Chapter-4
Analysis of Duloxetine hydrochloride and its related compounds in Pharmaceutical dosage forms and in-Vitro Dissolution studies by stability indicating UPLC
___________________________________________
4.1 Introduction of Duloxetine hydrochloride and survey of
analytical methods
Duloxetine hydrochloride is indicated for the treatment of major
depressive disorder (MDD). Its chemical designation is (+)-(S)-N-methyl-
β-(1-naphthyloxy)2-thiophenepropylamine hydrochloride. It is available
as 20 mg, 30 mg and 60 mg as capsules. It is a white to slightly
brownish white solid, which is slightly soluble in water. The molecular
formula of Duloxetine hydrochloride is C18H19NOS•HCl and molecular
weight of Duloxetine hydrochloride is 333.88
Fig: 4.1.F1: Chemical structure of Duloxetine hydrochloride
Molecular formula: C18H19NOS•HCl, Molecular weight : 333.88
Duloxetine is indicated for the acute and maintenance treatment of
major depressive disorder (MDD), as well as acute management of
generalized anxiety disorder. Also used for the management of
neuropathic pain associated with diabetic peripheral neuropathy, and
fibromyalgia. It has been used in the management of moderate to severe
stress urinary incontinence (SUI) in women. Duloxetine lacks affinity for
monoamine receptors within the central nervous system. It is a potent
inhibitor of neuronal serotonin and norepinephrine reuptake and a less
potent inhibitor of dopamine reuptake. The mechanism of action of
Duloxetine in SUI has not been determined, but is thought to be
associated with the potentiation of serotonin and norepinephrine activity
in the spinal cord, which increases urethral closure forces and thereby
reduces involuntary urine loss.
The aim of present study was to develop a new analytical method for
the determination of related substances and for quantitative estimation
of Duloxetine hydrochloride along with in vitro dissolution studies in
pharmaceutical dosage forms by liquid chromatography LC to establish
inherent stability of Duloxetine through stress studies under a variety of
ICH recommended test conditions. In the present study, the additional
benefit of in vitro dissolution studies is, Duloxetine degrades rapidly in
acidic pH conditions. As per regulatory requirement stability indicating
LC methods are required for acidic dissolution mediums. Hence
developed UPLC is useful for estimation of drug release in presence of
degradation impurities. Duloxetine has been determined in
pharmaceutical preparations by LC [1, 2], by spectrophotometric [3] and
by LCMS [4, 5, 6] methods. No liquid chromatographic method was
reported in major pharmacopeia’s like USP, EP, Japanese Pharmacopeia
and British Pharmacopeia. Literature survey reveals that no stability-
indicating liquid chromatographic method was reported for
determination of related substances and for quantitative estimation of
Duloxetine hydrochloride along with in vitro dissolution studies in
pharmaceutical dosage forms.
Hence a reproducible stability-indicating reverse phase UPLC method
was developed for the quantitative determination of Duloxetine
hydrochloride and its three impurities. The method was also applied for
study of in-vitro dissolution profiles in pharmaceutical dosage forms.
4.2. Development of a new analytical method for the determination
of related components and assay of Duloxetine hydrochloride in
pharmaceutical dosage forms by UPLC
4.2.1 Materials
Standards of Duloxetine Hydrochloride, Impurity-1, Impurity-2 and
Impurity-3, and Duloxetine 60 mg capsules were supplied by Dr. Reddy’s
laboratories limited, Hyderabad, India. The HPLC grade acetonitrile,
methanol, tertahydrofuran and analytical grade KH2PO4 and ortho
phosphoric acid were purchased from Merck, Darmstadt, Germany.
Water was prepared by using Millipore MilliQ Plus water purification
system.
4.2.2 Equipment
Acquity UPLCTM system (Waters, Milford, USA) we used consists of a
binary solvent manager, a sample manager and a photodiode array (PDA)
detector. The output signal was monitored and processed using
empower2 software. Cintex digital water bath was used for hydrolysis
studies. Photo stability studies were carried out in a photo stability
chamber (Sanyo, Leicestershire, UK). Thermal stability studies were
performed in a dry air oven (Cintex, Mumbai, India).
4.2.3 Chromatographic Conditions
LC was carried out on a Waters Aquity UPLC with photodiode array
detector. The output signal was monitored and processed using
empowers software. The chromatographic column used was Zorbax XDB
C-18, 50 mm x 4.6 mm i.d with 1.8 µm particles. The separation was
achieved on a gradient method. The mobile phase A contains a mixture of
0.01 M KH2PO4 (pH 4.0) buffer, tetrahydro furan and methanol in the
ratio 67: 23: 10 (v/v/v); respectively and the mobile phase B contains a
mixture of 0.01 M KH2PO4, (pH 4.0) buffer and acetonitrile in the ratio
60: 40 (v/v); respectively.
The flow rate of mobile phase was 0.6 mL min-1. The UPLC gradient
program was set as: time (min) / % solution B: 0/0, 6.0/0, 8.0/100,
13.0/100, 14.0/0 and 16.0/0. The column temperature was maintained at 40 ºC and the detection was monitored at a wavelength 236 nm. The
injection volume was 5 µL.
