Anda di halaman 1dari 4

ISSN: 0974-6943

Varsha Jadhav et al. / Journal of Pharmacy Research 2009, 2(4),694-697 Research Article
Available online through
www.jpronline.info
Validation of high performance liquid chromatographic method for the
determination 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. Jadhav
E-mail:drvmjadhav_bvcop@rediffmail.com
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 both
as bulk drug and formulations. A new sensitive and reproducible HPLC method was developed and validated for the determination of tibolone
in tablets. The separation was achieved by using a JASCO HPLC system 2000 series comprising of JASCO PU 2080 plus pump, JASCO UV
2075 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 phase
consisting of Methanol: Acetonitrile: Water (10:60:30). Flow rate was 1.0 ml with an average operating pressure of 200 kg /cm² and retention
time was found to be 6.8 ± 0.02min.The method was validated for accuracy, precision, linearity, reproducibility and robustness and statistical
comparison was performed by analysis of variance. The developed HPLC technique is precise, specific, accurate and stable. Statistical
analysis proves that the method is reproducible, selective and suitable to be applied for analysis of tibolone in commercial pharmaceutical
dosage form for routine quality control application.

Key words: Tibolone, RP-HPLC, Dosage form

INTRODUCTION

Tibolone [7α, 17α-7-methyl-17-hydroxyl-19-norpregn-5(10)- Injection volume : 20µL


en-20-yn-3-one], also called 7-methyl-norethynodrelor Org OD14, is Mobile phase : Methanol: Acetonitrile: Water (10:60:30)
a synthetic steroid used in the hormonal replacement therapy (HRT) Flow rate : 1 ml/min
for postmenopausal women. It has been used in Europe for almost 2 Detection : 205 nm
decades, primarily for the prevention of postmenopausalosteoporo- Column temperature : Ambient
sis and treatment of climacteric symptoms and provides addition Diluent : Mobile phase
clinical benefits with regard to breast tissue and sexual well-being. Retention time : 6.8 min
Literature survey revealed that various methods have been reported
for the estimation of Tibolone in biological matrices such as plasma Standard Stock Solution:
by HPLC, LC – MS. No method has been reported for estimation of
Tibolone in tablet dosage forms. The aim of this work was to develop About 100 mg of Tibolone was weighed and taken in a 100
a validated HPLC method for determination of Tibolone in bulk and ml volumetric flask, dissolved and diluted to make the diluent (1000
pharmaceutical dosage form. [1-2] mg/ml)

MATERIALS AND METHODS Preparation of Mobile Phase:

Instrument To optimize the HPLC parameters several mobile phase com-


positions were tried. Satisfactory peak symmetry was obtained with
The instrument used was a JASCO HPLC system 2000 se- mobile phase consisting of Methanol, Acetonitrile and Water in pro-
ries comprising of JASCO PU 2080 plus pump, JASCO UV 2075 plus portion of 10:60:30. The mobile phase was filtered through 0.45 cellu-
detector, Rheodyne manual injector fitted with 100 ìl sample loop. lose nitrate filter paper and degassed by ultrasonication for 20 min.
Data acquisition and treatment done using Borwin chromatography
software version 1.5. The analytical column used was hyqsil C-8 [250 Calibration Curve:
mm x 4.6 mm, 5 mm]
Appropriate aliquots of standard stock solution (1000 mg/
Chromatographic Condition ml) was diluted to 100 mg/ml in 10ml volumetric flask and resultant
solution was diluted up to the mark with mobile phase to obtain final
Column used : Hi Qsil C-8 [250 mm x 4.6 concentration of 1, 2, 4, 6, 8, 10 and 11 mg/ml. These solutions were
mm, 5 mm]
Journal of Pharmacy Research Vol.2.Issue 4.April 2009 694
ISSN: 0974-6943
Varsha Jadhav et al. / Journal of Pharmacy Research 2009, 2(4),694-697
Varsha Jadhav etal.,Validation of high performance liquid chromatographic method for the determination of Tibolone in
bulk and pharmaceutical dosage form

OH CH
CH3

H H

O CH3

Fig 1: Tibolone

Fig 2: Tibolone (formulation chromatogram)

Calibration Curve of Tibolone


y = 89577x
1500000 R2 = 0.9997
area of peak

1000000 A rea

500000 Linear (Area)

0
0 5 10 15
Tibolone conc.m c g / m l
Fig 2: Calibration curve of Tibolone
Table 1: Data for calibration curve Table 2: Linear regression data for calibration curve
Sr. Concentration Peak Parameters HPLC method
No. of Tibolone area
(mg/ml) Range 1-11 mg/ml
1. 1 97458.87 Correlation coefficient 0.9997
2. 2 187406.75 Slope 89577x
3. 4 358744.45
4. 6 538649.89
5. 8 709397.48
6. 10 899201.45
7. 11 984456.5
Table 3: Precision of proposed HPLC method
Concentration Concentration Of Tibolone (mg/ml) found on
of Tibolone (mg/ml) Inter-day Intra-day
Mean CV Mean CV

30 30.009 0.0315 29.99 0.2082


50 50.006 0.0450 49.96 0.1096

Journal of Pharmacy Research Vol.2.Issue 4 April 2009 695


Varsha Jadhav et al. / Journal of Pharmacy Research 2009, 2(4),694-697 ISSN: 0974-6943
Varsha Jadhav etal.,Validation of high performance liquid chromatographic method for the determination of Tibolone in
bulk and pharmaceutical dosage form
Table 4: Robustness testing of HPLC method

