7.1 introduction of tadalafil and survey of analytical...
TRANSCRIPT
7.1 Introduction of Tadalafil and survey of analytical methods:
Tadalafil (Cialis®) (Fig: 7.1) is used in oral treatment for erectile
dysfunction. Tadalafil is chemically (6R, 12aR)-2,3,6,7,12,12a-hexahydro-
2-methyl-6-(3,4-methylene dioxyphenyl) pyrazino (1′, 2′: 1,6) pyrido- (3,4-
b) indole-1, 4-dione. Tadalafil is a white crystalline solid that melts at
approximately 301-302°C. It is practically insoluble in water and very
slightly soluble in ethanol. The empirical formula of tadalafil is C22H19N3O4.
The molecular weight of Tadalafil is 389.4.
Fig: 7.1 Chemical structure of Tadalafil
NH
N
N
O
O
O
O
Molecular formula: C22H19N3O4
Molecular weight : 389.4
(6R, 12aR)-2, 3, 6, 7, 12, 12a-hexahydro-2-methyl-6-(3, 4-methylene
dioxyphenyl) pyrazino (1′, 2′: 1, 6) pyrido- (3, 4-b) indole-1, 4-Dione
It is widely recognized that enantiomers have distinict biological
interactions and thus potentially different pharmacokinetic,
pharmacological and /or toxicological effects (1, 2). To assure patient
safety and clinical efficacy, the pharmacological evaluation of stereo
isomers is an integral part of new drug development (3, 4). Analytical
169
methods to determine the enantiomeric purity of new investigational drugs
are often attained through a series of generic or screening methodologies
(5-9). Although many analytical techniques can be employed to achieve
this, the most widely used is liquid chromatography (LC) employing a
chiral stationary phase (CSP) (10-15).
Tadalafil is used in oral treatment for erectile dysfunction, is a selective
inhibitor of cyclic guanosine monophosphate (cGMP)-specific
phosphodiesterase type-5 (PDE-5). Through the inhibition on PDE-5, (R,
R)-tadalafil increases the concentration of cyclic guanosine
monophosphate (cGMP), producing smooth muscle relaxation and
increased blood flow to the corpus cavernosum, thereby enhancing erectile
response following appropriate sexual stimulation (1).
Few analytical methods have been reported for the estimation of (R, R)-
tadalafil in formulation using HPLC (17-19) and capillary electrophoresis
with UV detection (20, 21). A high throughput validated analytical method
for the quantitation of (R, R) - tadalafil in human plasma using LC–MS-MS
has been published (22). So far there are no analytical methods available
for separation and quantification of the (R, R)-tadalafil and its enantiomer
in bulk drugs and in pharmaceutical dosage forms. The novelty of this
work is, first time (R, R)-tadalafil and its enantiomer was separated and
quantified in both drug substance and in drug product by HPLC. In this
chapter a brief description on development and validation of a novel LC
170
method for separation and quantitative estimation of (R, R)-tadalafil from
its enantiomer.
7.2 Development and validation of a novel chiral RP-LC method for
the separation of (R, R)-Tadalafil and its enantiomer:
7.2.1 Materials:
Samples of (R, R)-tadalafil and its enantiomer (Fig: 7.2) was obtained
from Dr.Reddy’s Laboratories Ltd, Hyderabad, India. The received samples
were certified and of purity greater than 99.5%. Commercially available
Cialis, 20mg Tadalafil tablets were purchased. HPLC grade acetonitrile
and methanol were purchased from Merck, Darmstadt, Germany. High
pure water was prepared by using millipore Milli-Q plus water purification
system.
7.2.2 Equipment:
The LC system used for method development, forced degradation
studies and method validation was Agilent 1100 series system (Agilent
Technologies, 5301 Stevens Creek Blvd, Santa Clara, CA 95051, United
States) and Waters 2996 PDA system. (Waters Corporation, 34 Maple
Street, Milford, MA, 01757 USA). The output signal was monitored and
processed using Empower software (Waters Corporation) on Pentium
computer (Digital equipment Co). The mass studies for Tadalafil and its
enantiomer were carried out on a Quattro LC-MS/MS (Micromass,
Manchester, UK) using Masslynx software.
171
Fig: 7.2 Chemical structure of the enantiomer of (R, R)-tadalafil
NH
N
N
O
O
O
O
Molecular formula: C22H19N3O4
Molecular weight : 389.4
(6S, 12aS)-2, 3, 6, 7, 12, 12a-hexahydro-2-methyl-6-(3, 4-methylene
dioxyphenyl) pyrazino (1′, 2′: 1, 6) pyrido- (3, 4-b) indole-1, 4-Dione.
7.2.3 Sample preparation:
Stock solutions of (R, R)-tadalafil and its enantiomer (400 µg mL-1) were
prepared individually by dissolving appropriate amount in the diluent
(mobile phase was used as diluent - the mobile phase contains a mixture
of water, methanol and acetonitrile in the ratio of 55:40:5 (v/v). A Stock
solution of sample and impurity mixture was prepared (400 µg mL-1) in
diluent.
