267_30-34

7/17/2019 267_30-34

http://slidepdf.com/reader/full/26730-34 1/5

30Sharwaree Hardikar. et al . / I nternational J ournal of B iological & Pharmaceutical Research. 2013; 4(1): 30-34.

e- ISSN 0976 - 3651

Print ISSN 2229 - 7480

International Journal of Biological

&Pharmaceutical ResearchJournal homepage: www.ijbpr.com

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR

DETERMINATION OF CARBAMAZEPINE IN RAT PLASMA

Hardikar Sharwaree*, Undale Vaishali, Saindane Rupesh, Garud Mayuresh

Pune District Education Association’s Seth Govind Raghunath Sable College of Pharmacy,

Saswad, Dist. - Pune, India - 412 301.

ABSTRACTValidation of bioanalytical procedure is a regulatory requirement. Selective and sensitive analytical methods for the

quantitative evaluation of drugs (analytes) are critical for the successful conduct of preclinical studies and as a support to

formulation development. During the course of a typical product development program, a defined bioanalytical method

undergoes many modifications. In the present study, to cater the need of employment of bioanalytical method for

carbamazepine using rat plasma was developed and validated. Present bioanalytical method validation included all of the

procedures that demonstrated that, this method used for quantitative measurement of carbamazepine in rat plasma is reliable

and reproducible for the intended use. The fundamental parameters assessed for this validation were accuracy, precision,

selectivity, sensitivity and reproducibility.

Key Words: Carbamazepine, Rat plasma, Validation.

INTRODUCTIONCarbamazepine (CBZ) is a best-selling

antiepileptic, anticonvulsant drug. Its conventional dosage

forms yield variability in peak plasma concentrations from

4 to 32 hours. This is because of its erratic dissolution

particularly at gastric pH. Hence a lot of work is

undertaken by researchers to improve its dissolution since

it is pharmacodynamic as well as pharmacokinetic demand

of CBZ (Anonymous 1). Many of the RP-HPLC methods

for determination of CBZ in human and rabbit plasma are

reported in the literature. These are useful to researchers in

the later phase of product development. To assist

preliminary research work undertaken; there is need todevelop a method to assess concentration of CBZ in rat

plasma if rabbits are unavailable. Hence, the main

objective of the work was to develop and validate a RP-

Corresponding Author

Sharwaree Rajan Hardikar

Email: [email protected]

HPLC analytical method to determine CBZ concentration

in rat plasma.

MATERIALS AND METHODS

Apparatus

The high performance liquid chromatogram used

was Agilent 1120 Compact LC with UV Detector. Column

used was C-18 column. Other equipments used for this

research work were electronic analytical balance

(Schimadzu, Japan), Vortex mixer and table mount high

speed cold centrifuge.

ReagentsTest substance carbamazepine was obtained as a

gift sample from Cipla Ltd, Mumbai. Chromatographic

pure acetonitrile and water were used for mobile phase. All

other reagents used were of analytical pure grade.

Chromatographic conditi ons

The chromatographic column used was C-18

column with internal diameter of 2.1 and 150 mm of length

with average particle size of 5 µm. Mobile phase used was

IJBPR

http://www.ijbpr.com/




7/17/2019 267_30-34



water: acetonitrile (70:30). The column temperature was

kept at 25oC. Flow rate was maintained at 1 mL/min.

Aliquot of 20 µL was injected into column for HPLC

analysis. Monitoring was performed at 220 nm (Guidance

for Industry, 2001).

Solu tion PreparationStock solution of CBZ (200 µg/mL) was prepared

in a 25 mL amber glass volumetric flask. CBZ working

solutions of concentration 0.25, 0.5, 1.0, 2.5, 5, 10, 15, 20

µg/mL were prepared by spiking stock solution

appropriately into rat plasma obtained by procedure

mentioned below. They were all stored away from light

(Dzodić PL et al ., 2010).

