UV SPECTROPHOTOMETRIC ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF CLOMIPHENE CITRATE IN METHANOL

UV SPECTROPHOTOMETRIC ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF CLOMIPHENE CITRATE IN METHANOL

Abhishek Soni ^{* 1}, Amit Chaudhary ¹, Shivali Singla ² and Sachin Goyal ²

Department of Pharmaceutics ¹, School of Pharmacy, Abhilashi University, Chail Chowk, Mandi - 175028, Himachal Pradesh, India.

Himalyan College of Pharmacy ², Kala Amb, Sirmaur - 173030, Himachal Pradesh, India.

ABSTRACT: The present study describes simple, accurate, precise and cost effective UV-spectroscopic method for the estimation of clomiphene citrate in methanol as per ICH guidelines. The absorption maxima for clomiphene citrate in methanol was found to be 235nm and 294 nm. The drug follows linearity (linearity 1, linearity 2 & linearity 3) in the concentration range 3-40 μg/ml at 294 nm and 3-24 μg/ml at 235 nm with a correlation coefficient value of 0.9991, 0.9989, 0.9988 and 0.999, 0.9994, 0.9994 respectively. The accuracy of the method was checked by recovery experiment performed at three levels i.e., 80%, 100%, 120%. The percent recovery was found to be in the range of 90-120%. The low values of % RSD are indicative of accuracy and reproducibility of method. The % RSD <2 indicates that method is precise. The ruggedness of method was studied with the help of different analysts. No significant degradation of clomiphene citrate was showed in force degradation studies like heat-induced degradation the spectra for acid degradation, acid/base hydrolysis, oxidation and photo degradation. The above method was a rapid tool for estimation of clomiphene citrate in formulations.

Clomiphene citrate, Methanol, Method development

INTRODUCTION: Clomiphene citrate is a non steroidal compound. Clomiphene citrate has been used for induction of ovulation since 1962. It is the treatment of first choice in women with ovulatory disorders who are normally oestrogenized, i.e. predominantly those with polycystic ovaries (PCO). Clomiphene citrate has both oestrogenic and anti-oestrogenic properties. Acting as an anti-oestrogen, clomiphene citrate is thought to displace endogenous oestrogen from hypothalamic and pituitary oestrogen receptor sites ¹.

Chemical Name of Clomiphene Citrate: ^2-6

• 2-Cp-(2-chloro-1, 2 diphenylvinyl) phenoxy] triethylamine
• Ethanamine, 2- [4- (2- chloro- 1, 2-diphenyl-ethenyl)-phenoxyl-N, N-diethyl-, 2-hydroxy- 1,2,3-propanetricarboxylate.
• 2, 4, 2-chioro-1, 2-diphenylethenyl)-phenoxy]-N,N-diethylethanamine 2-hydroxy -1,2,3 propanetricarboxyIate.

Molecular Formula: C₂₆ H₂₈ CINO, C₆H₅ O₇

Molecular Weight: 598.1 gm

Dose: 50 mg daily for 5 consecutive days.

From the chemical point of view, clomiphene is a tri-phenylene derivative with structural similarities to diethylboestrol. Clomiphene has two isomeric forms, cis and trans, which in the current nomenclature correspond to E-Clomiphene (enclomiphene) and Z-Clomiphene (zuclomiphene) respectively. The action of E-Clomiphene has oestrogenic, whereas Z-Clomiphene is mainly anti-oestrogenic effects. The commercial preparation is a racemic mixture that contains 60% E-Clomiphene (enclomiphene) and 40% Z-Clomiphene (zuclomiphene).

FIG. 1: STRUCTURE OF E-CLOMIPHENE (I) AND Z-CLOMIPHENE (II)

EXPERIMENTAL:

Materials: Clomiphene citrate standard powder was kindly supplied from Unichem Laboratories Limited, India. All chemical and reagent used were obtained from Research Laboratory, Abhilashi University and were of analytical grade.

Instrument: The instrument used for the study was a UV-Visible Spectrophotometer (Shimadzu, UV-1800, Japan) having two matched quartz cells with 1cm path length.

Preparation of Working Standard Stock Solution Preparation: 10 mg of drug was weighed accurately and 10 ml methanol was transferred into a volumetric flask and sonicated for 5-10 min. This solution was further diluted with methanol, to obtain various dilutions from 3-60 µg/ml.