7.2.4 Preparation of Tablets Sample Solution:
Twenty tablets were individually weighed to get the average weight of
the tablets and powdered in mortar. A sample of the powdered tablets,
equivalent to 40 mg of (R, R)-tadalafil was transferred to 100 mL
volumetric flask. About 75 mL of mobile phase was added and kept on
rotatory shaker for 10 min to disperse the material completely and
172
sonicated for 10 min and diluted to 100 mL. The content was centrifuged
for 10 min at 3,000 rpm. The supernatant solution was collected and
filtered using 0.45 µm nylon 66-membrane filter.
7.2.5 Method development and optimization of chromatographic
conditions:
7.2.5.1 Selection of wavelength:
(R, R)-tadalafil and its enantiomer solutions were prepared in diluent at
a concentration of 100 ppm and scanned in UV spectro photometer; both
(R, R)-tadalafil and its enantiomer were having UV maxima at around 220
nm (Fig: 7.3). Hence detection at 220 nm was selected for method
development purpose.
Fig: 7.3 Typical UV spectrums of (R, R)-tadalafil and its enantiomer
and overlaid spectrum of both (R, R)-tadalafil and its enantiomer
220.2
285.1
AU
0.00
0.20
0.40
0.60
220.2
285.1
AU
0.00
0.20
0.40
0.60
0.80
1.00
nm220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
Wavelength (nm)
173
Absorbance
UV spectrum of (R, R)-tadalafil
UV spectrum of enantiomer of (R, R)-tadalafil
220.2
284.0
323.2 337.5 356.6360.7 368.3 382.7 394.7
220.2
285.1
nm220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00
Wavelength (nm)
7.2.5.2 Selection of column and mobile phase:
A mixture of (R, R)-tadalafil and its enantiomer was used during the
method development study. After a brief survey of existing analytical
methods it was thought to start the development activity initially on chiral
columns both in normal phase mode and reverse phase modes.
Accordingly, initial trials were carried in normal phase mode using
chiral columns. The selection of chiral columns was made based on
existing literature (1-7).
Different stationary phases were selected for method development
purpose. Chiralcel and Chiralpak columns were chosen for method
development purpose in normal phase mode. In these columns, the
stationary phase was derivatives of Polysaccharides such as cellulose and
amylase. These columns are suitable for mobile phase compositions
containing organic solvents and for normal phase type solvents.
174
Absorbance
Overlaid UV spectrum of both (R, R)-tadalafil & its enantiomer
1. Chiralcel OD-H (cellulose tris (3, 5-dimethylphenylcarbamate) on silica-
gel).
2. Chiralpak AD-H (amylose tris (3, 5-dimethylphenylcarbamate) on silica-
gel).
Prior to the analysis the entire HPLC system including the injector and
the injection loop was flushed with methanol, isopropyl alcohol, ethanol
and n-hexane solvent in series. As the HPLC system was equipped with
auto-sampler, this unit also flushed with above series of solvents. The
mobile phase chosen for initial method development purpose was a pre
sonicated mixture of n-hexane: isopropyl alcohol (90:10, v/v). The
chiralcel OD-H column was washed initially with n-hexane for about one
hour. After column flushing, column was equilibrated with the mobile
phase for one hour until a stable base line was achieved. No separation
was observed on chiralcel OD-H column. Another trial was carried on
polysaccharide type chiral column namely chiralpak AD-H. The above
column washing and stabilization steps were same as that of chiralcel OD-
H column.
The mobile phase chosen for initial method development purpose was a
pre sonicated mixture of n-hexane: isopropyl alcohol (90:10, v/v) and
there was no indication of separation. Elution of a single peak without any
resolution was observed. Different combinations of n-hexane, n-heptane
methanol, ethanol and isopropyl alcohol were tried in both the columns.
Resolution (Rs ~ 0.9) was observed on Chiral AD-H column with the
175
mobile phase composition of n-heptane: 2-Propanol: Ethanol: 50:30:20
(v/v/v). To improve resolution trifluoroacetic acid (TFA), triethylamine and
diethyl amine were introduced to the above mobile phase system and
trials were made. No fruitful results were obtained. Since the analysis on
normal phase columns is expensive and the life time of these columns is
short due to coated stationary phase and also these columns are
compatible to limited solvents only (limited solvent compatibility). Keeping
all these points in view gave up the further optimization trails on coated
type polysaccharide columns. Trails were taken in reverse phase to
minimize the cost of analysis.
To achieve separation between the enantiomers of Tadalafil a new
stationary phase which is feasible for amino acids separations was
selected, in which macrocyclic glycopeptides were linked through five
covalent bonds to a silica surface. CHIROBIOTIC-T (Fig: 7.4) was the
column selected for this purpose. In this column the macro cyclic
glycopeptide was teicoplanin. The separation mechanism in this column
was may be due to complex chiral environment, possibility for π - π
interactions, chiral hydrogen bonding sites, peptide binding sites and
multi-modal possibilities. This column was selected because of its unique
selectivity for underivatised α, β, γ or cyclic amino acids, N-derivatised
amino acids, alpha hydroxyl – carboxylic acids, acidic compounds
including carboxylic acids, phenols, neutral aromatic analytes and cyclic
aromatic and aliphatic amines. The column has a pH range from 3 to 7.