Treatment to plasma samples

2 mL blood was withdrawn from retro orbital

cavity of Male Sparge Dawlwy rats in 6 X 50 mm glass

test tubes containing disodium edetate. To the blood

sample, fixed amount of CBZ stock solution was spiked,

volume was adjusted to 5 mL by using distilled water andmixture was vortexed for 30 seconds and then centrifuged

at 4500 rpm for 15 minutes at 4oC. In order to extract the

drug form plasma sample, 0.5 mL of supernatant was

transferred to centrifuge tube and 1.5 mL of mixture of

chloroform and ethyl acetate (1:1) was added, vortexed and

centrifuged at 4500 rpm for 15 minutes at 4oC. Organic

layer was separated after centrifugation and dried at 60oC

in water bath. At last, 2000 µL acetonitrile was added to

centrifuge tube and 20 µL samples was injected for HPLC

analysis. Calibration curves were constructed by analysing

the extracted solutions obtained by procedure mentioned

above. The triplicates of extracted plasma samples with

varying concentrations of CBZ (0.25, 0.5, 1, 2.5, 5, 10, 15and 20 µg/ml) were analysed to construct the calibration

curve (Sachin Ramrao Patil et al ., 2012).

RESULTS AND DISCUSSION

Specif ici ty and Selectivi ty

In order to determine the specificity of current

method, the chromatogram of blank plasma and plasma

sample spiked with CBZ were compared. Figure 1A and

1B represents the chromatograph of blank and plasma

spiked by CBZ respectively. The retention time of CBZ

was found to be 6.37 minutes and no interference of any

plasma component was observed in this range.

Method Vali dation

L inearity and Sensitivity

Linearity of method was determined by

constructing the calibration curve in triplicate by plotting

area under the curve of peak obtained by analysing various

concentrations of CBZ. Figure 2 represents the calibration

curve of CBZ. The linearity range was found and

confirmed to be from 0.25 to 20.0 µg/mL. The regression

equation obtained was y= 4330000x +381373 with

correlation coefficient 0.998. The regression equations

obtained when the procedure was repeated three times are

given in table 1 as standard deviation of slope (Lm) and

intercept (Li) on the ordinate. The linearity of graph was

proved by high correlation coefficient for regression

equation. Limit of detection (LOD) and Limit of

quantitation (LOQ) were also determined from the slopesof calibration curves by using the equations given below.

LOD = 3.3 σ

S

LOQ = 10 σ

S

where σ = the standard deviation of the response

S = the slope of the calibration curve

The LOD and LOQ was calculated and reported in table 2.

Accur acy, Precision and Recovery

Accuracy study was carried out to determine the

closeness of the mean test results obtained by the method

to true value. Accuracy is determined by replicate analysisof samples containing known amounts of the analyte. In

the present work it was confirmed by using three different

concentrations as 5, 10 and 15 µg/mL in triplicate. Briefly,

to 10 µg/ml solution of CBZ prepared in extracted plasma,

three different concentrations of CBZ 5,10 and 15 µg/mL

of CBZ were spiked externally and recovery was

determined. Experiments were performed in triplicate.

Data for accuracy and recovery studies is reported in table

3.

The precision of an analytical method describes

the closeness of individual measures of an analyte when

the procedure is applied repeatedly to multiple aliquots of a

single homogeneous volume of biological matrix.Precision was assessed by repeated analysis of plasma

specimen containing therapeutic concentration of the drug

being investigated. Repeatability and intermediate

precision of method was confirmed by assessing three

concentrations of CBZ (5, 10 and 20 µg/mL) and results

are reported in table 4.Relative standard deviation(RSD)

less than 15% proved accuracy and precision of the

method.

Robustness

The robustness of an analytical procedure is a

measure of its capacity to remain unaffected by small, but

deliberate variations in method parameters and provides an

indication of its reliability during normal usage.

Robustness of current method was determined by changing

the wavelength of detection by 220± 2 nm and effect of

change in wavelength on retention time and AUC was

determined. Data for robustness study is shown in table 5.

