Ivacaftor
|
Injection |
Area of Tezacaftor |
Area of Ivacaftor |
|
Injection-1 |
172166 |
121879 |
|
Injection-2 |
171355 |
122615 |
|
Injection-3 |
174046 |
120359 |
|
Injection-4 |
171215 |
121879 |
|
Injection-5 |
170312 |
120042 |
|
Injection-6 |
173148 |
120142 |
|
Average |
172040.3 |
121152.7 |
|
Standard Deviation |
1371.4 |
1102.6 |
|
%RSD |
0.8 |
0.9 |
The %RSD should be not more
than 2. The %RSD for the Tezacaftor and Ivacaftor were found to be 0.8 and 0.9
respectively, which were within the limit. Hence method was rugged.
Accuracy:
The accuracy of an analytical
method is the closeness of test results obtained by that method to the true
value. Accuracy may often express as percent recovery by the assay of known
added amounts of analyte. Accuracy of the developed method was confirmed by
doing recovery study as per ICH guideline at three different concentration
levels 50%, 100%, 150% and the Values were measured. This performance was done
in triplicate. The results were shown in Table 6.
Table 6: Accuracy (recovery)
data for Tezacaftor and Ivacaftor
|
Sample |
%Concentration (at specification Level) |
Area |
Amount Added(mg) |
Amount Found(mg) |
% Recovery |
Mean % Recovery |
|
Tezacaftor |
50% |
86275 |
10 |
9.91 |
99.1 |
99.36 |
|
100% |
173130.3 |
20 |
19.89 |
99.45 |
||
|
150% |
259262.7 |
30 |
29.86 |
99.53 |
||
|
Ivacaftor |
50% |
60929.3 |
15 |
14.92 |
99.47 |
99.27 |
|
100% |
121422 |
30 |
29.74 |
99.13 |
||
|
150% |
181266.7 |
45 |
44.65 |
99.22 |
*Average of three
determinations
The percentage recovery for
each level should be between 98-102%. The mean percentage recovery for
Tezacaftor and Ivacaftor were found to be 99.29 and 99.65 respectively, which
were within the limits. Hence the method was accurate.
Limit of detection (LOD):
Detection limit of an
individual analytical procedure is the lowest amount of analyte in a sample
which can be detected but not necessarily quantitated, under the stated
experimental conditions. LOD of Tezacaftor and Ivacaftor were determined based
on signal-to-noise ratio. The results were shown in Table 7.
Table 7: Results of LOD
|
Drug name |
Baseline noise (µV) |
Signal obtained (µV) |
S/N ratio |
|
Tezacaftor |
45 |
134 |
2.98 |
|
Ivacaftor |
45 |
137 |
3.04 |
The S/N ratio value for LOD
solution shall be 3. The results obtained were within the limit.
Limit of quantification (LOQ):
Quantification limit of an
individual analytical procedure is the lowest amount of analyte in a sample
which can be quantitatively determined with suitable precision and accuracy.
LOQ of Tezacaftor and Ivacaftor were determined based on signal-to-noise ratio.
The results were shown in Table 8.
Table-8: Results of LOQ
|
Drug name |
Baseline noise (µV) |
Signal obtained (µV) |
S/N ratio |
|
Tezacaftor |
45 |
451 |
10.02 |
|
Ivacaftor |
45 |
448 |
9.96 |
The S/N ratio value for LOQ
solution shall be 10. The results obtained were within the limit.
The S/N ratio values for LOD
and LOQ were found to be 2.98 and 10.02 for Tezacaftor and 3.04 and 9.96 for
Ivacaftor respectively, which showed that the method was sensitive.
Robustness:
The Robustness of an
analytical method is a measure of its capacity to remain unaffected by small
but deliberate variations in method parameters and provides an indication of
its reliability during normal usage. The robustness of the proposed method was
determined by analysis of aliquots from homogenous lots by differing physical
parameters like flow rate (±0.1ml/min), mobile phase ratio
(±10%)
which may differ but the responses were still within the limits of the assay.
The results were shown in Table 9.
The Retention time, USP plate
count, USP tailing factor obtained for change of flow rate, variation in mobile
phase were found to be within the limit. Hence the method was robust.
Assay:
The developed and validated
RP-HPLC method was used to determine Tezacaftor and Ivacaftor in combined
dosage form.
Calculation:
% Assay was calculated by
using the following formula
Sample Area Weight of Standard
Dilution of Sample Average
weight Percentage Purity of Drug
% Assay= –––––––––– ×
–––––––––––––––– × –––––––––––––––– × –––––––––––– × –––––––––––––––––––––– ×
100
Standard Area Dilution of Standard Weight of
Sample Lable
Claim
100
Fig 6: Chromatogram of
Tezacaftor and Ivacaftor Sample for Assay
Table 9: Robustness data of
Tezacaftor and Ivacaftor
|
S. No |
Robustness Condition |
System Suitability Results of Tezacaftor |
System Suitability Results of Ivacaftor |
|||
|
USP Resolution |
USP Tailing |
USP Plate Count |
USP Tailing |
USP Plate Count |
||
|
1. |
Unaltered |
6.22 |
0.94 |
2494.14 |
1.19 |
3994.84 |
|
2. |
Flow rate (0.9 ml/min) |
5.81 |
0.92 |
1737.21 |
1.13 |
3737.21 |
|
3. |
Flow rate (1.1 ml/min) |
5.27 |
0.91 |
1711.26 |
1.09 |
3518.95 |
|
4. |
Mobile phase (46:45:9) |
6.51 |
1.02 |
2356.27 |
1.09 |
3664.51 |
|
5. |
Mobile phase (34:55:11) |
3.85 |
0.99 |
2975.63 |
1.16 |
3673.43 |
* Results for actual
flow rate and Mobile phase composition have been considered from Accuracy
standard.