176
The chirobiotic T column used in this study was a previously used
column (not a brand new column) in reverse phase mode and stored in
15% ethanol solution. This column was chosen for method development
purpose in reverse phase mode since the column was compatible with
aqueous mobile phases with buffers containing a limited percentage of
organic modifiers. Initially the entire chromatographic system was flushed
with methanol and then with warm milli-Q water. Before starting the
analysis flushed the column with milli-Q water and then with ethanol for
30 min at a flow rate of 0.5 mL min-1. After completion of analysis the
column was flushed with ethanol and stored in 15% ethanol (15 mL
ethanol in 85 mL water).
Phosphate buffers were not selected for method development on
chirobiotic T, as per literature suggestion. Initial trial was made with a
degassed mixture of water: methanol (60:40 v/v), elution of a single peak
was observed; no separation was observed between the enantiomers.
Second trial was made with water: methanol (50:50, v/v) as mobile phase.
A resolution (Rs) of about 1.2 was observed between the enantiomer of (R,
R)-tadalafil and (R, R)-tadalafil, but tailing of (R, R) - tadalafil was greater
than 2. One more trial was made with water: methanol (55:45 v/v)
composition. A resolution (Rs) of about 2.5 was observed between the
enantiomer of (R, R)-tadalafil and (R, R)-tadalafil, but tailing of (R, R)-
tadalafil was greater than 1.6. Since resolution was obtained with this
composition, this composition (water: methanol (55:45 v/v)) and
177
CHIROBIOTIC-T (250x4.6) mm with 5µm particle size column was taken
as base and optimisation of the method was carried out.
Fig.7.4 Proposed Structures of CHIROBIOTIC-T
(Macrocyclic Glycopeptide Teicoplanin):
A, B, C, D are inclusion pockets (weak)
7.2.5.3 Optimisation of the method:
In the optimization of the method trials were made by changing the
organic ratio. To decrease the tailing factor (R, R)-tadalafil, acetonitrile was
introduced into the mobile phase. When water: methanol: acetonitrile
(50:45:30, v/v/v) was used as mobile phase, elution of a single peak was
observed, no separation was observed between enantiomers, this might be
due to the higher concentration of acetonitrile. (Here higher organic
modifier (acetonitrile) concentration reduced the retention and enantio
178
OH
NHR
CH
2OH
HO
O
HNCOCH
3
HO
O
NH
2
O
HO
Cl
HHO
H
O
HN
NH
O
H
B
A
OCl
N
O
N
HO
HHO
C
OHOH
O
NHH
NH
O
HOOC
HD
HO
OH
CH
2OHO
HOH
O
CH2OH
HO
HO
OH
selectivity). Further trials were carried with lower concentration of organic
modifier. When water: methanol: acetonitrile (50:40:10, v/v/v) was used
as mobile phase, a resolution (Rs) of about 1.7 was observed between (R,
R)-tadalafil and its enantiomer. Tailing factor of (R, R)-tadalafil was 1.0. To
further improve the resolution, acetonitrile content was decreased in the
mobile phase to 5 % from 10%. When water: methanol: acetonitrile
(55:40:5, v/v/v) was used as mobile phase satisfactory resolution between
(R, R)-tadalafil and its enantiomer (Rs ~2.4) and tailing factor of (R, R)-
tadalafil (~1.1) was observed (Fig: 7.5).
The stationary phase and % of organic phase (both methanol and
acetonitrile) found to be crucial in getting the separation between (R, R)-
tadalafil and its enantiomer. Acetonitrile played a major role in controlling
the tailing of (R, R) Tadalafil.
The interference of excipients (hydroxy propyl cellulose, sodium lauryl
sulphate, micro crystalline cellulose, lactose monohydrate, and
croscarmellose cellulose and magnesium sterate) was also checked by
injecting sample solutions of excipients. There was no interference of
excipients with (R,R) Tadalafil peak and its enantiomer.
179
Fig: 7.5 Typical chromatograms of (R, R)-tadalafil spiked with its
enantiomer at 0.15% level & racemic mixture of Mixture of
enantiomer of (R, R)-tadalafil & (R, R)-tadalafil
AU
-0.020
-0.015
-0.010
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
SS
-Tad
alaf
il -
10.7
36
RR
-Tad
alaf
il -
12.7
66
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
SS
-Tad
alaf
il -
10.3
22
RR
-Tad
alaf
il - 1
2.47
6
X axis - Time in minutes Y axis - Absorbance AU
180
(b) Mixture of enantiomer of (R, R)-tadalafil & (R, R)-tadalafil
(a) 100% (R, R)-tadalafil + 0.15% its enantiomer Spiked
7.2.5.4 Optimized chromatographic conditions for the determination
of enantiomeric purity of Tadalafil:
Column : Chirobiotic-T, (250x4.6) mm with 5µm
particle size.