As the RSD of Retention time and AUC by

changing wavelength were below 15% it was concluded

that method is robust enough to be used to assess in vivo

bioavailability.

7/17/2019 267_30-34



Figure 1A. Chromatograph of blank rat plasma

Figure 1B. Chromatograph of CBZ spiked in rat plasma

Figure 2. Calibration curve of CBZ

7/17/2019 267_30-34



Table 1. Slope and Y-intercept of calibration curves

Slope of Line (Lm) Y-intercept (Li)

5.00 X 1006 72109

4.00 X 10 1.0 X 10

4.00 X 10 2.0 X 10

Table 2. LOD and LOQ of CBZSlope of lines Mean slope Standard Deviation of slopes LOD(µg/mL) LOQ(µg/mL)

5.00 X 10

4.33X 1006 577350.3 0.44 1.334.00 X 1006

4.00 X 10

Table 3. Data for accuracy study

% Recovery

CONC.

OF STOCK SOL.*

CONC

Of

SPIKED

SOL.*

TOTA

L

CONC

*

AUC

OBSERV

ED

CONC*

%

RECOVER

Y

MEAN

RECOVE

RY

SD RSD

10 5 15 69930325 16.1 107.33

102.3811 4.30 4.0110 10 20 86865763 20.05 100.2510 15 25 1.08x 10 24.89 99.56

*All conc. in µg/mL

Table 4. Repeatability and Intermediate Precision study

CONC. (µg/ml) AUC1 AUC2 AUC3 MEAN SD RSD

REPEATABILITY

5 21306544 20445255 20950929 20900909 432817.7 2.070808

10 48368568 46565564 48925992 47953375 1233772 2.572858

20 89302903 83749233 87049561 86700566 2793235 3.221703

INTERMEDIATE PRECISION

5 22388027 21306544 22053136 21915902 553648.1 2.526239

10 46512623 48368568 46697883 47193025 1022256 2.16611620 90298695 89302903 87091681 88897760 1641445 1.846441

Table 5. Data for Robustness study

WAVELE

N-

GTH(nm)

RETENTION

TIME(RT)(min)

MEAN

RT(min)

SD

OF RT

RSD

OF RTAUC

MEAN

AUC

SD

OF AUC

RSD OF

AUC

218 6.08

6.11 0.06 0.94

61540011

6413743

14053236 6.319611

219 6.06 64299617

220 6.06 70032643

221 6.16 65424292

222 6.18 59390594

CONCLUSION Simple reversed-phase HPLC method for analysis

of the carbamazepine in rat plasma had been developed

and validated. The optimized chromatographic conditions

gave optimum baseline separation. The method was found

to be linear, precise, accurate and sensitive; thus being

suitable for direct analysis of carbamazepine in the

laboratory. The method was found to be robust and will

enable quality-control analyst to perform preclinical

bioavailability studies.

ACKNOWLEDGMENTSThe authors are thankful to Tatyasaheb Kore

Institute of Pharmacy, Warna Nagar for the technical

support.

7/17/2019 267_30-34



REFERENCES

Anonymous 1. http://www.nlm.nih.gov/medlineplus/druginfo/meds/a682237.html

Dzodić PL, Zivanović LJ, Protić AD, Zećević ML, Jocić BM. Determination of carbamazepine and its impurities iminostilbene

and iminodibenzyl in solid dosage form by column high-performance liquid chromatography. J AOAC Int. 2010;

93(4): 1059-68.

Guidance for Industry: Bioanalytical Method Validation. Published by U.S. Department of Health and Human Services, Foodand Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM),

2001.

Sachin Ramrao Patil, Lokesh Kumar, Gunjan Kohli, and Arvind Kumar Bansal. Validated HPLC Method for Concurrent

Determination of Antipyrine, Carbamazepine, Furosemide and Phenytoin and its Application in Assessment of Drug

Permeability through Caco-2 Cell Monolayers. Sci Pharm. 2012; 80(1): 89 – 100.

267_30-34

Documents