chromotographic method

Journal of Pharmacy Research Vol.2.Issue 4.April 2009

ISSN: 0974-6943

Validation of high performance liquid chromatographic method for thedetermination of Tibolone in bulk and pharmaceutical dosage form

Varsha Jadhav*, Sachin Gholve and Vilasrao Kadam *Department of Quality Assurance, Bharati Vidyapeeth’s College of Pharmacy, Navi-Mumbai – 400614, MH, India*For correspondence:Dr. (Mrs.) V. M. JadhavE-mail:[email protected] Received on:01-12-2008; Accepted on :07-02-2009

ABSTRACT

To develop and validate a sensitive, selective, precise high performance liquid chromatographic (HPLC) method of analysis for tibolone bothas bulk drug and formulations. A new sensitive and reproducible HPLC method was developed and validated for the determination of tibolonein tablets. The separation was achieved by using a JASCO HPLC system 2000 series comprising of JASCO PU 2080 plus pump, JASCO UV2075 plus detector, Rheodyne manual injector fitted with 100 µl sample loop. Data acquisition and treatment done using Borwin chromatog-raphy software version 1.5. The analytical column used was thermo C8 (250 X 4.6mm; 5µm) at ambient temperature using a mobile phaseconsisting of Methanol: Acetonitrile: Water (10:60:30). Flow rate was 1.0 ml with an average operating pressure of 200 kg /cm² and retentiontime was found to be 6.8 ± 0.02min.The method was validated for accuracy, precision, linearity, reproducibility and robustness and statisticalcomparison was performed by analysis of variance. The developed HPLC technique is precise, specific, accurate and stable. Statisticalanalysis proves that the method is reproducible, selective and suitable to be applied for analysis of tibolone in commercial pharmaceuticaldosage form for routine quality control application.

Key words: Tibolone, RP-HPLC, Dosage form

Research Article

INTRODUCTION

Tibolone [7α, 17α-7-methyl-17-hydroxyl-19-norpregn-5(10)-en-20-yn-3-one], also called 7-methyl-norethynodrelor Org OD14, isa synthetic steroid used in the hormonal replacement therapy (HRT)for postmenopausal women. It has been used in Europe for almost 2decades, primarily for the prevention of postmenopausal osteoporo-sis and treatment of climacteric symptoms and provides additionclinical benefits with regard to breast tissue and sexual well-being.Literature survey revealed that various methods have been reportedfor the estimation of Tibolone in biological matrices such as plasmaby HPLC, LC – MS. No method has been reported for estimation ofTibolone in tablet dosage forms. The aim of this work was to developa validated HPLC method for determination of Tibolone in bulk andpharmaceutical dosage form.[1-2]

MATERIALS AND METHODS

Instrument

The instrument used was a JASCO HPLC system 2000 se-ries comprising of JASCO PU 2080 plus pump, JASCO UV 2075 plusdetector, Rheodyne manual injector fitted with 100 ìl sample loop.Data acquisition and treatment done using Borwin chromatographysoftware version 1.5. The analytical column used was hyqsil C-8 [250mm x 4.6 mm, 5 mm]

Chromatographic Condition

Column used : Hi Qsil C-8 [250 mm x 4.6mm, 5 mm]

Injection volume : 20µLMobile phase : Methanol: Acetonitrile: Water (10:60:30)Flow rate : 1 ml/minDetection : 205 nmColumn temperature : AmbientDiluent : Mobile phaseRetention time : 6.8 min

Standard Stock Solution:

About 100 mg of Tibolone was weighed and taken in a 100ml volumetric flask, dissolved and diluted to make the diluent (1000mg/ml)

Preparation of Mobile Phase:

To optimize the HPLC parameters several mobile phase com-positions were tried. Satisfactory peak symmetry was obtained withmobile phase consisting of Methanol, Acetonitrile and Water in pro-portion of 10:60:30. The mobile phase was filtered through 0.45 cellu-lose nitrate filter paper and degassed by ultrasonication for 20 min.

