1. INTRODUCTION:

Clomiphene Citrate, chemically known as 2-[4-(2-chloro-1, 2-diphenylethenyl) phenoxy]-N, N-diethylethanamine, 2-hydroxypropane-1,2,3-tricarboxylate(Figure 1), is a white to pale yellow powder, slightly soluble in water¹.

Figure 1 Structure of Clomiphene Citrate

It is used for the induction of ovulation in anovulatory women desiring pregnancy, particularly in cases of ovarian dysfunction.

It releases the gonadotrophins by direct action on the hypothalamic/pituitary axis and by reducing the inhibitory influence of endogenous estrogens². Clomiphene Citrate has both estrogenic and anti-estrogenic properties, but its precise mechanism of action has not been determined. Clomiphene Citrate appears to stimulate the release of gonadotropins, follicle-stimulating hormone (FSH), and leuteinizing hormone (LH), which leads to the development and maturation of ovarian follicle, ovulation and subsequent development and function of corpus luteum, thus resulting in pregnancy³. The drug is official in the United State Pharmacopeia and British Pharmacopeia. Literature survey reveals that Clomiphene Citrate was determined by spectrophotometric^4-8, array-type DNA glass slide⁹, HPLC^10-13, capillary electrophoresis¹⁴and NMR methods¹⁵. The isomers of clomiphene Citrate were also separated by HPLC¹⁶. The estimation of drug was also carried out in blood plasma¹⁷. It was observed that there is a paucity of reports on the estimation of Clomiphene Citrate in bulk and formulations using simple chromatographic system. Therefore, in the present investigation an attempt has been made to develop a simple, accurate, precise and sensitive RP-HPLC method for estimation of Clomiphene Citrate in bulk and their pharmaceutical dosage forms.

2. MATERIALS AND METHODS:

2.1 Chemicals:

The reagents used in this work were methanol (HPLC grade) and acetonitrile (HPLC grade), which were procured from Merck, India. The reference standard Clomiphene Citrate (Purity 99.9%) was received as a gift sample from Cipla Pvt. Ltd, Mumbai, India. The Fertomid tablet containing Clomiphene Citrate (50mg) was purchased from local pharmacy and the membrane filters were procured from Millipore, India. All other chemicals and reagents used were of HPLC grade and purchased from Merck, India.

2.2 Equipment:

The instruments used in the study were electronic balance (Contech CA Series), sonicator (Citizen CD-4820), UV-VIS Spectrophotometer (UV-1800 Spectral band width 2cm equipped with quartz cell at medium scanned speed). HPLC (Shimadzu, Kyoto, Japan) was monitored and integrated using Lab solution software. Additionally, syringe [Hamilton (Rheodyne-20μL)] and syringe filter [Himedia Syringe-driven filters (0.22μm)] were used.

2.3 Chromatographic conditions:

HPLC system was composed of an LC-20AD Prominence solvent delivery module, a manual rheodyne injector with a 20μL fixed loop, an SPD-M20A prominence PDA detector and DGU-20A5R degassing unit. Estimation was performed on a Shimadzu C₁₈ column ((250mm x 4.6mm i.d., 5µm) at ambient temperature. The mobile phase consisted of 900mL of HPLC grade methanol and 100mL of acetonitrile of HPLC grade. The mobile phase was sonicated for 10 min and filtered through 0.45μm membrane filter. The mobile phase flow rate was maintained at 1mL/min and eluents were monitored at 295nm. The samples were injected using a 20μL fixed loop. All determinations were performed at ambient temperature for a run time 20 min. The optimized chromatographic conditions are shown in Table.1.

Table 1 Optimized chromatographic conditions of Clomiphene Citrate

Parameters	Conditions
Stationary phase (column)	Shimadzu C₁₈ (250mmx4.5mm, 5µm)
Mobile phase ratio	Methanol: Acetonitrile (90:10v/v)
Detection Wavelength	295nm
Flow Rate	1mL/min
Injection Volume	20 µL
Column temperature	Ambient temperature
Run Time (min)	20
Retention time (min)	3.44

2.4 Method development:

2.4.1. Selection and preparation of mobile phase:

Various mobile phases containing methanol, acetonitrile, water and phosphate buffer (pH 3) in different ratios were tried with different flow rates. Good symmetrical peak was found with the mobile phase comprising methanol and acetonitrile in the ratio 90:10(v/v). Mobile phase was prepared by mixing 900mL of HPLC grade methanol with 100mL of acetonitrile of HPLC grade. The mobile phase was sonicated for 10 min and filtered through the 0.45µm membrane filter.

2.4.2. Preparation of standard stock solutions:

The standard stock solutions of 100μg/mL of the drug were prepared by dissolving 50mg of pure drug in the mobile phase in a 50mL volumetric flask and the volume was made up to the mark. Resulting solutions were further diluted with mobile phase to obtain a final concentration of 100μg/mL and stored under refrigeration.

