Download development and validation of rapid hplc

ANALELE UNIVERSITĂŢII “DUNĂREA DE JOS” GALAŢI
MEDICINĂ
FASCICULA XVII, no 2, 2012
ORIGINAL STUDY
DEVELOPMENT AND VALIDATION OF RAPID HPLC METHOD
FOR DETERMINATION OF ARIPIPRAZOLE IN BULK DRUG AND
PHARMACEUTICAL DOSAGE FORMS
Ashu M.1,
1
2
Shikha Parmar2
, Nagarajan K.
1
KIET School of Pharmacy, Ghaziabad, India
H.R.Institute of Pharmacy, Ghaziabad, India
[email protected]
ABSTRACT
A simple,selective, rapid, and economical reversed phase high performance liquid chromatography (RPHPLC) method for the determination of aripiprazole in the pharmaceutical dosage form has been developed and
validated.The separation and quantification were achieved on waters spherisorb 5µ ODS 24.6mm x 250mm
column using a mobile phase of buffer: acetonitrile : THF (30:60:10, v/v/v ) at a flow rate of 1.5 ml/min with
detection of analyte at 255 nm. The separation was achieved within 3.91± 0.1 min for aripiprazole sample. The
method showed good linearity in the range of 1-100 µg/ml. The intra and inter day R.S.D ranged from 0.831.18% The recovery (mean ±S.D.) of low, middle and high concentrations were 102 ± 0.82, 100.00 ± 1.05,
100.44 ± 0.75 respectively.
KEYWORDS: RP-HPLC, aripiprazole, Estimation, Bulk drug, Pharmaceutical dosage form.
(EPS) [1-3]. Although various bioanalytical methods
1. Introduction
for estimation of aripiprazole in human serum [4-6]
and spectrophotometric method for estimation of
Aripiprazole,7-(4-[4-(2,3-dichlorophenyl)-1-
aripiprazole in dosge form [7] have been reported in
piperazinyl]butoxy)-3,4-dihydro-2(1H)-quinolinone,
the literature, there is no HPLC method reported for
is a novel, atypical antipsychotic drug for treatment
the estimation of aripiprazole in pharmaceutical
of schizophrenia and schizoeffective disorders. It has
formulations. The present work describes a simple,
potent partial agonist activity at dopamine (D2)
precise and accurate RP-HPLC method for estimation
receptors, partial agonist activity at serotonin (5-
of aripiprazole in commercial dosage form. The
HT1A) receptors, and antagonist activity at 5HT2A
results of analysis were validated using International
receptors. As a result, aripiprazole can improve both
Conference on Harmonization (ICH) guidelines.
negative and positive symptoms of schizophrenia
with lower propensity for extrapyramidal symptoms
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ANALELE UNIVERSITĂŢII “DUNĂREA DE JOS” GALAŢI
FASCICULA XVII
in the Thin layer chromatogram. TLC results are
2. Material and methods
readily transferable to HPLC[10,11]. Mobile phase
Reagents. Acetonitrile (HPLC grade) was
tried for this purpose were acetonitrile: buffer (30:70,
procured from Qualigens, India, Milli-Q-water was
v/v), acetonitrile : buffer (50:50, v/v), methanol :
purchased from Rankem, India. Reference standards
water (70:30, v/v), methanol: water (50:50, v/v),
of
methanol: water: acetonitrile (35:30:35, v/v/v),
aripiprazole were procured from Mapro
methanol:acetonitrile:buffer
pharmaceutical Ltd, Mumbai, India .
(15:25:60,
v/v/v),
Instrumentation. The HPLC system used in the
methanol:acetonitrile:buffer (15:35:50, v/v/v) and
study was Waters, a rheodyne 7202 25µL Hamilton
buffer:acetonitrile:THF (30:60:10 v/v/v). Effect of pH
syringe, a dectector (Waters, UV-2489 UV/Vis
and flow rate was also seen on the resolution in all
detector). Separation was accomplished on a waters
the above used systems. The condition that gave the
spherisorb 5µ ODS 24.6mm x 250mm column. The
best resolution and symmetry was selected. Same
system was empowered by Compaq Presario SR
solvent system was used for the extraction of the drug
52401L desktop PC. The mobile phase was composed
from the formulation containing excipients which was
of
used for quantification.