Preparation of Working Standard: From above standard stock solution 5 ml was further diluted to 50 ml with Methanol followed by sonication for 5 minutes. The final strength was 100 µg/ml. The stock was used to prepare various concentration from 3-40 µg/ml by dilution with methanol.

Selection of Wavelength for Analysis of Clomiphene Citrate: Appropriate volume 1.2 ml of working stock solution of clomiphene citrate was transferred into 10 ml volumetric flask, diluted with methanol up to the mark to give a concentration 12 µg/ml. The resulting solution was scanned between 200-400 nm. Absorbance of these solutions was recorded at 294 nm & 235 nm against methanol as blank using UV-visible spectrophotometer (shown in Fig. 2).

FIG. 2: ABSORPTION MAXIMA OF CLOMIPHENE CITRATE

Validation of UV Spectroscopy Method: ^7-13

Standard Calibration Curve (Linearity): A calibration curve was plotted over a concentration range of 3-40 μg/ml for clomiphene citrate at 294 nm, 3-24 μg/ml for clomiphene citrate at 235 nm. Accurately measured volume of working stock solution of clomiphene citrate at 294 nm and 235 nm were transferred to separate series of 10 ml volumetric flask and diluted up to the mark with methanol to obtain a concentration series of 3-40 μg/ml. The absorbance of all solution was taken at their respective wavelength. The calibration curve was constructed by plotting concentration against absorbance where each reading was an average of three determinations. [Shown in Table 1, 2 and 3 and Fig. 3a-3c, Fig. 4a-4c, Fig. 5a-5b and Fig. 6a-6b].

TABLE 1: STANDARD CURVE DATA OF CLOMIPHENE CITRATE AT 294nm

Linearity of Clomiphene Citrate at 235nm

TABLE 2: STANDARD CURVE DATA OF CLOMIPHENE CITRATE AT 235nm

TABLE 3: MEAN LINEARITY OF CLOMIPHENE CITRATE

Precision: The term precision is defined by the ISO International Vocabulary of Basic and General Terms in Metrology (ISO-VIM) and ICH as the closeness of agreement between quantity values obtained by replicate measurements of a quantity under specified conditions. Assessing the precision implies expressing numerically the random error or the degree of dispersion of a set of individual measurements by means of the standard deviation, the variance, or the coefficient of variation.

Repeatability: It is in the concordance of a series of measurements of the same quantity when the experiments are conducted under same conditions (analyst, apparatus, instrument, and day) in a rapid succession. For this experiment, standard solution of clomiphene citrate at 294 and 235nm (15 µg/ml) was prepared and analyzed six times as per the proposed method.

Intermediate Precision: It is the concordance of a series of measurements of the same quantity when the experiments are conducted within the same laboratory under different conditions (analyst, apparatus, instrument, and day). Standard solution of clomiphene citrate at 294 and 235nm (15 µg/ml) was prepared and analyzed as per the proposed method [shown in Table 4].

TABLE 4: REPEATABILITY AND INTERMEDIATE PRECISION STUDY

Accuracy: Accuracy was determined by means of recovery experiments, by the determination of % mean recovery of sample at three different levels (80-120%). At each level, three determinations were performed. Percent mean recovery was calculated as shown in Table 5 at 294 nm & 235 nm. The accepted limits of recovery are 90% - 120% and all observed data are within the required range which indicates good recovery values and hence the accuracy of the method developed [shown in Table 5].

TABLE 5: ACCURACY STUDY OF CLOMIPHENE CITRATE AT 294nm

Ruggedness: Ruggedness was determined by carrying out analysis by two different analysts and the respective percentage recovery was noted and the results were indicated as % RSD [shown in Table 6].

TABLE 6: RESULTS OF RUGGEDNESS OF CLOMIPHENE CITRATE

Limit of Detection (LOD) and Limit of Quantitation (LOQ): The detection limit of an individual analytical procedure is the lowest amount of analyte in the sample which can be detected but not necessarily quantitated as an exact value. The Quantitation limit of an individual analytical procedure is the lowest amount of analyte in the sample which can be quantitatively determined with suitable precision and accuracy (shown in Table 7). The LOD and LOQ of the proposed method were determined by using calibration curve:

LOD = 3.3σ / s

LOQ = 10σ / s

Where σ is the standard deviation of the response (Y intercept) and S is the slope of the calibration curve [shown in Table 7].

