Simultaneous RP-HPLC Method for Estimation of Metformin Hydrochloride and Fenofibrate in Synthetic Mixture

 

Lata Kothapalli*, Shivaji Mare, Veeren Dewoolkar, Anupam Banerjee, Asha Thomas, RK Nanda and AD Deshpande

Department of Pharmaceutical Chemistry, Pad. Dr. D.Y.Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune-411 018, Maharashtra, India

*Corresponding Author E-mail: lpkothapallidy@yahoo.co.in

 

ABSTRACT

The proposed method is a simple, accurate, precise, specific and rapid method for the simultaneous estimation of Metformin hydrochloride and Fenofibrate in bulk and synthetic mixture using C18 column (Kromosil, 4.6mm x 25 cm, 5µm) with Acetonitrile: 0.02 M Ammonium acetate buffer (80: 20 v/v) as a mobile phase, at a flow rate of 1.0 ml/min and detection was done at 249.0 nm. The retention time for metformin hydrochloride and fenofibrate was found to be 2.63+ 0.011and 9.26+ 0.017 min. respectively. Linearity of metformin hydrochloride and fenofibrate were found in the range of 5–50 μg/ml and 0.8-8.0 μg/ml respectively. The percentage assay of metformin hydrochloride and fenofibrate was found between 98% to 102 %.The statistical parameters were found satisfactory.

 

KEYWORDS: Metformin hydrochloride (MET), Fenofibrate (FNB), RP-HPLC

 

 


INTRODUCTION:

Metformin hydrochloride (MET) chemically 1,1-dimethylbiguanide hydrochloride and it is an oral hypoglycemic agent, clinically used in the treatment of diabetes which facilitates glucose uptake level in skeletal muscle and reduces the free fatty acid oxidation1,2 . MET is official in IP3, EP4, BP5. Fenofibrate (FNB), chemically 1-methyethyl 2-[4-(chlorobenzoyl)phenoxy]-2- methylpropanoate is official in BP5, a febric acid derivative which activates peroxisome proliferators-activation receptors and reduces the total plasma cholesterol and used in treatment of  hyperlipoproteinaemias6. It is reported that the clinical trial with a combination therapy of 500 mg of MET and 80 mg FNB significantly reduced low-density lipoprotein-cholesterol levels when compared to either treatment alone. Co administration of MET and FNB to provide a pharmaceutical composition containing both active components, whilst maintaining the bioavailability of each of the two components equivalent to or superior to that obtained with metformin alone or with the fibrate alone7,8. The synthetic mixture of combination of both the drug were prepared in the ratio of 500:80(MET: FNB) by using excipients like microcrystalline cellulose, Polyvinylpyrrolidone and magnesium stearate which are required for tablet formulation.

 

Various methods such as, HPLC9-10, HPTLC11, UV12-13 spectrophotometry methods have been reported for individual drugs in formulation.  An attempt has been made to develop simple, rapid and accurate RP-HPLC method for simultaneous estimation of MET and FNB from its synthetic mixture.

 

MATERIALS AND METHODS

A HPLC Quaternary gradient system (Lachrom HPLC) consisting of L-7100 Merck Hitachi Pump, UV visible detector (L-7400), Rheodyne injection syringe with 20 µl was used for analysis.

 

Acetonitrile (HPLC Grade) and Ammonium acetate (AR grade) were purchased from Universal Laboratories, Mumbai and standard gift sample of pure MET and FNB from Emcure Pharmaceuticals Ltd. (R and D) Bhosari, Pune and Concept Pharmaceuticals Ltd., Aurangabad respectively.

 

Preparation of mobile phase

The mobile phase consisting of acetonitrile: 0.02M ammonium acetate in the ratio of 80:20 was prepared and sonicated for 15 min. and then it was filtered through a 0.45 µ membrane filter paper.

 

Preparation of standard stock solution of MET and FNB

About 10 mg of MET and FNB each was accurately weighed and transferred to 100 ml volumetric flasks respectively. It was dissolved in methanol and the solution was made up to the volume with methanol to obtain 100µg/ml of MET and FNB respectively.


TABLE 1:  Results of Analysis for Synthetic Mixture

Component

Amount      Present (mg)

*Amount Found (%)

*Standard Deviation (S.D.)

% Relative Standard

Deviation (% R.S.D.)

Standard Error (S.E.)

Metformin

500

99.10

0.7656

0.7725

0.3126

Fenofibrate

80

99.98

1.152

1.153

0.4705

* Mean of six determinations (n=6).

 

TABLE 2: Precision

Component

Repeatability

Intra – day

inter – day

*Mean

*S.D.

*Mean

*S.D.

*Mean

*S.D.

Metformin

100.23

1.091

99.92

0.0710

100.18

0.4038

Fenofibrate

100.31

0.8161

99.89

0.0611

99.93

0.4583

* Mean of six determinations (n=6).

