Rao et al. European Journal of Biomedical and Pharmaceutical Sciences
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A NEW ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF STABILITY
INDICATING RP-UPLC ASSAY FOR THE DETERMINATION OF LURASIDONE
HYDROCHLORIDE IN BULK AND TABLET DOSAGE FORMS.
Tirukkovalluri Siva Rao2*, Tene Sivaganesh
1,2, Kommula Ramasrinivas
1, Uppalapu Sesham Raju
1,
Pinninti Sateesh Kumar1
1Department of Analytical Chemistry, AurobindoPharma Ltd, Pydibhimavaram, India.
2Department of Inorganic & Analytical Chemistry, Andhra University, Visakhapatnam, 530003, India.
Article Received on 19/04/2018 Article Revised on 09/04/2018 Article Accepted on 30/05/2018
]
1. 0.INTRODUCTION
Lurasidone is a benzothiazol derivative (Figure 1)
belonging to second-generation antipsychotics class.It is
used for the treatment of schizophrenia and bipolar
depression. Although its mechanism of action is not fully
understood, it is believed that the efficacy of
LurasidoneHCl is mediated mainly through antagonist
activity at the dopamine D2, and the 5-
hydroxytryptamine (5HT, serotonin) receptors: 5-
HT2Aand 5-HT7.[1]
LurasidoneHcl demonstrated both
antipsychotic and antidepressant action. Despite its side
effects like higher rates of akathisia, Parkinsonism and
hyperprolactinemia, it has relatively lower risk for
developing sedation or overweight/ obesity.
LurasidoneHcl is available as tablets under the brand
name of Latuda. Several chromatographic methods have
been described for the quantification of Lurasidone Hcl
including spectrometry[2-5]
, TLC[6]
, HPTLC[7, 9]
,
GC/MS[8]
, HPLC[9-16]
and LC/MS.[17-22]
The goal of this
study was the development of a new UPLC method for
determination of LurasidoneHCl in its API bulk drug and
tablet dosage forms. Although there are some previous
HPLC methods developed for this purpose, they allow
analysis at a narrow concentration range and the
separation of stereoisomer’s not reported in any method.
Developed method has a wide concentration range, low
quantification and detection limit compare to all HPLC
and UV methods. It was validated according to the
international conference on harmonization guideline[23]
and applied for the analysis of bulk drug and tablet
dosage form.
SJIF Impact Factor 4.918 Research Article
ejbps, 2018, Volume 5, Issue 6 843-857.
European Journal of Biomedical AND Pharmaceutical sciences
http://www.ejbps.com
ISSN 2349-8870
Volume: 5
Issue: 6
843-857
Year: 2018
*Corresponding Author: Prof. Tirukkovalluri Siva Rao
Department of Inorganic & Analytical Chemistry, Andhra University, Visakhapatnam, 530003, India.
ABSTRACT
Method development, validation and stability indicating RP-UPLC method for Assay determination of
LurasidoneHcl in pharmaceutical products. This method has high degree of performance to separation and
quantification of Lurasidone even in the presence of its impurities. This efficient Separation can be achieved by
using new Waters Acquity CSH Phenyl hexyl with dimensions (100mm x 2.1 mm, 1.7μm). The buffer used in this
method KH2PO4 at pH 2.5 and Acetonitrile in the ratio of (65:35 v/v) with a flow rate of 0.5 ml/min and the
absorbance was monitored at 230 nm. The total run time was 3.0 min. The correlation coefficient of the method
shows good linear relationship with 0.9999. The limit of detection and quantification are determined for
LurasidoneHCl 0.01µg/ml and 0.03µg/ml. The signal to noise ratio has been observed 7 and 12 for LOD & LOQ
respectively. The precision of the method %RSD is less than 0.40% and the % recovery of LurasidoneHCl is
between 99.5 – 100.1 %.When the drug is subjected to different stress conditions and the resulting degradation
products obtained were not interfere during the determination of LurasidoneHCl.
KEYWORDS: Lurasidone HCl, RP-UPLC, Method development, Validation, Stability-indicating.