Calibration Curve:

Appropriate aliquots of standard stock solution (1000 mg/ml) was diluted to 100 mg/ml in 10ml volumetric flask and resultantsolution was diluted up to the mark with mobile phase to obtain finalconcentration of 1, 2, 4, 6, 8, 10 and 11 mg/ml. These solutions were

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Varsha Jadhav et al. / Journal of Pharmacy Research 2009, 2(4),694-697

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Journal of Pharmacy Research Vol.2.Issue 4 April 2009

ISSN: 0974-6943

CH3

OH

O

H

H

H

CH3

CH

Fig 1: Tibolone

Fig 2: Tibolone (formulation chromatogram)

C a l i b r a t i o n C u r v e o f T i b o l o n e

y = 8 9 5 7 7 xR 2 = 0 . 9 9 9 7

0

5 0 0 0 0 0

1 0 0 0 0 0 0

1 5 0 0 0 0 0

0 5 1 0 1 5

T i b o l o n e c o n c . m c g / m l

area

of p

eak

A r e a

L i n e a r ( A r e a )

Fig 2: Calibration curve of Tibolone

Sr. Concentration PeakNo. of Tibolone area

(mg/ml)1. 1 97458.872. 2 187406.753. 4 358744.454. 6 538649.895. 8 709397.486. 10 899201.457. 11 984456.5

Table 1: Data for calibration curve

Parameters HPLC method

Range 1-11 mg/mlCorrelation coefficient 0.9997Slope 89577x

Table 2: Linear regression data for calibration curve

Table 3: Precision of proposed HPLC method

Concentration Concentration Of Tibolone (mg/ml) found onof Tibolone (mg/ml) Inter-day Intra-day

Mean CV Mean CV

30 30.009 0.0315 29.99 0.208250 50.006 0.0450 49.96 0.1096

Varsha Jadhav etal.,Validation of high performance liquid chromatographic method for the determination of Tibolone inbulk and pharmaceutical dosage form


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Journal of Pharmacy Research Vol.2.Issue 4.April 2009

ISSN: 0974-6943

injected into chromatographic system and chromatograms were ob-tained and peak area ratio was determined for each concentration ofdrug solution. Calibration curve of Tibolone were constructed byplotting peak area ratio vs applied concentration of Tibolone andregression equation was computed. Similarly the sample solution waschromatographed and concentration of Tibolone in tablet sampleswas found out using regression equation. [11-12]

Method Validation:

As per the USP XXIII, system suitability tests for HPLCwere carried out on freshly prepared standard stock solution ofTibolone and the parameters studied and results obtained with 20 ulinjection volumes. The method was validated for accuracy, precision,specificity, detection limit, quantitation limit and robustness by fol-lowing procedures.[3-5, 9, 10]

Accuracy:

The accuracy of the method was determined by calculatingrecovery of Tibolone by method of standard addition. Known amountof Tibolone (1, 2, 4 mg/ml) was added to a pre quantified samplesolution and the amount of Tibolone was estimated by measuring thepeak area ratios and by fitting these values to the straight line equa-tion of calibration curve.[3]

Precision:

The intra day and inter day precision study of Tibolone wascarried out by estimating the corresponding responses 3 times on thesame day and on 3 different days (first, third and fifth day) for threedifferent concentrations of Tibolone (30 & 50 mg/ml) and the resultare reported in terms of relative standard deviation. The analyticaldata is given in table 5. [3-5]

Specificity:

Complete separation of Tibolone in mobile phase was no-ticed. The average retention time was found to be 6.8 respectively forsix replicates. The peaks obtained were sharp and have clear baselineseparation. [5]

Linearity:

Linearity of the method was investigated by serially dilut-ing the stock solution to give a concentration range of 1 to 11 mg/mland injected 20 ul with universal injector (Rheodyne). The flow ratewas maintained at 1.0 ml/min. Temperature of the column was keptambient and the effluent was monitored at 205 nm. Calibration curvewas constructed by plotting concentration against peak area as shownin fig 3. [3-5]

Robustness:

Robustness of the method was studied by changing thecomposition of organic phase ±5% and also by observing the stabil-ity of the drugs for 24 hours at 35ºC temp in mobile phase. [5]

Analysis of Formulation

Twenty tablets each containing 2.5 mg Tibolone were accu-rately weighed and average weight was calculated. A quantity of finepowder equivalent to 10 mg was weighed accurately and transferredin to 100 ml standard flask, dissolved and made up to the volume withmobile phase and filtered through membrane filter. Then 10mg/mlsolutions were injected in to the column and chromatogram was re-corded which is shown in fig 2.

