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PHARM ANALYSIS
Year : 2009  |  Volume : 1  |  Issue : 4  |  Page : 371-374 Table of Contents     

Determination of progesterone in capsules by high-performance liquid chromatography and UV- spectrophotometry


1 Department of Pharmaceutical Analysis, B.R. Nahata Contract Research Centre, Mandsaur, India
2 Department of Pharmaceutics, College of Pharmacy, IPS Academy, Indore, India

Date of Web Publication25-Jan-2010

Correspondence Address:
D Maliwal
Department of Pharmaceutical Analysis, B.R. Nahata Contract Research Centre, Mandsaur
India
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DOI: 10.4103/0975-1483.59330

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   Abstract 

The rapid, simple, and accurate chromatographic (high-performance liquid chromatography) and spectrophotometric method for the determination of progesterone in capsule was elaborated. Methanol was found to be a suitable extraction solvent. The samples were chromatographed on Linchrocart C18 column and UV detection at 254 nm. The elution was achieved isocratically with a mobile phase of methanol - water (80:20, v/v). The method was validated for precision, linearity, accuracy, and limit of detection.

Keywords: Analysis in capsules, progesterone, reversed-phase chromatography, UV-spectrophotometry


How to cite this article:
Maliwal D, Jain P, Jain A, Patidar V. Determination of progesterone in capsules by high-performance liquid chromatography and UV- spectrophotometry. J Young Pharmacists 2009;1:371-4

How to cite this URL:
Maliwal D, Jain P, Jain A, Patidar V. Determination of progesterone in capsules by high-performance liquid chromatography and UV- spectrophotometry. J Young Pharmacists [serial online] 2009 [cited 2014 Apr 24];1:371-4. Available from: http://www.jyoungpharm.in/text.asp?2009/1/4/371/59330


   Introduction Top


Progesterone, pregn-4-ene-3, 20-dione [Figure 1], is a C-21 steroid hormone involved in the female menstrual cycle. The therapeutic dose is 100-200 mg of progesterone. Progesterone is widely used in hormone-therapy and therefore it is necessary to establish a simple and accurate method for its identification and quantitative determination in pharmaceuticals. The literature shows that gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), [1],[2],[3],[4],[5] and high performance liquid chromatography (HPLC) [6],[7],[8] have been reported for the simultaneous determination of progesterone, 17-hydroxyprogesterone and other 3-keto steroids. However, no single method has been developed for progesterone in single component form. In the present study, new, simple and selective HPLC and UV spectrophotometric methods were elaborated for the determination of progesterone in commercial dosage forms.

Experimental details

Reagents

progesterone was purchased from Tanya Biotech (Punjab, India). The marketed formulation used was Prolin 100 capsule, which contain 100 mg of Progesterone. Methanol for chromatography obtained from Merck Ltd, New Delhi, India, was used. Water used was of HPLC grade water from Merck Ltd, New Delhi, India. All the other reagents were of analytical grade.

Instrumentation

Liquid chromatography
: A Merck Hitachi HPLC system consisting of a L-7110 pump, and a L-7400 variable wavelength detector (UVVIS) was used. Manual injections were made using a Rheodyne injectable valve (20 µl loop). The detector wavelength was set at 254 nm. The chromatographic separations were performed at ambient temperature on a Linchrocart C18 ((250 × 4.0 mm), 5ìm). The mobile phase was a mixture of methanol and water (80:20, v/v), filtered, and flowing at the rate of 1 ml/min. The data were collected and analyzed with Winchrom software.

Spectrophotometry: A thermospectronic model of Elico India SL-159 UV/VIS spectrophotometer with 1 cm matched quartz cells was used for the spectrophotometric method.

Solutions: Stock solutions (1.0 mg/ml) of progesterone were prepared by dissolving appropriate amount in methanol: water. These solutions were stable for 48 h at room temperature. The working solutions of 0.01 mg/ml for progesterone were prepared by diluting the stock solutions with the methanol: water.

Method 2 Chromatographic Method

Preparation of the calibration curve of the drug

From stock solutions of progesterone 1 ml was taken and diluted up to 10 ml. From this solution 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 ml volume was transferred to 10 ml volumetric flasks and make up the volume up to 10 ml with mobile phase, gives a standard drug solution of 5, 10, 20, 25, 30 µg/ml concentration. Each of the standard drug solutions was injected three times and the mean peak area of drug was calculated and plotted against the concentration of the drug. The regression equation was found out by using this curve.

Assay in dosage form

Twenty capsules were accurately weighed, and the average capsule mass was calculated. To a fine powder the amount equivalent to 100 mg (after a declaration) of progesterone was diluted with mobile phase in 100 ml volumetric flasks. Further 0.1 ml of this solution was taken and diluted up to 10 ml to obtain a final concentration of 10 µg/ml of progesterone The sample was injected using a 20 ìl fixed loop into the column. All measurements were repeated five times for each concentration. The results are shown in [Table 1].

Method 2 Spectrophotometry

The absorptivity of progesterone in methanol: water was examined in the range 200-400 nm and the ëmax value (position of maximum absorbance of a peak) was recorded. The spectrum exhibits a maximum at 254 nm.