Rao et al. European Journal of Biomedical and Pharmaceutical Sciences
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(S) N
(R)
O
O
(R)(R)
N
N
SN
(Endo, 1R, 2S)-4-[2-(4-Benzo[d]isothiazol-3-yl-piperazin-1-ylmethyl)-cyclohexylmethyl]-4-aza-tricyclo[5.2.1.02,6]decane-3,5-dione
(S)
( R)
(S) N
(R)
O
O
(R)(R)
N
N
SN
(R)
(S)
LURASIDONE
(S) N
(R)
O
O
( S)( S)
N
N
SN
(R)
( S)
[(3aR, 4S, 7aS)(1S, 2S)-STEREO ISOMER
(S) N
(R)
O
O
(R)(S)
N
N
SN
(R)
(S)
[(3aR, 4S, 7aS)(1S, 2R)-STEREO ISOMER
(S) N
(R)
O
O
(R)(S)
N
N
SN
(R)
(S)
[(3aR, 4S, 7aS)(1R, 2S)-STEREO ISOMER
(S) N
(R)
O
O
(R)(R)
N
N
SN
( S)
(R)
[(3aS, 4S, 7aR)(1R, 2R)-STEREO ISOMER Fig: 1.
Table: 1.
Product Name Lurasidone Hydrochloride
Chemical Name
(3aR,4S,7R,7aS)-2[(1R,2R)-2-[4-(1,2-Benzisothiazol-3-Yl)- Piperazin-1-
Ylmethyl]Cyclohexyl Methyl]Hexahydro-4,7-Methano-2h-Isoindole-1,3-Dione,
Hydrochloride
CAS Reg.No: 367514-88-3
Molecular Formula C28H36N4O2S. HCl
Molecular Weight 529.14
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2.0. EXPERIMENTAL
2.1. Chemicals and reagents
Samples of Lurasidone HCl were contributed from
Aurobindo pharma Ltd, Pydibhimavaram, India. HPLC
grade Acetonitrile, AR grade KH2PO4 and
Orthophosphoric Acid were purchased from Merck india
Ltd, Mumbai. High pure water was prepared by using
Millipore Milli-Q water purification system. New Waters
Acquity CSH Phenyl hexyl (100 x 2.1 mm, 1.7 μm)
column (Part no. # 186005407), waters AcquityBEH
(50mm×2.1mm, 1.7μm), waters Acquity HSS
T3(100mm×2.1mm,1.8μm) and waters Acquity HSS
C18(100mm×2.1mm,1.8μm) was procured from Waters
India Ltd, Bangalore.
2.2. Instrumentation (Apparatus)
Acquity UPLC system equipped with an LC pump
(model ACQ-BSM) used for method development and
validation itscontain, an online degasser, auto sampler
(model ACQ-SM) with thermostat, and detector (TUV)
(model ACQ-TUV). The data was acquired, monitored
and processed by using Empower3 software. Design
expert version 9 (Stat-Ease Inc., Minneapolis, USA) was
used for the optimizing chromatographic conditions. The
buffers pH was monitored by using Metrohm 780 p
H
meter and weights taken by using the Sartorius
CPA225D balance. In this research work we have
attempted various chromatographic columns for method
development. After number of trails we have been
selected Waters Acquity CSH Phenyl hexyl (100 x 2.1
mm, 1.7 μm) column based on its performance.
2.3. Chromatographic conditions
The chromatographic separations were performed by
using New Waters Acquity CSH Phenyl hexyl (100 x 2.1
mm, 1.7 μm) (Part no. # 186005407). The pH
of the
buffer KH2PO4was adjusted to pH
2.5 by addition of
orthophosphoric Acid whereas the mobile phase contains
buffer (pH 2.5) and Acetonitrile in the ratio of (65:35).
The flow rate of the mobile phase is 0.5 mL/min. The
column temperature was maintained at 50°C and the
absorption was measured at 230nm. The total Run time
of the method was found to be 3.0min and the injection
volume was 6µL. Water and Acetonitrile in the ratio of
(50:50 v/v) used as a diluent. Mobile phase and diluent
Filter through 0.22 finer porosity membrane filter. The
chromatographic conditions were given in table-2.
Table: 2
Instrument : RP-UPLC make by Waters
Mode of analysis : Isocratic
Flow rate : 0.5 mL/min
Detector wave length : 230 nm
Column temperature : 500C
Injection volume : 6.0 µL
Column : WatersAcquityCSHPhenylhexyl(100x2.1mm, 1.7μ)
Run time : 3.0 min
Sample Manager Temp : Ambient
Inference: Results from the table-2 indicated that the
eluting run time was completed within 3 minutes
compared with other HPLC methods for assay
determination in the literature. Moreover the injection
volume of the sample required for RP-UPLC method is 6
µL is sufficient but in the case of HPLC methods it needs
about 20 µL.
