DEVELOPMENT AND VALIDATION OF A NOVEL CLEANING VALIDATION AND ASSAY METHOD FOR SIMULTANEOUS ESTIMATION OF ROSUVASTATIN AND FENOFIBRATE BY RPHPLC

DEVELOPMENT AND VALIDATION OF A NOVEL CLEANING

VALIDATION AND ASSAY METHOD FOR SIMULTANEOUS

ESTIMATION OF ROSUVASTATIN AND FENOFIBRATE BY

RP-HPLC

Nazareth Celina* and Bodke Anushka

Department of Pharmaceutical Chemistry, P.E.S’s Rajaram and Tarabai Bandekar College of

Pharmacy, Farmagudi, Goa, India- 403401.

ABSTRACT

A new, simple, precise, accurate and reproducible RP-HPLC method

for cleaning validation and assay for simultaneous estimation of

Rosuvastatin and Fenofibrate in tablet dosage form was developed and

validated. The chromatography was carried out on

AGILENT-ZORBAX RP-Cyano column (250mm×4.6mm,5µm) in a isocratic

mode utilizing Potassium dihydrogen phosphate buffer (pH 2.5 with

OPA): Methanol : Acetonitrile (45:33:22, v/v/v) as a mobile phase, at a

flow rate of 1.5ml/min. Detection was carried out at 252 nm. Retention

times of Rosuvastatin and Fenofibrate were 3.6 and 10.01 mins,

respectively. The Beer Lambert’s law was obeyed in the concentration

range 0.5-1.5µg/mL for both Rosuvastatin and Fenofibrate. The mean

% recoveries at 100% and LOQ level were 96.7% and 99.0% for

Rosuvastatin and 90.2% and 89.5% for Fenofibrate, respectively.

KEYWORDS: RP-HPLC, Rosuvastatin, Fenofibrate, Cleaning Validation.

INTRODUCTION

Rosuvastatin “fig 1” is a competitive inhibitor of HMG-CoA reductase. HMG-CoA

reductase, catalyses’ the conversion of HMG-CoA to mevalonate, an early rate-limiting step

in cholesterol biosynthesis. Rosuvastatin acts in the liver. The decreased hepatic cholesterol

concentration stimulates the up regulation of hepatic low density lipoprotein (LDL) receptors

which increases hepatic uptake of LDL and inhibits hepatic synthesis of very low density

lipoprotein (VLDL). The overall effect is a decrease in plasma LDL and VLDL levels.[1,5]

Volume 7, Issue 3, 1454-1465. Research Article ISSN 2277– 7105

Article Received on 21 Dec. 2017,

Revised on 11 Jan. 2018, Accepted on 31 Jan. 2018

DOI: 10.20959/wjpr20183-10949

*Corresponding Author

Nazareth Celina

Department of

Pharmaceutical Chemistry,

P.E.S’s Rajaram and Tarabai

Bandekar College of

Pharmacy, Farmagudi, Goa,

Fenofibrate “fig 2”, a fibrate, exerts its therapeutic effects through activation of peroxisome

proliferator activated receptor alpha (PPARα). This results in increased lipolysis and

elimination of triglyceride-rich particles from plasma by activating lipoprotein lipase and

reducing production of apoprotein C-III. This results in decrease in triglycerides producing an

alteration in the size and composition of LDL from small, dense particles, to large buoyant

particles. These larger particles have a greater affinity for cholesterol receptors and are

catabolized rapidly.[1,5]

N N

CH₃

O OH

N C H₃

S O O

CH₃

OH OH F

Fig. 1: Rosuvastatin.

Chemically Rosuvastatin [C22H28FN3O6S] is

(3R,5S,6E)-7-[4-(4-fluorophenyl)-2-(N-methylmethanesulfonamido)-6-(propan-2-yl)pyrimidin-5-yl]-3,5-dihydroxyhept-6-enoic acid

with molecular weight 481.538 and Fenofibrate [C20H21ClO4]is Isopropyl

2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoate. The combination of Rosuvastatin and

Fenofibrate is used to treat hyperlipidemias, mixed dyslipidemia, hypercholesteriamia and

hypertriglyceridemia.[1,5]

CH₃ O

Cl

O

CH₃ O

O

CH₃ CH₃

Cleaning validation is done to confirm reliability of a cleaning procedure and thus refers to

generating documented evidence that an approved cleaning procedure will provide equipment

that is suitable for processing of pharmaceutical products or active pharmaceutical

ingredients (APIs). Cleaning validation is of critical importance and thus must be strictly

followed by manufacturers of pharmaceutical products and APIs.[6,7]

A review of literature indicated that many UV Spectrophotometric, RP-HPLC and LCMS

methods have been reported for the assay of Rosuvastatin and Fenofibrate either alone or in

combination.However, no cleaning validation method for the above drug combination has

been reported. Considering the importance and need of cleaning validation in pharmaceutical

manufacturing it was thought worthwhile to develop a cleaning validation method for

simultaneous determination of Rosuvastatin and Fenofibrate. The new method could further

be used for assay of tablet formulation.