Mobile phase : Water, methanol and acetonitrile (55:40:5)
(v/v/v).
Flow rate : 1.0 mL min-1
Column temperature : 27 °C
Wavelength of detection : 220 nm (UV detection technique)
Injection volume : 20µL
Run time : 30 min
Diluent : Mobile Phase
7.2.5.5 Optimized chromatographic conditions for the determination
of molecular weights of Tadalafil and its enantiomer by LCMS:
Column : Chirobiotic-T, (250x4.6) mm with 5µm
particle size.
Mobile phase : Water, methanol and acetonitrile (55:40:5)
(v/v/v).
Flow rate : 1.0 mL min-1
Column temperature : 27 °C
Injection volume : 5µL
Run time : 20 min
Diluent : Mobile Phase
181
Quattro micro Tune Parameters :
Source (ES-)
Capillary Voltage : 3.6 (kV)
Cone Voltage : 65.0 (v)
Extractor : 2.00 (v)
RF Lens : 0.1 (v)
Source Temp : 120° C
Desolvation Temp : 250° C
Cone gas flow : 250mL min-1
Analyser
LM1 Resolution : 15.0
HM1 Resolution : 15.0
Ion energy 1 : 0.5
Entrance : 50
Collision : 2
Exit : 50
Multiplier : 650 (v)
Mass Lynx v 4.1 software (waters) was used to calculate the molecular
formulas of the de protonated molecules according to the accurate mass
data.
182
7.2.5.6: Mass spectral data of Tadalafil and its enantiomer:
Mass spectral data (Table: 7.1) was recorded using Electro spray
ionization technique (ES - technique) and the values obtained for (M-H)
confirm the molecular weights (M) of the (R, R)-tadalafil and Enantiomer of
(R, R)-tadalafil (Fig: 7.6-Fig: 7.7).
Table: 7.1 Mass spectral data of (R, R)-tadalafil and its enantiomer
Compound Name Molecular weight (M) (M-H) value
(R, R)-tadalafil 389.4 388.3
Enantiomer of
(R, R)-tadalafil389.4 388.3
183
Fig: 7.6 Typical ES (-) Mass spectrum of (R, R)-tadalafil
184
185
186
Fig: 7.7Typical ES (-) Mass spectrum of
enantiomer of (R, R)-tadalafil
187
7.2.6 Validation of Analytical method and its results:
The developed and optimized HPLC method was taken up for
validation. The analytical method validation was carried out in accordance
with ICH guidelines [23, 24].
7.2.6.1 System suitability test:
System suitability testing is an integral part of analytical procedure.
The tests are based on the concept that the equipment, electronics,
analytical operations and samples to be analyzed constitute an integral
system that can be evaluated as such. System suitability test parameters
to be established for a particular procedure depend on the type of
procedure being validated (8).
To the (R, R)-tadalafil standard and enantiomer of (R, R)-tadalafil was
spiked at 0.15% level with respect to the concentration of (R, R)-tadalafil
and injected for five times into HPLC system. Resolution between
enantiomer of (R, R)-tadalafil and (R, R)-tadalafil, tailing factor, theoretical
plates for enantiomer of (R, R)-tadalafil and (R, R)-tadalafil, RSD% for the
areas of enantiomer of (R, R)-tadalafil and (R, R)-tadalafil was calculated.
System suitability results were tabulated (Table: 7.2 and Fig: 7.8).
188
Fig: 7.8 Blank, (R, R)-tadalafil Sample and system suitability
chromatograms
AU
-0.020
-0.015
-0.010
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
189
(a) Blank
(b) (R, R)-tadalafil sample
(c) (R, R)-tadalafil spiked with 0.15% of its enantiomer
AU
-0.020
-0.015
-0.010
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
RR
-Tad
alaf
il - 1
2.54
4
AU
-0.020
-0.015
-0.010
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
SS
-Tada
lafil
- 1
0.736
RR
-Tada
lafil -
12.
766
X axis - Time in minutes Y axis - Absorbance AU
Table: 7.2 System suitability results
190
AU
-0.020
-0.015
-0.010
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
RR
-Tad
alaf
il - 1
2.54
4
AU
-0.020
-0.015
-0.010
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
Time in Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
SS
-Tada
lafil
- 1
0.736
RR
-Tada
lafil -
12.
766
X axis - Time in minutes Y axis - Absorbance AU
Table: 7.2 System suitability results
191
Name Retention time in
min
Resolution (RS )
Tailing factor (T)
No. of Theoretical plates (N)
RSD% forfor area
Enantiomer of (R, R)-tadalafil 10.7 - 1.06 12586 0.4
(R, R)-tadalafil 12.7 2.45 1.01 13427 0.6
7.2.6.2 Limit of quantification (LOQ) and limit of detection (LOD):
LOQ and LOD was established for enantiomer of (R, R)-tadalafil based
on signal to noise ratio method.
(a) Limit of quantification (LOQ):
The limit of quantitation (LOQ) of an analytical procedure is the lowest
amount of analyte in a sample, which can be quantitatively determined
with suitable precision and accuracy. The quantitation limit is a
parameter of quantitative assays for low levels of compounds in sample
matrices, and is used particularly for the determination of impurities.