Table: 3
S. No. NAME RT
1 Lurasidone HCl ~2.0
2.4. Preparation of standard and sample solutions
Preparation of standard solution
Weighed and transferred accurately 50 mg of
LurasidoneHCl working standard into a 100 mL clean,
dry volumetric flask add 50 mL of diluent and sonicate
to dissolve. Make up to volume with diluent. Diluted 5
mL of this solution to 50 mL with diluent. Filter through
0.22 finer porosity membrane filter.
Preparation of sample solution
Weighed and transferred accurately 50 mg of
LurasidoneHCl sample into a 100 mL clean, dry
volumetric flask add 50 mL of diluent and sonicate to
dissolve. Make up to volume with diluent. Diluted 5 mL
of this solution to 50 mL with diluent. Filter through
0.22 finer porosity membrane filter.
2.5. Calculation of Lurasidone HCl Assay
The samples assay was calculated by the following
equation
Where AT is peak average area due to LurasidoneHCl in
the sample preparation, AS is Average peak area due to
Lurasidone HCl in the standard. WS is the weight of
Lurasidone HCl standard taken in mg, WT is the Weight
of the sample taken in mg, P is the Potency of the
Lurasidone HCl working/reference standard. W.C is the
water content of the Lurasidone HCl.
).100(
100
5
50100
50
5
100 CWP
WT
WS
AS
AT
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3.0. RESULTS AND DISCUSSION
3.1.0. Method development and Optimization of RP-
UPLC conditions
The RP-UPLC conditions were optimized by using trials
with different columns, several mobile phase
compositions; flow rate and pH
were studied. The
Analytical method development and validation play an
important role in the determination of Assay in
pharmaceutical products. In this method less quantity of
solvents are used and total consumption of solvents is not
more than 0.525ml per run time. One of the principles of
green chemistry is prevention of waste. Further
advantages of RP-UPLC method is able to increase the
speed, sensitivity and resolution compared to HPLC
methods. By considering above aspects, RP-UPLC
instrument is a most suitable technique for the assay
determination of Lurasidone HCl.
3.1.1. Selection of stationary phase
Based on the structure, molecular weight of API and
impurities present in the products C18 columns like
Waters BEH C18 column were initially screened for the
separation. But these columns failed to provide
acceptable separation and peak shape. For also several
other HSS C18 columns with other stationary phases are
screened for separation but a remarkable selectivity was
achieved with New Waters Acquity CSH Phenyl hexyl
(100mm x 2.1 mm, 1.7 μm) partial size was finalised.
This column was not yet used in any other methods.
Waters AcquityBEH C18 column; The trifunctionally
bonded BEH Column particle gives a widest usable pH
range i.e’s 1-12range, superior low pH stability and ultra
low column bleed for high sensitivity applications.
Waters Acquity CSH Phenyl hexyl column; this
column are used to provide an alternative selectivity and
are a valuble tool for method development. The
trifunctionally bonded C6phenyl ligond is a robust and
low bleed sorbents that selectively retains poly aromatic
compounds through Π-Π interactions.
Table: 4 Stationary phases Information.
Brand Acquity UPLC Particle size(dp) 1.7μm
%Carbon Load 14 Particle Substrate Hybrid
Bonding Technology Phenyl-Hexyl Pore size 130Å
Chemistry Phenyl Silanol Activity Low
Endcapped yes Surface Area 185
ID 2.1 mm Technology CSH
Length 100mm USP Classification L11
Particle Shape Spherical Units in package 1/pkg
Mode Reversed-phase pHrange 1-11
3.1.2. Selection of Mobile phase
As one of the objectives of the method is to develop a
different buffer, i.e’s Ammonium acetate buffer with pH
9.0, diammonium hydrogen orthophosphate with pH3.3
and potassium dihydrogen orthophosphate+water buffers
were evaluated. It is observed that in potassium
dihydrogen ortho phosphate buffer is a promising
candidate for efficient separation of impurities. The
organic modifiers, methanol and acetonitrile were used at
different composition conditions. Based on the results,
acetonitrile was finalised as a organic modifier.
3.1.3. Selection of Diluent
LurasidoneHCl was practically insoluble in water and
slightly soluble in methanol. But It is soluble in
acetonitrile+ water composition and stable for at least 24
hrs at 25°C. Hence, acetonitrile + Water (50:50 v/v) was
selected as a diluent.