Fig: 4.2.F1: Chemical structures of Duloxetine hydrochloride
impurities
Impurity-1
Molecular formula: C18H19NOS•HCl
Molecular weight : 333.88
Impurity-2
Molecular formula: C18H19NOS•HCl
Molecular weight : 333.88
Impurity-3
Molecular formula: C10H8O
Molecular weight : 144.17
4.2.4 Sample preparation
A stock solution of Duloxetine hydrochloride (2.0 mg mL-1) was
prepared by dissolving an appropriate amount of drug in diluent (methanol: water 90:10 (v/v); respectively). Working solutions containing
200 and 20 μg mL-1 were prepared from this stock solution for
determination of related substances and for assay determination,
respectively. A mixed stock solution (0.5 mg mL-1) of the impurities (denoted Impurity-1 to Impurity-3) was also prepared in methanol.
4.2.5 Preparation of tablets sample solution
Duloxetine hydrochloride is a capsule conatins pellets containing 60 mg of Duloxetine. Twenty capsules of Duloxetine (60 mg) were
individually weighed to get the average weight of the pellets. An amount
of pellets equivalent to 20 mg of Duloxetine was transferred to 100 mL volumetric flask, added 70 mL of methanol: water 90:10, v/v (diluent)
sonicated for 20 min and diluted to 100 mL (200 µg mL-1). The resulting
solution was centrifuged at 3,000 RPM for 15 min (supernatant solution was used for purity evaluation). 10 mL of supernatant solution was
transferred and diluted to 100 mL with diluent (20 µg mL-1). This
solution was filtered using 0.45 µm nylon 66-membrane filter and used
for the assay analysis.
4.2.6 Generation of stress samples
One lot of Duloxetine hydrochloride capsules (60 mg) were selected for
stress testing. From the ICH Stability guideline: “stress testing has to be
carried out on a single lot of material. Different kinds of forced
degradation conditions (i.e., heat, humidity, acid, base, oxidative and
light) were employed on one lot of Duloxetine capsules based on the
guidance available from ICH Stability Guideline (Q1AR2). The details of
the stress conditions used are as follows:
a) Acid hydrolysis:
Drug solution in 1N HCl was exposed at 60°C for 3 h.
b) Base hydrolysis:
Drug solution in 1N NaOH was exposed at 60°C for 3 h
c) Oxidative stress:
Drug solution in 6% H2O2 was exposed at 30°C temperature for 3 h.
d) Water hydrolysis:
Drug solution in water was exposed at 60°C for 3 h.
e) Thermal stress:
Pellets were subjected to dry heat at 60°C for 10 days.
f) Photolytic degradation:
The photo degradation was carried out by exposing the Duloxetine
hydrochloride capsules pellets in solid state to light providing an overall
illumination of not less than 1.2 million lux hours and an integrated near
ultraviolet energy of not less than 200 W h/m2, which took about 10
days period in photo stability chamber.
4.3 Method development and optimization of chromatographic
conditions
Forced degradation studies were performed to develop a stability
indicating UPLC method for the quantitative determination and purity
evaluation of Duloxetine hydrochloride capsules. Stressed samples
obtained during forced degradation studies including the samples of
impurity-1, impurity-2 and impurity-3 were utilized in the UPLC method
development.
4.3.1 Selection of wavelength
Impurities and Duloxetine hydrochloride solutions were prepared in
diluents and scanned in UPLC PDA detector; all the impurities and
Duloxetine are having UV maxima at about 236 nm (Fig: 4.3.F1). Hence
detection at 236 nm was selected for method development purpose.
Fig: 4.3.F1: Typical UV spectrums of Duloxetine and its impurities.
4.3.2 Column Selection
The main target of the chromatographic method was to get the
separation among impurities namely impurity-1, impurity-2 and
impurity-3 and degradation products (mainly at 0.87 RRT) from
Duloxetine peak and elution of Duloxetine symmetrical peak. For initial
screening trials the mobile phase used was a mixture of 0.01 M KH2PO4
(pH 4.0) buffer, tetrahydro furan and methanol in the ratio 67: 23: 10
(v/v/v); respectively, the flow rate was 0.6 mL min-1. Inertsil ODS-3,
50 mm × 2.1 mm, 2μm particles, Zorbax XDB C-18, 50 mm x 4.6 mm i.d
with 1.8 µm particles and Waters Aquity BEH C18, 50 x 2.1 mm; 1.7μm
columns were used with isocratic elution.
Ab
s
or
b
a
nc
e
Overlay spectra of Duluxetine
and its impurities
For Inertsil ODS-3, 50 mm × 2.1 mm, 2μm particles column,
impurity-2 was co-eluted with Duloxetine peak and also impurity-3 was
eluted as a broad peak. For Waters Aquity BEH C18, 50 x 2.1 mm; 1.7
μm column, impurity-2 was co-eluted with Duloxetine peak and the
resolution between Duloxetine and degradation product at 0.87 RRT was
poor. For Zorbax XDB C-18, 50 mm x 4.6 mm with 1.8 µm particles
column, impurity -1, degradation product at 0.87 RRT, impurity-2 and
impurity-3 were well separated from Duloxetine peak, but impurity-3
was strongly retained (RT was at about 29 min). Hence Zorbax XDB C-
18, 50 mm x 4.6 mm with 1.8 µm particles column was chosen for
further method development.
