fenofibrate bioanalytical method development and...

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FENOFIBRATE

Bioanalytical method development and Validation using HPLC

5.1 DRUG PROFILE Fenofibrate

Fenofibrate is an antilipidemic agent which reduces both cholesterol and

triglycerides in the blood.1

Chemistry 2 Molecular Formula : C20H21ClO4 Chemical Name : Propan-2-yl-2-{4-[(4-chlorophenyl) carbonyl]

phenoxy} -2-methylpropanoate

Molecular Weight : 360.831

Description : Fenofibrate occurs as a white or almost white

crystalline powder.

Solubility : It is slightly soluble in ethanol (95%), very soluble in

methylene chloride and practically insoluble in water

Storage : Store below 30°C (86°F) and protect from light.

It is official in British Pharmacopoeia.3

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Mechanism of action

Fenofibrate is an antilipidemic agent, which lowers plasma TG by activating

lipoprotein lipase thus increasing very low density lipoprotein (VLDL) catabolism

with consequent increase in high density lipoprotein (HDL) levels.

Fenofibrate is an antilipidemic agent, which acts by activation of peroxisome

proliferator activated receptor-a (PPARa). Most of the triglyceride-rich particles are

eliminated from plasma by activating lipoprotein lipase and reducing production of

apoprotein C-III due to lipolysis. The resulting decrease in triglycerides produces a

change in the size and composition of LDL from small, dense particles, to big

buoyant particles. These bigger particles have a greater affinity for cholesterol

receptors and are catabolised rapidly.4, 5

Indications

It is indicated for the treatment to reduce elevated LDL-C Total-C,

Triglycerides and Apo B, and to increase HDL-C in adult patients with mixed

dyslipidemia and primary hypercholesterolemia.6

Adverse Effects

Severe stomach pain, Nausea, Vomiting, Unusual weakness or fever, Joint

pain, Indigestion, Bloating, Rash.

Dosage and Administration

The recommended dosage is 200-300mg capsule once daily with food.

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Contraindications 7

1. It is contraindicated in patients who are hypersensitive to the product.

2. Patients with severe hepatic and renal impairment.

3. Patients with Unexplained persistent liver function abnormality and primary

biliary cirrhosis.

4. Pre-existing gall bladder disease.

5. In Pregnancy and lactation.

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5.2: LITERATURE REVIEW: Fenofibrate

Fathy M et al (2011)8 reported two new selective, precise and accurate

methods for the determination of Fenofibrate in the presence of its basic degradation

product. In the first method Fenofibrate was determined using an algorithm bivariate

calibration derivative method. In the second method (HPLC), separation was

performed on RESTEK Pinnacle II phenyl column (5 µm, 250 × 4.6 mm) and

Pinnacle II phenyl (5 µm, 10 × 4 mm) guard cartridge using a mobile phase used

was of methanol-0.1% phosphoric acid (60:40, v/v) at a flow rate 2.0 ml/min, and

the column oven temperature was set at 50°C. The UV detector was time

programmed at 302 nm and 289 nm for the internal standard and Fenofibrate.

Madureira TV et al (2010)9 reported a simple analytical method using

solid-phase extraction followed by a high-performance liquid chromatography with

diode array detection (HPLC-DAD) analysis, compounds included six

pharmaceuticals (Carbamazepine, Diazepam, Fluoxetine, Propranolol,

Sulfamethoxazole and Trimethoprim) and the active metabolite of Fenofibrate.

Briefly, this method consisted of the preconcentration of water samples (2 L) on 500

mg Oasis HLB cartridges and HPLC analysis using a RP18 analytical column in a

gradient mode with a flow rate of 1.0 ml/min. Thus, it is concluded that this method

can be successfully applied for screening pharmaceuticals in polluted estuarine

areas.

Bhavesh D et al (2009)10 reported a rapid, specific and sensitive ultra-

performance liquid chromatography tandem mass spectrometry method for the

determination of Fenofibrate in human plasma. Extraction process was one-step

liquid-liquid extraction coupled with an Acquity UPLC(TM) BEH C18 column (50 x

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2.1 mm, i.d., 1.7 micron) with isocratic elution technique with a flow rate of 0.2

ml/min with Mefenamic acid (internal standard), multiple reaction-monitoring mode

using the electrospray ionization technique.

