INTRODUCTION:

Norethisterone is 17α -hydroxy-19-nor-17α -4-pregnen-20- yn-3-one¹ (Fig.1). The 19-nor-progestogen norethisterone is used as a progestogen component in contraceptives and in continuous- and sequentialcombined Hormone Replacement Therapy (HRT) in postmenopausal women.Metabolism of norethisterone in HRT target tissues may play arole in its biological response. However,both progestogenic and antiprogestogenic effects of norethisteronehave been described on uteroglobin and progesterone receptor expressionin the uterus of progesterone-treated prepubertal rabbits². Moreover, estrogenic activity of norethisteronein rat uterus is mediated through 5-reduced metabolites of norethisterone³. Conversion of norethisteronein e.g., uterus into more estrogenic metabolites may affect theestrogen/ progestogen balance that is the key feature of a combinedHRT. A literature survey revealed that several analytical methods were reported for the determination of norethsterone in biological fluids including liquid chromatography tandem mass spectrometry (LC–MS–MS)⁴, Voltammetry and quantification of the contraceptive drug norethisterone in bulk form and pharmaceutical formulation⁵,

Establishment of an HPLC identification system for detection of counterfeit steroidal drugs⁶, Highly Sensitive Analysis of Norethisterone in Human Serum by LC Coupled with Atmospheric Pressure Photoionization Tandem Mass Spectrometry⁷. The aim of the present work is to develop and validate an accurate, specific, economical and reproducible UV Spectrophotometric method for determination for norethisterone as bulk drug. Drug was found to be freely soluble in methanol hence this solvent was chosen for proceeding study.

MATERIALS AND METHOD:

All the absorbance measurements were made on Systronics – 2201 UV/Visible double beam spectrophotometer with 10mm matched cells. Whatman filter no. 41 was used to filter solutions. The norethisterone standard was provided by Cadila, Ahmedabad. All chemicals were of analytical grade. Methanol was purchased from Merck Ltd, Mumbai. Commercially available two formulations [primolut-N (Zota), Noritis (Cadila)] were procured from local market.

Optimization of Parameters:

It was fond that norethisterone has λmax at 270 nm by scanning of diluted solution. The selection criteria was based on the minimum strength, which gives the maximum absorbance.

Preparation of Standard Solution:

Stock standards of norethisterone was prepared by dissolving 50 mg of the compound in 100 ml of methanol to give stock concentrations of 500 µg ml. 50 mg of norethisterone accurately weighed was dissolved in about 50 ml of methanol and diluted with 100ml of the same solvent. 1ml of this solution was again diluted to 100 ml.

Fig. 1. Chemical Structure Norethisterone

Fig.2. Absorption Maximum of Norethisterone

Table 1: Summary of Optical, Regression and Validation Parameters.

Linearity range (µg/ml)	0.5-3 μg/ml
λmax	270 nm
Beer’s law limit (µg/ml)	0.5-10 μg/ml
Regression equation	Y = 0.332 × -0.002
Slope	0.332
Intercept	0.002
Corelation coefficient	0.999
Accuracy (%recovery)	100-100.09
LOD (µg/ml)	0.01987
LOQ (µg/ml)	0.06024

Preparation of calibration curve:

Suitable aliquots of the standard solution of norethisterone (1.0-6.0ml) were taken into 10 ml volumetric flasks, and volume was then made up to the mark with methanol to prepare a series of standard solutions containing 0.5-3.0 µg/ml. Absorbance of the complex was measured at 270nm against blank. A calibration curve was constructed by plotting absorbance of analyte versus nominal concentration using the weighted least (1/x) regression mode.

Estimation of norethisterone in tablets:

Twenty tablets were taken and their weight was calculated. The average weight of the tablets were found out, The quantity of powder equivalent to 50 mg of northisterone were transferred to 100ml volumetric flask and mixed with 50 ml of methanol and the solution was sonicated for 10 minutes. Then the volume was made up to 100 ml with the same solvent. The solution was filtered through Whatman filter paper no. 41. From the filtrate, 1 ml was transferred to 100 ml volumetric flask and the volume was made up to 100 ml with methanol, 10 ml was transferred to 50 ml volumetric flask and the volume was made up to 50 ml with methanol. The reference standard of norethisterone was also prepared in the same way at each concentration level and absorbance was noted at 270 nm against blank.

Fig.3. Calibration Curve for Norethisterone

Table: 2. Precision of Proposed Method

Concentration (μg/ml)	Concentration found on
	Intra-day		Inter-day
	S.D	%RSD	S.D	%RSD
1 2 3	0.001260 0.001291 0.001722	0.6666 0.1945 0.1722	0.002449 0.002179 0.002291	0.7558 0.3286 0.2308

Table 3: Analysis of Norethisterone in Tablet Formulations

S. No

Brand

Label claim

(mg/tablet)

Amount found

(mg/tablet)

% Recovery

% RSD

Primolut-N

Noritis

4.907

4.965

98.15

99.3

0.0342

0.0279

METHOD VALIDATION:

Accuracy of the method was determined by the recovery studies in the tablets formulation of the norethisterone. Recovery studies were carried out by addition of known quantities of standard drug solution to pre-analyzed sample at three different concentrations. Also the experiment was repeated three times in a day to determine intra-day precision and on three different days to determine inter-day precision. The percentage relative standard deviation (%RSD) was calculated at each concentration level. The values of method validation are given in Table 2. The proposed method obeys beer’s law in the concentration range of 0.5-3µg/ml. In this method, the correlation coefficient (r²) was found to be 0.999, the slope was 0.332 and intercept 0.002. Limit of detection (LOD) and limit of quantitation (LOQ) were calculated by repeating the blank measurements six times at 270nm. LOD and LOQ for norethisterone were found to be 0.01987 (µg/ml) and 0.06024 (µg/ml) respectively. It can be concluded that the developed method is sensitive.

RESULTS AND DISCUSSION:

The proposed method is simple and precise and do not suffer from any interference due to common excipients of tablets. Beer’s law is obeyed in concentration range of 0.5-3.0 µg/ml. Method were validated in terms of accuracy and precision. The accuracy of the methods was proved by performing recovery studies in the commercially available formulations. Values greater than 99% indicate that proposed method is accurate for the analysis of drug. The precision of the method was checked in terms of Inter-day and Intra-day, where methods were repeated on three different day and also repeated on three different time periods in same day. The results given in Table 2 shows % RSD of less than 1% at each level clearly indicate that the method is precise enough for the analysis of the drug. Summary of Optical, Regression and Validation Parameters are shown in Table 1. Commercial formulation of norethisterone tablets was successfully analyzed by the proposed and reference methods. The values obtained by the proposed and reference methods are presented in Table-3.

REFERENCES:

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Received on 27.10.2009 Modified on 29.11.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 172-174