TABLE 7: LOD AND LOQ

Robustness: Robustness is the ability to provide accurate and precise results under a variety of conditions. In order to measure the extent of method robustness, the most critical parameters were interchanged while keeping the other parameters unchanged and in parallel, the chromatographic profile was observed and recorded. The studied parameter was change in wavelength. The results for robustness study in Table 8 indicated that the small change in the conditions did not significantly affect the determination of Clomiphene citrate [shown in Table 8].

TABLE 8: ROBUSTNESS (CHANGE IN WAVELENGTH) FOR 15 µg/ml AT 294nm & 235nm

Forced Degradation Studies at 294nm & 235nm:

Effect of Acid and Base Hydrolysis: Sample solution containing 1 ml aliquot of API was transferred into a 10 ml of amber volumetric flask, then mixed with 1 ml of 0.1M HCl as well as 0.1M NaOH separately and left to stand for 1 h, 2 h, 4 h at 60 °C ± 2 °C after heating on bath samples were neutralized (NaOH sample with 1 ml 0.1M HCl and HCl sample with 1 ml 0.1M NaOH) and diluted up to 10 ml with mobile phase. All three samples were injected in triplicate after the neutralizing procedure and spectra were run as described previously [shown in Table 9 & Fig. 7a & b].

Effect of Oxidation: Sample solution containing 1 ml aliquot of API was transferred into a 10 ml amber volumetric flask, then mixed with 1 ml of 1% (v/v) hydrogen peroxide and left to stand for 1 hr, 2 h and 4 h at 60 °C ± 2 °C after heating on bath samples were diluted up to 10 ml with mobile phase. All three solutions were injected in triplicate and spectra were run as described previously [shown in Table 9 & Fig. 7c].

Effect of Photo Degradation: Photolytic degradation was studied by placing API solution in a clear volumetric flask and exposing it to direct UV light for 1 h, 2 h and 4 h.

The resultant solution was injected in triplicate and the spectra were run as described previously [shown in Table 9 & Fig. 7d].

Heat-Induced Degradation: One milliliter aliquot of a sample solution containing API was transferred to a 10 ml amber volumetric flask and then heated for 1 h, 2 h and 4 h at 60 °C ± 2 °C. The resultant each of solution were diluted in mobile phase up to 10 ml and injected in triplicate and the spectra was run as described previously [shown in Table 9 & Fig. 7e].

TABLE 9: FORCE DEGRADATION STUDY AT 294 nm & 235 nm

RESULTS AND DISCUSSION: The method was validated according to ICH guidelines with respect to Linearity, accuracy, precision, ruggedness, limit of detection, limit of quantitation, robustness and force degradation studies. The calibration curve for the methods were linear over concentration range 3-60 μg/ml for clomiphene citrate at 294nm, 3-24 μg/ml for clomiphene citrate at 235nm. The determination of coefficients (r²) was 0.9991, 0.9989, 0.9988 & 0.999, 0.9994, 0.9994 respectively. The methods were found to be precision and as the % RSD value for repeatability and Intermediate day were found to be less than ±2%. The accepted limits of accuracy (recovery) were found to be 90% - 120% and all observed data are within required range which indicates good recovery value.

The result of, robustness indicted the small change in the condition did not significantly affect the determination of CC. The LOD and LOQ were found to be 0.229µg/ml, 0.694 µg/ml and 0.126 µg/ml, 0.382 µg/ml respectively. In forced degradation studies as acid/base hydrolysis, oxidation, photo degradation, heat-induced degradation the spectra for acid degradation of API do not showed any significant degradation or no additional peak of sample after 1 h, 2 h and 4 h at different process.

CONCLUSION: A simple, accurate, precise and cost effective UV-spectroscopic method has been developed for the estimation of clomiphene citrate. The proposed method is successfully applied for estimation of drug in any formulation. The method can be used for the routine quality control analysis of clomiphene citrate.

ACKNOWLEDGEMENT: I am thankful to my research supervisor Dr. Sachin Goyal, and research co-supervisor Dr. Amit Chaudhary. I wish to express my deep-rooted gratefulness towards, Dr. Shivali Singla for her support, edifying, enlightening discussions and timely suggestions during my research work.

It is truly said, “Teacher occupies the place of God, whose indebtedness is impossible to return”.

CONFLICTS OF INTEREST: The authors declare that there is no conflict of interests regarding the publication.

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    How to cite this article:

    Soni A, Chaudhary A, Singla D and Goyal S: UV spectrophotometric analytical method development and validation of clomiphene citrate in methanol. Int J Pharm Sci & Res 2020; 11(5): 2229-37. doi: 10.13040/IJPSR.0975-8232.11(5).2229-37.

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