Korany MA et al (2008)11 reported a differential pulse polarographic and

square wave voltammetric techniques by reduction at a dropping-mercury working

electrode versus Ag/AgCl reference electrode for determination of Etofibrate,

Fenofibrate and Atorvastatin in their pharmaceutical preparations and human plasma

optimum conditions such as pH, scan rate and pulse amplitude were studied.

Kadav AA et al (2008)12 reported a UPLC method for simultaneous

determination of Atorvastatin, Fenofibrate and their impurities in tablets. The

chromatographic separation was carried on acquity UPLC BEH C18 column (1.7

micron, 2.1 mmx100 mm) with gradient elution technique, mobile phase was

acetonitrile and ammonium acetate buffer (pH-4.7; 0.01 M) at flow rate of 0.5

ml/min and detection by UV was at 247 nm. Total run time was 3.0 min within

which main compounds and six other known and major unknown impurities were

separated. All the validation parameters were done for Atorvastatin, Fenofibrate and

their known impurities.

Straka RJ et al (2007)13 reported a RP-HPLC method in combination with

anion-exchange solid-phase extraction, which rapidly and accurately determines

steady-state Fenofibrate serum concentrations. Isocratic conditions were used for

Chromatographic separation by using ultraviolet detection at 285 nm.

Concentrations were found to be linear over the range of 0.5 to 40.0 mg/L for

Fenofibric acid analysis. Accuracies were found from 98.65% to 102.4%.

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Alaa EG et al (2005)14 reported two spectrophotometric and HPLC methods

for the estimation of Fenofibrate, Vinpocetine and their hydrolysis products. The

resolution of drugs and hydrolysed products were estimated by using numerical

spectrophotometric methods such as partial least squares (PLS-1) and principal

component regression (PCR) applied to obtained UV spectra, and graphical methods

as first derivative of ratio spectra (1DD) or first (1D) and second (2D) derivative

spectrophotometry for Vinpocetine and Fenofibrate. HPLC method was developed

using ODS column with mobile phase consisting of acetonitrile-water (80:20 v/v,

pH-4.0) with λmax at 287 nm for Fenofibrate and a mobile phase consisting of

acetonitrile-10 mM KH2PO4, containing 0.1% diethylamine (60:40 v/v, pH 4.6) with

λmax at 270 nm for Vinpocetine.

Shihabi ZK (2004)15 reported capillary electrophoresis method to measure

Fenofibrate in capsules based on micellar electro kinetic capillary chromatography

with UV detection at 280 nm by employing a borate buffer containing sodium

dodecyl sulfate (SDS).

Meikle AW et al (2003)16 reported a simple HPLC-MS/MS method which

was evaluated on two patients who are suffering from Cushing syndrome who had

elevated Urinary free cortisol (UFC) excretion, which is usually done for diagnosis

of Cushing syndrome. Regular laboratory testing was inconsistent with the diagnosis

of Cushing syndrome which raised doubts. By identifying a probable cause of

analytical interference from Fenofibrate (Tricor), medication taken by the patients.

Fenofibrate peak was overlapped with the HPLC peak of cortisol and produced an

MS/MS transition overlapping the major transition of cortisol. HPLC-MS/MS

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method using multiple mass transitions, rather than a single transition, allows

accurate quantitation of urinary cortisol in patients taking Fenofibrate.

Lassner A et al (2001)17 reported a sensitive HPLC method for the

estimation of Fenofibric acid (FA), which is an active form of Fenofibrate in serum.

An easy one-step extraction procedure was carried for the isolation of FA from

human serum samples by using a mixture of n-hexane and ethylacetate (90: 10 v/v).

The recovery was 84.8% of the total Fenofibric acid in serum. The Seperation was

done isocratically on RP-HPLC using acetonitrile and 0.02M phosphoric acid

(55:45) at a flow rate of 1.0 ml/min, UV detection was found at 287 nm.