To establish limit of quantification for enantiomer of (R, R)-tadalafil, a
series of solutions with different known concentrations were prepared and
injected into the chromatographic system. For each injection signal to
noise ratio was monitored. Precision and accuracy studies were carried
out at that concentration where the S/N was about 10. Based on the
results the concentration was confirmed as limit of quantification (LOQ)
(Table: 7.3).
Table: 7.3 LOQ value of the Enantiomer of (R, R)-tadalafil
192
S.No Impurity name Concentration Signal / Noise ratio
1 Enantiomer of (R, R)-tadalafil 0.032 µg mL-1 10.2
(b) Limit of detection (LOD):
The detection limit of an individual analytical procedure is the lowest
amount of analyte in a sample, which can be detected but not necessarily
quantitated as an exact value.
To establish limit of detection for enantiomer of (R, R)-tadalafil a series
of solutions with different known concentrations were prepared and
injected into the chromatographic system. For each injection signal to
noise ratio was monitored. The concentration where the S/N was about 3
was chosen as limit of detection (LOD) value (Table: 7.4).
Table: 7.4 LOD value of the enantiomer of (R, R)-tadalafil
S.No Impurity name Concentration Signal / Noise ratio
1 Enantiomer of(R, R)-tadalafil 0.013 µg mL-1 2.7
7.2.6.3 Precision at Limit of Quantification level:
193
The precision at limit of quantification level was checked by injecting
six individual preparations of enantiomer of (R, R)-tadalafil at its LOQ level
with respect to test concentration (i.e. 400 µg mL-1). The RSD % for area of
enantiomer of (R, R)-tadalafil for six consecutive determinations was 4.5
(Table: 7.5).
Table: 7.5 Precision results of enantiomer of (R, R)-tadalafil at LOQ
level.
Preparation Area of enantiomer of (R, R)-tadalafil
1 1579
2 1542
3 1406
4 1584
5 1511
6 1478
Average 1516.7
SD 67.6
RSD% 4.5
95% Confidence interval of mean {1462.6,1570.8}
7.2.6.4 Accuracy at Limit of Quantification level:
Standard addition and recovery experiments were conducted to
determine accuracy of the developed method for the quantification of
enantiomer of (R, R)-tadalafil in [R, R)-tadalafil sample at LOQ level.
The recovery study for enantiomer of (R, R)-tadalafil was carried out in
triplicate at LOQ level of the (R, R)-tadalafil target analyte concentration
(400 µg mL-1). Prepared three different solutions containing enantiomer of
194
(R, R)-tadalafil at the limit of quantification level and injected each
solution once. Prepared the sample solution for three times from the same
homogeneous sample (at the analyte concentration i.e. 400 µg mL-1) and
injected each solution once. Prepared three different sample solutions
containing enantiomer of (R, R)-tadalafil at the limit of quantification level
and injected each solution once, calculated % Recovery.
The percentage recovery of enantiomer of (R, R)-tadalafil was calculated
(Table: 7.6). The method showed consistent and high absolute recovery at
LOQ level with a mean absolute recovery of 100.2% for drug substance
and 100.8 for drug product. The obtained absolute recovery was normally
distributed around the mean with uniform RSD values. The method was
found to be accurate with low % bias (< 1.0).
Table: 7.6 Recovery at LOQ level
S.No Impurity nameDrug Substance
Mean recovery (%)(n = 3 )
Drug ProductMean recovery (%)
(n = 3 ) RSD%
1 Enantiomer of(R, R)-tadalafil 100.2 100.8 0.8
7.2.6.5 Precision:
The precision of an analytical procedure expresses the closeness of
agreement (degree of scatter) between a series of measurements obtained
195
from multiple sampling of the same homogeneous sample under the
prescribed conditions. It was usually expressed as RSD%. Precision is a
measure of the degree of reproducibility of the analytical method under
normal operating circumstances. The precision of the analytical method is
determined by assaying a sufficient number of aliquots of a homogenous
sample to be able to calculate statistically valid estimates of SD or RSD
(CV). Precision includes repeatability, intermediate Precision and
reproducibility. Repeatability is the precision of a method under the
same operating conditions over a short period of time (Determining RSD).
Intermediate Precision expresses within-laboratories variations, different
days, different analysts, different equipment etc. Reproducibility
expresses the precision between laboratories (collaborative studies).
The precision of the developed method was evaluated initially by
performing system precision (Table: 7.7), then by injecting six individual
preparations of (R, R)-tadalafil (400 µg mL-1) spiked with 0.15% of
enantiomer of (R, R)-tadalafil with respect to Nateglinide analyte
concentration. The RSD% for % of enantiomer of (R, R)-tadalafil, for six
consecutive determinations was respectively as below (Table: 7.8).