3.1.4. Experimental design for optimising flow rate,
buffer concentration and column temperature
In initial method development trials with one factor at a
time (OFAT) variation revealed that the flow rate and
column temperature and composition of organic modifier
had significant impact on selectivity. Since optimising
the chromatographic parameters with OFAT approach
consumes lot of time and does not provide the design
space, a design of experiments (DoE) was used for
optimising these chromatographic parameters. The
design space defines the experimental region in which
changes to method parameters will not significantly
affect the quality and results. As working within the
design space is not considered as a change, the scientist
can have freedom to operate the method at different
chromatographic condition.
Based on the analysis, it was understood that, to obtain
good analyte peak shape, column temperature and flow
rate are monitored at 50°C and 0.5 mL/min respectively.
Rao et al. European Journal of Biomedical and Pharmaceutical Sciences
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Table: 5 Optimized chromatographic conditions.
Instrument : RP-UPLC make by Waters
Mode of analysis : Isocratic
Flow rate : 0.5 mL/min
Detector wave length : 230 nm
Column temperature : 500C
Injection volume : 6.0 µL
Column : WatersAcquityCSHPhenylhexyl (100x2.1mm, 1.7μ)
Run time : 3.0 min
Sample Manager Temp : Ambient
3.2.0. METHOD VALIDATION
The developed analytical method was subjected to
validation with respect to various parameters such as
linearity,limit of quantification (LOQ), limit of detection
(LOD), accuracy, precision, recovery studies, specificity
and reproducibility as per the ICH guidelines.
3.2.1. SYSTEM SUITABILITY
In the optimized RP-UPLC conditions, system suitability
parameters were evaluated for Lurasidone
HCl(Fig2).Tailing factor for Lurasidone HCl was not
more than 2.0. The USP plate count for Lurasidone HCl
is not less than 2000. % RSD of the six injections was
not more than 1.0%.The resolution between
LurasidoneHCland sterioisomer-1 is not less than 2.0 and
also resolution between LurasidoneHCland sterioisomer-
2 is not less than2.0.The resultsare summarised in
Table: 6.
System suitability chromatogram
Fig: 2.
Table: 6
Parameters LurasidoneHCl
Limit Initial SST Bracketing SST
Tailing factor 1.28 1.28 NMT 2.0
USP Plate count 4430 4464 NLT 2000
Retention time (min) 1.99 1.99 ~2.0
Resolution between LurasidoneHCland
sterioisomer-1 2.70 2.58 NLT 2.0
Resolution between LurasidoneHCland
sterioisomer-2 2.56 2.51 NLT 2.0
3.2.2. SPECIFICITY
The specificity of the proposed method was
demonstrated by interference study. It was found that
presence of some common exicipients did not
interferences at the retention time of LurasidoneHCl.
Thus the developed method can be successfully applied
for determination of LurasidoneHCl in bulk drug and
tablet dosage form.
3.2.3. Blank and Impurity interference
The blank, sample enriched with impurities were
prepared and injected in RP-UPLC. No interference was
observed at any of the peaks of interested in blank.
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Blank chromatogram
Fig:3
3.2.4. Forced degradation/ Stress study
In order to determine whether the analytical method and
assay were stability-indicating, LurasidoneHCl bulk drug
and tablet dosage form was stressed under various
conditions to conduct forced degradation studies.
Lurasidone HCl was soluble in acetonitrile+water in the
composition of (50:50). All solutions for forced
degradation studies were prepared to yield concentration
50μg/g of LurasidoneHCl.
Acid degradation (5N HCl)
Solution of Lurasidone HCl (50μg/g) for acid
degradation study was prepared using 5N hydrochloric
acid solution and the resultant solution was 1 hours to
facilitate acid degradation of Lurasidone HCl.
Alkali degradation (5N NaOH)
Solution of Lurasidone HCl (50μg/g) for base
degradation study was prepared by using 5N NaOH
resultant solution was 1 hours to facilitate alkali
degradation of Lurasidone HCl.
Oxidation (30 % H2O2)
Solution of Lurasidone HCl (50μg/g) for peroxide
degradation study was prepared using 30% H2O2
resultant solution was 1 hour to facilitate peroxide
degradation of Lurasidone HCl.
Thermal degradation
LurasidoneHCl bulk drug was kept in hot oven for
24hours at 80°C to determine the effects of thermal
degradation on the stability of Lurasidone HCl by using
the analytical method.