2.4.3. Preparation of calibration curve:

Aliquots of standard stock solutions were transfered in a 10mL volumetric flask and diluted up to the mark with methanol in such a way that the final concentrations of the drug were in the range of 10-50µg/mL⁵. Triplicate injections of 20µL were made and analysed by HPLC under the conditions as described above. Calibration curve was constructed by plotting the peak area on the y-axis against respective concentration of the drug on the x-axis.

2.4.4 System suitability tests:

The system suitability tests were performed by collecting data from five replicate injection of standard drug solution. The resolution, number of theoretical plates and peak asymmetry were calculated for the standard solutions^18-21.

2.4.4 Analysis of Pharmaceutical Formulation:

Tablet powder (297mg) equivalent to 50mg of Clomiphene Citrate was accurately weighed and transferred into 50ml volumetric flask and dissolved in sufficient volume of mobile phase and then volume was made up to the mark to get sample solution (1000 μg/mL). This solution was further diluted with mobile phase to a concentration of 30μg/mL. A volume of 20μL of the solution was injected into the HPLC system.

2.5. Method validation:

The developed method was validated by evaluating specificity, accuracy, precision, linearity, detection limit, quantification limit, robustness and ruggedness. Coefficient of variation and relative errors of less than 2% were considered acceptable except detection limit and quantification limit²².

2.5.1. Specificity^23,24:

The specificity of the method was determined by recording the chromatogram of mobile phase, standard solution and sample solution having 30µg/mL concentrations to determine the interference of excipient in the sample.

2.5.2. Accuracy^25,26:

The solutions for accuracy study were prepared by spiking pure drug into the tablet powder by following method. Accurately weighed quantity of tablet powder equivalent to 25mg of Clomiphene Citrate and 25mg of standard Clomiphene Citrate was taken into 50ml volumetric flask, mobile phase was added to it. Content in flask was sonicated for 10 min and the volume was adjusted to 50ml with mobile phase. From this 1mL portion was diluted upto 10mL with mobile phase to get a concentration of 100µg/mL. From above solution 3mL portion was diluted up to 10mL with mobile phase to get 30µg/mL. The area of final solutions were measured at 295nm. This procedure was repeated for six times. The accuracy of the method was determined in terms of recovery of Clomiphene Citrate.

2.5.3. Precision²⁷:

The precision of the method was ascertained from the peak area obtained by actual determination of six replicated of fixed amount of the drug (30µg/mL). The precision of the assay was also determined in terms of intra-day and inter-day variation in the peak areas of the set of drug solutions on three different days. The intra-day and inter-day variation in the peak area of the drug solution was calculated in terms of relative standard deviation (RSD).

2.5.4. Linearity:

A stock solution of Clomiphene Citrate of 100µg/ml was prepared with methanol. From it, various working standard solutions were prepared in the range of 10-50µg/mL and injected into HPLC. It was shown that the selected drug had linearity in the range of 10-50µg/mL. The calibration plot was generated by replicate analysis (n=3) at all concentration levels and the linear relationship was evaluated.

2.5.5. Detection limit and quantification limit²⁸:

The limit of detection (LOD) and limit of quantification (LOQ) were established based on the calibration curve parameters, according to the following formulas:

LOD=3.3SD/slope

LOQ=10SD/slope

2.5.6. Robustness:

Robustness of the proposed method for Clomiphene Citrate was carried out by the slight variation in flow rate. The percentage recovery and RSD were noted for Clomiphene Citrate.

2.5.7. Ruggedness:

The test solutions were prepared 30µg/ml as per test method and injected under variable conditions. Ruggedness of the method was studied by different analyst. The degree of reproducibility of the test results were determined as a function of the assay variables.

3. RESULTS:

3.1 Method development:

3.1.1 Chromatographic determination:

A number of HPLC chromatographic systems were investigated to optimize the determination of Clomiphene Citrate. Retention time for Clomiphene Citrate function of stationary phase (Shimadzu C₁₈ reversed-phase column), the mobile phase and other optimized chromatographic conditions are shown in Table 1. The results of system suitability tests are shown in Table 2. In system suitability R.S.D of area for standard Clomiphene citrate was found to be 0.060%. Retention time, number of theoretical plate and tailing factor were found to be 3.454min, 2501, 1.008 respectively. The values obtained demonstrated the suitability of the system for the analysis of these drugs.

3.1.2. Calibration curve and analysis of pharmaceutical formulation:

The coefficient of determination (R²), slope and intercept for Clomiphene Citrate were 0.999, 20321 and 60021, respectively. The retention time for Clomiphene Citrate was 3.45min. Calibration curve and representative chromatogram of Clomiphene Citrate at 295nm are shown in Figure 2 and 3, respectively. Percentage assay of Clomiphene citrate in marketed tablets was found to 99.5 % w/w. Therefore it passes the assay limit.