Table: 7.7 Results of System precision study
196
Table: 7.8
Results of precision study
S.No Area of (R, R)-tadalafil peak
Injection-1 19851236
Injection-2 19754982
Injection -3 19646981
Injection -4 19685966
Injection -5 19589103
Injection -6 19658931
Mean 19697867
SD 92586.0
RSD % 0.47
Preparation Retention time % enantiomer of(R, R)-tadalafil
1 10.736 0.148
2 10.736 0.150
3 10..735 0.149
4 10.736 0.151
5 10.736 0.149
6 10.735 0.152
Average 10.736 0.150
SD 0.0004 0.0013
RSD% 0.004 0.897
95% Confidence interval of mean {10.735,10.736} {0.149,0.151}
197
Results showed insignificant variation in measured response which
demonstrated that the developed method was repeatable with RSD s below
1.5%.
Intermediate precision study was performed by injecting six
individual preparations of (R, R)-tadalafil (400 µg mL-1) spiked with 0.15%
of % enantiomer of (R, R)-tadalafil with respect to (R, R)-tadalafil analyte
concentration over different days, different instruments, different columns
and with different analysts. Reproducibility of the method was checked by
performing the precision study in a different laboratory (Table: 7.9).
Table: 7.9 Results of Intermediate precision and reproducibility
S.No Parameter Variation
RSD % for% enantiomer of (R, R)-tadalafil
Resolutionbetween
enantiomer of(R, R)-tadalafil &(R, R)-tadalafil
198
1 Different System
(a) Waters 2695Alliance system(b) Agilent 1100
series VWD system
1.21.1
2.42.4
2 Different Column
B.No (a): US10017459
B.No (b): US10017562
1.21.0
2.42.5
3 Different Analyst
(a) Analyst-1(b) Analyst-2
1.20.9
2.42.4
4 Different Laboratories
(a) Lab-1(b) Lab-2
1.20.8
2.42.4
7.2.6.6 Linearity:
The linearity of an analytical method is its ability (within a given range)
to obtain results, which are directly proportional to the concentration
(amount) of analyte in the sample (8). Linearity experiments were carried
out by preparing the (R, R)-tadalafil sample solution containing
enantiomer of (R, R)-tadalafil from LOQ to 200% ((LOQ), 0.015, 0.0375,
0.075, 0.1125, 0.15, 0.1875, 0.225 and 0.3%) with respect to their
specification limit (0.15%). Each solution was injected thrice (n=3) into
HPLC and calculated the average area at each concentration.
Calibration curve was drawn by plotting average area of the
enantiomer of (R, R)-tadalafil on Y-axis and concentration on X-axis (Fig:
7.9) which showed linear relation ship with a regression coefficient of
199
greater than 0.999 for enantiomer of (R, R)-tadalafil (Table: 7.10). At all
concentration levels, standard deviation for peak area was significantly
low and RSD % was below 2.0. Analysis of residuals indicated that the
residuals were normally distributed around the mean with uniform
variance across all concentrations (Fig: 7.10) suggesting the
homoscedastic nature of data. Selected linear model with univariant
regression showed minimum % bias indicating goodness of fit which was
further supported by the low standard error of estimate and mean sum of
residual squares.
Table: 7.10 Results of the linearity experiments
% Level w.r.t specification limit(i.e. 0.15%)
Enantiomer of (R, R)-tadalafil (Average peak Area)
5.33 1623
10 3200
25 7869
50 15698
75 24125
100 32025
125 39856
150 48265
200 64236
Correlation Coefficient (r) 0.9999
Slope 321.74
Intercept -143.85
200
% Y-intercept -0.004
Fig 7.9: Linearity graph for enantiomer of (R, R)-tadalafil
Linearity plot for Enantiomer of (R, R)-tadalafil
y = 321.737x - 143.846R2 = 0.9999
500
10500
20500
30500
40500
50500
60500
70500
0 50 100 150 200 250
% Concentration
Ave
rage
Are
a
Fig: 7.10 Residual plot for the enantiomer of (R, R)-tadalafil
Residual plot for Enantiomer of (R, R)-tadalafil
-3202.5
-2202.5
-1202.5
-202.5
797.5
1797.5
2797.5
0 2 4 6 8 10
Order of Residuals
Resi
dual
s
Table: 7.11 Residual summary of enantiomer of (R, R)-tadalafil
201
Con (%)Mean area response achieved
Response calculated thru
Trend line equation
Residual (Response practical -Response
theoretical)
Residual square
5.33 1623 1571.0 -52.0 2701.5
10 3200 3073.6 -126.5 15989.6
25 7869 7899.7 30.6 939.4
50 15698 15943.2 245.2 60098.5
75 24125 23986.7 -138.3 19140.7
100 32025 32030.2 5.2 26.5
125 39856 40073.7 217.7 47371.5
150 48265 48117.2 -147.8 21859.6
200 64236 64204.2 -31.8 1014.4
Residual sum of squares 169141.84
Trend line equation y = 321.737x - 143.846
7.2.6.7 Accuracy:
The accuracy of an analytical procedure expresses the closeness of
agreement between the value, which is accepted either as a conventional
true value or an accepted reference value and the value found. Standard
addition and recovery experiments were conducted to determine accuracy
of the present method for the quantification of enantiomer of (R, R)-
tadalafil in bulk drug samples and drug product of (R, R)-tadalafil.
