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Original Article

Quantification of clebopride in human plasma (in-vitro)by RP-HPLC method

Kalaichelvi Ramalingama,b,*, Jayachandran Ekambaram c

aDepartment of Pharmaceutical Analysis, K.C. Reddy Institute of Pharmaceutical Sciences, Jangamguntla Palem, Medikonduru Mandal,

Guntur 522 348, Andhra Pradesh, IndiabDepartment of Science and Technology, Jawaharlal Nehru Technological University Hyderabad, Kukatpally, Hyderabad 500 085,

Andhra Pradesh, IndiacDepartment of Pharmaceutical Chemistry, S.C.S. College of Pharmacy, Harapanahalli 583131, Devanagari Dist, Karnataka, India

a r t i c l e i n f o

Article history:

Received 1 February 2013

Accepted 27 February 2013

Available online 26 March 2013

Keywords:

RP-HPLC

Clebopride malate

Liquideliquid extraction

Human plasma

* Corresponding author. Department of ScieHyderabad 500 085, Andhra Pradesh, India.

E-mail addresses: rkselvi123@rediffmail.c0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.02.023

a b s t r a c t

Aim: There is no any analytical method for the analysis of clebopride malate in human

blood plasma using liquideliquid extraction (LLE) by reverse phase high-performance

liquid chromatography (RP-HPLC) using UV detector. This study aims to determine

clebopride in human blood plasma (in-vitro) using a RP-HPLC method.

Methods: In the present RP-HPLCmethod drug was extracted from clebopride spiked human

plasma and analyzed using a Nucleosil C18 column, 25 cm � 4.6 mm, particle size 5 mm,

mobile phase consist of 10:80:10 v/v/v 10 mM ammonium formate buffer pH 5.5, acetoni-

trile and methanol with a flow rate of 0.6 mL/min, detection performed at 283 nm.

Results: The developed method was validated by performing selectivity, stability, accuracy,

precision, repeatability, linearity and the suitability of the system. The linearity was

obtained in the range of 1e60 ng/mL with correlation coefficient (r2) 0.998. LOD and LOQ

values were 0.0125 and 0.0378 ng/mL respectively.

Conclusion: Successfully the clebopride was extracted from drug spiked human plasma and

analyzed by RP-HPLC method. The developed method is sensitive and can be used for

pharmacokinetic studies of drug.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction functional gastrointestinal disorders. Detailed investigation at

Clebopride (Fig. 1), 4-amino-N-(1-benzylpiperidin-4-yl)-5-

chloro-2-methoxybenzamide, is a dopamine antagonist drug

with antiemetic and prokinetic properties used to treat

nce and Technology, Jaw

om, rkselvi1983@gmail.c2013, JPR Solutions; Publi

several centers has demonstrated its encouraging antiemetic,

gastrokinetic and anxiolytic properties.1e3 Literature survey

denotes that the drug can be estimated by thin-layer chro-

matography and high-performance liquid chromatography,4,5

aharlal Nehru Technological University Hyderabad, Kukatpally,

om (K. Ramalingam).shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

Fig. 1 e Structural of clebopride malate.

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 1 6 7e1 7 1168

UV spectrophotometry6 gas chromatography-mass spec-

trometry and radioimmunoassay in both animals7 andman.8,9

In the presentwork, an attempt has beenmade to develop and

validate a simple RP-HPLC method for the analysis of cle-

bopride from human plasma.

2. Materials and method

2.1. Materials

Shimadzu HPLC system equipped with SPD-20A prominence

UVeVIS detector, Manual Rheodyne injector (with 20 mL loop

size), pump (Shimadzu LC2010 Series), Spinchrom software,

the HPLC column Nucleosil C18, 25 cm � 4.6 mm, 5 mm, an

Elico UV/Visible double beam spectrophotometer SL-164,

Digital pH meter, ultrasonic bath, an analytical balance

(Shimadzu-BL 220H) sensitivity of 0.1 mg, filters vacuum

unit with 0.22 mm pore filter were used. Clebopride was

purchased from commercial supplier in India. Human

plasma was obtained from healthy volunteer and stored in

freezer.