Preparation of the calibration curve of the drug

From stock solutions of progesterone, dilutions were made to give a standard drug solution of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 g/ml concentration. Each of the standard drug solutions was injected three times and the mean absorbance of drug was calculated and plotted against the concentration of the drug. The regression equation was found out by using this curve and the calibration curve was obtained.

Assay in dosage form

Twenty capsules were accurately weighed and the average capsule mass was calculated. To a fine powder and amount equivalent to 100 mg (after a declaration) of progesterone was diluted with mobile phase in 100 ml volumetric flasks. Finally, the diluted sample of 10 µg/ml was taken and absorbance was measured by using the spectrophotometer at 254 nm. The concentration of progesterone was found out by using regression equation.


   Results and Discussion Top


A reversed-phase isocratic procedure was proposed as a suitable method for the analysis of Progesterone in capsules. A mixture of methanol: water (80:20, v: v) at a flow rate of 1 ml/min was found to be an appropriate mobile phase allowing adequate and rapid separation of analyte (retention time 6.39 min). As shown in [Figure 2], the substances were eluted forming well-shaped, symmetrical single peak, well separated from the solvent front. For quantitative applications, a linear calibration curve was obtained over the working concentration range of 5-30 µg/ml. The parameters of the calibration graph were y =0.3992x 2 0.0914, correlation coefficient r, 0.9997. The results indicate a good linear proportionality between the detector response and the concentration of progesterone. Methanol was chosen for the extraction from capsule because it is an excellent solvent for the analyte and is suitable for the reversed phase made of chromatography. The selectivity of the chosen chromatographic system was also ascertained. Excipients showed no interferences with the determination of progesterone and the internal standard. The precision of the elaborated methods is given in [Table 2]. In order to verify the accuracy of the described methods, recovery studies were carried out by analyzing model mixtures of progesterone. The recovery of progesterone was evaluated from 50 to 150% of the labeled tablet amount. The accuracy of the methods is given in [Table 3]. The described HPLC method of determination of progesterone in tablets is precise, sensitive, and accurate. The advantages of the proposed method are its short analysis time and a simple procedure for sample preparation. The results of the determinations of progesterone showed good precision and accuracy of the spectrophotometric method. When Student's t-test at the 95% confidence level was applied to compare the results obtained by the HPLC and the spectrophotometric method, the calculated t values did not exceed the tabulated one.


   Conclusion Top


The rapid, simple, and fairly reliable proposed methods were employed for the determination of progesterone in tablets. The satisfying recoveries and low coefficients of variation confirm the suitability of both proposed methods for the routine analysis of progesterone in tablets.

 
   References Top

1.Wang, Ya-Qi Cai, Bin He, Chun-Gan Yuan, Da-Zhong Shen. Determination of estrogens in water by HPLC-UV using cloud point extraction. China-Jpn-Korea Environ Anal Chem Symp 2006;70:47-51.   Back to cited text no. 1      
2.Ishibashi M, Takayama H, Nakagawa Y, Harima N. Diethylhydrogensilyl-cyclic diethylsilylene derivatives in gas chromatography/mass spectrometry of hydroxylated steroids. V. Analysis of cortisol and 6 beta-hydroxycortisol in human urine. Chem Pharm Bull 1988;36:845-8.  Back to cited text no. 2      
3.Ichimiya H, Egestad B, Nazer H, Baginski ES, Clayton PT, Sjövall J. Bile acids and bile alcohols in a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency: effects of chenodeoxycholic acid treatment. J Lipid Res 1991;32:829-41.  Back to cited text no. 3      
4.Setchell KD, Suchy FJ, Welsh MB, Zimmer-Nechemias L, Heubi J, Balistreri WF. Delta 4-3-oxosteroid 5 beta-reductase deficiency described in identical twins with neonatal hepatitis. A new inborn error in bile acid synthesis. J Clin Invest 1988;82:2148-57.  Back to cited text no. 4      
5.Meng LJ, Sjövall J. Method for combined analysis of profiles of conjugated progesterone metabolites and bile acids in serum and urine of pregnant women. J Chromatogr B Biomed Sci Appl 1997;688:11-26.  Back to cited text no. 5      
6.Kawasaki T, Tsuji A. Fluorescence high-performance liquid chromatography for determination of ketosteroids in biological fluids using dansyl hydrazine: application to clinical analysis 1980;100:925-32.  Back to cited text no. 6      
7.Kawasaki T, Maeda M, Tsuji A. Determination of 17-hydroxycorticosteroids in urine by fluorescence high-performance liquid chromatography using Dns-hydrazine as a pre-column labeling reagent. J Chromatogr 1982;232:1-11.  Back to cited text no. 7  [PUBMED]    
8.Katayama M, Nakane R, Matsuda Y, Kaneko S, Hara I, Sato H. Determination of progesterone and 17-hydroxyprogesterone by high performance liquid chromatography after pre-column derivatization with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza- s-indacene-3-propionohydrazide. Analyst 1998;123:2339-42.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]  


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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