 

TABLE 3:  Recovery Studies and Its Statistical Validation Data

Level of % Recovery

% *Mean Recovery

*Standard

Deviation (S.D.)

% Relative Standard Deviation (% R.S.D.)

Standard Error (S.E.)

MET

FNB

MET

FNB

MET

FNB

MET

FNB

80

99.69

99.45

0.5533

0.3647

0.5550

0.3667

0.3194

0.2106

100

98.96

100.25

0.6158

0.7798

0.6222

0.7778

0.3555

0.4502

120

99.35

98.77

0.9059

0.4545

09118

0.4602

0.5230

0.2624

*Mean of three determinations (n=3). MET – Metformin hydrochloride. FNB – Fenofibrate.

 

TABLE 4: Robustness Data of MET and FNB

Chromatographic changes

Flow Rate (ml/min)

Level

RT

Tailing factor

% Content found

MET

FNB

MET

FNB

MET

FNB

0.8

-2

3.23

12.38

1.4

1.3

99.55

98.40

1.0

0

2.62

9.26

1.3

1.2

100.04

99.83

1.2

+2

2.13

8.44

1.2

1.0

99.54

99.96

*Mean  ±  S.D

2.66 ± 0.5511

10.03 ± 2.079

1.3 ± 0.10

1.17 ± 0.1518

99.71 ± 0.2858

99.40 ± 0.8656

% of ACN

in the mobile phase (v/v)

Level

RT

Tailing factor

% Content found

MET

FNB

MET

FNB

MET

FNB

78

-2

2.66

10.80

1.5

0.8

98.90

101.88

80

0

2.62

9.26

1.3

1.2

99.65

99.38

82

+2

2.58

8.83

1.6

1.2

101.65

101.56

*Mean  ±  S.D

2.62 ± 0.040

9.63 ± 1.036

1.47 ± 0.1528

1.06 ± 0.2309

100.07 ± 1.422

100.94 ± 1.360

Temperature

Level

RT

Tailing factor

% Content found

MET

FNB

MET

FNB

MET

FNB

28

-1

2.64

10.00

1.4

1.2

100.03

100.26

29

0

2.62

9.26

1.3

1.2

99.89

100.02

30

+1

2.58

10.10

1.5

1.2

99.90

99.05

*Mean  ±  S.D

2.61 ± 0.0305

9.79 ± 0.4588

1.4 ± 0.1

1.2 ± 0.0

99.94 ± 0.0781

99.78 ± 0.6407

*Mean of three determinations (n=3). MET – Metformin hydrochloride. FNB – Fenofibrate.

 

TABLE 5: Validation and System Suitability Parameters

Parameter

MET

FNB

Linearity range (μg/ml)

5-50

0.8-8.0

*Slope + S.D

504711± 1383.9

418941 ± 1547.90

*Intercept + S.D

84942± 585.35

21520± 56.58

*Regression coefficient (r2) + S.D

0.9997 ±  ­­0.01

0.9994 +0.01

*Retention time (min.) + S.D.

2.62 ± 0.011

9.26 ± 0.0173

Tailing factor

1.33

1.25

Resolution factor

------

6.64

*Mean of three determinations (n=3). MET – Metformin hydrochloride. FNB – Fenofibrate.

 


Preparation of mixed standard sample solution:

The standard stock solution of each drug was suitably diluted with the mobile phase. The solution was kept in an ultrasonic bath for 20 min and filtered through 0.22 µ membrane filter paper. The sample solution was prepared in the ratio 6.25:1 of MET and FNB.

 

Chromatographic conditions:

The mobile phase consisting of Acetonitrile: 0.02M Ammonium acetate in the ratio of 80:20 was pumped by the dual plunger reciprocating pump (L-7100 Lachrom, Hitachi) at a flow rate of 1.0 ml/min.and the separation was carried out on a C18 column (Kromasil, 4.6 mm x 25 cm, 5 µm). The column temperature was maintained at 290C. The sample was injected through a Rheodyne injector and was analyzed by variable wavelength detector (L-7400) set at 249.0nm (Isobestic point). The data was acquired, stored and analyzed (programmed with Winchrom software).

 

 


Fig. 1: Separation of Metformin and Fenofibrate from Synthetic Mixture (A.) Metformin hydrochloride. (B.) Fenofibrate.