Streel B et al (2001)18 reported a fully automated RP-HPLC with UV

detection method for the estimation of Fenofibrate in plasma, which involves the

solid-phase extraction (SPE) of the analyte from plasma on disposable extraction

cartridges (DECs). The cartridge was conditioned with methanol and pH-7.4

phosphate buffer first then filled with octadecyl silica and Sulindac (internal

standard) along with diluted plasma of 0.8ml volume was applied on the DEC. The

liquid chromatographic (LC) separation of the analytes was carried on a Nucleosil

RP8 stationary phase (5 micron), mobile phase used is a mixture of methanol and

0.04 M phosphoric acid (60:40 v/v) and UV detection was at 288 nm.

Lacroix PM et al (1998)19 reported an HPLC method for the estimation of

drug content, HPLC and NMR methods for related compounds in Fenofibrate raw

materials. By HPLC method 11 known and six unknown impurities were resolved

from the drug. Chromatographic separation was carried on Waters Symmetry ODS

column (100 x 4.6 mm, 3.5 micron), a mobile phase consisting of acetonitrile/water/

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trifluoroacetic acid 700/300/l (v/v/v) with a flow rate of 1.0 ml/min with UV

detection at 280 nm. An NMR method for related compounds was developed which

is suitable for 12 known and several unknown impurities, 400 MHz or greater NMR

is required.

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5.3. EXPERIMENTAL: METHOD DEVELOPMENT 5.3.1. MATERIALS AND METHODS

Materials

Fenofibrate and Nevirapine were obtained from CIPLA Pharmaceuticals,

Mumbai. HPLC grade acetonitrile, ethylacetate and methanol were purchased from

SD fine chemicals, Mumbai, India. Analytical Grade ammonium acetate was

purchased from SD fine chemicals, Mumbai, India. Pooled drug free expired human

plasma was donated by Red Cross Society, Warangal.

Instrumentation

The HPLC system consisted of Alliance Waters 2695 with dual λ

Absorbance UV detector. Separation was carried out on Inertsil C18 column

(4.6x250mmx5µm). A vortex-mixer (Remi), ultrasonic bath (Bransonic),

a model C-30 centrifuge (Remi) and a model cool safe 110-4/scan speed 32 were

used for sample preparation.

Chromatographic conditions

The HPLC system consisted of Alliance waters 2695 with dual λ Absorbance

UV detector. The wavelength of detection as set at 295nm. Separation was carried

out on Inertsil C18 column (4.6x250mmx5µm) using 60:40 v/v ammonium acetate

buffer and acetonitrile as mobile phase at a flow rate of 1.0 ml/min. The mobile

phase was filtered through nylon millipore (0.2µm) membrane filter, purchased from

pall life sciences, Mumbai and degassed with Ultrasonicator prior to use.

Chromatography was carried out at room temperature 250c .

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Preparation of Stock solutions

Stock solutions of Fenofibrate (500 µg/ml) and Nevirapine (1000 µg/ml)

internal standard were prepared in Methanol. Further dilutions were carried out in

60% acetonitrile.

Preparation of calibration standards

Calibration standards were prepared freshly by spiking drug free plasma with

Fenofibrate stock solution to give the concentrations of 0.3, 0.6, 1.2, 2.5, 5.0, 10.0

and 20.0µg/ml respectively; they were stored at -200c till the time of analysis.

Preparation of quality control standards

Quality control standards were prepared at three levels namely LQC, MQC

and HQC. These standards were prepared freshly by spiking drug free plasma with

Fenofibrate stock solution to give the concentrations of 1.0, 8.0 and 16.0µg/ml

respectively, they were stored at -200c till the time of analysis. Detailed procedure

for the preparation is shown in Table 46.

Sample preparation method

Drug free human plasma (500µl) was spiked with appropriate volume of

drug stock. To the above prepared sample, 50µl of Nevirapine (50µg/ml) was added

as an IS. The sample was vortex mixed for 4 min to assure complete mixing.

Analytes were extracted by adding with 3.0ml of ethyl acetate followed by vortex

mixing for 4 min and centrifugation at 2000 rpm/min in a cooling centrifuge for

15min at 40C. The organic phase containing analytes were separated and analytes

were obtained as dried residues after drying in lyophilizer. The analyte residue

obtained was reconstituted with 250µl of mobile phase and analysed using HPLC

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system according to parameters optimized. Detailed procedure for the preparation is

shown in Table 42. Calibration data is shown in Table-43, calculated concentrations

of calibrations standards and mean concentrations of calibration standards data were

showed in Tables 44 and 45.

