Development and Validation of Isocratic RP-HPLC Method for Raloxifene Hydrochloride in Bulk and Pharmaceutical Formulation.

 

P. VenkataSuresh*, G.V. Srujana, G. Lavanya, K.M.L. Manoja, M. Hadassah, B. Srilekha, S. Vasudha Lakshmi and N.Rama rao

Dept. of Pharmaceutical Analysis, Chalapathi Institute of Pharmaceutical Sciences, Lam, Guntur-34, India.

*Corresponding Author E-mail: sureshpharma78@gmail.com

 

ABSTRACT:

A simple, sensitive, rapid, robust and reproducible method for the determination of Raloxifene hydrochloride in bulk and pharmaceutical formulation (Tablets) was developed using reverse phase high performance liquid chromatographic method (RP-HPLC). The RP-HPLC analysis was performed isocratically on Equisil-ODS (octadecylsilane; C18), 5, 4.6 x 250 mm analytical column using a mobile phase consisting of 0.02M potassium dihydrogen ortho Phosphate buffer (pH adjusted to 3.00 0.01 with ortho phosphoric acid after addition of 0.1% triethyl amine), Methanol, Acetonitrile in the Ratio of 40:30:30 (v/v/v) with a flow rate of 1mL/min. The analyte was monitored with UV detector at 282 nm. The developed method Raloxifenehydrochloride elutes at a retention time of 2.70 min. The proposed method is having linearity in the concentration range from10 to70 g/mL of Raloxifenehydrochloride. The present method was validated with respect to system suitability, linearity, precision, limit of detection (LOD) and limit of quantification (LOQ), accuracy (recovery), ruggedness, robustness, stability, forced degradation studies (specificity).The proposed method can be readily utilized for bulk drug and pharmaceutical formulations.

 

KEYWORDS: HPLC, Raloxifene hydrochloride.

 


INTRODUCTION:

Raloxifene hydrochloride chemically known as [6-Hydroxy-2-(4-hydroxyphenyl) benzo[b]thien-3-yl]-[4-(2-piperidinyl)ethoxy] phenyl] hydrochloride. Raloxifene hydrochloride is a selective estrogen receptor modulator (SERM) of the benzo thiophen class; it is approved for the prevention and treatment of osteoporosis in postmenopausal women. The results of several large clinical trials have shown that raloxifene reduces the rate of bone loss at both distal sites and in the spinal column and may increase bone mass at certain sites1. Adverse effects include deep vein thrombosis, pulmonary embolism and legcramps2. Raloxifene hydrochloride is listed in Merck Index3.

 

Literature survey reveals various analytical methods such as Spectrophotometric4, Capillary electro phoresis5, few HPLC methods in bulk6, formulations7-8 and in human plasma by LC-MS/MS9 methods for estimation of raloxifene hydrochloride in biological fluids.

 

The present study was aimed at developing a simple, sensitive, rapid, reproducible, precise and accurate isocratic RP-HPLC method for the analysis of Raloxifene hydrochloride in bulk and pharmaceutical dosage forms using UV detector at 282 nm.

 

EXPERIMENTAL

Chemicals and solvents: Raloxifenehydrochloride was procured as gift sample from Dr. Reddys labs LTD (Hyderabad, India). HPLC grade water, acetonitrile and methanol was procured from Merck Ltd (Mumbai, India). All other chemicals were used analytical reagent grade (AR grade) like potassium di hydrogenorthophosphate procured from Qualigens Fine chemicals (Mumbai, India), triethylamine and orthophospharic acid procured from SD Fine chemicals Ltd. (Hyderabad, India).

 

Chromatographic conditions: Shimadzu HPLC (Spinchrom CFR version 2.4.01.093 data handling system) with LC- 20AT prominence liquid chromatography and SPD-20A prominence UV/Visible detector was employed for present study. The chromatography determination performed by using a mobile phase fixed composition of 0.02M potassium dihydrogen ortho Phosphate buffer (pH adjusted to 3.000.01 with orthophosphoricacid after addition of 0.1%triethyl amine), Methanol, Acetonitrile in the Ratio of 40:30:30 (v/v/v) and a stationary phase of stainless steel column Equisil-C18 Column (250 mm long, 4.6 mm internal diameter filled with Octadecylsilane (C18); chemically bonded to porous silica particles of 5 m particle size).The chromatography run time was maintained up to 8.0 min with flow rate at 1.0 mL/min and the eluent was monitored at 282 nm.