buffer: acetonitrile:THF (30:60 :10, v/v). The
mobile phase was filtered through 0.45 µm Millipore
Calibration Curve. Different concentrations
HVLP filter and degassed by sonication before use. A
(1-100 µg/ mL) were made for the preparation of
flow rate of 1.5 ml/min was set .Detection was
calibration curve from the stock solution. The mobile
carried out at 255 nm. The retention time was 3.9±0.1
phase after filtration through a 0.45 µm membrane
minutes (Fig.2). The validation of the proposed
filter was delivered at 1.5 mL/ min for column
method was also carried out according to ICH
standardization, and baseline was continuously
guidelines.
monitored during the process. The UV scan of
optimize
the
aripiprazole was performed between 200-400nm and
effect
of
wavelength of detection was selected at 255 nm. The
chromatographic variables such as mobile phase, pH,
prepared dilutions were injected serially and areas
flow rate and solvent ratio were studied. Various
under the peaks were calculated for each dilution. The
solvent systems were tried for the development of a
stability of drug in solution during analysis was
suitable
of
determined by repeated analysis of samples during
in bulk drug and pharmaceutical
the course of experimentation on the same day and
formulations.The more physical and chemical data
also after 48 h storage of drug solution at laboratory
that are available for each sample, the more chance
bench conditions and in the refrigerator.
Method
chromatographic
HPLC
aripiprazole
Development.
conditions,
method
for
To
the
determination
there is of selecting the right mobile phase system,
Method Validation.
thus ensuring success right from the start [8] Every
Linearity. The concentrations of aripiprazole
successful separation adds to the analyst’s wealth of
within 1-100 µg/ mL were prepared from stock
experience. To a limited extent, the choice of a
solution and areas under peak were obtained.
Precision. Precision was considered at two
method can be done by an expert system[9]
The best mobile phase strength for a specific
levels i.e. repeatability and intermediate precision.
separation problem can be determined by thin-layer
Repeatability of sample application was determined
chromatography. It is represented by that solvent or
as intraday variation whereas intermediate precision
solvent mixture which gives an Rf value od about 0.3
was determined by carrying out inter-day variation
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ANALELE UNIVERSITĂŢII “DUNĂREA DE JOS” GALAŢI
FASCICULA XVII
for the determination of aripiprazole at three different
µg/mL of aripiprazole was prepared. Standard
concentration levels of 25, 50 and 75 µg/mL.
solution in the concentration range of about 75 µg/ml
Accuracy as recovery. Accuracy of the method
were also prepared.
was studied by recovery experiments. About 5.5 mg
of placebo and 25, 50, and 75 mg of aripiprazole,
were transferred into a 100 mL
3. Results and discussions
volumetric flask.
About 50 ml of mobile phase were added, sonicated
Method Development. The proposed HPLC
for 10 min and shaken for 5 min. The volume was
procedure was optimized with a view to develop a
made up to the mark with mobile phase and mixed.
suitable analytical method. Mobile phases tried for
Solution was filtered through a 0.45 µm Millipore
this purpose were acetonitrile: buffer (30:70),
HVLP filter and the filtrate collected by discarding
acetonitrile: buffer (50:50), methanol: water (70:30),
few ml of filtrate. The final concentrations of
methanol: water (50:50), methanol: water: acetonitrile
accuracy were 25, 50, and 75 µg/mL of aripiprazole,
(35:30:35). The chromaotogram
respectively.
Buffer:Acetonitrile:THF (30:60:10, v/v/v) solvent
obtained with
Specificity. A Synthetic mixture containing 50
system was found to have very good symmetry with
mg of aripiprazole, and 30 mg each of starch, lactose,
lowest Rt (3.9 ± 0.01 ) and sharp well defined peak.
magnesium stearate, and avicel, which are present as
Therefore the mixture of Buffer:Acetonitrile:THF
excipients in the pharmaceutical dosage form, was
(30:60:10, v/v/v)
accurately weighed and transferred to a 50 mL
drug was stable for a period of 48 h at laboratory
volumetric flask. The mixture was shaken well with
temperature and under refrigerator temperature in
30 mL of methanol and then diluted to volume with
Buffer:Acetonitrile:THF (30:60:10) mixture.
was chosen as mobile phase. The
methanol, a final solution containing 75 µg/ mL
Method Validation.
aripiprazole was prepared.
Linearity. The linearity range of aripiprazole
Robustness. Robustness was carried out to
solutions was obtained as 1 -100 µg/mL. The slope of
evaluate the influence of small but deliberate
regression was 15601 and regression coefficient was
variations in the chromatographic conditions for the
0.999 (Table I).
determination of aripiprazole. Robustness of the
method was determined by changing the flow rate
Table I. The results of linear regression for
aripiprazole.
(0.8 & 1.2 mL/min ) , mobile phase ratio ( ±10% ) ,
pH (±10% ), and temperature (±10%)
Parameter
Value
1-100
Assay of commercial dosage form. Accurately
Concentration range, µg/ml
Slope of regression
Intercept
Correlation coefficient
weighed quantity of Arip 75 purchased from Sun
Pharmaceutical, India equivalent to about 50 mg of
aripiprazole was transferred in to a 250 ml volumetric
flask. About 100 ml of mobile phase was added, the
Precision.
solution was sonicated for 20 min with continuous
of
repeatability
and
intermediate precision were expressed in terms of %
shaking at 30ºC. Volume was made up with mobile
RSD and are shown in Table II. The low value of
phase. The solution was filtered through a 0.45µm
RSD indicates good repeatability of the proposed
HVLP filter paper by discarding first few ml of the
filtrate.
Results
15601
48378
0.999
method.
A solution of final concentration of 75
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ANALELE UNIVERSITĂŢII “DUNĂREA DE JOS” GALAŢI
FASCICULA XVII
Accuracy as recovery. The values of drug
showed that percentage recoveries were high and
recovered, mean recovery and % RSD are shown in
RSD values were low, which confirms the method is
Table 3, which indicate satisfactory accuracy of the
suitable for routine determination of aripiprazole in
proposed method.
its pharmaceutical preparation. Figure 1.
Table IV. Specificity of method
Table II. Intra-day and inter-day precision of the
method (n=5)
Concentration
(µg/mL)
Intra-day
precision
Mean
RSD,
%
25.22
1.13
50.42
1.91
75.57
0.89
25.00
50.00
75.00
Added, µg/ mL
75.00
75.00
75.00
75.00
Inter-day precision
Mean
RSD, %
25.62
50.77
75.83
1.18
1.35
0.83
Mean
RSD
Table III. Recovery values obtained for the
determination of Aripiprazole
Recovery
level, %
Set
Drug
added
(mg)
Drug
reccovered
mg
%
1
2
25.00
25.00
25.3
25.5
101.2
102.0
100
3
1
2
3
25.00
50.00
50.00
50.00
25.7
50.6
49.8
49.6
102.8
101.2
99.60
99.20
150
1
2
3
75.00
75.00
75.00
75.5
74.7
75.8
100.66
99.60
101.06
Mean
recovery
Changed
Parameters
Temperature
%
RSD
102
0.80
50
Flow Rate
1.058
Mobile
phase ratio
100.44
75.78
0.625
Table V. Robustness of the method (n=5)
pH
100
Recovered, µg/ mL Recovery, %
75.92
101.22
75.81
101.08
76.45
99.92
74.94
0.754
% RSD
Normal
conditions
0.65
Normal
conditions
0.63
Normal
conditions
0.64
Normal
conditions
0.64
-5°C
+5°C
0.20
-0.2 unit
1.51
+0.2 unit
0.18
-10%
0.15
+10%
0.44
-2%
0.24
+2%
0.09
0.17
Table VI. Results of assay of aripiprazole ( µg/ mL)
in pharmaceutical dosage form ( n=3)
Specificity. The specificity of the method was
tested by calculating the percentage recovery of each
Added
Recovered
Recovery, %
75
76.43.
101.43
75
75.23
101.23
75
74.97
99.97
component in the presence of possible interfering
materials such as starch, lactose, magnesium stearate,
and avicel. The results are presented in Table IV,
which shows that separation of analytes from the
excipients was complete.
Robustness of the method. There was no
significant
change
in
the
retention
time
of
Mean
75.54
RSD
0.778
aripiprazole by changing the flow rate, pH, mobile
phase ratio and temperature. Low value of % RSD
Shows a typical chromatogram obtained from
indicates the robustness of the method as shown in
analysis of a Arip 75 branded formulation. Figure 2.
Table V.
shows chromatogram of a blank sample and Figure 3.
Application of method to assay Aripiprazole in
pharmaceuticals.
shows a chromatogram of placebo formulation.
The method was used for the
determination of aripiprazole in
The stability of aripiprazole in solution was
Arip 75 (Sun
checked by determining the percentage deviation of
Pharma, India). The results obtained (Table VI)
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ANALELE UNIVERSITĂŢII “DUNĂREA DE JOS” GALAŢI
FASCICULA XVII
the amounts present in solution after 48 hr at room
4. Conclusions
temperature in comparison with the amount at zero
time. The results obtained after 48 hr showed no
The proposed HPLC method in this study
significant variation; the percentage deviation was
using
less than 2% of the initial amount. This is indicative
preparation procedures is particularly appropriate for
of good stability of each component in the mixture
the routine analysis of aripiprazole in pharmaceutical
over a period of 48 hr.
dosage form. This method has the advantages of
common
simplicity,
reagents
precision,
and
accuracy,
simple
sample
sensitivity
and
quantification of aripiprazole compared with other
reported methods and can be employed for its assay
in dosage form with a single injection. The selectivity
of
the
chosen
chromatographic
systems
was
ascertained. Excipients showed no peak in the range
of the retention times corresponding to the analyte.
References
1.Frederique L, Kayssa D, Khalid T, Linda K, Sophie B, Pascal
P and Marie-L. P. Development and validation of a highperformance liquid chromatography method using diode array
detection for the simultaneous quantification of aripiprazole and
dehydro-aripiprazole in human plasma. J. Chromatogr. B ,2008,
867: 15-19
2. Jordon S, Koprivica V, Dunn R, Tottori K, Kikuchi T and
Altar C.A. In vivo effects of aripiprazole on cortical and striatal
dopaminergic and serotonergic function. Eu.r J. Pharmacol. 2004,
483: 45-53
3. Katrin MK, Matthias JM, Gerald Z, Alois S, Arian M,
Jaroslav M and Christoph H.. Therapeutic Monitoring of
Aripiprazole
by
HPLC
with
column-Switching
and
Spectrophotometric Detection. Clinical Chemistry, 2005, 51: 17181721
4. Mallikaarjun S, Salazar DE, Bramer SL. Pharmacokinetics,
tolerability, and safety of aripiprazole following multiple oral
doping in normal healthy volunteers. J.Clin Pharmacol. 2004,
44:179-87
5. Kalaichelvi R, Thangabalan B, Srinivasa DR and
Jayachandran E. UV Spectrophotometric determination of
Aripiprazole in bulk and Pharmaceutical formulation. Eur. J.
chem.,2009, 6, S 87-S90
6. Shapiro DA., Renock S, Arrington E, Chiodo LA., Liu LX.
and Sibley DR. Aripiprazole, a novel atypical antipsychotic drug
with
a
unique
and
robust
pharmacology.
Neuropsychopharmacology ,2003, 28:1400-11
7. Xiao-cong Z, Feng W, Ping X, Rong HZ and Huan DL. LCESI-MS foe rapid and sensitive determination OF Aripiprazole in
human plasma. Chromatogr. 2006, 64: 387-391
8.B.Serkiz.The use of gas chromatography - tandem mass
spectrometry (gc-msn) for optimisation of targeted research on
residues in matrices of animal origin . LC GC Int. 1997, 10: 310.
9. M.Peris, An overview of recent expert system applications in
analytical chemistry Crit.Rev.Anal.Chem,1996, 26: 219-237.
10. W.Jost, H.E.Hauck, F.Eisenbeiss, Kontakte. Fundamentals
of Thin-Layer Chromatography. Merk, 1984,3/84, 45.
11. P.Renold, E.Madero, T.Maetzke. Automated normal-phase
preparative high-performance liquid chromatography as a
substitute for flash chromatography in the synthetic research
laboratory. J.Chromatogr. A. 2001, 908: 143-148.
Figure 1. Typical HPLC Chromatogram of Arip MT
Figure 2. HPLC Chromatogram of Blank
Figure 3. HPLC chromatogram of Placebo
formulation
53