5.4 EXPERIMENTAL: METHOD VALIDATION 5.4.1 VALIDATION PARAMETERS Specificity

A solution containing 0.3µg/ml Fenofibrate was injected onto the column

under optimized chromatographic conditions to show the separation of Fenofibrate

from the impurities from the plasma. The specificity of the method was checked for

the interference from plasma.

Linearity and Range

Six samples of each calibration standard were analysed. Wide range

calibration was determined by solutions containing 0.3µg/ml to 20.0µg/ml. The

spiked concentration and their respective peak area ratios with respective to internal

standards were subjected to least squares regression. After examining the percent

deviation, a proper model was chosen. The slope, Y-intercept, and coefficient of

determination (r2) were obtained from linear regression equation.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

The limit of quantitation refers to the lowest amount of an analyte in a

sample that can be quantitatively determined with suitable precision and accuracy.

There are different approaches to determine the LOD and LOQ. Typically the

concentration level that generates a signal-to-noise (S/N) of 3.3 is regarded as the

LOD and the concentration level that generates S/N = 10 is regarded as the LOQ.

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Here LOD was calculated using the formula 3.3 times σ/s, similarly LOQ was

calculated using the formula 10 times σ/s where “σ” is the standard deviation of the

intercept obtained for calibration curve and “s” is the slope of the calibration curve.

Precision and accuracy

Intraday precision and accuracy was determined by analyzing quality control

standards (1.0, 8.0 and 16.0µg/ml) and LLOQC standards (0.15µg/ml) five times a

day randomly, interday precision and accuracy were determined from the analysis of

each quality control standards (1.0, 8.0 and 16.0µg/ml) and LLOQC standards

(0.3µg/ml) once on each of five different days.

Ruggedness

The ruggedness of the method was determined by analysing spiked control

samples of (n=6) medium concentrations i.e. 8.0µg/ml using two different columns.

Recovery studies

The percent mean recoveries were determined by measuring the responses of

the extracted plasma Quality control samples at HQC, MQC and LQC against

unextracted Quality control samples at HQC, MQC and LQC.

For calculation of recovery of the compound, spiked control samples were

prepared at low, medium and high concentrations i.e. 1.0, 8.0 and 16.0µg/ml

concentrations. The samples were processed as mentioned above and the

concentration of the compound was determined from the regression of the analytical

standard calibration curve. Recovery was calculated by comparing the observed

concentrations in spiked samples to that of respective unextracted samples.

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Stability studies

The stability of Fenofibrate was determined by measuring concentration

change in control samples overtime under set conditions.

Freeze-thaw stability study of Fenofibrate was carried out by subjecting

samples to three freeze and thaw cycles. Samples before study and after study were

analysed by developed method. Similarly Stock solution stability study of

Fenofibrate (Stability after ‘0’ hrs), Stock solution stability study (stability after

‘8’ hrs), Bench top stability study of Fenofibrate (Stability after ‘10’ hrs) and Inter

injection stability study of Fenofibrate were carried out by subjecting samples to

study conditions.

5.4.2 DATA ANALYSIS 20

Data analysis was done using MS-Excel package for calculating mean,

standard deviation and % relative standard deviation.

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5.5. RESULTS AND DISCUSSION

Under the chromatographic conditions employed, the sample showed sharp

peaks for drug and internal standard with good resolution. The retention time for the

Fenofibrate was found to be 6.672±0.05 minutes and the retention time for

Nevirapine (IS) was 5.213±0.03 minutes. . The results of validation parameters are

shown in Figure 31. The method developed was validated for specificity, accuracy,

precision, linearity, range and stability as per USFDA guidelines.

The specificity of the method was proven by the absence of peaks near the

retention time of the drug as well as the internal standard (Figure 32).

The calibration function was developed for Peak area ratio Vs Concentration

(in µg/ml) and it was linear over the concentration range of 0.3 to 20.0µg/ml. The

regression line equation for the analysis was y= 0.058x - 0.008 with a coefficient of

correlation (r2) = 0.999. The chromatogram of calibration standards and calibration

curve were shown in Figures 33 to 39.

The LOD is calculated using the formula 3.3 times σ/s, similarly LOQ is

calculated using the formula 10 times σ/s where “σ” is the standard deviation of the

intercept obtained for calibration curve and “s” is the slope of the calibration curve.