 

Preparation of stock solutions:

Preparation of standard stock solution: About 100 mg of pure sample of raloxifene hydrochloride was accurately weighed and dissolved in methanol in a 100 mL standard flask to get a working standard concentration of 1 mg/mL (final concentration equal to 1000 g/mL).

 

Preparation of sample stock solution: Each brand of 20 tablets (Brand.1-FIONA, Dr. Reddys Labs, India; Brand.2-ESSERM, Torrent pharmaceuticals, Ahmadabad, India) were accurately weighed and powdered. Sample of powdered tablets equivalent to 100 mg of raloxifene hydrochloride was taken and transferred to 100 mL volumetric flask. About 70 mL of methanol was added and sonicated for 30 min with intermittent shaking. Sample stock was filtered through 0.45 filter paper in to another 100 mL volumetric flask and diluted up to the mark (100 mL) with methanol (final concentration equal to 1000 g/mL).

 

Assay of pharmaceutical dosage forms: After completion of validation each tablet brand sample stock solution (1000 g/mL) was further diluted to working concentration (60 g/mL) within range of calibration curve and injected 20 l of the sample solution. The assay procedure was repeated six times by individually weighing of each brand tablet powder. The amount of drug in each tablet was calculated from the calibration curve. The sample chromatogram was recorded in Fig.1 and assay values are tabulated in Table.3.

 

Method validation:10-13

System suitability: System suitability study was carried out as specified in USP. Injected 30 g/mL concentration for performing system suitability these parameters include column efficiency (N), Capacity factor (K'), Resolution (Rs), Tailing factor, HETP (mm). Suitability parameters were tabulated in Table.1.

 

Table.1: System suitability parameters

System suitability parameters

Value

Tailing factor

1.1201

Theoretical plates(n)

4579

Capacity factor(K')

2.14

HETP(mm)

0.0221

Peak width at half height in min (W05)

0.09

Resolution (Rs)

9.1022

Retention time(min)

2.70

 

Linearity: Aliquots of standard raloxifene hydrochloride stock solution(1000 g/mL) in the concentration range of 0.1 to 0.7 ml were transferred in 10mL volumetric flask and make up to mark with mobile phase to get final calibration curve standard concentrations of 10,20,30,40,50,60 and 70 g/mL Each calibration curve standard solution was injected thrice in to HPLC system. The calibration graphs were obtained by plotting the peak area of drug on y-axis and the concentration (g/mL) on X-axis. The method is having good linearity (r2 = 0.9992).

 

Precision: To study day to day variations on the developed method 20, 40 and 60 g/mL standard solutions were subjected to intra-day and inter-day precision. The low % RSD values indicate that this method is having good precision and is not affected by day to day variations. The results were given in Table-2.

 

Limit of Detection (LOD) and Limit of Quantification (LOQ): The LOD and LOQ of were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines. The Limit of detection was found to be 0.03 g/m and Limit of quantification was found to be 0.09 g/mL.

 

Accuracy (Recovery study): Accuracy of the method was studied by recovery experiments. The recovery was should performed at three levels 80,100 and 120% as per ICH guide lines. The present recovery experiments were performed at three levels concentrations (48, 60, 72 g/mL) by adding known amount of pure drug was added to the previously analyzed pharmaceutical sample solution (sample solution of known concentration 60 g/mL).

 

Forced degradation study (specificity): To check the developed method is specific and stability indicating, the drug was subjected stressed conditions like treatment with 0.1M HCL, 0.1M NaOH, 3%H2O2, 50C heat and Ultra violet light. The treated samples were analyzed by the proposed chromatographic conditions and additional peaks were resolved from the drug peak.

 

Ruggedness: Method ruggedness was checked by varying the lot number and manufactures of reagents (Orthophosparic acid, triethylamine), solvents (HPLC grade water, Methanol, Acetonitrile) procured from Qualigens Fine chemicals (Mumbai, India) and column (Inersil ODS -250 x 4.6 mm; 5 m). The effect of changes was observed on chromatographic parameters like retention time, tailing factor, resolution, peak area and theoretical plates. Results obtained show no significant variation in above parameters indicating ruggedness of method.

 

Robustness: Performed experiments by slightly varying the experimental conditions like the proportions of the mobile phase (2% on total proportion), concentration of buffer pH (0.023 M with pH-3.0 0.5 units), column temperature (20C 5C), flow rate (1.0 ml/min 0.2 ml) and wavelength (282 nm 2 nm) of detection. The effect of changes was observed on chromatographic parameters like retention time, tailing factor, resolution, peak area and theoretical plates. Results obtained show no significant variation in above parameters indicating robustness of method.

