Development and validation of Ropinirole hydrochloride and its related compounds by UPLC in API and pharmaceutical dosage forms

Article abstract of DOI:10.1002/jccs.201000052

A novel stability-indicating gradient reverse phase ultra performance liquid chromatographic (RP-UPLC) method was developed for the determination of purity of Ropinirole in presence of its impurities and forced degradation products. The method was developed using Waters Aquity BEH 100 mm, 2.1 mm, 1.7 μm C-8 column with mobile phase containing a gradient mixture of solvent A and B. The eluted compounds were monitored at 250 nm. The run time was within 4.5 min which Ropinirole and its four impurities were well separated. Ropinirole was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Ropinirole was found to degrade significantly in oxidative and base stress conditions and stable in acid, water, hydrolytic and photolytic degradation conditions. The degradation products were well resolved from main peak and its impurities, thus proved the stability indicating power of the method. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also suitable for the assay determination of Ropinirole in pharmaceutical dosage forms and dissolution studies.

Full text of DOI:10.1002/jccs.201000052

348
Journal of the Chinese Chemical Society, 2010, 57, 348-355
Development and Validation of Ropinirole Hydrochloride and its Related
Compounds by UPLC in API and Pharmaceutical Dosage Forms
Ch. Krishnaiah,a,b* M. Vishnu murthy,a A. Raghupathi Reddy,a Ramesh Kumara and K. Mukkantib
aDr. Reddy’s Laboratories Ltd. IPDO, Bachupally, Hyderabad-500072, A. P, India
bDepartment of Chemistry, J.N.T. University, Kukatpally, Hyderabad-500072, A. P, India
A novel stability-indicating gradient reverse phase ultra performance liquid chromatographic (RP-
UPLC) method was developed for the determination of purity of Ropinirole in presence of its impurities
and forced degradation products. The method was developed using Waters Aquity BEH 100 mm, 2.1 mm,
1.7 mm C-8 column with mobile phase containing a gradient mixture of solvent A and B. The eluted com-
pounds were monitored at 250 nm. The run time was within 4.5 min which Ropinirole and its four impuri-
ties were well separated. Ropinirole was subjected to the stress conditions of oxidative, acid, base, hy-
drolytic, thermal and photolytic degradation. Ropinirole was found to degrade significantly in oxidative
and base stress conditions and stable in acid, water, hydrolytic and photolytic degradation conditions. The
degradation products were well resolved from main peak and its impurities, thus proved the stability indi-
cating power of the method. The developed method was validated as per International Conference on Har-
monization (ICH) guidelines with respect to specificity, linearity, limit of detection, limit of quantifica-
tion, accuracy, precision and robustness. This method was also suitable for the assay determination of
Ropinirole in pharmaceutical dosage forms and dissolution studies.
Keywords: UPLC; Ropinirole hydrochloride; Forced degradation; Validation; Stability-indicat-
ing.
INTRODUCTION
Though high-performance liquid chromatography
dro-2H-indol-2-one, is potent anti Parkinson’s disease
drug. Parkinson’s disease caused by deficit of Dopamine
(one of the brain’s chief chemical messengers). Though the
literature cited some of the references on impurity profile
of Ropinirole with capillary electrophoresis and assay de-
termination by HPLC method of Ropinirole hydrochloride
tablets.1-6 However, there are no reports available on stabil-
ity indicating analytical related substances method for
Ropinirole hydrochloride API and dosage forms. It is,
therefore, felt necessary to develop a new stability indicat-
ing method for the related substance determination and
quantitative estimation of Ropinirole. We intend to opt for
a faster chromatographic technique, UPLC- for the said
study. An attempt was made on determining whether UPLC
can reduce analysis times without compromising the
resolution and sensitivity.
(HPLC) is a well-established reliable technique used in
controlling the quality and consistency of active pharma-
ceutical ingredients (API’s) and dosage forms, it is often a
slow technique because of the complexity of some of the
samples, it could still be improved.
Ultra-performance liquid chromatography (UPLC) is
a new category of separation technique based upon well-
established principles of liquid chromatography, which uti-
lizes sub-2 mm particles for stationary phase. These parti-
cles operate at elevated mobile phase linear velocities to af-
fect dramatic increase in resolution, sensitivity and speed
of analysis. Because of its speed and sensitivity, this tech-
nique is gaining considerable attention in recent years for
pharmaceutical and biomedical analysis. In the present
work, this technology has been applied to the method de-
velopment and validation study of related substance and
assay determination of Ropinirole hydrochloride bulk drug
and dosage forms.
Hence a reproducible stability-indicating RP UPLC
method was developed for the quantitative determination
of Ropinirole hydrochloride and its four impurities namely
imp-A, imp-B, imp-C, and imp-D (Fig. 1). The method was
developed using Waters (sub-2 micron columns) Aquity
Ropinirole, 4-[2-(Diproplyamino)ethyl]-1,3-Dihy-
* Corresponding author. E-mail: krishnaiahch@drreddys.com; krishna_charu@yahoo.co.in