The calculated LOD and LOQ are shown in Table 47.

A system suitability test was carried out by injecting six aqueous mixtures of

Fenofibrate and Nevirapine (IS). The following parameters were observed for

repeated injections (n=6) of both Fenofibrate and Nevirapine (IS) (Table 48).

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The intraday and interday precision and accuracy of the method was found to

be 0.09 to 5.03% and 100.00 to 100.20% respectively for the quality control

samples. This is within the acceptance limits of precision is 15% and accuracy is 85

to 115% (Table 49). The limit of quantitation was found to be 0.3µg/ml at such

concentration the inter day precision was found to be 0.07 to 0.29 and the accuracy

was 100% to 100% respectively for the quality control samples, which are within

the acceptance limits of precision is 20% and accuracy is 80 to 120% (Table 50).

The results for ruggedness on different columns were shown in Table 52.

The percent mean recovery for Fenofibrate in LQC, MQC and HQC was

62.7%, 64.2 % and 65.3% respectively (Tables 53 to 55.).

Stability was assessed by comparing against the freshly thawed quality

control samples. The percent mean stability for HQC and LQC were 100.00% and

100.00% respectively, which is within the acceptance limits of 85 to 115%. Plasma

Quality control samples of Fenofibrate were found to be stable for at least one

month. The results were shown in the Table 56. Results for Stock solution stability

study (Stability after ‘0’ hrs), Stock solution stability study (Stability after ‘8’ hrs),

Bench top stability (Stability after ‘10’ hrs) and Inter injection stability study were

shown in Tables 57 to 60.

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Table 42: Preparation of Fenofibrate calibration standards in Plasma

Fenofibrate Concentration

(µg/ml) Drug stock

solution (µl)* Blank plasma (µl)

FFB CS1 0.3 12 988

FFB CS2 0.6 24 976

FFB CS3 1.25 50 950

FFB CS4 2.5 100 900

FFB CS5 5.0 200 800

FFB CS6 10.0 400 600

FFB CS7 20.0 800 200

* indicates drug stock solution from 10 µg/ml

Table 43: Calibration data of Fenofibrate

Concentration (µg/ml)

Peak Area Ratio of Peak Area Fenofibrate Internal Standard

0.3 5761 522497 0.011

0.6 11745 554621 0.021

1.25 25760 566244 0.045

2.5 78951 542818 0.145

5.0 158518 551221 0.287

10.0 323629 547925 0.59

20.0 625822 540980 1.156

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Table 44: Calculated concentrations of Fenofibrate calibration standards

Fenofibrate Actual

Concentration (µg/ml)

Calculated concentrations

1 2 3

FFB CS1 0.3125 0.307 0.317 0.332

FFB CS2 0.625 0.617 0.673 0.547

FFB CS3 1.25 1.242 1.255 1.234

FFB CS4 2.5 2.546 2.564 2.564

FFB CS5 5.0 4.973 4.813 5.067

FFB CS6 10.0 10.05 10.11 10.303

FFB CS7 20.0 20.297 19.404 20.146

Table 45: Mean concentrations of Fenofibrate calibration standards

Fenofibrate Concentration

(µg/ml) Mean of calculated

concentrations SD % RSD

FFB CS1 0.3125 0.3186 0.013 3.95

FFB CS2 0.625 0.6123 0.063 10.31

FFB CS3 1.25 1.243 0.011 0.85

FFB CS4 2.5 2.558 0.010 0.41

FFB CS5 5.0 4.951 0.128 2.59

FFB CS6 10.0 10.155 0.132 1.30

EPR CS7 20.0 19.949 0.478 2.40

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Table 46: Preparation of Fenofibrate quality control standards in Plasma

Fenofibrate Concentration (µg/ml)

Drug stock solution (µl)*

Blank plasma (µl)

FFB LQC 1.0 40 960

FFB MQC 8.0 320 680

FFB HQC 16.0 640 360

* indicates drug stock solution from 10 µg/ml

Table 47: Calibration curve parameters for Fenofibrate

1 2 3 Mean SD

Slope 0.0246 0.0255 0.0247 0.0249333 0.000493

Intercept 0.000322 0.00148 0.000448 0.00075 0.000635

r2 0.999 0.997 0.998 - -

LOD 0.084 µg/ml - - - -

LOQ 0.254 µg/ml - - - -

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Table 48: HPLC System suitability parameter (Precision) for Fenofibrate and Nevirapine (IS)