MATERIALS AND METHODS

Equipment

HPLC of Agilent 1260 make with auto sampler, auto injector and UV detector of Agilent

make was used for the study. Rosuvastatin and Fenofibrate APIs were obtained as gift

samples from Vergo Pharma Research Laboratories, Verna, Goa. The tablet dosage

formulation LIPIROSE-F 10 marketed by Mankind Pharma Ltd. containing 10 mg

Rosuvastatin and 145 mg Fenofibrate was purchased from local pharmacy. All chemicals

used and solvents used were of AR/HPLC grade of Rankem/ Merck.

Chromatographic Conditions

PARAMETERS CHROMATOGRAPHIC CONDITIONS

Mobile Phase Phosphate buffer (pH 2.5): Methanol:

Acetonitrile (45:33:22, v/v/v)

Runtime 15 min

Injection Volume 20µL

Flow Rate 1.5 mL/min

COT 35°C

Column Agilent ZorbaxCYANO (250mm×4.6mm,5µm)

Wavelength 252 nm

Standard Stock Solution

i. Cleaning Validation

Standard stock solutions of Rosuvastatin and Fenofibrate (100 µg/mL) were prepared

separately in methanol. From this, mixed standard solution was prepared by taking 1 mL

from each standard stock and diluting it to 100 mL with 50% methanol with final

concentration of 1 µg/mL. The chromatographic system was subjected to system suitability.

ii. Assay

Standard stock solutions of Rosuvastatin and Fenofibrate were prepared in methanol having

concentration of 100µg/mL and 1450 µg/mL respectively. The working mixed standard

solution was prepared by diluting 5 mL from each standard stock to 50 mL with diluent II, to

get final concentration of 10µg/mL and 145 µg/mL of Rosuvastatin and Fenofibrate

respectively.

Sample Solution

The test samples were prepared by crushing 10 tablets and to this 70 ml of diluent I was

added and sonicated for about 10 mins. Volume was made up to mark with diluent I. The

solution was centrifuged for 10 mins at 5000 rpm. A volume of 5 ml of supernatant was

transferred to a 50 ml volumetric flask and diluted to mark using diluent II and mixed

thoroughly.

Placebo Solution

Placebo sample was prepared in similar way as sample solution except that placebo powder

was used in place of tablet powder.

Validation

Both the methods were validated for specificity, linearity, accuracy, precision, LOQ, LOD

and robustness as per ICH guidelines.

In case of cleaning validation method, Specificity was evaluated to determine that swabs used

showed no interference in the method. Linearity was performed from LOQ level to 150%.

Accuracy was established by performing recovery studies at 100% and LOQ level

concentration of the drugs. The precision was determined by injecting six replicates of

Rosuvastatin and Fenofibrate mixed standard solution at LOQ level. The limit of detection

Noise ratio” method. The concentration which gave a Signal to Noise ratio of about 3 for

limit of detection and about 10 for limit of quantitation was derived. The solutions were

prepared and injected and response was seen. Robustness was demonstrated by bringing

about deliberate changes in mobile phase composition, pH of Buffer in mobile phase, flow

rate and column temperature and calculating its impact on system suitability parameters.

For the assay method, specificity was performed in presence of placebo solution. Linearity

was established considering the tablet strength i.e. at 0.5 µg/mL-15µg/mL for Rosuvastatin

and 0.5 µg/ mL -215µg/mL for Fenofibrate. Accuracy was performed at three levels i.e., at

50%, 100%, 150%. Precision was established by performing repeatability studies and % RSD

was calculated.

RESULT AND DISCUSSIONS

Preliminary experiments were carried out to achieve the best chromatographic conditions for

the simultaneous determination of both the drugs. Several column types and mobile phases

along with other parameters were tried. The spectrum scans using a PDA detector showed

λmax of Rosuvastatin as 242 nm and of Fenofibrate as 287 nm. The Isosbestic point was determined and λ of 252 nm was chosen as the wavelength of analysis as seen in “fig 3”. The

optimised mobile phase composition was Phosphate buffer (pH 2.5 with OPA): Methanol:

Acetonitrile (45:33:22, v/v/v) at a flow rate of 1.5 ml/min.

Fig. 3: Overlain spectra of Rosuvastatin and Fenofibrate.

The mixed standard solution was injected six times, and the system suitability parameters

were verified. The results are depicted in table 1. As seen, the system suitability parameters

typical chromatogram of Rosuvastatin and Fenofibrate is shown in “fig. 4”. The retention

time of Rosuvastatin and Fenofibrate was found to be 3.6 min and 10.01 min respectively

with resolution of 23.67.

Table 1: Summary of System Suitability Testing.

System Suitability parameters Observed values Acceptance

criteria

ROS FEN

% RSD (n=6) 0.655896 0.921821 NMT 2.0%

Average of Theoretical plates 7480.8 11157.33 NLT 2000

Average of Tailing factor 1.243 1.165 NMT 2.0

Resolution 23.67 NLT 1.5

Fig. 4: A representative chromatogram of Rosuvastatin and Fenofibrate.