Table 4.3.T1: Experimental results on different stationary phases
Column Parameter Imp-
1 DUL*
Imp-
2
Imp-
3 Remarks
Inertsil ODS-3,
50 mm × 2.1 mm,
2μm particles
Retention
time 2.53 4.03 - 10.93
Imp-2 and
unknown (0.87
RRT) peeks were co-
eluting with
Duloxetine peak
USP Tailing 1.5 1.7 - 0.9
Zorbax XDB C-18,
50 mm × 4.6 mm,
containing 1.8μm particles
Retention time
2.56 3.83 5.00 28.6 Imp-1,
unknown (0.87
RRT), Imp-2
and Duloxetine peaks are well
separated from
each other. But Imp-3
USP Tailing 1.1 1.7 1.3 1.1
retention time
was about 29
min
Waters Aquity BEH C18, 50 x
2.1 mm; 1.7μm
column
Retention
time 1.42 2.26 - 9.39
Imp-2 peak was
co-eluting with
Duloxetine and unknown peak
at RRT 0.87 is
partially merged
with Duloxetine peak
USP Tailing 0.9 1.8 - 1.6
*Duloxetine
4.3.3 Effect of pH
To get better separation of impurities namely impurity-1, impurity-2,
impurity-3 and degradation products (mainly the unknown impurity at
0.87 RRT) from Duloxetine and to elute the impurities and Duloxetine as symmetrical peaks, different pH buffers from pH range 4.0 to 7.0 (pH 4.0
and 7.0) was studied. For pH 4.0 study, the mobile phase was “0.01 M
KH2PO4 buffer, tetra hydro furan and methanol in the ratio 67: 23: 10 (v/v/v), pH adjusted to 4.0 with ortho phosphoric acid”. For pH 7.0
study, the mobile phase was “0.01 M ammonium acetate buffer, tetra
hydro furan and methanol in the ratio 67: 23: 10 (v/v/v), pH adjusted to
7.0 with aqueous ammonia solution”. For above all pH study column used was Zorbax XDB C-18, 50 mm × 4.6 mm, 1.8 μm and flow rate was
0.6 mL min-1
At pH 7.0, Peak distortion of Duloxetine peak was noticed and co-
elution of impurity-2 with Duloxetine was observed and also impurity-3
was strogly retained (RT was about 19 min) in the column. At pH 4.0,
impurity-1, unknown (0.87 RRT), impurity-2 and Duloxetine peaks are
well separated from each other. But impurity-3 was strongly retained (RT
was about 29 min). Since impurity-3 was strongly retained when by pH
decreased from 7.0 to 4.0, no further reduction in the pH was studied.
Hence pH 4.0 was selected for method pH. To elute impurity-3 faster,
elution mode was switched to gradient elution mode from isocratic mode.
4.3.4 Effect of Column Temperature
The effect of column oven temperature was studied from 25° C to
40°C, the symmetry of Duloxetine peak was 1.8 at 25°C. When
temperature was increased to 40°C, the symmetry of Duloxetine peak
was improved to 1.4. Hence column temperature was chosen at 40°C.
4.3.5 Effect of Organic solvent
When “mixture 0.01 M KH2PO4 buffer, tetra hydro furan and
methanol in the ratio 67: 23: 10 (v/v/v), pH adjusted to 4.0 with ortho
phosphoric acid” was used as mobile phase (solvent A) in isocratic
elution, the compound impurity-3 was strongly retained in the column.
To elute impurity-3 faster, elution mode was changed to gradient mode
with solvent B (mixture of 0.01 M KH2PO4, (pH 4.0) buffer and
acetonitrile in the ratio 60: 40 (v/v); respectively).
When acetonitrile was used as a solvent in solvent A (buffer: tetra
hydro furan: acetonitrile: 67: 23: 10, v/v/v) instead of methanol, the
resolution between Duloxetine and impurity-2 and the resolution
between unknown impurity at RRT 0.87 and Duloxetine was poor.
Ratio’s of methanol and tetrahydrofuran in solvent A were optimized to
achieve good resolution between Duloxetine and impurity-2, and between
unknown impurity (at RRT 0.87) and Duloxetine peaks.
When methanol was used as a solvent in solvent B (buffer: methanol:
60:40, v/v) instead of acetonitrile, the retention time of impurity-3 was
shifted towards higher side (13 min) due to high interactions of impurity-
3 with column (lipophilicity). To improve the retention time of impurity-3,
the strength of the solvent was increased by replacing methanol with
acetonitrile. Different mixtures of buffer and acetonitrile were applied to
improve the retention time and symmetry of impurity-3. When 40%
acetonitrile was used as a solvent in solvent B retention time of impurity-
3 was improved (9.8 min) and symmetry of impurity-3 was also
improved.
Fig. 4.3.F1: Typical chromatograms from method development trials
4.3.6 Optimized chromatographic conditions for the determination
of related substances and assay of Duloxetine
Column : Zorbax XDB C-18, 50 mm × 4.6 mm,
containing 1.8 μm particles.
Elution : Gradient.
Mobile phase-A : Buffer, tetra hydro furan and methanol in the
ratio 67: 23: 10 (v/v/v).
Mobile phase-B : Buffer and acetonitrile in the ratio 60: 40 (v/v)
Flow rate : 0.6 mL min-1
Column temperature : 40°C
Wavelength of detection : 236 nm
Injection volume : 5 L
Run time : 16 min
Diluent : methanol: water 90:10 (v/v)
Gradient Program : Time (min)/ % solution B: 0.01/0, 6.0/0,
8.0/100, 13.0 /100, 14.0/0 and 16.0/0
Buffer : 0.01 M KH2PO4 buffer pH adjusted to 4.0 with
orthophosporic acid Retention time : Duloxetine about 4.15 min
Relative Retention Time : Impurity-1 - about 0.65
Impurity-2 - about 1.31
Impurity-3 - about 2.36
The interference from excipients gelatin, hypromellose, hydroxypropyl
methylcellulose acetate succinate, sodium lauryl sulfate, sucrose, sugar
spheres, talc, titanium dioxide, and triethyl citrate was also checked by injecting sample solutions of excipients. There was no interference found
at retention times of impurities (impurity-1, impurity-2 and impurity-3)
and Duloxetine peak.