Name Replicates RT Peak area

Fenofibrate (n=6)

1 7.059 673863

2 7.036 648921

3 7.019 669539

4 7.047 659253

5 7.051 670216

6 7.039 668539

Mean 7.041 665055.16

SD 0.0139 9278.25

% RSD 0.19 1.39

Internal Standard (n=6)

1 5.271 29251

2 5.239 27639

3 5.277 25453

4 5.204 26402

5 5.253 28493

6 5.228 29026

Mean 5.245 27710.66

SD 0.0274 1518.52

% RSD 0.5 5.4

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Table 49: Intra-day accuracy and precision for Fenofibrate

Fenofibrate QC ID LQC MQC HQC

Actual conc.(µg/ml) 1.0 8.0 16.0

Intraday

1 1.062 8.527 17.05

2 1.076 8.447 17.09

3 1.068 8.975 17.075

4 1.073 8.046 17.069

5 1.073 9.131 17.087

Mean 1.070 8.625 17.074

± SD 0.0055 0.4344 0.0160

% RSD 0.5142 5.0372 0.0938

% Accuracy 100.00 100.20 100.00

Table 50: Inter-day accuracy and precision for Fenofibrate

Fenofibrate QC ID LQC MQC HQC

Actual conc.(µg/ml) 1.0 8.0 16.0

Interday

1 1.049 8.176 16.495

2 1.055 8.177 16.481

3 1.05 8.184 16.514

4 1.055 8.181 16.493

5 1.049 8.191 16.508

Mean 1.051 8.181 16.498

± SD 0.0031 0.0060 0.0130

% RSD 0.2976 0.0740 0.0789

% Accuracy 100.00 100.00 100.00

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Table 51: Precision & Accuracy of LLOQC Standard

QC ID LLOQC Actual conc.(µg/ml) 0.3125

1 0.307

2 0.323

3 0.319

4 0.317

5 0.332

Mean 0.3196

±SD 0.0090

%RSD 2.8471

% Accuracy 100.06

Table 52: Ruggedness of the method developed for

Fenofibrate (on different columns)

MQC

Replicates

Column 1

(area ratio)

Column 2

(area ratio)

1 0.869 0.886

2 0.873 0.886

3 0.878 0.864

4 0.884 0.882

5 0.862 0.842

6 0.852 0.855

Mean 0.870 0.869

± SD 0.011 0.018

% RSD 1.32 2.12

% Accuracy 100.06

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Table 53: Recovery study for Fenofibrate from human plasma - LQC

Fenofibrate

Replicates

LQC

Unextracted

(area ratio)

Extracted

(area ratio)

%Recovery

1 0.099 0.062 62.63

2 0.098 0.063 64.29

3 0.098 0.064 65.31

4 0.098 0.058 59.18

5 0.098 0.061 62.24

6 0.099 0.062 62.63

Mean 0.098 0.062 62.712

±SD 0.001 0.002 2.092

%RSD 0.53 3.35 3.34

Table 54: Recovery study for Fenofibrate from human plasma -MQC

Fenofibrate

Replicates

MQC

Unextracted

(area ratio)

Extracted

(area ratio)

%Recovery

1 0.867 0.554 63.90

2 0.867 0.556 64.13

3 0.867 0.549 63.32

4 0.866 0.569 65.70

5 0.867 0.574 66.21

6 0.867 0.542 62.51

Mean 0.867 0.557 64.296

±SD 0.0004 0.012 1.410

%RSD 0.05 2.17 2.19

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Table 55: Recovery study for Fenofibrate from human plasma - HQC

Fenofibrate

Replicates

HQC

Unextracted

(area ratio)

Extracted

(area ratio)