2.2. Preparation of mobile phase

Mobile phase was a mixture of 10 mM Ammonium formate

buffer pH 5.5, acetonitrile and methanol (10:80:10) v/v/v.

The mixture was filtered using 0.22 mm milipore filter with

vacuum assistance and sonicated by ultrasonic bath for

15e20 min.

2.3. Preparation of standard solution

A stock solution was prepared by dissolving accurately

weighed 100 mg of clebopride in 100mL of methanol to yield a

final concentration of 1 mg/mL, sonicated for 5 min, allowed

to equilibrate to room temperature.

Table 1 e System suitability data.

Drug USP plate count Tailing factor (T) R

M

Clebopride

20 ng/mL

4467 1.41

2.4. Preparation of linearity standards

The stock solution (1000 mg/mL of clebopride) was diluted

suitably and spiked with human blank plasma to get

1e60 ng/mL of drug.

2.5. Extraction of clebopride from plasma

200 mL of each calibration standardswere pipetted into a series

of Ria vial tube and vortexed briefly. 3mL of mixture of diethyl

ether: dichloromethane (50:50 (v/v)) was added to each Ria

vial and caped. All calibration samples were vortexed for

approximately for 3 min and centrifuged at 4000 rpm for

approximately 5 min at 10 �C. The organic layer (2.0 mL) was

quantitatively transferred to a 4mL glass tube and evaporated

to dryness at 40 �C under a stream of nitrogen. Then, the dried

extract was reconstituted in 200 mL ofmobile phase and a 20 mL

aliquot was injected into the chromatographic system using

Hamilton syringe.

3. Results and discussion

3.1. Optimization of HPLC conditions

The drug was estimated at 283 nm using UV detector to

maximize the signal of compound and minimize the signal of

plasma interferents. The ratio of mobile phases was opti-

mized by several trials to get good resolution and symmetric

peak shape for the clebopride. The developed HPLC method

was optimized by monitoring chromatographic parameters

including retention time, column efficiency (HETP) of the

various variations of composition, and flow rate of mobile

phase. Efficiency values (N) showed the results of �4400, this

suggested that the sharp peak produced enough. The system

suitability parameters are given in Table 1.

3.2. Validation

The developed method was evaluated for linearity,

selectivity, accuracy and precision, stability during various

stress conditions including bench top stability, freeze thaw

stability, stability of stock solutions and dilution integrity and

recovery.

3.2.1. SelectivityBlank plasma was tested for endogenous interference.

Selectivity was evaluated by extracting different blank

plasma samples. The absence of interfering peaks at the same

etention time (min), (n ¼ 6) Peak area, (n ¼ 6)

ean � S.D %RSD Mean � S.D %RSD

3.4 � 0.010 0.2987 404 � 4.49 1.112

Fig. 2 e A typical chromatogram of blank plasma.

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 1 6 7e1 7 1 169

retention time of clebopride was considered as evidence

for selectivity of the method. The typical chromatograms for

the blank plasma and sample are given in Figs. 2 and 3

respectively.

3.2.2. LinearityCalibration curve was plotted by taking concentration of an-

alyte in X axis and detector response in Y axis. The developed

method was linear in the concentration range of 1e60 ng/mL

with the correlation coefficient value of 0.998. Slope and

intercept of the linearity curve (Fig. 4) was found to be 20.23

and 0.919 respectively.

Fig. 3 e A typical chromatogram

3.2.3. RecoveryRecovery of clebopride was evaluated by comparing the de-

tector response of clebopride in three quality control samples

(LQC, MQC and HQC) with the response of same in equivalent

methanolic solutions (Table 2).

3.2.4. Precision and accuracyFor precision and accuracy studies, samples were prepared at

three concentration levels, low (LQC),medium (MQC) and high

(HQC) quality controls, corresponding to 2, 20 and 50 ng/mL of

clebopride respectively with six replicates each. Precision and

accuracy was evaluated at inter and intraday (Table 3).

of clebopride in plasma.

Fig. 4 e Linearity curve of clebopride in plasma.

Table 3e Intraday and interday precision and accuracy ofclebopride.