4.4 Degradation behavior
UPLC studies on Duloxetine under different stress conditions
suggested the following degradation behavior.
4.4.1 Degradation in Acidic solution
Duloxetine underwent degradation in 1N HCl on heating at 60°C for 3
h. Major degradation was observed with formation of impurity-1,
impurity-3 and unknown impurity at RRT 0.87 (3.392 RT) (Fig: 4.4.F1(a) to Fig: 4.4.F1(b)).
Fig: 4.4.F1 (a): Typical UPLC chromatogram of acid hydrolysis
Fig: 4.4.F1 (b): Peak purity plot of Duloxetine under Acid
hydrolysis
Purity Angle Purity
Threshold Purity Flag Peak Purity
0.072 0.271 No Pass
4.4.2 Degradation in Basic solution:
When Duloxetine HCl pellets were exposed to 1 N NaOH at 60°C for 3 h, minor degradation was observed with formation of impurity-3 (Fig:
4.4.F2 (a) to Fig: 4.4.F2 (b)).
Fig: 4.4.F2 (a): Typical UPLC chromatogram of alkali hydrolysis
Fig: 4.4.F2 (b): Peak purity plot of Duloxetine under alkali
hydrolysis
Purity Angle Purity
Threshold Purity Flag Peak Purity
0.182 0.322 No Pass
4.4.3 Oxidative conditions
Duloxetine HCl pellets were exposed to 6% Hydrogen peroxide at 30
ºC for 3 h. Duloxetine has shown no significant sensitivity towards the
treatment of Hydrogen peroxide (Fig: 4.4.F3 (a) to Fig: 4.4.F3 (b)).
Fig: 4.4.F3 (a): Typical UPLC chromatogram of oxidative degradation
Fig: 4.4.F3 (b): Peak purity plot of Duloxetine under oxidative
degradation
Purity Angle Purity
Threshold Purity Flag Peak purity
0.160 0.341 No Pass
4.4.4 Degradation in Neutral (Water) solution
When Duloxetine HCl pellets were exposed to water at 60°C for 3 h,
major degradation products were observed with the formation of
impurity-1, impurity-3 and unknown impurity at RRT 0.87 (3.177 RT)
(Fig: 4.4.F4 (a) to Fig: 4.4.F4 (b)).
Fig: 4.4.F4 (a): Typical UPLC chromatogram of Water hydrolysis
Fig: 4.4.F4 (b): Peak purity plot of Duloxetine under water hydrolysis
Purity Angle Purity
Threshold Purity Flag Peak purity
0.198 0.274 No Pass
4.4.5 Photolytic conditions
When the drug pellets was exposed to light for an overall illumination
of 1.2 million lux hours and an integrated near ultraviolet energy of 200-
watt hours/square meter (w/mhr), no degradation of the drug was
observed (Fig: 4.4.F5 (a) to Fig: 4.4.F5 (b)).
Fig: 4.4.F5 (a): Typical UPLC chromatogram of Photo degradation
Fig: 4.4.F5 (b): Peak purity plot of Duloxetine under photo
degradation
Purity Angle Purity
Threshold Purity Flag Peak purity
0.112 0.251 No Pass
4.4.6 Thermal Degradation
Duloxetine HCl is stable for heat when drug pellets were exposed to
dry heat at 60°C for 10 days (Fig: 4.4.F6 (a) to Fig: 4.4.F6 (b)).
Fig: 4.4.F6 (a): Typical UPLC chromatogram of thermal degradation
Fig: 4.4.F6 (b): Peak purity of Duloxetine under thermal degradation
The unknown impurity (0.87 RRT) which was formed in acid
hydrolysis and aqueous hydrolysis was investigated with LCMS study.
The mass of unknown impurity was 298.4 and the mass of Imp-2 (4-(3-
methylamino)-1-(thiophen-2-yl) propyl) naphthalin-1-ol) was 298 and the UV spectra of Impurity-2 was similar to that of unknown impurity.
Hence, based LCMS data and UV spectra, the possible chemical name of
the unknown impurity could be ‘2-(3-methylamino)-1-(thiophen-2-yl) propyl) naphthalin-1-ol. The proposed structure of unknown impurity
was positional isomer of Impurity-2.
Fig: 4.4.F7: Typical UV spectrum of unknown impurity at RRT 0.87
Purity Angle Purity
Threshold Purity Flag Peal purity
0.112 0.250 No Pass
Fig: 4.4.F8: LC-Mass spectrum of unknown impurity at RRT 0.87
Peak purity test performed for the Duloxetine peak using photodiode
array (PDA) detector, data confirmed the purity of the peak for all the
stressed samples. Assay of all stressed samples were calculated using
qualified standard of Duloxetine Hydrochloride. Considering the purities
from the respective chromatograms of stressed samples, mass balance
(%assay + % degradents + % impurities) was calculated for each stress
sample. The mass balance of stressed samples was more than 99.1%.
This clearly demonstrates that the developed UPLC method was found to
be specific for Duloxetine in presence of its impurities (impurity-1,
impurity-2 and impurity-3) and degradation products.