 

 


Assay of synthetic mixture:

The synthetic mixture of both drugs in combination was prepared in the ratio 500:80(MET:FNB) using most commonly used excipients like magnesium stearate (10%) and  M.C.C (Q.S.) required for tablet dosage form. From this mixture amount equivalent to 25 mg of MET and 4 mg FNB was weighed and transferred to 50 ml volumetric flask and dissolved in methanol and content was kept for sonication for about 30 min.Finally  the volume was made up to the mark with methanol. The solution was then filtered through a 0.22 µ membrane filter to give stock solution containing 500μg/ml of MET and 200μg/ml of FNB. Suitable aliquots of the solution were further diluted with mobile phase to obtain sample solutions within the concentration range for the two drugs. A 20 µl volume of each sample solution was injected into sample injector of HPLC six times under the chromatographic conditions as described above. The area under the curve of each peak was measured at 249.0 nm. The amount of each drug present in the sample solutions was determined using the prepared calibration curves of standard MET and FNB respectively. Results are given in Table 1.

 

Precision:

From the standard stock solutions, mixed standards containing MET and FNB in the ratio of 6.25:1 were prepared. Also sample solution was diluted to mixtures containing metformin and fenofibrate in the ratio 6.25:1. Standard and sample solutions (n=6) were injected using a universal Rheodyne injector with injection volume of 20µl. From the peak area of metformin and fenofibrate present in the pure mixture, the amount of each drug present in sample (n=6) was determined. The repeatability and intermediate precision (intraday and interday) were determined and result of which are given in Table 2.

 

Linearity:

Aliquots of standard stock solution of MET and  FNB  stock solution were taken  in 10 ml volumetric flasks and diluted up to the mark with mobile phase in such a way that final concentration of MET and  FNB   were in the range of 5-50 µg/ml for MET and 0.8-8.0µg/ml for FNB respectively. Triplicate injection of 20µl were made for each concentration of each drug separately and chromatographed under the conditions as described above. Evaluation of two drugs was preformed with the UV detector set at 249nm and peak area was recorded. The plot of peak areas Vs respective concentration of MET and FNB was found to be linear. Each standard solution was injected six times into the column at a flow rate of 1.0 ml/min in the range of 5-50µg/ml and 0.8-8.0µg/ml with coefficient of correlation (r2) 0.9997 and 0.9994 for MET and FNB respectively.

Specificity:

The specificity of the RP-HPLC method was determined by complete separation of MET and FNB as shown in Fig. 1 with parameters like retention time (tR), resolution (RS) and tailing factor (T). Here tailing factor for peaks of MET and FNB was less than 2 % and resolution was satisfactory. The average retention time ± standard deviation for MET and FNB were found to be 2.63 ± 0.011 and 9.26 ± 0.017 respectively for six replicates. The peaks obtained for MET and FNB were sharp and have clear baseline separation.

 

Accuracy:

Recovery studies were carried out by addition of standard drug solution. A known amount of standard MET and FNB corresponding to 80 %, 100 %, and 120 % of the label claim was added to preanalysed sample of synthetic mixture separately.  From the amount of drug found, percentage recovery was calculated as per the International Conference on Harmonization (ICH) Guidelines14.  The recovery studies were carried out three times, at each level of recovery. The results of studies along with its statistical validation data are given in Table 3.

 

Robustness studies:

Robustness studies were performed by carrying out deliberate variations of the analytical parameters and effect of the same on the responses such as retention time of the drugs, tailing factor, assay results was examined. Following three factors were selected for changes: flow rate changed (1.0 + 0.2 ml/min), concentration of acetonitrile (80 + 2) and temperature (29 + 1 oC). The solution   containing 20µg/ml of MET and 3.2µg/ml of FNB were injected into the column. A number of analyses (n=3) were conducted at three level of the factor (-, 0, +). Results are given in Table 4.

 

RESULTS AND DISCUSSION:

The goal of this study was to develop a rapid and sensitive HPLC method for the analysis of MET and FNB in synthetic mixture using the most commonly employed C18 column with UV detection.

 

The mobile phase consisted of Acetonitrile and 0.02 M Ammonium acetate (80: 20 v/v). The retention times for MET and FNB were 2.63 and 9.26 min. respectively (Fig. 1). The peak areas of both the drugs were reproducible as indicated by RSD value which is less than 2%. When the calibration curve of concentration of MET and FNB and its respective peak areas were plotted, a good linear relationship was observed between the concentration and their respective peak areas in the range of 5-50µg/ml for MET and 0.8-8.0µg/ml for FNB. The results of the assay, recovery studies and its statistical validation data indicate high degree of precision and accuracy of the proposed method. The results of the validation and system suitability parameter are given in Table 5.

 

Hence it can be concluded that the developed RP-HPLC method can be employed successfully for the estimation of MET and FNB in both bulk and synthetic mixture.

 

ACKNOWLEDGEMENTS:

The authors thank to Emcure Pharmaceuticals Ltd., Pune for supplying gift samples of metformin hydrochloride  and Concept Pharma, Aurangabad (India) for fenofibrate.

 

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Received on 14.02.2009       Modified on 23.04.2009

Accepted on 19.05.2009      © RJPT All right reserved

Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 694-697