Photo stability (Sun light)
Lurasidone HCl bulk drug was exposed to sunlight to
determine the effects of light irradiation on the stability
of LurasidoneHCl in the solid state. Approximately 200
mg of Lurasidone HCl bulk drug was spread on a glass
dish in a layer that was less than 2 mm thick. All samples
for photo stability testing were placed indirect sunlight
exposed for 48hours. The bulk drug were removed from
the sun light and the contents of bulk drug equivalent to
100 mg of LurasidoneHCl was accurately weighed and
transferred to volumetricflask to make the final
concentration 50μg/g of Lurasidone HCl. The proposed
method evaluates the ability to separate the impurities of
Lurasidone HCl from its degradation products shown in
Figure: 4 to 9. The results and system suitability
parameter are summarised in Table: 7
Undegraded Sample chromatogram. Sample chromatogram of peroxide degradation
Fig:4 Fig:5
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Sample chromatogram of Thermal degradation Sample chromatogram of Photo degradation
Fig:6 Fig:7
Sample chromatogram of Acid degradation Sample chromatogram of Base degradation
Fig:8 Fig:9
Table:7
Parameters LurasidoneHCl Limit
Initial SST Bracketing SST
Tailing factor 1.35 1.34 NMT 2.0
USP Plate count 4699 4614 NLT 2000
Retention time (min) 1.99 1.99 ~2.0
Resolution between LurasidoneHCland
sterioisomer-1 2.76 2.65 NLT 2.0
Resolution between LurasidoneHCland
sterioisomer-2 2.72 2.58 NLT 2.0
Degradation study
S.No Condition Time % Degradation
% Assay %Degradation 1 Undegraded sample Fresh 99.9% NA 2 5N HCL added sample 60min 35.3% 64.7% 3 5N NaOH added sample 60min 38.1% 61.9% 4 30% H2O2 added sample 60min 49.8% 50.2% 5 Thermal 80°C heated sample 24 hrs 99.6 0.4% 6 Photo Degradation sample 48 hrs 99.3 0.7%
Inference: Based on the above results from the table it is
revealed that in the HCl, Oxidative and NaOH
degradation process high degradation products are
obtained than that of the all undegarded samples.
3.2.5. LINEARITY
3.2.6. Linearity for LurasidoneHCl Assay
The calibration curve was plotted over the concentration
range of 0.03 to 75.3μg/g of Lurasidone HCl. The
calibration curves were prepared by plotting the peak
area versus the concentration and analyzed through linear
regression (Figure-10). The linearity was observed in the
Rao et al. European Journal of Biomedical and Pharmaceutical Sciences
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expected concentration range, demonstrating its
suitability for analysis.The system suitability and results
are summarised in Table: 8.
Table: 8 Linearity (Correlation coefficient)
Parameters LurasidoneHCl
Limit Initial SST Bracketing SST
Tailing factor 1.28 1.28 NMT 2.0
USP Plate count 4430 4464 NLT 2000
Retention time (min) 2.02 2.02 ~2.0
Resolution between LurasidoneHCland
sterioisomer-1 2.70 2.58 NLT 2.0
Resolution between LurasidoneHCland
sterioisomer-2 2.56 2.51 NLT 2.0
Precision
Injection-01 1208217
%RSD NMT 1.0%
Injection-02 1208194
Injection-03 1207300
Injection-04 1206664
Injection-05 1206113
Injection-06 1205253
Mean 1206957
SD 1178
RSD 0.10
S.No Concentration(μg/ml) Response(Area)
1 75.33 1761924
2 60.3 1436463
3 50.22 1207821
4 40.2 958124
5 25.11 598262
6 15.1 350948
7 5.022 115780
8 2.511 66112
9 1.0044 33180
10 0.5022 11801
11 0.2511 10328
12 0.062775 2714
13 0.030388 1374
14 0.010506 844
Slope 23611.2340199
Intercept 3773.6523776
Correlation 0.99988
LurasidoneHCl Linearity Calculation sheet
Fig:10
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Inference: Based on correlation coefficient values from
table-8 were within the Acceptance criteria.
3.2.7. LIMIT OF DETECTION AND LIMIT
QUANTIFICATION
The limit of detection (LOD) and limit of quantitation
(LOQ) were established for LurasidoneHClby diluting
the standard stock solution. The concentration at
0.01µg/g, 0.030µg/g LOD and LOQ for LurasidoneHCl.
The signal to noise ratios was found to more than 7 and
12 respectively for the analyte. Hence these
concentrations were finalised as LOD and LOQ
concentrations. Further precision was found to be 2.67%
at LOD level for analyte and 4.06 at LOQ level the
results are summarised in Table: 9. Overlay
chromatograms of LOD & LOQ are representing below.
Table: 9 LOD and LOQ Precision.