4.5 Analytical method Validation, results and discussion
The developed and optimized UPLC method was taken up for
validation. The analytical method validation was carried out in
accordance with ICH guidelines [7, 8].
4.5.1 System Suitability Test
Duloxetine standard, impurity-1, impurity-2 and impurity-3 were
spiked at 0.30% level with respect to the concentration of Duloxetine in
diluent and injected for five times into UPLC system. Resolution between
impurities and Duloxetine, tailing factor for impurities and Duloxetine,
RSD% for the areas of impurities and Duloxetine was calculated. Good
resolution was obtained between impurities and Duloxetine (Fig: 4.5.F1).
System suitability results were tabulated in Table 4.5.T1.
Fig: 4.5.F1: Typical UPLC chromatogram of system suitability
Table 4.5.T1: System Suitability results
Compound
(n=5)
Resolution (Rs) USP
Tailing factor (T)
RSD%
for area
Impurity-1 -- 1.1 0.4
Duloxetine 3.7 1.1 0.3
Impurity-2 2.5 1.3 0.8
Impurity-3 22.3 1.1 0.5
n = Number of determinations
4.5.2 Precision
Assay method precision (repeatability) study was evaluated by
carrying out six independent assays of Duloxetine hydrochloride test
sample against qualified reference standard and RSD of six consecutive
assays was 0.15% (Table 4.5.T2). Results showed insignificant variation
in measured response which demonstrated that the method was
repeatable with RSDs below 0.2%.
Table 4.5.T2: Precision results of the assay method
Preparation % Assay
1 99.9
2 99.8
3 100.1
4 99.7
5 99.9
6 99.7
Mean 99.9
SD 0.1516
% RSD 0.15
95% Confidence interval of mean 99.8 to 100.0
The precision of the related substance method was evaluated by
injecting six individual preparations of Duloxetine (0.2 mg mL-1) spiked with 0.30% of impurity-1, impurity-2 and impurity-3 with respect to
Duloxetine analyte concentration. The % RSD for area of impurity-1,
impurity-2 and impurity-3 for six consecutive determinations was below
2.0% (Table 4.5.T3).
Results showed insignificant variation in measured response which
demonstrated that the related substances method was repeatable with
RSDs below 2.0%.
Table 4.5.T3: Precision results of the RS method
Preparation Impurity-1 Impurity-2 Impurity-3
1 22350 16070 46126
2 22377 15886 46126
3 22772 15902 45210
4 22925 15932 45415
5 22715 16048 45525
6 22151 15652 43827
Mean 22548 15915 45340
SD 298.7 149.8 814.4
% RSD 1.3 0.9 1.8
Intermediate precision for assay and related substances method was
performed by carrying out six independent assays of Duloxetine
hydrochloride test sample against qualified reference standard and
calculated %RSD of six consecutive assays. Related substances method
was performed by injecting six individual preparations of Duloxetine (0.2
mg mL-1) spiked with 0.30% impurity-1, impurity-2 and impurity-3 with
respect to Duloxetine analyte concentration over different days, different
instruments and with different analysts (Table 4.5.T4).
Table 4.5.T4: Results of repeatability and Intermediate precision
S.No
Parameter
Variation
%RSD
for Assay
%RSD
for Related Substances
1
Repeatability
(a) Analyst-1
(b) Waters Acquity
UPLC system with PDA detector.
(c) Day-1
<0.50%
< 2.0%
2
Intermediate
precision
(a) Analyst-2
(b) Waters Acquity
UPLC system with TUV detector.
(c) day-2
<0.50%
< 2.0%
4.5.3 Limit of quantification (LOQ) and limit of detection (LOD)
LOQ and LOD were established for Duloxetine and for impurity-1,
impurity-2 and impurity-3 based on signal to noise ratio (S/N) method.
4.5.3.1 Limit of quantification (LOQ)
The quantification limits were established by S/N methodology and
established concentration values are reported in Table 4.5.T5.
Table 4.5.T5: LOQ values for Duloxetine and its impurities
S.No Impurity
name Concentration
Signal to noise
ratio
1 Duloxetine 0.080 µg mL-1 9.6
2 Impurity-1 0.026 µg mL-1 10.4
3 Impurity-2 0.054 µg mL-1 9.5
4 Impurity-3 0.028 µg mL-1 9.8
4.5.3.2 Limit of detection (LOD)
The detection limits were established by S/N methodology and
established concentration values are reported in Table 4.5.T6.
Table 4.5.T6: LOD values for Duloxetine and its impurities
S.No Impurity name Concentration Signal to noise
ratio
1 Duloxetine 0.020 µg mL-1 3.1
2 Impurity-1 0.010 µg mL-1 3.3
3 Impurity-2 0.020 µg mL-1 2.5
4 Impurity-3 0.010 µg mL-1 3.4
4.5.4 Precision at Limit of quantification
The precision of the related substance method was also checked by
injecting six individual preparations of impurity-1, impurity-2 and
impurity-3 at their LOQ level with respect to Duloxetine analyte concentration. Results showed insignificant variation in measured
responses which demonstrate that the method was repeatable at LOQ
level with RSD below 2.3 % (Table 4.5.T7).