Estimation of Ropinarole API and Pharmaceutical Dosage Forms
J. Chin. Chem. Soc., Vol. 57, No. 3A, 2010 349
BEH 100 mm, 2.1 mm, 1.7 mm C-8 column, the run time
laboratories limited, Hyderabad, India. Commercially avail-
able Ropitor tablets were used for the dosage form analysis.
The HPLC grade acetonitrile, analytical grade K2HPO4,
acetonitrile, ammonium acetate and ortho phosphoric acid
were purchased from Merck, Darmstadt, Germany. Water
was prepared by using Millipore MilliQ Plus water purifi-
cation system.
was within 4.5 min and gradient elution mode.
EXPERIMENTAL
Materials and reagents
Active pharmaceutical ingredient standard, all impu-
rity standards and samples were supplied by Dr. Reddy’s
Chromatographic conditions
LC was carried out on a Waters Aquity UPLC with
photodiode array detector. The output signal was moni-
tored and processed using empowers software. The chro-
matographic column used Acquity UPLC BEH C-8 100
mm, 2.1 mm, and 1.7 mm particle size. The separation was
achieved on a gradient method. The mobile phase A con-
tains a mixture of 0.01 M K2HPO4 (pH 7.2) buffer, aceto-
nitrile in the ratio 90:10 (v/v); respectively and the mobile
phase B contains a mixture of 0.01 M K2HPO4, (pH 7.2)
buffer and acetonitrile in the ratio 50:50 (v/v), respectively.
The flow rate of mobile phase was 0.27 mL min-1. The
UPLC gradient program was set as: time (min)/% solution
B: 0.01/55, 1.7/55, 2.9/98, 3.5/98, 3.6/55 and 4.5/55. The
column temperature was maintained at 40 °C and the detec-
tion was monitored at a wavelength 250 nm. The injection
volume was 1.0 mL.
LC-MS/MS conditions
LC-MS/MS system (Agilent 1200 series liquid chro-
matograph coupled with Applied Biosystems 4000 Q Trap
triple quadrupole mass spectrometer with Analyst 1.4 soft-
ware, MDS SCIEX, USA) was used for the unknown com-
pounds formed during forced degradation studies. Inertsil
C8, 250 ´ 4.6 mm, 5 mm column (GL Sciences, USA) was
used as stationary phase. 0.01 mM ammonium acetate
(Merck, Darmstadt, Germany) was used as buffer. Buffer
and acetonitrile in the ratio 90:10, v/v; was used as solvent
A and Water and acetonitrile in the ratio 50:50, v/v; was
used as solvent B. The gradient program (T/% Solvent B)
was set as 0.01/20, 5/20, 20/40, 30/55, 40/80, 50/80, 52/20,
60/20. The mixture was degassed in an ultrasonic bath be-
fore use. Solvent A was used as diluent. The flow rate was
1.0 mL min-1. The analysis was performed in positive elec-
tro spray positive ionization mode. Ion Source voltages
was 5000 V. Source temperature was 450 °C. GS1 and GS2
are optimized to 30 and 35 psi respectively. Curtain gas
flow was 20 psi.
Preparation of Stock Solutions/Preparation of Sample
Solution/Stress Studies
Fig. 1. Chemical structures of Ropinirole and its impu-
rites.
A stock solution of Ropinirole hydrochloride (0.05