Parameters LurasidoneHCl
Limit Initial SST Bracketing SST
Tailing factor 1.30 1.28 NMT 2.0
USP Plate count 4409 4379 NLT 2000
Retention time (min) 2.03 2.02 ~2.0
Resolution between
LurasidoneHCland sterioisomer-1 2.56 2.57 NLT 2.0
Resolution between
LurasidoneHCland sterioisomer-2 2.55 2.50 NLT 2.0
Precision
Injection-01 1202740
%RSD NMT 1.0%
Injection-02 1204250
Injection-03 1207175
Injection-04 1207828
Injection-05 1206549
Injection-06 1205919
Mean 1205744
SD 1914
RSD 0.16
LurasidoneHCl LOQ (%RSD NMT 10.0%) LOD (%RSD NMT 33.0%)
Injection-1 1280 710
Injection-2 1181 739
Injection-3 1243 755
Injection-4 1214 765
Injection-5 1205 730
Injection-6 1315 753
Average 1240 742
Standard Deviation 50 20
%RSD 4.06 2.67
Overlay Chromatogram of LOD
Fig-11
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Overlay Chromatogram of LOQ
Fig-12
3.2.8. ACCURACY
3.2.9. Accuracy for Assay of LurasidoneHCl
The accuracy of the method was determined by
calculating recoveries of Lurasidone HCl by the standard
addition method. Known amounts of standard solutions
of Lurasidone HCl (80, 100, and 120 % level) were
added to previously analyzed sample solutions of bulk
drug. The percentage of recoveries was calculated. The
percentages of recoveries were between 98.0 to102.0.The
results are summarised in the following Table: 10.
Table: 10 Accuracy and SST parameters.
Parameters LurasidoneHCl
Limit Initial SST Bracketing SST
Tailing factor 1.28 1.29 NMT 2.0
USP Plate count 4408 4392 NLT 2000
Retention time (min) 1.98 1.98 ~2.0
Resolution between LurasidoneHCland
sterioisomer-1 2.58 2.56 NLT 2.0
Resolution between LurasidoneHCland
sterioisomer-2 2.56 2.54 NLT 2.0
Precision
Injection-01 1216350
RSD (NMT 1.0%)
Injection-02 1219336
Injection-03 1215581
Injection-04 1215100
Injection-05 1214995
Injection-06 1216947
Mean 1216385
SD 1628
%RSD 0.13
% of Drug Added Amount Added
(W/W)
Amount Recovered
(W/W)
% Recovery
(Criteria 98.0 to 102.0)
LurasidoneHClat 80% 80.3 80.0 99.7
LurasidoneHClat 80% 80.4 80.0 99.5
LurasidoneHClat 80% 80.2 80.2 100.0
LurasidoneHClat 100% 100.2 100.2 100.1
LurasidoneHClat 100% 100.6 99.5 99.5
LurasidoneHClat 100% 100.3 99.9 99.9
LurasidoneHClat 120% 120.4 120.0 99.7
LurasidoneHClat 120% 120.4 120.2 99.9
LurasidoneHClat 120% 120.5 120.2 99.7
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Inference: Results from the table-10, it is illustrated that
recovered concentration of spiked samples was found to
be within the acceptance criteria i.e. 98.0 to 102.0.
3.2.10. PRECISION
The precision of the method was demonstrated by system
precision and method precision.
3.2.11. System precision
System precision for assay was demonstrated by
injecting standard solution under the same operating
conditions. The peak areas of Lurasidone HCl were
measured and the % RSD was found to be 0.19%.The
results are summarised in Table: 11.
Table: 11
Parameters Lurasidone HCl
Limit Initial SST Bracketing SST
Tailing factor 1.29 1.30 NMT 2.0
USP Plate count 4366 4674 NLT 2000
Retention time (min) 2.06 1.98 ~2.0
Resolution between Lurasidone HCl and
sterioisomer-1 2.62 2.60 NLT 2.0
Resolution between Lurasidone HCl and
sterioisomer-2 2.49 2.52 NLT 2.0
System precision
S.No Area Criteria % RSD
Injection-1 1225499
(NMT1.0%)
Injection-2 1224730
Injection-3 1225963
Injection-4 1223581
Injection-5 1224343
Injection-6 1223344
Mean 1224577
SD 10.36
%RSD 0.08
Inference: Results from the table-11, it is illustrated
%RSD of standard was found to be within the acceptance
criteria i.e. NMT 1.0%
3.2.12. Method precision
Method precision for analyte was demonstrated by
preparing six samples at spec level. These solutions were
injected along with a standard solution of Lurasidone
HCl prepared at spec level. The relative standard
deviation all six preparations results was found to be
0.23%.The results are summarised in Table: 12.