Table 4.5.T7: Precision results of the RS method at LOQ level
Preparation Impurity-1 Impurity-2 Impurity-3
1 1035 1496 2143
2 1073 1558 2155
3 1032 1489 2171
4 1089 1466 2206
5 1051 1531 2185
6 1033 1492 2133
Mean 1052.1 1503.6 2165.5
SD 23.92 31.87 27.28
% RSD 2.3 2.1 1.3
4.5.5 Accuracy at LOQ level
The recovery studies for impurity-1, impurity-2 and impurity-3 were
carried out in triplicate at LOQ level of the Duloxetine target analyte
concentration (200 µg mL-1). The percentage recovery of impurity-1, impurity-2 and impurity-3 was calculated (Table 4.5.T8). The method
showed consistent and high absolute recoveries at LOQ level with mean
absolute recovery ranging from 94.8 % to 98.5%.
Table 4.5.T8: Recovery at LOQ level
S.No
Impurity name
Mean recovery
(%)(n = 3 )
%RSD
1 Impurity-1 98.5 2.7
2 Impurity-2 94.8 2.2
3 Impurity-3 98.4 2.7
4.5.6 Linearity
4.5.6.1 Linearity of the assay method
The linearity of the assay method was established by injecting test
sample at 10%, 50%, 75%, 100%, 125% and 150% of Duloxetine assay
concentration (i.e.100 µg mL-1). Each solution was injected into UPLC
system (Table 4.5.T9). Calibration curve obtained by least square
regression analysis between peak area and the concentration showed
(Fig: 4.5.F2 and Table 4.5.T10) linear relationship with a regression
coefficient of 0.999. The best fit linear equation obtained was y =
30204.95 x con + 2701.18.
Table 4.5.T9: Linearity results of the assay method
Concentration
(µg mL-1)
Peak area
0.2 6062
10 303123
15 462023
20 607522
25 760123
30 903890
Correlation
Coefficient(r) 0.99992
Slope 30204.95
Intercept 2701.18
Fig: 4.5.F2: Linearity plot for Assay Method
Table 4.5.T10: Residual summary of Linearity results of Duloxetine
% of impurity
[w.r.t 200
µg/ml]
Mean area
Response chieved
Response calculated
thru Trend
line equation
Residual
(Response
practical -Response
theoretical)
Residual
square
0.2 6062 6684.55 623 387572.7
10 303123 302693.06 -430 184850.8
15 462023 453717.80 -8305 68976280.7
20 607522 604742.55 -2779 7725337.7
25 760123 755767.30 -4356 18972142.9
Residual sum of squares
192492369.67
Trend line equation y = 30204.95 x con + 2701.18
4.5.6.2 Linearity of the related substances method
Linearity experiments were carried out by preparing the Duloxetine sample solutions containing impurity-1, impurity-2 and impurity-3 from
LOQ to 150% (i.e. LOQ, 50, 100, 125 and 150%) with respect to their
specification limit 0.30% (Table 4.5.T11).
Calibration curve was drawn by plotting average area of the impurity (impurity-1, impurity-2 and impurity-3) on the Y-axis and concentration
on the X-axis (Fig: 4.5.F3 to Fig: 4.5.F5) which showed linear relation
ship with a regression coefficient of greater than 0.998 for all impurities.
Analysis of residuals indicated (Table 4.5.T12 to Table 4.5.T14) that the residuals were normally distributed around the mean with uniform
variance across all concentrations suggesting the homoscedastic nature
of data.
Table 4.5.T11: Linearity results of the RS method
S.No
% of impurity
w.r.t 200
µg/ml
Impurity-1
(Peak area)
Impurity-2
(Peak area)
Impurity-3
(Peak area)
1 LOQ 1037 1468 2155
2 0.15 12724 8523 21822
3 0.30 22620 15247 44727
4 0.375 27642 19151 56901
5 0.45 32773 22000 64105
Correlation
coefficient (r) 0.998439 0.998326 0.998558
Intercept 897.8831 656.335 374.1549
Slope 71666.6 48469.53 145724.8
Fig: 4.5.F3: Linearity plot for impurity-1
Linearity graph of Impurity-1
y = 71667x + 897.88
R2 = 0.9969
0
5000
10000
15000
20000
25000
30000
35000
0 0.1 0.2 0.3 0.4 0.5
% impurity w .r.t test concentration
Mean
are
a
Fig: 4.5.F4: Linearity plot for impurity-2
Linearity graph of Impurity-2
y = 48470x + 656.33
R2 = 0.9967
0
5000
10000
15000
20000
25000
0 0.1 0.2 0.3 0.4 0.5
% impurity w .r.t test concentrationM
ean
are
a
Fig: 4.5.F5: Linearity plot for impurity-3
Linearity graph of Impurity-3
y = 145725x + 374.15
R2 = 0.9971
0
10000
20000
30000
40000
50000
60000
70000
0 0.1 0.2 0.3 0.4 0.5
% impurity w .r.t test concentration
Mea
n a
rea
Table 4.5.T12: Residual summary of Linearity results of impurity-1
% of
impurity [w.r.t 200
µg/ml]
Mean
area Response
chieved
Response calculated
thru Trend
line equation
Residual (Response
practical -
Response theoretical)
Residual square
0.013 1037 1829.5 793 628133.9
0.15 12724 11647.9 -1076 1158048.1
0.30 22620 22397.9 -222 49344.4
0.375 27642 27772.9 131 17124.1
0.45 32773 33147.9 375 140515.8
Residual sum of squares
1993166.36
Trend line equation y = 71667x + 897.88
Table 4.5.T13: Residual summary of Linearity results of impurity-2
% of
impurity
[w.r.t 200
µg/ml]
Mean area
Response achieved
Response
calculated
thru Trend line
equation
Residual
(Response
practical -Response
theoretical)
Residual
square
0.027 1468 1965.0 497 247021.2
0.15 8523 7926.8 -596 355497.2
0.30 15247 15197.2 -50 2480.7
0.375 19151 18832.4 -319 101500.9