350 J. Chin. Chem. Soc., Vol. 57, No. 3A, 2010
Krishnaiah et al.
mg mL-1) was prepared by dissolving an appropriate
amount of drug in acetonitrile: water 20:80 (v/v); respec-
tively. Working solutions containing 0.05 and 0.005 mg
mL-1 were prepared from this stock solution for determina-
tion of related substances and for assay determination, re-
spectively. A mixed stock solution (0.0005 mg mL-1) of the
impurities (denoted Imp-A to Imp-D) was also prepared in
diluent.
inter-day variation (n = 18). Different analysts prepared
different solutions on different days. The RSD (%) of peak
area was calculated for each impurity.
The precision 2.5, 5.0 and 7.5 mg mL-1 of the assay
was evaluated by performing six independent assays of a
test sample of Ropinirole hydrochloride and comparison
with a reference standard. The RSD (%) of the six results
was calculated.
Twenty Ropinirole hydrochloride 4 mg tablets were
weighed and transferred the pellets in to a clean, dry mor-
tar. Pellets equivalent to 50 mg drug was dissolved in 100
mL of methanol to give a solution containing 0.5 mg mL-1.
1.0 mL of this solution was diluted to 10 mL with aceto-
nitirle: water 20:80 (v/v), to give a solution containing 0.05
mg mL-1 and filtered through a 0.22 mm Nylon membrane
filter. The solution obtained was analyzed by UPLC.
Forced degradation of Ropinirole hydrochloride was
performed to provide an indication of the stability-indicat-
ing properties and specificity of the method. The stress
conditions used for the degradation study included light
(10 days), heat (80 °C, 10 days), acid hydrolysis (1 M HCl,
24 h), base hydrolysis (1 M NaOH, 1 h), aqueous hydroly-
sis (24 h), and oxidation (6% H2O2 5 h).
By injecting a linear (0.05% to 0.5% w.r.t to test con-
centration) solutions of known concentrations, based on
the Standard Deviation (s) of the Response and the Slope
(S) of the calibration plot, using the formulae LOD = 3.3
s/S and LOQ = 10 s/S. LOD and LOQ for Ropinirole and
for the four impurities were estimated respectively. Preci-
sion was also determined at LOQ level and % RSD was
calculated for the peak area for each impurity and for
Ropinirole.
The accuracy of an analytical procedure expresses the
closeness of agreement between the true value and the value
found. The accuracy of the assay method for Ropinirole hy-
drochloride was evaluated in triplicate at three concentra-
tions, 2.5, 5.0 and 7.5 mg mL-1 on drug product and recov-
ery was calculated for each added concentration.
For impurities, recovery was determined in triplicate
for 37.5, 75.0 and 112.5 ng mL-1 of the analyte concentra-
tion (0.05 mg mL-1) on drug product and recovery of the
impurities was calculated.
The purity of peaks obtained from stressed samples
was checked by use of the PDA detector. The purity angle
was within the purity threshold limit obtained in all stressed
samples and demonstrates the analyte peak homogeneity.
Assay of stressed samples was performed by comparison
with reference standards and the mass balance (% assay +
% impurities + % degradation products) for stressed sam-
ples was calculated.
Detector response linearity for imp-A to imp-D was
assessed by injecting eight separately prepared solutions
covering the range LOQ–200% (LOQ, 0.015, 0.0375,
0.075, 0.1125, 0.15, 0.1875, 0.225, 0.2625 and 0.30%) of
the normal sample concentration (0.05 mg mL-1). The cor-
relation coefficients, slopes and Y-intercepts of the calibra-
tion curve were determined.
Method Validation
The described method has been extensively validated
(Sensitivity, Linearity, Accuracy, Precision, Solution Sta-
bility and Mobile Phase Stability and Robustness) for assay
and related substances by UPLC determination.7
The robustness of an analytical procedure is a mea-
sure of its capacity to remain unaffected by small, but de-
liberate variations in method parameters and provides an
indication of its reliability during normal usage.
The repeatability of the related-substance method was
checked by six-fold analysis of 0.05 mg mL-1 Ropinirole
hydrochloride spiked with 37.5, 75.0 and 112.5 ng mL-1 of
each of the four impurities. The RSD (%) of peak area was
calculated for each impurity.
To determine the robustness of the method the experi-
mental conditions were deliberately changed. The resolu-
tion of Ropinirole hydrochloride and the four impurities
were evaluated. The mobile phase flow rate was 0.27 mL
min-1; to study the effect of flow rate on resolution it was
changed to 0.24 and 0.30 mL min-1. The effect of pH was
studied at pH 7.1 and 7.3 (instead of pH 7.2). The effect of
column temperature was studied at 35 °C and 45 °C (in-
stead of 40 °C).
Inter- and intra-day variation and analyst variation
was studied to determine intermediate precision of the pro-
posed method. Intra-day precision was determined by six-
fold analysis of 0.05 mg mL-1 Ropinirole hydrochloride
spiked with 0.15% of each of the four impurities. The same
protocol was followed for three different days to study

