ORIGINAL ARTICLES
3.4. Chromatographic conditions
3.5.6. Robustness
The chromatographic separation was achieved on an Zorabax XDB C18,
50 mm ꢀ 4.6 mm, 5 mm column using as mobile phase a mixture of aqu-
eous 0.1% trifluoroacetic acid, methanol, tetrahydrofuran (60 : 20 : 20, v/v/v).
The mobile phase was filtered and degassed through a nylon membranefil-
ter (pore size 0.45 mm). The flow rate of the mobile phase was 0.8 ml/min.
The column temperature was maintained at 25 ꢁC and detection was set at
a wavelength of 230 nm. The injection volume was 20 mL. The test con-
centration for the related substance analysis was 2.0 mg ꢂ mLꢃ1. The stand-
ard and the test dilutions were prepared in mobile phase.
To determine the robustness of the method developed, the experimental
conditions were deliberately altered and the resolution between duloxetine
HCl, imp-A, imp-B, imp-C, imp-D and imp-E was recorded.
The flow rate of the mobile phase was 1.0 mL ꢂ minꢃ1. To study the effect
of flow rate on the resolution, flow was changed by 0.2 units from 0.8 to
1.2 mL ꢂ minꢃ1. The effect of the column temperature on resolution was
studied at 20 and 30 ꢁC instead of 25 ꢁC. The effect of the percent organic
solvent concentration on resolution was studied by varying acetonitrile
from ꢃ5 to þ5% and the pH of the mobile phase was studied by varying
pH by ––0.2 to þ0.2, while other mobile phase components were held
constant.
3.5. Validation of the method
3.5.1. Specificity
Specificity is the ability of the method to measure the analyte response in
the presence of its potential impurities. The specificity of the LC method
developed for duloxetine HCl was determined in the presence of its impu-
rities, namely imp-A, imp-B, imp-C, imp-D and imp-E. Stress studies were
performed for duloxetine HCl bulk drug to provide an indication of the
stability indicating property and specificity of the proposed method. Forced
degradation under stress conditions of UV light (254 nm), heat (60 ꢁC),
acid (0.5 N HCl), base (0.5 N NaOH) and oxidation (3.0% H2O2) was
used to evaluate the ability of the proposed method to separate duloxetine
HCl from its degradation products. For heat and light studies, the study
period was 48 h, whereas for the acid, base and oxidation studies it was
48 h. Peak purity for the samples under stress conditions was investigated
for the duloxetine HCl peak using a PDA detector.
3.5.7. Solution stability and mobile phase stability
The solution stability of duloxetine HCl and its impurities in the related
substance method was investigated by leaving spiked sample solutions in
tightly capped volumetric flasks at room temperature for 24 h. The con-
tents of imp-A, imp-B, imp-C, imp-D and imp-E were determined every
6 h up to the study period. The mobile phase stability was also investi-
gated for 48 h by injecting the freshly prepared sample solutions every 6 h.
Contents of imp-A, imp-B, imp-C, imp-D, imp-E and imp-F were checked
in the test solutions.
The % R.S.D. for the assay of duloxetine HCl was calculated during the
mobile phase and solution stability experiments.
Acknowledgements: The authors wish to thank all their collegues of the
R & D Department, Inogent Laboratories Private Ltd., Hyderabad, India,
for their support in carrying out this investigation. The authors would also
like to thank Dr. G. Omprakash, Inogent Laboratories Pvt. Ltd, Hyderabad,
for his valuable suggestions.
3.5.2. Precision
The precision of the related substance method was checked by injecting
six individual preparations of duloxetine HCl (2.0 mg ꢂ mLꢃ1) spiked with
0.15% of imp-A, imp-B, imp-C, imp-D and imp-E with respect to duloxe-
tine HCl analyte concentration. The % R.S.D of the area for each of imp-
A, imp-B, imp-C, imp-D and imp-E was calculated.
The intermediate precision of the method was also evaluated using differ-
ent analysts and different instruments in the same laboratory.
References
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and characterisation of a phenolic impurity in a commercial sample of
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3.5.3. Limit of detection (LOD) and limit of quantification (LOQ)
The limit of detection (LOD) and limit of quantification (LOQ) were deter-
mined by the slope method by injecting a series of dilute solutions with
known concentrations. A precision study was also carried out at the LOQ
level by injecting six individual preparations of imp-A, imp-B, imp-C,
imp-D and imp-E and calculating the % R.S.D. of the area.
EMEA, Europa (2005) Cymbalta: Scientific Discussion.
International Conferences on Harmonization Q2 (R1) (2005) Validation of
analytical procedures: Text and methodology.
3.5.4. Linearity
International Conferences on Harmonization Q3A (R2) (2006) Draft Re-
vised Guidance on Impurities in New Drug Substances.
Linearity test solutions for the assay method were prepared from duloxe-
tine HCl stock solutions at five concentration levels from 50 to 150 % of
assay analyte concentration (125, 187.5, 250, 312.5 and 375 mg ꢂ mLꢃ1).
The peak area versus concentration data were analysed by least-squares
linear regression.
Linearity test solutions for the related substance method were prepared by
diluting stock solutions to the required concentrations. The solutions were
prepared at six concentration levels from LOQ to 200% (0.5 mg ꢂ mLꢃ1) of
the specification level (LOQ, 0.075, 0.10, 0.15, 0.2, 0.25 and 0.3%). The
peak area versus concentration data were analysed by least-squares linear
regression.
Jansen PJ, Oren PL, Kemp CA, Maple SR, Baertschi SW (2000) Charac-
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enteric polymers hydroxypropyl methylcellulose acetate succinate and
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3.5.5. Accuracy
The accuracy of the assay method was evaluated in triplicate at three con-
centration levels, 125, 250 and 375 mg ꢂ mLꢃ1 (50%, 100% and 150%).
The percentage recovery was calculated from the slope and Y-intercept of
the calibration curve obtained in the linearity study.
The accuracy study for impurities was carried out in triplicate at 0.075%,
0.10%, 0.125%, 0.15%, 0.2%, 0.25% and 0.3% of the duloxetine HCl ana-
lyte concentration (0.5 mg ꢂ mLꢃ1). The percentage recoveries for impurities
were calculated from the slope and Y-intercept of the calibration curves.
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