%Recovery

1 1.573 1.048 66.62

2 1.572 1.057 67.24

3 1.574 0.993 63.09

4 1.572 0.992 63.10

5 1.569 1.082 68.96

6 1.566 0.992 63.35

Mean 1.571 1.027 65.394

±SD 0.003 0.040 2.546

%RSD 0.19 3.89 3.89

Table 56: Freeze-thaw Stability of Quality Control Standards

Stability after 3 freeze thaw cycles

Fenofibrate LQC HQC

Actual conc.(µg/ml) 1.0 16.0

1 0.975 17.473

2 0.983 17.516

3 0.983 17.657

4 0.956 17.48

5 0.982 17.473

6 0.98 17.628

Mean 0.976 17.537

± SD 0.0104 0.0831

% RSD 1.0735 0.4740

% Accuracy 100.00 100.00

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Table 57: Stock solution stability study of Fenofibrate (Stability at ‘0’ hrs)

Replicates SSS(0hrs) LQC SSS(0hrs) HQC

1 0.267 4.696

2 0.265 4.649

3 0.272 4.724

4 0.276 4.745

5 0.268 4.73

6 0.271 4.724

Mean 0.270 4.711

± SD 0.004 0.034

% RSD 1.47 0.73

Table 58: Stock solution stability study of Fenofibrate (Stability after ‘8’ hrs)

Replicates SSS(8hrs) LQC SSS(8hrs) HQC

1 0.262 4.412

2 0.265 4.457

3 0.263 4.459

4 0.266 4.449

5 0.261 4.454

6 0.262 4.501

Mean 0.263 4.455

± SD 0.002 0.028

% RSD 0.74 0.64

% Accuracy 97.53 94.57

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Table 59: Bench top stability study of Fenofibrate (Stability after 10 Hours)

Time (h) 10.00(h)BTS

Fenofibrate LQC HQC


1 0.983 17.404

2 0.978 17.439

3 0.985 17.58

4 0.99 17.342

5 0.977 17.338

6 0.981 17.348

Mean 0.982 17.408

± SD 0.0048 0.0931

% RSD 0.4889 0.5352

% Accuracy 100.00 100.00

Table 60: Inter injection stability study of Fenofibrate

Fenofibrate LQC HQC


1 1.091 17.394

2 1.09 17.347

3 1.095 17.468

4 1.094 17.298

5 1.089 17.295

6 1.093 17.291

Mean 1.092 17.348

± SD 0.0023 0.0708

% RSD 0.2167 0.4081

% Accuracy 100.00 100.00

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CHROMATOGRAMS OF DEVELOPED METHOD

Figure 31: Chromatogram showing Retention a time of Aqueous Mixture consists of Fenofibrate (5µg/ml) and Nevirapine (5µg/ml).

Figure 32: Chromatogram of Blank plasma sample showing no interference at the RT of Fenofibrate and Nevirapine

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Figure 33: Chromatogram of Fenofibrate calibration standard-1(FFB CS1)


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Figure 40: Calibration curve of Spiked concentrations (FEB CS1-CS7)

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Figure 41: Chromatogram of Fenofibrate Low Quality Control (FFB LQC)

Figure 42: Chromatogram of Fenofibrate Middle Quality Control (FFB MQC)

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Figure 43: Chromatogram of Fenofibrate High Quality Control (FFB HQC)

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5.6. SUMMARY

A Simple, rapid, selective and sensitive HPLC method was developed and

validated for the determination of Fenofibrate from human plasma. The drug was

extracted with ethyl acetate. Fenofibrate was measured in plasma using a validated a

HPLC method with UV detector at 295nm chromatographic peaks were separated

on 5µm Intensil, C18 column (4.6x250mmx5µm) using 60:40 v/v 20mM ammonium

acetate buffer with initial pH and acetonitrile as mobile phase at a flow rate of 1.0

ml/min. The chromatograms showed good resolution and no interference from

plasma. The retention time of Fenofibrate and Nevirapine (IS) were approximately

6.6±0.05 min and 5.2±0.03 min respectively. The mean recovery from human

plasma was found to be above 62%. The method was linear over the concentration

range of 0.3 to 20.0µg/ml with a coefficient of correlation (r2) 0.999. Both intraday

and interday accuracy and precision data showed good reproducibility. This method

was successfully applied to pharmacokinetic studies.