Accuracy of the developed method was established at 50, 75, 100 and
150% of the enantiomer of (R, R)-tadalafil specification limit (0.15%) with
respect to analyte concentration (400 µg mL-1).
Quantification of enantiomer of (R, R)-tadalafil:
202
Prepared the sample solution three times with respect to analyte
concentration (400 µg mL-1) and injected into the chromatographic system.
The enantiomer of (R, R)-tadalafil is absent in both bulk sample and drug
product.
a. Accuracy at 50% impurity specification level:
Prepared test solution in triplicate (n=3) containing enantiomer of (R,
R)-tadalafil at 0.075% level to the limit of enantiomer of (R, R)-tadalafil
with respect to (R, R)-tadalafil concentration (400 µg mL-1). Each solution
was injected once into chromatographic system. % Mean recovery of
enantiomer of (R, R)-tadalafil was calculated from this solution using the
area of impurity standard at 0.15% level with respect to analyte (Table:
7.12).
Table: 7.12 Recovery at 50% level
S.No Impurity NameDrug substance
Mean recovery (%)(n = 3 ) RSD%
Drug productMean recovery (%)
(n = 3 ) RSD%
1 enantiomer of(R, R)-tadalafil 98.0 0.7 97.8 0.9
b. Accuracy at 75% impurity specification level:
Prepared test solution in triplicate (n=3) containing enantiomer of (R,
R)-tadalafil at 0.1125% level to the limit of enantiomer of (R, R)-tadalafil
with respect to (R, R)-tadalafil concentration (400 µg mL-1). Each solution
was injected once into chromatographic system. %Mean recovery of
enantiomer of (R, R)-tadalafil was calculated from this solution using the
203
area of impurity standard at 0.15% level with respect to analyte (Table:
7.13).
Table: 7.13 Recovery at 75% level
S.No Impurity NameDrug substance
Mean recovery (%)(n = 3 ) RSD%
Drug productMean recovery (%)
(n = 3 ) RSD%
1 enantiomer of(R, R)-tadalafil 98.7 0.7 99.4 0.3
c. Accuracy at 100% impurity specification level:
Prepared test solution in triplicate (n=3) containing enantiomer of (R,
R)-tadalafil at 0.15% level to the limit of enantiomer of (R, R)-tadalafil with
respect to (R, R)-tadalafil concentration (400 µg mL-1). Each solution was
injected once into chromatographic system. %Mean recovery of
enantiomer of (R, R)-tadalafil was calculated from this solution using the
area of impurity standard at 0.15% level with respect to analyte (Table:
7.14).
Table: 7.14 Recovery at 100% level
S.No Impurity NameDrug substance
Mean recovery (%)(n = 3 ) RSD%
Drug productMean recovery (%)
(n = 3 ) RSD%
1 enantiomer of(R, R)-tadalafil 100.8 0.4 100.5 0.5
d. Accuracy at 150% impurity specification level:
Prepared test solution in triplicate (n=3) containing enantiomer of (R,
R)-tadalafil at 0.225% level to the limit of enantiomer of (R, R)-tadalafil
204
with respect to (R, R)-tadalafil concentration (400 µg mL-1). Each solution
was injected once into chromatographic system. %Mean recovery of
enantiomer of (R, R)-tadalafil was calculated from this solution using the
area of impurity standard at 0.15% level with respect to analyte (Table:
7.15).
Table: 7.15 Recovery at 150% level
S.No Impurity NameDrug substance
Mean recovery (%)(n = 3 ) RSD%
Drug productMean recovery (%)
(n = 3 ) RSD%
1 enantiomer of(R, R)-tadalafil 100.8 1.2 102.5 1.0
The mean absolute recovery of enantiomer of (R, R)-tadalafil in drug
substance was ranged from 98.0 to 100.8%. The mean absolute recovery
of enantiomer of (R, R)-tadalafil in drug product of (R, R)-tadalafil was
ranged from 97.8% to 102.5%. This recovery study indicated that the
method was suitable for determination of enantiomer of (R, R)-tadalafil in
drug substance and drug product.
7.2.6.8 Solution stability and mobile phase stability:
The solution stability for enantiomer of (R, R)-tadalafil was carried out
by leaving both unspiked and spiked sample solution in tightly capped
volumetric flask at room temperature on a laboratory bench for 48h.
Content of enantiomer of (R, R)-tadalafil was determined for every 6h
interval and compared with freshly prepared solution at each time point.
205
Mobile phase stability was also carried out for 48h by injecting the
freshly prepared sample solutions for every 6h interval. Content of
enantiomer of (R, R)-tadalafil was checked in the test solutions. Mobile
phase prepared was kept constant during the study period.
No significant change was observed in the content of enantiomer of (R,
R)-tadalafil during solution stability and mobile phase stability
experiments. The solution stability and mobile phase stability experiments
data confirms that sample solutions prepared in diluent and mobile phase
used during the study was stable up to the study period of 48h.