The recorded chromatogram was
shown in Fig 6 and results were shown in Table 10.
Table 10: Results of Assay for
Tezacaftor and Ivacaftor
|
Drug Name |
Label Claim (mg) |
% Assay |
|
Tezacaftor |
100 |
99.69 |
|
Ivacaftor |
150 |
100.21 |
The % assay should be within
range of 98-102%. The % assay of Tezacaftor and Ivacaftor were found to be
99.69 and 100.21respectively.
FORCED DEGRADATION STUDIES:
The International Conference
on Harmonization (ICH) guidelines entitled stability testing of new drug
substances and products requires that stress testing be carried out to
elucidate the inherent stability characteristics of the active substance. The
specificity of the method was demonstrated through forced degradation studies
conducted on the sample using acid, alkaline, oxidative, thermal and photolytic
degradations. The sample was exposed to these conditions and the percentage
degradation was calculated.
Hydrolytic degradation under
acidic condition:
To 3ml of stock solution of
Tezacaftor and Ivacaftor, 3ml of 0.1N HCl was added and refluxed for 30mins at
60°C. Then the solution was cooled and neutralized with 0.1N NaOH and made up
the volume to 10ml with diluent. The solution was filtered with 0.45m syringe filter and
placed in vial. Then injected into the system and the chromatogram was recorded
to assess the stability of the sample.
Hydrolytic degradation under
alkaline condition:
To 3ml of stock solution of
Tezacaftor and Ivacaftor, 3ml of 0.1N NaOH was added and refluxed for 30mins at
60°C. Then the solution was cooled and neutralized with 0.1N HCl and made up
the volume to 10ml with diluent. The solution was filtered with 0.45m syringe filter and
placed in vial. Then injected into the system and the chromatogram was recorded
to assess the stability of the sample.
Oxidative (or) Peroxide
degradation:
To 3ml of stock solution of
Tezacaftor and Ivacaftor, 1ml of 30% w/v of hydrogen peroxide (H2O2)
was added. The solution was kept at 60°C for 30mins. The solution was filtered
with 0.45m syringe filter and placed in vial. Then injected into
the system and the chromatogram was recorded to assess the stability of the
sample.
Thermal induced degradation:
Tezacaftor and Ivacaftor drug
substances were taken in Petri dish and kept in Hot air oven at 105°C for 6
hours. Then the solution was prepared to achieve final concentration 60µg/ml of
Tezacaftor and 90µg/ml of Ivacaftor. The prepared solution was filtered with
0.45m syringe filter and placed in vial. Then sample was
injected into HPLC and the chromatogram was recorded to assess the stability of
the sample.
Photo degradation:
The photochemical stability of
the drug substance was studied by exposing the Tezacaftor and Ivacaftor drug
substances to UV Light by keeping the Petri dish in UV Chamber for 7days or
200Watt hours/m2 in photo stability chamber. Then the
solution was prepared to achieve final concentration 60µg/ml of Tezacaftor and
90µg/ml of Ivacaftor. The prepared solution was filtered through 0.45m syringe filter and
placed in vial. Then sample was injected into HPLC and the chromatogram was
recorded to assess the stability of the sample.
Calculation:
% Degradation was calculated
by using the following formula
Area of unstressed - Area of stressed
% Degradation =
––––––––––––––––––––––––––––––––– × 100
Area
of unstressed
The recorded chromatograms
were shown in Fig 7-11 and results were shown in Table 11.
The results showed that
Tezacaftor was more sensitive towards alkaline degradation and Ivacaftor was
more sensitive towards photo degradation. Degradation of drug up to 10% is generally accepted. The % degradation results of Tezacaftor
and Ivacaftor were within limits.
Fig 7: Chromatogram of Acid
degradation
Fig 8: Chromatogram of Alkaline
degradation
Fig 9: Chromatogram of Peroxide
degradation
Fig 10: Chromatogram of Thermal
degradation
Fig 11: Chromatogram of Photo
degradation
Table 11: Results for Stability
studies of Tezacaftor and Ivacaftor
|
S. No |
Stress condition |
Tezacaftor |
Ivacaftor |
||
|
Area |
% Drug Degraded |
Area |
% Drug Degraded |
||
|
1 |
Unstressed |
173721 |
- |
121552.3 |
- |
|
2 |
Acid |
168367 |
3.08 |
115862 |
4.68 |
|
3 |
Alkaline |
158372 |
8.84 |
113974 |
6.23 |
|
4 |
Peroxide |
163774 |
5.73 |
114812 |
5.55 |
|
5 |
Thermal |
167377 |
3.65 |
111938 |
7.91 |
|
6 |
Photo |
163129 |
6.10 |
111378 |
8.37 |
CONCLUSION:
The developed RP-HPLC method
was simple, precise, accurate, sensitive, reliable and cost effective for the
simultaneous estimation of Tezacaftor and Ivacaftor in bulk and pharmaceutical
dosage form. The developed method was stability indicating and can separate
degradants. Therefore this RP-HPLC method can be used for the routine analysis
of these drugs in bulk, pharmaceutical formulations and also for stability
studies in research institutions, quality control department in industries,
testing laboratories.
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Received on 12.09.2019 Modified on 18.11.2019
Accepted on 10.01.2020
©Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2020; 10(1):19-26.
DOI: 10.5958/2231-5675.2020.00005.8