Table 4: Robustness testing of HPLC method

Factors Chromatographic changes Retention time Tailing factor

% of acetonitrile in mobile phase586264Different columnHipersilHi QsilAcetonitrile from different lotsFirst lotSecond lotFlow rate0.951.001.05

~ 6.705~ 6.854~ 6.930

~ 6.869~ 6.834

~ 6.832~ 6.856

~ 6.750~ 6.841~ 6.701

1.3381.3241.390

1.2981.302

1.3041.336

1.2961.3261.330

Table 5: Analysis of formulation by proposed HPLC methodBrand of the tablet Labeled amount (mg) Observed amount (mg) Purity (%)AAA 2.5 2.47 99%

Varsha Jadhav etal.,Validation of high performance liquid chromatographic method for the determination of Tibolone inbulk and pharmaceutical dosage form


696

Journal of Pharmacy Research Vol.2.Issue 4 April 2009

ISSN: 0974-6943

Source of support: Nil, Conflict of interest: None Declared

RESULTS AND DISCUSSION

A rapid and reliable isocratic RP-HPLC method for determi-nation of Tibolone has been developed and validated. This chro-matographic assay fulfilled all the requirements to be identified as areliable and feasible method including accuracy, linearity and precisedata. The developed HPLC technique is precise, specific, accurateand stable. Statistical analysis proves that the method is reproduc-ible, selective and suitable to be applied for analysis of Tibolone incommercial pharmaceutical dosage form for routine quality controlapplication. [6-8]

ACKNOWLEDGEMENTS

The authors are grateful to Cipla Ltd, Mumbai, for providinggift sample of pure Tibolone. The author would like to express theirgratitude to Dr. V.J. Kadam for providing research facilities.

REFERENCES

1. Ming Zuoa,b, Ming-jie Gaoa, Zhen Liua, Lei Caia, Geng-Li Duana,p-Toluenesulfonyl isocynate as a novel derivatization reagent to

Varsha Jadhav etal.,Validation of high performance liquid chromatographic method for the determination of Tibolonein bulk and pharmaceutical dosage form

enhance the electrospray ionization and its application in the de-termination of two stereo isomers of 3-hydroxy-7-methyl-nor-ethynodrel in plasma, Journal of Chromatography B, 814 (2005)331–337.

2. Marcel E. de Gooyer, Hendrika M. Oppers-Tiemissen, DirkLeysen,Herman A.M. Verheul, Helenius J. Kloosterboer, Tiboloneis not converted by human aromatase to 7a-methyl-17a-ethynylestradiol(7a-MEE): Analyses with sensitive bioassays forestrogens and androgens and with LC-MSMS, Steroids 68 (2003)235–243.

3. General Chapter 1225, Validation of compendial methods, UnitedStates Pharmacopeia 30, National Formulary 25, Rockville, Md.,USA, The United States Pharmacopeial Convention, Inc., (2007).

4. Validation of analytical procedure and methodology adopted ICHguidelines, 1996

5. Robert A Nash, Alfred H Wachter, pharmaceutical process valida-tion, 3rd ed, volume 129, pp 507-523.

6. Hokanson, G.C. A life cycle approach to the validation of analyti-cal methods during pharmaceutical product development, part I:the initial validation process Pharm tech pp 118-130

7. Huber L, validation of hplc methods, Biopharm 12:64/66 (march1999).

8. Lloyd R Snyder, Joseph J Kirkland, Joseph L Glajch, practicalhplc method development, 2nd ed, a wiley- intersciences publica-tion.

9. Remington: The Science and Practice of pharmacy, edn.19, p.965.10. James Swarbrich, James C Boylan, Encyclopedia of Pharmaceuti-

cal Technology – Volume 16, Marcel Dekker INC, New York.11. Hokanson G. C., A life cycle approach to the validation of analyti-

cal methods during pharmaceutical product development, Part I:The initial validation process, Pharm. Tech., Sept. 1994, pp. 118–130.

12. Green J. M., A practical guide to analytical method validation,Anal. Chem. News & Features, 1 May 1996, pp. 305A–309A.


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