7.2.6.9 Robustness:
The capability of the method to remain unaffected by small but
deliberate variations in the method parameters was study in order to
anticipate the problems, which may arise during the regular application of
the developed method (23). To determine the robustness of the developed
method, experimental conditions were deliberately altered and the
resolution between enantiomer of (R, R)-tadalafil and (R, R)-tadalafil was
evaluated.
a. Flow rate:
The flow rate of the mobile phase was 1.0 mL min -1 . To study the effect
of flow rate on the resolution 0.2 units of flow changed from 1.0 mL min-1
(i.e. 0.8 mL min-1 to 1.2 mL min-1 and Fig 7.11).
206
b. Column Temperature:
The effect of change of column temperature on resolution was studied
at 22°C to 32°C instead of 27°C, while the other mobile phase components
were held constant as stated in chromatographic conditions (Fig 7.12).
c. Mobile phase composition:
The effect of change in percent of methanol on resolution was studied
by varying from -10% to +10% of 40% of methanol (i.e. 36 % to 44% of
total volume), while the rest composition was held constant as stated in
chromatographic conditions (Fig 7.13).
In all these conditions (flow rate, column temperature, mobile phase
composition the resolution between the enantiomer of (R, R)-tadalafil and
(R, R)-tadalafil was always greater than 2.2 illustrating the robustness of
the method (Table: 7.16).
Table: 7.16 Robustness results of the method
S.No Parameter VariationResolution between
enantiomer of (R, R)-tadalafil &[R, R)-tadalafil
1 Temperature (a) At 22°C(b) At 32°C
2.312.43
2 Flow rate (a)At 0.8 mL min-1
(b)At 1.2 mL min-12.362.21
3 %Methanol (a) At 90%(b) At 110%
2.412.22
Fig: 7.11 Robustness study - Effect of Temperature
207
2.432.31 2.39
2.44 2.42
1.0
1.5
2.0
2.5
3.0
3.5
4.0
21.0 23.0 25.0 27.0 29.0 31.0 33.0
Temperature of Column in°C
Res
olut
ion
Fig: 7.12 Robustness study - Effect of Flow
2.212.312.442.412.36
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0.7 0.8 0.9 1.0 1.1 1.2 1.3
Flow rate mL/min
Res
olut
ion
Fig: 7.13 Robustness study - Effect of Mobile phase composition
208
2.222.312.41 2.43
2.44
1.0
1.5
2.0
2.5
3.0
3.5
4.0
85 90 95 100 105 110 115
Mobile phase composition (%Methanol)
Reso
lutio
n
7.3 Stability study of (R, R)-tadalafil bulk drug as per ICH Conditions
(Q1AR2):
One manufacturing lot of (R, R)-tadalafil drug substance was placed for
stability study in chambers maintained at ICH defined conditions. Long
term (25°C+ 2°C/ 60% RH + 5%RH) and accelerated (40°C+ 2°C/ 75% RH
+ 5%RH) stability study was carried out for (R, R)-tadalafil bulk drug and
content of enantiomer of (R, R)-tadalafil was monitored in the stability
samples using the developed HPLC method conditions. The analysis of
stability samples was carried up to 12 months period. The enantiomer of
(R, R)-tadalafil was absent in drug substance sample considered for
stability studies. The enantiomer of (R, R)-tadalafil was not observed in
both long term and accelerated stability conditions. The developed HPLC
209
method performed satisfactorily for the quantitative evaluation of stability
samples.
7.4 Summary and conclusions of the present study:
A new chiral RPLC method was developed for chiral separation of (R,
R)-tadalafil and its enantiomer in drug substance and drug product under
ICH recommended conditions. (R, R)-tadalafil and its enantiomer was
baseline resolved (Rs > 2.2) on Chirobiotic-T column. The method is found
to be linear from LOQ to 200%. The method is precise, accurate, rugged
and robust. Both (R, R)-tadalafil and its enantiomer was stable in sample
solution for a study period of 48h. Mobile phase was stable for a study
period of 48 h. The method was completely validated showing satisfactory
data for all the method validation parameters tested (Table 7.18). Thus the
developed method can be employed for the quantitative determination of
(R, R)-tadalafil and enantiomer of (R, R)-tadalafil in drug substance, drug
product and in-process materials.
Table: 7.17 Summary of the Validation results
Validation parameter Enantiomer of (R,R)-tadalafil
Precision (RSD %)
Retention timeArea
0.0040.89
210
LOD-LOQ
Limit of detection (µg mL-1)Limit of quantification (µg mL-1)
Precision at LOQ (RSD %)Accuracy at LOQ (%Recovery)
Drug substanceDrug product
0.0130.0324.5
100.2100.6
Linearity
Calibration range (%)Calibration points
Correlation coefficientCalibration equation
Slopeintercept
5.33-2009
0.9999321.737x-143.85
321.74-143.85
Accuracy (%Recovery)
Drug substanceDrug product
98.0-100.897.8-102.5
Robustness Resolution betweenenantiomer of (R, R)-tadalafil and[R, R)-tadalafil is greater than 2.2
Solution Stability/ Mobile phase Stability
Stable up to 48h
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