Estimation of Ropinarole API and Pharmaceutical Dosage Forms
J. Chin. Chem. Soc., Vol. 57, No. 3A, 2010 351
The stability of Ropinirole hydrochloride and its im-
purities in solution was determined by leaving test solu-
tions of the sample and reference standard in tightly capped
volumetric flasks at room temperature for 48 h during
which they were assayed at 24 h intervals. Stability in the
mobile phase was determined by analysis of freshly pre-
pared sample solutions at 24 h intervals for 48 h and com-
pared the results with those obtained from freshly prepared
reference standard solutions. The RSD (%) of the assay and
related substances results was calculated for both the mo-
bile phase and solution-stability experiments.
Table 1. Accuracy of Ropinirole impurities
Amound added Amount recovered Recovery
(mg mL-1)
Level
(mg mL-1)
(%)
At 50% (n = 3)
Impurity-A
Impurity-B
Impurity-C
Impurity-D
0.0375
0.0376
0.0375
0.0374
0.0365
0.0368
0.0370
0.0350
097.3
097.9
098.7
093.6
At 100% (n = 3)
Impurity-A
Impurity-B
Impurity-C
Impurity-D
0.0740
0.0760
0.0750
0.0740
0.0790
0.0720
0.0780
0.0720
106.8
094.7
104.0
097.3
At 150% (n = 3)
Impurity-A
Impurity-B
Impurity-C
Impurity-D
RESULTS AND DISCUSSION
0.1125
0.1124
0.1127
0.1124
0.1120
0.1110
0.1135
0.1098
099.6
098.8
100.7
097.7
Method Development and Optimization
pKa of Ropinirole durg substances is 9.5. The main
objective of the chromatographic method is to separate crit-
ical closely eluting impurities Imp-A and Imp-B; Imp-D
and Ropinirole as a symmetrical peak. The blend solution
containing 50 mg mL-1 Ropinirole and 1 mg mL-1 of each
four impurities are prepared in the diluent. All the impuri-
ties of Ropinirole were subjected to separation by reversed-
phase LC on a Waters Aquity BEH C18, 50 ´ 2.1 mm, 1.7
mm column with pH 3.0, 0.01 M di potassium hydrogen
orthophosphate buffer - acetonitrile in 90:10 ratio as a sol-
vent A and pH 3.0, 0.01 M potassium dihydrogen ortho-
phosphate buffer and acetonitrile in 50:50 ratio as solvent
B. Two compounds viz., between Imp-D and Ropinirole
was not satisfactory level. Using different C18 UPLC col-
umns (Inertsil ODS-3, 50 mm ´ 2.1 mm, 2 mm particles and
Zorbax XDB C-18, 50 mm ´ 2.1 mm, containing 1.8 mm
particles, 0.3 mL min-1) - using different buffer pH (3.0 and
7.2) conditions. But at all above conditions, separations
among the impurities are not satisfactory level. It is found
that use of buffer prepared by adjusting the pH of 0.01 M di
potassium hydrogen phosphate to 7.2 with diluted potas-
sium hydroxide (solvent A: buffer and acetonitrile in the
ratio 90:10; v/v and solvent B: buffer and acetonitrile in the
ratio 50:50 v/v) with gradient elution (time (min)/% Sol-
vent B: 0.01/55, 1.7/55, 2.9/98, 3.5/98, 3.6/55 and 4.5/55),
Waters Aquity BEH C8, 100 ´ 2.1 mm, 1.7 mm column with
pH 7.2 gives enabled separation for Imp-D and Ropinirole,
but the resolution (Rs) between Imp-A and Imp-B is not up
to satisfactory level. When column oven temperature is in-
creased from ambient to 40 °C, gives enabled separation
and all pair compounds (Rs > 2.5). Eluted analyte retention
time was about 2.1 mins. Interference from the excipients
(placebo) was also checked, no interference was observed
with impurity peaks and Ropinirole analyte (drug sub-
stances) peak.
System suitability parameters were evaluated for
Ropinirole and its four impurities. USP tailing factor for all
four impurities and Ropinirole is found less than 1.1. USP
resolution of Ropinirole hydrochloride and four potential
impurities were greater than 2.0 for all pairs of compounds.
Validation of the Method
The described method has been extensively validated
(Sensitivity, Linearity, Accuracy, Precision, Solution Sta-
bility and Mobile Phase Stability and Robustness) for pu-
rity by HPLC determination.
RSD (%) in the study of the repeatability of the assay
of Ropinirole hydrochloride was within 0.8%. The % RSD
of peak area for the four impurities namely Imp-A, Imp-B,
Imp-C and Imp-D in the study of the repeatability was 2.5,
3.7, 7.2 and 4.4%, respectively. RSD (%) results of Ropini-
role hydrochloride and its impurities for intermediate pre-
cision (intra and inter day repeatability) are within 7.2%.
These results confirmed the method was highly precise.
The limit of detection for Ropinirole, Imp-A, Imp-B,
Imp-C and Imp-D were 0.5, 0.25, 0.9, 1.1 and 0.35 ng mL-1
respectively. The limit of quantification for Ropinirole,
Imp-A, Imp-B, Imp-C and Imp-D were 1.75, 0.9, 2.95, 3.6
and 1.1 ng mL-1 respectively. RSD (%) for precision at the
LOQ concentration for Ropinirole, Imp-A, Imp-B, Imp-C
and Imp-D were, 0.9%, 2.3%, 2.1%, 3.8% and 5.3%, re-
spectively.