The Specificity of the method was established by analyzing the prepared swab solutions. The

method was found specific as swab analysis showed no interference “fig.5”. The Beer

Lambert’s range for the drugs was established by plotting calibration curves “fig. 6” and “fig.

7”. Linearity data has been summarized in table 2. As seen, the Beer Lambert’s range was

established in the concentration range of LOQ-1.5µg/ml for both Rosuvastatin and

Fenofibrate with correlation coefficient (r2) = 0.999. Accuracy was performed at LOQ and

100% level. The mean % recovery at 100% level was found to be 96.7% and 90.2% for

Rosuvastatin and Fenofibrate respectively. The mean % recovery at LOQ level was found to

be 99.0% and 89.0% for the two drugs respectively (table 3). The method was found precise

with % RSD less than 2.0%. The LOD and LOQ values were calculated based on the

Signal-to-Noise ratio. The LOD was obtained as 0.006µg/ml for Rosuvastatin and 0.015µg/ml for

Fenofibrate and the LOQ was obtained as 0.02µg/ml for Rosuvastatin and 0.05µg/ml for

Fenofibrate which showed that method is sensitive (table 4 and table 5). The method was

found robust as minor changes in chromatographic conditions, yielded insignificant changes

Fig. 5: A representative chromatogram of swab interference.

Fig. 6: Calibration curve for Rosuvastatin.

Fig. 7: Calibration curve for Fenofibrate.

Table 2: Linearity data for Rosuvastatin and Fenofibrate.

Parameters Rosuvastatin Fenofibrate

Linearity range 0.02 µg/ml-1.63 µg/ml 0.05µg/ml-1.69 µg/ml

Slope 472739.268105 401383.061383

Intercept 7570.80000 -433.00000

Correlation Coefficient 0.999 0.999

Table 3: Accuracy at 100% level and LOQ level.

Table 4: Signal to noise ratio for Rosuvastatin and Fenofibrate.

Signal (S) Height Ratio (S/N)

Rosuvastatin 83677 610.781

Fenofibrate 27916 203.766

Noise (N) 137 --

Table 5: LOQ and LOD for Rosuvastatin and Fenofibrate.

Components

Limit of Quantitation (LOQ)

Limit of Detection (LOD) Concentration,

in µg/mL

Concentration, in µg/mL

Rosuvastatin 0.02 0.006

Fenofibrate 0.05 0.015

Table 6: Summary of results for Robustness.

Parameter Level Rosuvastatin Fenofibrate

%RSD TF %RSD TF

pH 2.5 1.1 1.3 1.2 1.2

2.7 0.8 1.3 0.9 1.3

Flowrate(ml/min) 1.3 0.6 1.4 0.8 1.4

1.7 0.3 1.2 0.6 1.2

COT (°C) 30 0.3 1.2 0.9 1.3

40 0.3 1.3 0.9 1.3

% Methanol 90% 1.3 1.3 1.3 1.3

110% 1.3 1.3 0.9 1.3

% Acetonitrile 90% 0.9 1.3 0.7 1.4

110% 0.4 1.4 0.4 1.4

The assay method developed was applied for evaluation of tablet formulation. The linearity

range for assay was found to be 0.5-15µg/ml for Rosuvastatin and 0.5-215µg/ml for

Fenofibrate with r2 = 0.999 for both the drugs as depicted in table 7. The method was found

specific as there was no interference due to excipients or mobile phase. The method was

accurate as the mean % recoveries were found within 90.0% - 110.0% at 50%, 100%, 150%

level. The % assay was found to be within the acceptance criteria of 90.0% - 110.0%.

Sample No. (Swab no)

% Recovery

100% Level LOQ Level

ROS FEN ROS FEN

1 95.7 90.0 98.6 90.0

2 96.4 92.1 98.7 89.1

3 98.1 88.4 98.7 89.3

Table 7: Linearity data for Rosuvastatin and Fenofibrate for assay method.

Parameters Rosuvastatin Fenofibrate

Linearity range 0.5µg/ml-15µg/ml 0.5µg/ml-215µg/ml

Slope 501660 388457

Intercept 40328 83842

Correlation Coefficient 0.9998 1

Linear Regression Equation 501660x+40328 388457x+83843

Table 8: Accuracy data for Rosuvastatin and Fenofibrate for assay method.

Accuracy Levels

Average % Recovery Rosuvastatin

Average % Recovery Fenofibrate

50% 99.84 99.76

100% 98.33 98.54

150% 98.89 99.63

CONCLUSION

A novel method for cleaning validation was developed. The method is simple, cost effective,

specific, accurate, precise, reproducible and robust. The target of developing a common

chromatographic method for both cleaning validation and assay of formulation has been

achieved. These methods can be used to estimate Rosuvastatin and Fenofibrate as

independent components as well as in combination in quality control procedures.

ACKNOWLEDGMENT

The authors would like to thank Vergo Pharma Research Laboratories Ltd. Verna-Goa for

providing gift samples of the drugs and facilities to carry out this research work.

CONFLICT OF INTEREST

Authors declare no conflict of interest.

ABBREVIATIONS USED

Std: Standard

ICH: International Conference on Harmonization

ROS: Rosuvastatin

FEN: Fenofibrate

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