Concentration(ng/mL)

Accuracy(% nominal)

(n ¼ 6)

Precision (%CV)

Interday(n ¼ 18)

Intraday(n ¼ 6)

2 81.94 4.94 3.55

20 89.83 1.02 0.92

50 87.27 1.14 0.98

Table 4 e Validation parameters.

Parameters Results

Selectivity Pass

System suitability Pass

LOD (ng/mL) 0.0125

LOQ (ng/mL) 0.0378

Accuracy and precision Pass

Intra-day precision Pass

Linearity (ng/mL) 1e60

Short term stock stability Pass

Long term stock stability Pass

Long term plasma stability Pass

Bench top stability Pass

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 1 6 7e1 7 1170

3.2.5. Stability3.2.5.1. Bench top stability in plasma. Six aliquots each of the

low and high quality control samples were kept at room

temperature (25 � 5 �C) after spiking into plasma. After

completion of 6 h the samples were extracted and analyzed

against the concentration of freshly prepared one. Percent

changes (Bias) for clebopride concentration for spiked sam-

ples over stability testing period of 6 h at room temperature

(25� 5 �C)was�6.3% to�2.2% as compared to nominal values.

3.2.5.2. Stock solution stability. The short-term stock

solutions stability of analyte was evaluated at room tem-

perature (25 � 5 �C) for at least 06 h. Long-term stability of

analyte was evaluated at refrigerated temperature (2e8 �C)for 35 days for analyte by comparing instrument response of

the stability samples to that of comparison samples. Percent

change (Bias) in clebopride area response over the stability

testing period of 06 h at 25 � 5 �C was �2.1%. Percent change

(Bias) in clebopride area response over the stability testing

period of 35 day at 2e8 �C was �1.3%. The results are

within �l0%.

3.2.5.3. Freeze and thaw stability. The freeze and thaw sta-

bility of analyte was determined after at least three freeze and

thaw cycles. At least six aliquots at each of low and high

quality control samples were stored at �20 � 5 �C and

Table 2 e Recovery study.

S. no LQC response

Extracted Un extracted Extracte

1 32 34 400

2 29 36 398

3 29 35 403

4 32 36 406

5 25 36 405

6 29 37 409

Mean 29.333 35.667 403.500

SD 2.582 1.033 4.037

% CV 8.802 2.896 1.001

% Recovery 82.24

subjected to three freeze thaw cycles at an interval of 8e16 h.

After the completion of third cycle the samples were analyzed

and stability of samples were compared against freshly pre-

pared calibration curve samples. Percent change (Bias) in

clebopride concentration over the stability testing period after

three freeze thaw cycles was �6.54% to �2.52%. The results

are within �15%.

3.2.5.4. Dilution integrity. Sample having final concentration

about two times of higher calibration curve standard was

prepared in plasma. Then the samples were diluted 5 times

and 10 times with analyte free control human plasma to meet

their actual concentrations in the calibration curve range. The

samples were extracted and results were compared with

nominal concentration. % Accuracy and precision of dilution

integrity samples for 1/5th dilutionswere 97.90% and 1.4% and

MQC response HQC response

d Un extracted Extracted Un extracted

450 1002 1153

455 1026 1155

459 1032 1168

461 1024 1171

450 1011 1159

448 1019 1173

453.833 1019.000 1163.167

5.345 10.918 8.589

1.178 1.071 0.738

88.91 87.61

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 1 6 7e1 7 1 171

for l/10th dilutions were 97.56% and 1.49%. The results are

within �15%. All the results for validation parameters are

summarized in Table 4.

4. Conclusion

Optimization of HPLC conditions and clebopride extraction

fromblood plasma by liquideliquid extraction have been done

and analyzed by HPLC UV detector. The developed method

was validated by selectivity, repeatability, linearity, detection

limit, quantification limit, precision, accuracy, and suitability

of the system. The method can be used to analyze clebopride

in human blood plasma, so that the results obtained can be

directly used to test the bioavailability and to test its

bioequivalence.

Conflicts of interest

All authors have none to declare.

Acknowledgments

The authors express their sincere thanks to the management,

K.C. Reddy Institute of Pharmaceutical Sciences and Chala-

pathi Institute of Pharmaceutical sciences, Guntur for

providing the necessary facilities to carry out the research

work.

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