Factors Chromatographic changes Retention time Tailing factor


% of acetonitrile in mobile phase 1.338
~ 6.705
58 1.324
~ 6.854
62 1.390
~ 6.930
64
Different column ~ 6.869 1.298
Hipersil ~ 6.834 1.302
Hi Qsil
Acetonitrile from different lots
First lot ~ 6.832 1.304
Second lot ~ 6.856 1.336
Flow rate
0.95 ~ 6.750 1.296
1.00 ~ 6.841 1.326
1.05 ~ 6.701 1.330

Table 5: Analysis of formulation by proposed HPLC method


Brand of the tablet Labeled amount (mg) Observed amount (mg) Purity (%)
AAA 2.5 2.47 99%

injected into chromatographic system and chromatograms were ob-


tained and peak area ratio was determined for each concentration of Specificity:
drug solution. Calibration curve of Tibolone were constructed by
plotting peak area ratio vs applied concentration of Tibolone and Complete separation of Tibolone in mobile phase was no-
regression equation was computed. Similarly the sample solution was ticed. The average retention time was found to be 6.8 respectively for
chromatographed and concentration of Tibolone in tablet samples six replicates. The peaks obtained were sharp and have clear baseline
was found out using regression equation. [11-12]
separation[5]
.

Method Validation: Linearity:

As per the USP XXIII, system suitability tests for HPLC Linearity of the method was investigated by serially dilut-
were carried out on freshly prepared standard stock solution of ing the stock solution to give a concentration range of 1 to 11 mg/ml
Tibolone and the parameters studied and results obtained with 20 ul
injection volumes. The method was validated for accuracy, precision, and injected 20 ul with universal injector (Rheodyne). The flow rate
specificity, detection limit, quantitation limit and robustness by fol- was maintained at 1.0 ml/min. Temperature of the column was kept
lowing procedures.[3-5, 9, 10] ambient and the effluent was monitored at 205 nm. Calibration curve
was constructed by plotting concentration against peak area as shown
Accuracy: in fig 3. [3-5]

The accuracy of the method was determined by calculating Robustness:


recovery of Tibolone by method of standard addition. Known amount
of Tibolone (1, 2, 4 mg/ml) was added to a pre quantified sample Robustness of the method was studied by changing the
solution and the amount of Tibolone was estimated by measuring the composition of organic phase ±5% and also by observing the stabil-
peak area ratios and by fitting these values to the straight line equa- ity of the drugs for 24 hours at 35ºC temp in mobile phase. [5]
tion of calibration curve.[3]
Analysis of Formulation
Precision:
Twenty tablets each containing 2.5 mg Tibolone were accu-
The intra day and inter day precision study of Tibolone was rately weighed and average weight was calculated. A quantity of fine
carried out by estimating the corresponding responses 3 times on the powder equivalent to 10 mg was weighed accurately and transferred
same day and on 3 different days (first, third and fifth day) for three in to 100 ml standard flask, dissolved and made up to the volume with
different concentrations of Tibolone (30 & 50 mg/ml) and the result mobile phase and filtered through membrane filter. Then 10mg/ml
are reported in terms of relative standard deviation. The analytical solutions were injected in to the column and chromatogram was re-
data is given in table 5. [3-5] corded which is shown in fig 2.

Journal of Pharmacy Research Vol.2.Issue 4.April 2009 696


ISSN: 0974-6943
Varsha Jadhav et al. / Journal of Pharmacy Research 2009, 2(4),694-697
Varsha Jadhav etal.,Validation of high performance liquid chromatographic method for the determination of Tibolone
in 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-
RESULTS AND DISCUSSION ethynodrel in plasma, Journal of Chromatography B, 814 (2005)
331–337.
A rapid and reliable isocratic RP-HPLC method for determi- 2. Marcel E. de Gooyer, Hendrika M. Oppers-Tiemissen, Dirk
Leysen,Herman A.M. Verheul, Helenius J. Kloosterboer, Tibolone
nation of Tibolone has been developed and validated. This chro- is not converted by human aromatase to 7a-methyl-17a-
matographic assay fulfilled all the requirements to be identified as a ethynylestradiol(7a-MEE): Analyses with sensitive bioassays for
reliable and feasible method including accuracy, linearity and precise estrogens and androgens and with LC-MSMS, Steroids 68 (2003)
235–243.
data. The developed HPLC technique is precise, specific, accurate
3. General Chapter 1225, Validation of compendial methods, United
and stable. Statistical analysis proves that the method is reproduc- States Pharmacopeia 30, National Formulary 25, Rockville, Md.,
ible, selective and suitable to be applied for analysis of Tibolone in USA, The United States Pharmacopeial Convention, Inc., (2007).
commercial pharmaceutical dosage form for routine quality control 4. Validation of analytical procedure and methodology adopted ICH
guidelines, 1996
application. [6-8] 5. Robert A Nash, Alfred H Wachter, pharmaceutical process valida-
tion, 3rd ed, volume 129, pp 507-523.
ACKNOWLEDGEMENTS 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
The authors are grateful to Cipla Ltd, Mumbai, for providing 7. Huber L, validation of hplc methods, Biopharm 12:64/66 (march
gift sample of pure Tibolone. The author would like to express their 1999).
8. Lloyd R Snyder, Joseph J Kirkland, Joseph L Glajch, practical
gratitude to Dr. V.J. Kadam for providing research facilities. hplc 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-
REFERENCES cal methods during pharmaceutical product development, Part I:
The initial validation process, Pharm. Tech., Sept. 1994, pp. 118–
1. Ming Zuoa,b, Ming-jie Gaoa, Zhen Liua, Lei Caia, Geng-Li Duana, 130.
p-Toluenesulfonyl isocynate as a novel derivatization reagent to 12. Green J. M., A practical guide to analytical method validation,
Anal. Chem. News & Features, 1 May 1996, pp. 305A–309A.

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

Journal of Pharmacy Research Vol.2.Issue 4 April 2009 697