Table: 12
Method precision
Lurasidone HCl Assay result % RSD Criteria % RSD
Sample-01 99.6
0.23% NMT 1.0%
Sample-02 99.3
Sample-03 99.5
Sample-04 99.8
Sample-05 99.2
Sample-06 99.3
Inference: Results from the table-12, it is illustrated that
%RSD of results precision was found to be within the
acceptance criteria i.e. NMT 1.0%
3.2.13. Intermediate precision (ruggedness):
Intermediate precision for analyte was demonstrated by
preparing six different samples at spec level by different
analyst and different day. These solutions were injected
Rao et al. European Journal of Biomedical and Pharmaceutical Sciences
www.ejbps.com 854
along with a standard solution of Lurasidone HCl
prepared at spec level. The relative standard deviation of
analyte content obtained from all six preparations results
was found to be 0.41%. The system suitability
parameters and results are summarised in Table: 13.
Table: 13
Parameters LurasidoneHCl
Limit Initial SST Bracketing SST
Tailing factor 1.29 1.29 NMT 2.0
USP Plate count 4371 4386 NLT 2000
Retention time (min) 1.97 1.98 ~2.0
Resolution between LurasidoneHCland
sterioisomer-1 2.58 2.55 NLT 2.0
Resolution between LurasidoneHCland
sterioisomer-2 2.52 2.52 NLT 2.0
System Precision
Injection-01 1209608
%RSD (NMT 1.0%)
Injection-02 1212759
Injection-03 1214233
Injection-04 1210823
Injection-05 1213025
Injection-06 1211585
Mean 1212006
SD 1667
%RSD 0.14
Intermediate precision
Lurasidone HCl Assay result % RSD Criteria % RSD
Sample-01 100.2
0.33% NMT 1.0%
Sample-02 99.7
Sample-03 100.6
Sample-04 99.8
Sample-05 99.9
Sample-06 99.9
Cumulative RSD for precision
Analyst-01 Analyst-02 Cumulative (12 Results) Criteria % RSD
0.23% 0.33% 0.40% NMT 1.0
Inference: Sample Results from the table-12, 13 it is
observed that cumulative RSD’s were found to be within
the limit i.e. NMT1.0%.
3.3. FILTER COMPATIBILITY
Filter compatibility to the sample is concluded from the
recovery study indicated that there is no absorption of
these component to filter.
3.4. SOLUTION STABILITY
The standard and samples solutions were kept on room
temperature at 25°c and injected the aged samples (Every
one hour) into the RP-UPLC. The peak areas
corresponding to Lurasidone HCl were measured.
Calculated the similarity factor and found that the values
are below 1.0% RSD. Thus indicates the sample and
standard solutions are stable for at least 24 hrs when
stored on room temperature condition. System suitability
parameters are shown below table-14.
Table-14
Parameters LurasidoneHCl
Limit Initial SST Bracketing SST
Tailing factor 1.29 1.28 NMT 2.0
USP Plate count 4341 4412 NLT 2000
Retention time (min) 1.99 1.99 ~2.0
Resolution between Lurasidone
HCl and sterioisomer-1 2.65 2.50 NLT 2.0
Resolution between Lurasidone
HCl and sterioisomer-2 2.57 2.49 NLT 2.0
Rao et al. European Journal of Biomedical and Pharmaceutical Sciences
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3.5. METHOD ROBUSTNESS
Method robustness was performed by applying small
changes in the ratio of mobile phase, injection volume,
and column temperature, pH and flow rate. The results of
change in ratio of mobile phase, column temperature,
wavelength, and injection volume are shown in Table 15.
The flow rate 0.45– 0.55 mL/min, buffer pH 2.40 to
2.60,wavelength 227 to 233nm and Column temperature
45°C to 55°C and Study of the mobile phase composition
61.5:38.5, 68.5:31.5. Based on this, the method is proved
to be robust and can easily be implemented in quality
control laboratories for the regular analysis of
Lurasidone HCl samples with great confidence.
Table-15.