 


Table.2: Intraday and inter day precision values

Concentration of raloxifene hydrochloride(g/mL)

 

Concentration of Raloxifene hydrochloride (g/mL) found

%RSD (Relative standard deviation found )

Intra-day Mean(n=6)

Inter-day Mean(n=6)

Intra-day(%CV) Mean (n=6)

Inter-day(%CV) Mean(n=6)

20

40.02

39.99

0.32

0.41

40

50.04

50.01

0.51

0.45

60

59.96

59.87

0.22

0.37

*Mean of Six determinations

 

Fig: 1. Chromatogram for Raloxifene hydrochloride at 282 nm

 

Table.3: Assay of dosage forms

Brand name

Labeled claim (mg/tab)

Weight of sample taken

Amount found

%CV

%Assay

Brand.1- FIONA (Dr.Reddys labs)

60

10.03

60.04

0.414

99.93

Brand.2-ESSERM, (Torrent)

60

10.01

59.97

0.359

100.05

*Mean of Six determinations

 


Stability: Freshly prepared sample solution (accurately weighed tablet powdered equivalent to 10 mg/10 ml) was stored at normal laboratory conditions (18 to 20c) and observed short term stability (one day) and long term stability (after fifteen days) at fixed concentration of 30g/mL. Each stability (long and short term stability) was evaluated by repeatedly six injections of same concentration as per proposed method. Stability study (short term and long term) was also performed for freshly prepared buffer (0.02 M potassium dihydrogen ortho Phosphate buffer; pH adjusted to 3.00 0.01 with ortho phosphoric acid after addition of 0.1%triethyl amine) and Mobile phase (Buffer : Methanol, acetonitrile;40:30:30 (v/v/v) at normal laboratory conditions (18 to 20C).

 

RESULTS AND DISCUSSION:

The objective of present study was to develop a rapid and sensitive HPLC method for the analysis of Raloxifene hydrochloride in bulk and formulations using most commonly employed RP-HPLC method with C‑18 column and UV detection at 282nm.Mobile phase consisting of 0.0 2M potassium dihydrogen ortho Phosphate buffer (pH adjusted to 3.00 0.01 with ortho phosphoric acid after addition of 0.1%triethyl amine), Methanol, Acetonitrile in the Ratio of 40:30:30 (v/v/v) with a flow rate of 1 ml/min, run time 8.0 min and retention time was 2.70 min had given immaculate results when compare to other methods. The coefficient of variation for system suitability experiment was observed to be less than 1% for column efficiency (N), Capacity factor (K'), Resolution (Rs),Tailing factor, HETP (mm) indicating suitability of the system. A plot of peak area against concentration (g/mL) gave a linear regression (r2=0.9992) over the concentration range 10 to 70 g/mL. The proposed method was also validated for intra and inter day variation and %CV was found to be less than 1%. The lowest level of quantification was observed to be 0.090g/mL which indicates the sensitivity of method. The accuracy of HPLC method was assessed by adding known amount of the drug to pre analyzed sample solution. There was a high % recovery (100.10%) indicating that proposed method is highly accurate. Treating the samples with Acid, Alkali, oxidizing agents which confirm that the method was the developed method is specific and stability indicating method. No interfering peaks were observed in the chromatogram indicating that excipients used in tablets formulation did not interfere with the estimation of drug by proposed method. Robustness results obtained show no significant variation in parameters (retention time, tailing factor, resolution, peak area and theoretical plates) indicating robustness of method. Results obtained from stability experiment reveal that sample solution, buffer and mobile phase were stable for purpose of routine analysis under normal laboratory conditions.

 

CONCLUSION:

The developed method was found to be simple, sensitive, accurate, precise, and reproducible it can be used for routine quality control analysis of Raloxifene Hydrochloride in bulk and pharmaceutical formulations.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Dr. Reddys labs Ltd; Hyderabad for providing the gift sample of Raloxifene Hydrochloride. We would also like to thank Chalapathi institute of pharmaceutical sciences for providing all the facilities to complete our work successfully.

 

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Received on 22.06.2010 Modified on 03.07.2010

Accepted on 14.07.2010 RJPT All right reserved

Research J. Pharm. and Tech. 4 (1): January 2011; Page 146-149