Table 2 System Suitability Parameters

Sr. No.	Conc. (µg/mL)	t_R (min)	Area	No. of Theoretical Plates	Tailing Factor
1	30	3.442	668259	2500	1.0
2	30	3.432	667248	2503	0.998
3	30	3.410	668149	2502	1.01
4	30	3.44	668120	2501	1.03
5	30	3.45	667998	2502	1.003
	Mean	3.434	667954	2501	1.008
	(±)S.D	0.015	405.88	1.14	0.013
	%R.S.D	0.44	0.060	0.045	1.29

3.2 Method validation:

3.2.1 Accuracy, precision and linearity:

The accuracy of the method was determined and indicated by the % recovery. The results are shown in Table 2. Intra-day and inter-day precision data of RP-HPLC method for Clomiphene Citrate are shown in Table 3 and 4, respectively. In linearity the coefficient of determination (R²) for Clomiphene Citrate was 0.999 as shown in Figure 2 and Table 5.

Table 3 Accuracy study of Clomiphene Citrate

Sr. No.	Sample Weight + Pure drug added (mg)	Conc. (µg/mL)	Area at 295nm		% Recovery
Sr. No.	Sample Weight + Pure drug added (mg)	Conc. (µg/mL)	Standard	Sample	% Recovery
1	148.52 + 25	30	668259	668560	99.95
2	148.50 + 25	30	668256	668458	99.96
3	148.51 + 25	30	668387	668498	99.98
4	148.52 + 25	30	668195	668560	99.94
5	148.5 + 25	30	668248	668454	99.96
6	147.98 + 25	30	668251	668630	99.94
			Mean		99.95
			SD		0.0151
			%R.S.D		0.0151

Table 4 Intra-day precision of Clomiphene Citrate (n=3)

Conc. ((µg/mL))	Time (Hr)	Peak Area	Total Average	(±) S.D	%R.S.D
15	0	385749	380862	4478.92	1.175
	4	376952
	8	369887
30	0	663908	656967	7006.40	1.066
	4	657098
	8	649897
45	0	952076	946263	7450.16	0.787
	4	948850
	8	937865
Mean % R.S.D					1.00

Table 5 Inter-day precision of Clomiphene Citrate (n=3)

Time (Day)	Conc. (µg/mL)	Average Area	Total Average	(±)S.D	%R.S.D
15	1	380862	376907	6018.23	1.59
	2	379878
	3	369981
30	1	656967	648312	8993.3	1.38
	2	648954
	3	639015
45	1	946263	935287	10633.33	1.13
	2	934567
	3	925033
Mean % R.S.D					1.36

Table 6 linearity of Clomiphene Citrate (n=3)

Sr. No.	Conc. (µg/mL)	Avg. Area	(±) S.D	% R.S.D
1	10	274187	617.33	0.225
2	20	435650	617.33	0.141
3	30	663908	1008.5	0.151
4	40	851491	655.92	0.077
5	50	1086816	6088.35	0.560

3.2.2 LOD and LOQ:

The LOD and LOQ of Clomiphene Citrate were found to be 0.1µg/mL and 0.32µg/mL, respectively.

3.2.3 Robustness and ruggedness:

Robustness of the method was studied by deliberate variations of the analytical parameter such as flow rate (1±0.2mL/min). The results are given in Table 6. Ruggedness of the method was carried out by different analysts. The results are displayed in Table 7.

Table 8 Ruggedness of Clomiphene Citrate

Sr. No.	Conc (µg/mL)	Analyst I	Analyst II	% Amount Found
1	30	667845	667845	99.93
2	30	665543	665643	99.59
3	30	669276	668276	100.15
4	30	662551	662851	99.14
5	30	664917	664591	99.5
6	30	668963	668963	100.1
Mean		666515	666361	99.73
SD		2630.45	2392	0.3933
% R.S.D		0.394	0.358	0.394

4. DISCUSSION:

In the present investigation a RP-HPLC method was developed and validated for estimation of Clomiphene Citrate in tablet dosage form. To develop RP-HPLC Method for the estimation of Clomiphene Citrate in tablet dosage form, mobile phase Methanol: ANC (90:10 v/v), was selected at a flow rate 1.0mL/min and estimation was carried out using column, Shimadzu C18 (250mm, 4.5mm, 5µm). Wavelength of detection was selected at 295 nm. Then these samples were analyzed by RP-HPLC method with set parameters and results were analysed and validated. The percentage assay of marketed tablets was found to be 99.5%w/w. Result indicated that the method was linear in concentration range of 10-50µg/mL with R² value 0.999, and regression line equation was found to be y =20411x+50881. Precision and repeatability results showed % RSD of less than 2%, LOD was found to be 0.1µg/ml, LOQ was found to be 0.32µg/ml and tailing factor was less than 2.

5. CONCLUSION:

The validated HPLC methods employed here proved to be simple, specific, accurate, and precise. