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*Corresponding author e-mail: christine_elmaraghy@hotmail.com
© 2017 NSP
Natural Sciences Publishing Cor.
J. Pharm. Appl. Chem., 3, No. 1, 57-61 (2017) 57
http://dx.doi.org/10.18576/jpac/030108
Validated HPLC Method for Simultaneous Determination of Aripiprazole and Co-
Administered Clonazepam in Spiked Human Plasma
Christine M. El-Maraghy 1,*, Hesham Salem 2, Sawsan M. Amer 3and Marianne Nebsen 3, 4
1Analytical Chemistry department, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), 11787
6th October city, Egypt.
2Pharmaceutical Analytical Chemistry department, Faculty of Pharmacy, Deraya University, Minia, Egypt.
3Analytical Chemistry department, Faculty of Pharmacy, Cairo University, Kasr-El Aini Street, 11562Cairo, Egypt.
4Pharmaceutical Analytical Chemistry department, Faculty of Pharmacy & Drug Technology, Heliopolis University, 3
Cairo Belbeis desert road, 2834El- Horria, Cairo, Egypt.
Received: 5 Aug. 2016, Revised: 22 Oct. 2016, Accepted: 25 Oct. 2016.
Published online: 1 Jan. 2017.
Abstract: A sensitive and selective high performance liquid chromatographic method was developed and validated for the
simultaneous quantification of Aripiprazole and co-administered Clonazepam in spiked human plasma. Aripiprazole,
Clonazepam and the internal standard Citalopram were extracted from plasma and analyzed on a reversed-phase C18
column using a mobile phase consisting of acetonitrile and 0.05M potassium dihydrogen orthophosphate (pH= 3.6) in a
ratio of (40: 60, v/v). The ultraviolet (UV) detection was at 220 nm. The method was linear over concentration range (20-
1000 ng mL-1) for both drugs. The method is very simple and allows obtaining a very good recovery of the analytes.
Keywords: Aripiprazole; Clonazepam; plasma; HPLC; co-administered.
1 Introduction
Aripiprazole (ARI) (Fig.1), chemically is 7-(4-[4-(2,3-
dichlorophenyl)-1-piperazinyl]butoxy)-3,4-dihydro-2(1H)
quinolinone, molecular formula: C23H27Cl2N3O2 and
molecular weight: 448.39 [1]. ARI has been approved by
FDA at the end of 2002 [2]. It is considered to act as a
partial dopamine D2-receptor agonist, partial serotonin (5-
HT1A) receptor agonist and (5-HT2A) receptor antagonist
[3, 4]. It is used in the management of schizophrenia and is
also under investigation for bipolar disorder [1].
Clonazepam (CLO) (Fig.2) is a benzo diazepine derivative,
chemically it is 5-(o-chlorophenyl)-1,3-dihydro-7-nitro-2H-
1,4-benzodiazepin-2-one [1]. CLO is a highly potent
anticonvulsant, amnestic and anxiolytic. It has shown itself
to be useful as a short-term adjunct to SSRI treatments in
clinical depression and obsessive compulsive disorder, and
this combination is superior to SSRI treatments alone [5].
The literature survey revealed analytical methods for the
estimation of ARI with other drugs or in presence of its
main metabolite in biological fluids, including HPLC
methods [6-11] and capillary electrophoresis [12].
Fig. 1. Chemical structure of Aripiprazole
Fig. 2. Chemical structure of Clonazepam
2 Experimental
2.1 Apparatus
La chrom, Merck Hitachi series chromatographic system
equipped with pump L-7110 and wavelength UV–VIS
Journal of Pharmaceutical and Applied Chemistry
An International Journal
58 C. El-Maraghy et al.: Validated HPLC Method …
© 2017 NSP
Natural Sciences Publishing Cor.
detector L-7420 was used. Column X terra® RP-C18column
(150 mm×4.6 mm, 5 µm particle size i.d.) was from (waters
corporation), Ultra-sound sonicator (Crest Ultrasonics, New
York), pH meter equipped with combined glass electrode
for pH adjustement (Jenway 3510,UK).
2.2 Reagents and materials
All chemicals used throughout the work were of analytical
grade and solvents were of HPLC grade; acetonitrile,
methanol and orthophosphoric acid (Riedel-dehaen, Sigma-
Aldrich, Germany), potassium dihydrogen orthophosphate
(ADWIC, Egypt).
2.3 Samples
Authentic samples: Aripiprazole (purity 99.87%) was
kindly supplied by Bristol-Myers Squibb Company, Cairo,
Egypt. Clonazepam (CLO) (purity 99.67%) was supplied
by EIPICO (Egyptian International Pharmaceutical Industry
Company), Egypt. The internal standard (I.S) Citalopram
(purity 99.87%) was supplied by SEDICO Company,
Egypt. The human plasma used was obtained from the
holding company for biological products and vaccines
(VACSERA, Egypt).
2.4 Standard solutions
Stock standard solutions (0.1 mg mL-1) of ARI and CLO
were prepared, daily in methanol. Working standard
solutions of ARI and CLO (10.0 µg mL-1) were prepared by
suitable dilution of the stock solutions, respectively.
Aliquots equivalent to (0.2, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 mL)
were transferred from the ARI and CLO working solutions,
separately and were diluted in 10-mL volumetric flasks
with methanol to reach a concentration of (0.2-10.0 µg mL-
1). The internal standard solution was of concentration (0.1
mg mL-1).
2.5 Sample preparation
0.7 mL of the frozen plasma was thawed at room
temperature and was spiked with a fixed volume (100 µL)
of the serially diluted solutions of ARI and CLO to obtain
concentrations of (20-1000 ng mL-1) and 100 µL of the I.S
was added to give a concentration of (500 ng mL-1).
2.6. Drug extraction
In this proposed method, several extracting solvents were
tried; hexane, acetonitrile, methanol, perchloric acid, ethyl
acetate and diethyl ether but the recoveries of the two drugs
were 70-75%. A mixture of acetonitrile: methanol (ratio
1:1) was found to be optimal for precipitation of plasma
proteins and for obtaining good recoveries of the drugs
(recoveries were above 85%). Two mL of (acetonitrile:
methanol, 1:1) was added to the spiked plasma then the
mixture was vortexed for 2 min and centrifuged for 10 min
at 4500 rpm. The upper layer was transferred to another
clean tube and then filtered through a 0.45μm Millipore
syringe filter. 10-μL of the supernatant was injected onto
the liquid chromatographic system for analysis.
2.7 Chromatographic conditions
Using RP-C18 column(150 mm×4.6 mm, 5 µm particle size
i.d.) and the mobile phase as a mixture of acetonitrile and
0.05M potassium dihydrogen orthophosphate (pH= 3.6
adjusted using orthophosphoric acid) in a ratio of (40: 60,
v/v). The analysis was done under isocratic condition at a
flow rate 1 mL min-1 and at room temperature using UV
detector at 220 nm.
2.8 Method validation
The method described above was validated with regard to
linearity, sensitivity, accuracy, precision, specificity, and
stability according to “The Guidance for Industry
Bioanalytical Method Validation” published by the Food
and Drug Administration [13].
2.8.1 Linearity and sensitivity
The analytical range to be validated was chosen on the
basis of the expected plasma concentrations of the studied
ARI and CLO drugs [14, 15]. The calibration curves were
done for ARI and CLO in the biological sample. The
calibration curves should consist of a blank sample (matrix
sample processed without I.S), a zero sample (matrix
sample processed with I.S), and six to eight samples
covering the expected range, including LLOQ that were
prepared by adding the required volume of working
solutions of ARI and CLO drugs to blank plasma. The
plasma samples were subjected to the sample preparation
procedure and injected into the LC. The calibration curves
of ARI and CLO were plotted using the peak area ratio of
ARI and CLO to the I.S versus the nominal concentration.
Six calibration curves models were determined by
calculating the linear regressions (correlation coefficient, r).
Sensitivity was defined by the lower limit of quantitation
(LLOQ), which was the concentration of ARI and CLO at
which the signal to noise (S/N) ratio was greater than 5
with acceptable accuracy and precision. These values were
set as the lowest concentrations in calibration curves. The
calibration models were accepted if the recoveries were
within ± 20% at the lower limit of quantification (LLOQ)
and within ± 15% at all other calibration levels and if at
least 2/3 of the standards met this criterion, including
highest and lowest calibration levels.
2.8.2 Precision and accuracy
The intra- and inter- day precision (expressed as RSD %)
and accuracy expressed as (recovery %) were determined
by analysis three replicates of each concentration of ARI
and CLO on the same day to determine the intra-day
accuracy and precision of the method. To confirm the inter-
J. Pharm. Appl. Chem., 3, No. 1, 57-61 (2017)/ http://www.naturalspublishing.com/Journals.asp 59
© 2017 NSP
Natural Sciences Publishing Cor.
day precision three replicates of each concentration were
analyzed at three separate days.
2.8.3 Specificity
Specificity is the ability of an analytical method to
differentiate and quantify the analyte in the presence of
other components in the sample [13]. The specificity of the
method was investigated by analyzing six blank plasma
samples. Each blank sample was tested for interference
using proposed extraction procedure and the response of the
endogenous compounds at the retention times of the studied
drugs in plasma samples and by a separation of the two
peaks of ARI and CLO with acceptable resolution factor.
2.8.4 Extraction recovery
The recoveries of ARI, CLO and I.S from plasma were
evaluated by comparing the mean peak areas of triplicate
analyses of three unextracted samples of drugs in methanol
to mean peak areas of the same concentrations with
prepared spiked plasma samples.
2.8.5 Drug stability in biological fluid
Drug stability in a biological fluid is a function of the
storage conditions, the chemical properties of the drug, the
matrix, and the container system. The stability of ARI and
CLO drugs in human plasma (expressed as recovery % ±
RSD) was investigated in four ways:
2.8.5.1 Short term stability (STS)
Three aliquots of each of the low, medium and high
concentrations should be thawed at room temperature and
kept at this temperature for 4 hours (based on the expected
duration that samples will be maintained at room
temperature in the study) and analyzed.
2.8.5.2 Post-preparative stability (PPS)
The stability of processed samples, including the resident
time in the autosampler, should be determined. In this
study, three replicates were prepared, and kept at room
temperature for approximately 24 h.
2.8.5.3 Freeze-thaw stability (FTS)
Three aliquots at each of the low, mid and high
concentrations were stored at (-20°C) for 24 h and thawed
at room temperature. When completely thawed, the samples
were refrozen for 24 h under the same conditions. The
freeze–thaw cycle were repeated three times, and then
analyzed.
2.8.5.4 Long-term stability (LTS)
Long-term stability was determined by storing five aliquots
of each of low, mid and high concentrations of the studied
drugs at -20°C for 30 days.
The concentrations of all the stability samples were
compared to the mean values for the standards at the
appropriate concentrations (low, medium and high samples)
from the first day of long-term stability testing.
3 Results and Discussion
An accurate, sensitive, selective and validated HPLC
method was developed for simultaneous determination of
ARI and a common co-administered mood stabilizer drug;
Clonazepam in human plasma.
3.1 Method optimization
Fig.3. Chromatogram of blank plasma
Fig.4. Chromatogram of extracted ARI, CLO and IS from
human plasma.
The chromatographic conditions, especially the
composition of mobile phase, were optimized to achieve a
good resolution and symmetric peak shapes for the two
drugs and the internal standard, as well as a short analytical
time. Initially a mixture of acetonitrile: 0.05 M potassium
dihydrogen orthophosphate, pH=3.6 was used as a mobile
phase with a ratio of (60: 40 v/v). The resolution between
the peaks of internal standard and ARI was less than 1.5
and the I.S peak was attached to plasma peak. Increasing
the buffer ratio (polarity increase) to 60% resulted in
increasing the retention of I.S and ARI slightly away from
the plasma peak. The mobile phase used for the
simultaneous determination of ARI and CLO was mixture
of acetonitrile and potassium dihydrogen orthophosphate,
pH (3.6) in a ratio of (40: 60 v/v). The chromatograms for
the plain plasma and the mixture of ARI (tR= 5.16), CLO
(tR= 7.98) and I.S (tR= 3.71) are shown in Fig. 3 and 4,
respectively. Upon utilizing the above conditions for the
60 C. El-Maraghy et al.: Validated HPLC Method …
© 2017 NSP
Natural Sciences Publishing Cor.
determination of ARI and CLO in six different plasma
sources, the absolute peak overcome the matrix effect and
gave reproducible results. Calibration graphs were obtained
by plotting the peak area ratio of ARI and CLO to that of
the I.S versus the nominal concentration.
Linearity range was found to be 20-1000 ng mL-1 using the
following regression equations:
For ARI: A =0.0009 C + 0.0592 r = 0.9995
For CLO: A =0.020 C – 0.0593 r = 0.9996
Where, A represents the peak area ratio, C is the
concentration in ng mL-1 and r is the correlation coefficient.
3.2 Method validation
3.2.1 Linearity and lower limit of quantification
(LLOQ)
Calibration curves were linear in the concentration range of
20-1000 ng mL-1 for ARI and CLO. The validation
parameters are listed in Table 1. The coefficient of
determination (r2) of the calibration curves was ≥ 0.999.
The lowest limit of quantification for ARI and CLO was
determined to be 20 ng mL-1, with a signal to noise ratio
(S/N) of 5.5.
Table 1. Validation parameters for the proposed HPLC
method, for determination of ARI and CLO in spiked
human plasma.
Validation Parameters
ARI
CLO
Linearity range (ng mL-1)
20-1000
20-1000
Correlation coefficient (r)
0.9995
0.9996
Slope
0.0009 ±
0.018
0.020 ±
0.012
Standard error of the slope
1.265×10-5
26.06×10-5
Intercept
0.0592
-0.0593
Standard error of the intercept
0.007113
0.1465
Standard deviation of residuals
from line
0.01146
0.2360
LLOQ (ng mL-1)
20.0
20.0
a Average of six separate determinations.
3.2.2 Precision and accuracy
The intra- and inter-day precision and accuracy of five
concentrations of ARI and CLO drugs are summarized in
Table 2. For ARI, The intra- and inter-day RSDs were less
than 2.82 and 2.94, respectively. The intra-day accuracy
ranged from 87.43 to 97.48 %. For CLO, The intra- and
inter-day RSDs were less than 2.31and 1.89, respectively.
The intra-day accuracy ranged from to 89.64 to 97.54 %.
The above values show that the method is accurate and
precise.
3.2.3 Specificity
Specificity was assessed to show that the intended analytes
are measured and that their quantitation is not affected by
the presence of the biological matrix. Retention times of
ARI, CLO and the IS were 5.16 ± 0.10 min, 7.98 ± 0.12
min and 3.71 ± 0.12 min, respectively. No significant
interference from endogenous peaks was observed at these
retention times.
3.2.4. Extraction recovery
Absolute recovery was calculated by comparing peak areas
obtained from freshly prepared sample extracted with
unextracted standard solutions of the same concentration.
Recovery data was determined in triplicates at three
concentrations (low, medium, high) as recommended by the
FDA guidelines 13. The good recovery of ARI and CLO
from plasma using the (acetonitrile: methanol) mixture
proved that this extraction method reliably eliminated
interfering material from plasma. The mean percent
recovery values of ARI and CLO at low, medium and high
quality control levels are listed in Table 3. While the mean
percent recovery of the IS at a concentration of 500 ng mL-
1 was 91.6 with an acceptable precision (RSD < 8%).
3.2.5 Drug stability in biological fluid
The stability of ARI and CLO drugs under various
conditions is shown in Table 4. All results were within the
acceptance criteria of ± 15% deviation from the nominal
concentration.
Table 2. Precision and accuracy of the proposed HPLC
method for determination of ARI and CLO in spiked
human plasma
ARI
Accuracy
CLO
Accuracy
Intra-day
precision
Taken
conc.
(ng mL-
1)
RSD
(n=5)
Recovery
% a
Taken
conc.
(ng mL-
1)
RSD
(n=5)
Recovery
% a
50
2.82
87.43
50
0.67
89.64
200
0. 74
93.65
200
1.36
90.58
500
1.46
96.32
500
1.02
94.00
700
1.17
97.48
700
0.97
95.37
900
1.35
95.14
900
2.31
97.54
Inter-day
precision
Taken
conc.
(ng mL-
1)
RSD
(n=5)
Taken
conc.
(ng mL-
1)
RSD
(n=5)
50
2.45
50
1.32
200
2.13
200
0.36
500
2.66
500
0.42
700
1.87
700
1.21
900
2.94
900
1.89
a Average of three separate determinations.
Table 3. Results of recovery of ARI and CLO in human
plasma, by the proposed extraction procedure
Concentration (ng mL-1)
ARI
CLO
Recovery a
RSD a
Recovery a
RSD a
20
89.43
4.43
86.36
2.86
400
91.82
3.12
88.38
5.76
800
90.76
4.16
88.74
4.28
a Average of three separate determinations.
J. Pharm. Appl. Chem., 3, No. 1, 57-61 (2017)/ http://www.naturalspublishing.com/Journals.asp 61
© 2017 NSP
Natural Sciences Publishing Cor.
Table 4. Stability study of ARI and CLO in human plasma
by the proposed HPLC method
Stability
parameters
ARI
CLO
Plasma
conc.
(ng mL-1)
Recovery
%
± RSD a
Plasma
conc.
(ng mL-1)
Recovery
%
± RSD a
Short term
(4 h, room
temperature)
20
95.87 ±
2.54
20
98.43 ±
0.87
400
97.32 ±
2.76
400
96.52 ±
1.55
800
93.21 ±
2.21
800
96.32 ±
1.81
Post-preparative
(24 h, room
temperature)
20
94.32±
3.86
20
97.51 ±
1.96
400
92.43 ±
3.53
400
94.85 ±
3.15
800
92.38 ±
4.43
800
95.71 ±
2.87
Freeze – thaw
(three cycles)
20
88.30 ±
2.45
20
95.63 ±
3.21
400
91.75 ±
2.03
400
97.92 ±
1.47
800
89.58 ±
1.67
800
94.22 ±
2.85
Long term
(– 20°C, 30 days)
20
87.74 ±
2.87
20
92.65 ±
2.22
400
87.53 ±
4.36
400
90.84 ±
2.85
800
90.75 ±
4.85
800
88.73 ±
4.23
a Average of three separate determinations.
4 Conclusion
A sensitive, accurate, precise and validated HPLC method
was developed for simultaneous determination of ARI and
co-administered clonazepam drug in human plasma. The
method is economical and fast (simple extraction
procedure) and it could be used in the future bioequivalence
studies and as an alternative procedure for routine
therapeutic drug monitoring of patients treated with
Aripiprazole and Clonazepam combination.
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