5.7 CONCLUSION

Various methods reported in literature were studied. In the present study a

simple, rapid, specific, rugged, accurate, precise and stable method was developed

for the estimation of Fenofibrate in human plasma. The calibration curve developed

is y= 0.058x - 0.008µg/ml with r2=0.999. The percent mean recovery for

Fenofibrate in LQC, MQC and HQC was 62.7%, 64.2 % and 65.3% respectively.

The method is accurate, precise and rugged with % RSD < 15% and 20% when

tested at MQC, HQC and LQC levels respectively. The stability was assessed at

different levels. The results of the Freeze-thaw stability, Bench top stability and

Inter injection stability studies showed that the compound under analysis is stable

167

under test conditions. Hence the method developed can be used for estimation of

Fenofibrate present in human plasma.

168

5.8. BIBLIOGRAPHY 1. 05/08/2011/www.drugbank.ca/drugs/DB01039.

2. Neil Maryadele JO. Merck Index., 2006; 14: 1743.

3. British Phrmacopoeia. Norwish.2004; I: 805.

4. Hardman J, Goodman-Gilman A, Limbird L.The pharmacological basis of

Therapeutics 1996; 9: 994.

5. Foye.O. William. Principles of Medicinal chemistry 1989; 3: 490.

6. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology 2003; 5:310-311.

7. 22/08/2011/www.mims.com/USA/drug/info/fenofibrate.

8. Fathy M, Salamaa M, Mohamed W, Nassar I, Mohie M, Sharaf E, Din K, Khalid

AM, Mohamed YK.Determination of fenofibrate and the degradation product

using simultaneous UV-derivative spectrometric method and HPLC. Amer. J.

Anal. Chem 2011; 3: 332-343.

9. Madureira TV, Rocha MJ, Cass QB, Tiritan ME. Development and optimization

of a HPLC-DAD method for the determination of diverse pharmaceuticals in

estuarine surface waters. J. Chrom. Sci 2010; 3: 176-182.

10. Bhavesh D, Shah S, Shiva P. Determination of fenofibric acid in human plasma

by ultra performance liquid chromatography-electrospray ionization mass

spectrometry: application to a bioequivalence study. Biomed. Chrom 2009; 9:

922-928.

11. Korany MA, Hewala II, Abdel-Hay KM. 2008. Determination of etofibrate,

fenofibrate and atorvastatin in pharmaceutical preparations and plasma using

differential pulse polarographic and square wave voltammetric technique.

J. AOAC 2008; 5:1051-1058.

169

12. Kadav AA, Vora DN. Stability indicating UPLC method for simultaneous

determination of atorvastatin, fenofibrate and their degradation products in

tablets. J. Pharm. Biomed. Anal 2008; 1: 120-126.

13. Straka RJ, Burkhardt BT, Fisher JE.Determination of fenofibric acid

concentrations by HPLC after anion exchange solid-phase extraction from

human serum. Ther. Drug Monitoring 2007; 2: 197-202.

14. Alaa EG, Samy E, Mostafa K,Mesbah G,Hadad M. Spectrophotometric and

liquid chromatographic determination of fenofibrate and vinpocetine and their

hydrolysis products. IIFarmaco 2005; 5: 425-438.

15. Shihabi ZK.Fenofibrate and fenofibricacid analysis by capillary electrophoresis

2004; 10: 1648-1651.

16. Meikle AW, Findling J., Kushnir M.M, Rockwood AL Nelson GJ, Terry

AH.2003. Pseudocushing syndrome caused by fenofibrate interference with

urinary cortisol assayed by HPLC.J.Clinic.Endocri.Metabol 2003; 8: 3521-3524.

17. Lassner A, Banditt P, Trager U.Rapid and simple method for detection of

fenofibric acid in human serum by high performance liquid chromatography.

Pharmazie 2001; 1: 50-51.

18. Streel B, Hubert P,Ceccato A. Determination of fenofibric acid in human plasma

using automated solid-phase extraction coupled to liquid chromatography.

J. Chrom., 2001; 2: 391-400.

19. Lacroix PM, Dawson BA, Sears RW, Black DB, Cyr TD, Ethier JC. Fenofibrate

raw materials: HPLC methods for assay and purity and an NMR method for

purity. J. Pharm. Biomed. Anal., 1998; 3: 383-402.

20. Bolton S.Pharmaceutical Statistics: practical and clinical application. Marcel

Dekker., 1994; 3: 216-264

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