Method Robustness
Ch
rom
ato
gra
ph
ic P
ara
met
er
Co
nd
itio
n
Ret
enti
on
tim
e(R
T)
~2
.0
Resolution
Th
eore
tica
l p
late
s(N
) N
LT
20
00
Ta
ilin
g(T
) N
MT
2.0
Res
ult
(NL
T 9
8.0
TO
10
2.0
)
Lurasidone
HCl&Sterioiso
mer-1
Lurasidone
HCl&Steriois
omer-2
Init
ial
Aft
er
Init
ial
Aft
er
Wavelength(nm) 227 1.97 2.66 2.62 2.64 2.59 4561 1.33 100.0
233 1.97 2.65 2.60 2.62 2.62 4492 1.32 100.2
Temperature(°C) 45°C 1.97 2.59 2.61 2.67 2.67 4397 1.34 99.4
55°C 1.96 2.61 2.62 2.60 2.60 4558 1.31 99.8
Acetonitrile (%) 61.5:38.5 1.41 2.12 2.10 2.07 2.04 4341 1.41 99.7
68.5:31.5 2.5 3.19 2.84 3.12 3.15 4993 1.30 99.9
Flow rate(ml/min) 0.45 2.18 2.67 2.65 2.63 2.63 4519 1.35 100.0
0.55 1.81 2.61 2.63 2.59 2.60 4395 1.29 100.6
pH variation 2.40 1.96 2.69 2.56 2.71 2.60 4433 1.32 100.0
2.60 1.97 2.65 2.66 2.66 2.62 4458 1.32 99.9
Inference: Solvent composition is more critical based on
above data. Measure the mobile phase composition
accurately.
4.0. BATCH ANALYSIS
20 tablets of Lurasidone HCl with brand name of Sun
pharmaceuticals tablets was purchased and analyzed with
equal quantity of 3samples. Each tablet having around
135mg it contains about 40mg of Lurasidone HCl API.
20 tablets were taken and crush the samples with the help
of mortar and analysed with optimum chromatographic
conditions. But the sample solution slight haziness
observed it’s filtered with 0.22µ filter paper and
analysed. The SST parameters and results are
summarized in table-16.
DETAILS OF TABLET
Marketed By
Sun Pharma laboratories limited,
Andheri (E), Mumbai-400059,
B.No:DT1610036B,
Unit: II.
Table-16.
Parameters Lurasidone HCl
Limit Initial SST Bracketing SST
Tailing factor 1.29 1.29 NMT 2.0
USP Plate count 4376 4386 NLT 2000
Retention time (min) 1.96 1.99 ~2.0
Resolution between Lurasidone HCl and sterioisomer-1 2.54 2.58 NLT 2.0
Resolution between Lurasidone HCl and sterioisomer-2 2.57 2.52 NLT 2.0
TABLETS ANALYSIS RESULTS
Lurasidone HCl Assay result % RSD Criteria % RSD
Tablet-01 100.0
0.28% NMT 1.0% Tablet-02 100.2
Tablet-03 100.6
Rao et al. European Journal of Biomedical and Pharmaceutical Sciences
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5.0. DISCUSSION
The purpose of the present work was to develop a short,
robust, precise RP-UPLC method for the accurate
quantisation of Lurasidone HCl Assay. This method was
developed for drug substance and drug product also. As
mentioned in the introduction section, several reports are
available for quantification of Lurasidone HCl but
present method was highly sensitive.The developed
method was successfully validated for drug substance
and drug product as per the ICH guideline. The proposed
method is much superior to reported methods in terms of
solvent consumption, run time, instrumental technique
(RP-UPLC), selectivity, and applicability to Assay
analysis.
6.0. APPLICATION TO PHARMACEUTICAL
INDUSTRY
This work will help industry to develop, manufacture and
launch the product in a fast and economical way which
in turn reduces the cost of the medicine and help the
patient to avail quality, innovative and affordable
medicine.
7.0. CONCLUSION
A stability indicating RP-UPLC Assay method has been
developed for determination of Lurasidone HCl.
Developed method was proved to be robust using the
experimental design, this method can be successfully
implemented in the quality control lab for the routine
analysis of this product. Further this RP-UPLC method
was successfully validated as per ICHQ2 (R1) guideline
and proved to be precise, linear, sensitive, accurate, and
robust. This method is short and simple, hence
implementation of this method in quality control and
analytical development labs can yield high throughput.
As low amounts of solvents are required, implementation
of this method will be eco friendly. This was the first
RP-UPLC Assay method that can accurately quantitative
the Lurasidone HCl Assay in drug substance and drug
product.
ACKNOWLEDGEMENTS
The authors are heartily thankful to acknowledge
Aurobindo Pharma Ltd and Andhra University for
providing all the facilities to carry out the research work.
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