352 J. Chin. Chem. Soc., Vol. 57, No. 3A, 2010
Krishnaiah et al.
Fig. 2a. Ropinirole spiked chromatogram.
Fig. 2b. Water hydrolysis degradation chromatogram.
Fig. 2c. Oxidative degradation chromatogram.

Estimation of Ropinarole API and Pharmaceutical Dosage Forms
J. Chin. Chem. Soc., Vol. 57, No. 3A, 2010 353
Fig. 2d. Base degradation chromatogram.
Fig. 2e. Tablet chromatogram.
Fig. 2f. Acid degradation chromatogram.
Fig. 2. Blend chromatogram of Ropinirole and its impurities.

354 J. Chin. Chem. Soc., Vol. 57, No. 3A, 2010
Krishnaiah et al.
Recovery of Ropinirole hydrochloride from pharma-
ceutical dosage forms ranged from 98.2 to 101.3%. Recov-
ery of the four impurities from pharmaceutical dosage forms
ranged from 93.6 to 106.8 (Table 1).
tion between Imp-A and Imp-B was greater than 2.5 for all
flow rates (0.23, 0.30 mL min-1), column temperature (35,
45 °C) and pH (7.1, 7.3) variation conditions.
RSD (%) for assay of Ropinirole hydrochloride dur-
ing solution stability and mobile phase stability experi-
ments was within 1.2%. No significant changes in the
amounts of the four impurities were observed during solu-
tion stability and mobile phase experiments when per-
formed using the related substances method. The results
from solution stability and mobile phase stability experi-
ments confirmed that standard solutions and solutions in
the mobile phase were stable for up to 48 h during assay
and determination of related substances.
The linearity calibration plot for the assay method
was obtained over the calibration ranges tested, i.e. 2.5-7.5
mg mL-1 (n = 3) and correlation coefficient obtained was
greater than 0.999. The result shows that an excellent cor-
relation existed between the peak area and concentration of
the analyte.
Linear calibration plot for the related substance method
was obtained over the calibration ranges tested, i.e. LOQ to
0.30% (n = 3) for impurities. The correlation coefficient
obtained was greater than 0.998. The above result shows
that an excellent correlation existed between the peak area
and the concentration of imp-A, imp-B, imp-C and imp-D.
In all the deliberate varied chromatographic condi-
tions (flow rate, column temperature and pH variation), all
analytes were adequately resolved and elution orders re-
mained unchanged. Resolution (Rs) between Imp-D and
Ropinirole hydrochloride was greater than 2.0 and resolu-
All forced degradation samples were analyzed at an
initial concentration 50 mg mL-1 of Ropinirole with UPLC
conditions mentioned in chromatographic section using
PDA detector to ensure the homogeneity and purity of
Ropinirole peak. Significant degradation of Ropinirole was
observed in basic and oxidative hydrolysis conditions lead-