0.45 22000 22467.6 468 218670.8
Residual sum of squares
925170.77
Trend line equation y = 145725x + 374.15
Table 4.5.T14: Residual summary of Linearity results of impurity-3
% of
impurity [w.r.t
200
µg/ml]
Mean area Response
achieved
Response
calculated thru
Trend line
equation
Residual
(Response practical -
Response
theoretical)
Residual
square
0.027 2155 2414.3 259 67237.3
0.15 21822 22232.9 411 168814.2
0.30 44727 44091.6 -635 403752.0
0.375 56901 55020.9 -1880 3534615.1
0.45 64105 65950.3 1845 3405133.5
Residual sum of squares
7579552.06
Trend line equation y = 145725x + 374.15
4.5.7 Accuracy
4.5.7.1 Accuracy of the assay method
Accuracy of the assay method was established by injecting three preparations of test sample using Duloxetine hydrochloride capsules 60
mg at 50%, 100% and 150% of analyte concentration (20 µg mL-1). Each
solution was injected trice (n=3) into UPLC system and the mean peak
area of Duloxetine peak was calculated. Assay of test solution was
determined against three injections (n=3) of qualified Duloxetine standard. The method showed consistent and high absolute recoveries at
all three concentration (50,100 and 150%) levels with mean absolute
recovery ranging from 99.6 % to 101.2% (Table 4.5.T15). The obtained absolute recoveries were normally distributed around the mean with
uniform RSD values.
4.5.7.2 Accuracy of the RS method
Accuracy of the related substances method established at 50%, 100% and 150% of the impurities specification limit (0.20%). Test solutions
were prepared in triplicate (n=3) with impurities (impurity-1, impurity-2
and impurity-3) at 0.15%, 0.30% and 0.45% level w.r.t. analyte concentration (0.2 mg mL-1). Mean % recovery of impurities calculated in
the test solution using the area of impurities standard at 0.30% level
with respect to analyte (Table 4.5.T15).
Table 4.5.T15: Recovery results
Amount
spiked
% Recovery
Mean ± % RSD
Duloxetine Impurity-1 Impurity -2 Impurity -3
50%
101.2±0.12 100.5±1.4 93.3±1.7 99.7±1.3
100 %
99.6±0.11 101.6±1.8 96.8±2.1 98.6±2.1
150 %
100.4±0.15 104.5±1.2 96.2±0.6 98.6±2.6
The related substances method showed consistent and high absolute
recoveries of all five impurities at all three concentration (50,100 and 150%) levels with mean absolute recovery ranging from 93.3 % to 104.5%
in drug product.
4.5.8 Solution state stability
The solution state stability of Duloxetine in diluent in the assay method was carried out by leaving the test solutions of sample in tightly
capped volumetric flasks at room temperature for two days. The same
sample solution were assayed for every 24 hours interval up to the study period, each time freshly prepared reference standard was used to
estimate the assay of test sample. The % RSD of assay of Duloxetine
during solution stability experiments was with in 1.0%.
The solution stability of Duloxetine in the related substance method
was carried out by leaving sample solution in tightly capped volumetric flask at room temperature for two days. Content of impurity-1, impurity-
2, and impurity-3 were checked for every 24 hours interval up to the
study period. No significant change was observed in the impurity content during solution stability experiments from the initial values up to the
study period. Hence Duloxetine sample solutions are stable for at least
48 hours in the developed method.
Mobile phase stability of the developed method was established by injecting fresh samples for 48 hours. Fresh sample solution was assayed
for every 24 hours interval up to the study period, each time freshly
prepared reference standard was used to estimate the assay of sample. The % RSD of assay of Duloxetine during solution stability experiments
was with in 1.0.
Table 4.5.T16: Solution stability results of the assay method
S.No Interval % Assay
Solution stability
% Assay
Mobile phase stability
1 0 h 99.7 99.5
2 24 h 99.2 99.0
3 48 h 98.7 99.4
% RSD 0.50 0.26
4.5.9 Robustness
To determine the robustness of the developed method experimental
conditions were purposely altered and the resolution between impurity-1
and Duloxetine and between Duloxetine and impurity-2 were evaluated.
In each of the deliberately altered chromatographic condition (flow rate
0.5 mL min-1 and O.7 mL min-1, varying column temperature from 35°C
to 450C), varying pH of mobile phase by −1 to +1 units), the resolution
between impurity-1, Duloxetine, impurity-2 and impurity-3 was greater
than 2.4 and tailing factor for Duloxetine and its impurities was less
than 1.3, illustrating the good robustness of the method.
4.5.10 Mass balance
The stressed samples of Duloxetine hydrochloride drug product were
assayed against the qualified reference standard and the results of mass
balance obtained were very close to 99%. The results of mass balance obtained in each condition are presented below (Table 4.5.T17).