ing to the formation of imp-B; imp-C and imp-D respec-
tively (Fig. 2). This was confirmed by co-injecting imp-B,
Table 2. Summary of forced degradation results
#Assay (%w/w *Mass balance
RS by UPLC
Degradation condition
Time
on anhydrous (%assay+Deg.
Remarks/observation
% degradation
basis)
Products)
HCl-1 M (Acid hydrolysis)
NaOH-1M (Base hydrolysis)
24 hrs
1 hr
99.2%
99.8
0.2%
4.9%
No significant degradation
observed
94.8%
99.2
Impurity-B and C
degradation products were
formed
Water hydrolysis
24 hrs
5 hrs
99.5%
91.8%
99.8
99.1
0.3%
7.9%
No significant degradation
observed
Oxidation by H2O2 (6.0%)
Impurity-B, C and D
degradation products were
formed.
Thermal (80 °C) solid
10 days
10 days
99.5%
99.8%
99.9
99.9
0.1%
0.1%
No degradation observed.
No degradation observed.
UV at 254 nm
At ´ Ws ´ P ´ 100
# % Assay by HPLC (w/w) =
(on anhydrous basis)
As ´ Wt ´ (100-WC)
Where:
At: Average area due to sample preparation
As: Average area due to reference standard preparation
Wt: Weight of the sample in mg
Ws: Weight of the reference standard in mg
P: Potency of the Reference standard
WC: Water content
* Mass balance: % assay + % sum of all compounds + % sum of all degradants

Estimation of Ropinarole API and Pharmaceutical Dosage Forms
J. Chin. Chem. Soc., Vol. 57, No. 3A, 2010 355
imp-C and imp-D standard to these degraded samples and
by LC-MS/MS analysis. LC-MS/MS analysis was per-
formed as per experimental conditions and mass of the im-
purity was 309, 277 and 275 which was corresponding to
the mass of imp-B, imp-C and imp-D. Ropinirole was found
to be stable under hydrolytic, acidic, heat and photolytic
(10 days) degradation conditions.
The method is stability-indicating and can be used for rou-
tine analysis of production samples, it may be useful for
study of in vitro dissolutions pharmaceutical dosage forms
and to check the stability of samples of Ropinirole hydro-
chloride.
ACKNOWLEDGEMENTS
Assay studies were carried out for stress samples (at 5
mg mL-1) against Ropinirole qualified reference standard.
The mass balance (% assay + % sum of all compounds + %
sum of all degradants) results were calculated for all stressed
samples and found to be more than 99%. The purity and as-
say of Ropinirole was unaffected by the presence of its im-
purities and degradation products and thus confirms the
stability-indicating power of the developed method. Re-
sults from force degradation studies were presented in Ta-
ble 2.
The Authors wish to thank the management of Dr.
Reddy’s group for supporting this work.
Received March 23, 2010.
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CONCLUSION
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