Table 4.5.T17: Mass balance of the assay method
Stress condition Time
~%
Degradation
% Assay of
active substance
Mass balance
(%Assay +
%impurities
+% Degradation
products)
Acid hydrolysis
(1 N HCl,60°C ) 3 h 40.22 % 58.9 99.1
Base hydrolysis
(1 N NaOH, 60°C )
3 h 2.04 % 97.6 99.6
Oxidation (6% H202) 3 h 0.21% 99.6 99.8
Water hydrolysis
(60°C) 3 h 14.22% 84.6 99.3
Thermal (60° C) 10 days 0.13% 99.5 99.6
Light (photolytic
degradation) 10 days 0.21% 99.9 100.1
4.6 In vitro Dissolution Studies by developed stability indication
UPLC method
Dissolution tests are one of the tests most used in the
characterization of drugs and in the quality control of dosage forms. It
was introduced in the 1960’s and it is evolved into a test that
pharmaceutical manufacturers hope will better predict the in vivo
performance of drug products. In addition, the reliability and
discriminatory capabilities of dissolution methods for delayed release
products has gained much attention in recent years. The most widely
used dissolution tests for delayed release products use 900 or 1000 mL
of an acid medium followed by 900 or 1000 mL of aqueous buffer
medium with USP apparatus I (basket) or apparatus II (paddle) at
rotation speeds of 100 or 50 RPM, respectively. It is desirable to have an
in vitro dissolution method that is sensitive to formulation factors that
affect the dissolution process and in consequence bioavailability. In our
study, release characteristics of Duloxetine hydrochloride capsules were
studied according to the FDA dissolution method (Basket) at 37 C ± 0.5
using 1000 mL of 0.1N HCl (2 h) followed by 1000 mL of 6.8 phosphate
buffer (pH6.8) at 100 RPM rotation speed [9]. In vitro dissolution was
conducted on Duloxetine hydrochloride 60 mg test product and on
Duloxetine hydrochloride 60 mg innovator (Cymbalta) product. At
scheduled time intervals, 1 mL of sample was withdrawn and replaced
with fresh medium. The final solution was filtered through 0.45 µm nylon
membrane filter and then analyzed by the proposed UPLC method.
The dissolution characteristics of Duloxetine hydrochloride are given
Table 4.6.T1 and Fig: 4.6.F1. Analyzing the in-vitro dissolution profile of
test and innovator, it could be concluded that test showed the faster
release rate than that of innovator. Initially at 15 min and at 30 min,
dissolution rate of test product slightly faster than the innovator product,
but from 45 min to 90 min both test and innovator products were
releasing in a similar way. The release of the total amount of Duloxetine
was achieved at 90 min for innovator product; test product was released
completely at 45 min in the medium. Based these observations from test
and innovator products, it could be concluded that initial time points up
to 30 min were critical for in- vitro dissolution mapping. This result was
in agreement with the literature and the dissolution method mentioned
above would be appropriate to simulate the in vitro release of Duloxetine
hydrochloride for innovator and test products.
Table 4.6.T1: Results of In-vitro release profile of Duloxetine
hydrochloride 60 mg capsules for test and Innovator
Time in
minutes
% Dissolution
Test product Innovator
15 54 44
30 91 72
45 93 89
60 93 93
90 93 93
Fig: 4.6.F1: In-vitro release profile of Duloxetine hydrochloride 60
mg capsules for test and Innovator
4.7 Summary and Conclusions
A new reverse phase ultra performance liquid chromatographic
method was developed for assay and impurities estimation Duloxetine
hydrochloride with stability-indicating power for pharmaceutical dosage forms. The proposed developed method is capable of separating all
Duloxetine hydrochloride impurities in presence degradation impurities
formed under forced degradation studies and also proposed method can be employed for assay determination of Duloxetine hydrochloride in
pharmaceutical dosage forms. The column used for development of this
method was Zorbax XDB C-18, 50 mm × 4.6 mm, 1.8 µm column. The detection wavelength was to monitor eluted components was 236 nm.
Duloxetine hydrochloride capsules pellets were exposed to degradation
conditions of acid, oxidative, base, hydrolytic, photolytic and thermal
degradation. The degradation impurities were well separated from Duloxetine peak and its related compounds, showed the stability-
indicating nature and power of the developed method. The developed
UPLC method was also applied for in-vitro dissolution studies for test and innovator products. The developed method was validated as per ICH
guidelines for precision, accuracy, linearity, specificity, limit of detection,
limit of quantification, system suitability and robustness and found to be precise, accurate, linear rugged, robust and specific. Thus the developed
and validated liquid chromatographic method can be used for stability
analysis and production samples analysis of Duloxetine hydrochloride in pharmaceutical dosage forms.
Table 4.7.T1: Summary of analytical method validation
Test Parameter
Related Substance method
Assay
method
Imprity-1 Impurity-2 Impurity-3
Precision (RSD)
1.3 0.9 1.8 0.15
LOD
(µg mL-1) 0.010 0.020 0.010 0.020
LOQ
(µg mL-1) 0.026 0.054 0.028 0.080
Precision
at LOQ(%RSD) 2.3 2.1 1.3 -
Linearity
(Corre coefficient) 0.9984 0.9983 0.9985 0.9999
Accuracy % at
100%
(Drug product)
101.6 96.8 98.6 99.6
Robustness the resolution between Impurity-1, Duloxetine, Impurity-2 and Impurity-3 was greater than 2.4
Solution
Stability
Standard and test solutions were stable for 48 hours at room temperature
Mobile phase
Stability
Mobile phase was stable for 48 hours at room temperature
Specificity
For all stress conditions, purity angle is less than
the purity threshold and there is no purity flag observed for purity results
References:
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[3] M.M. Kamila, N. Mondal and L.K. Ghosh, Pharmazie. 62 (2007)
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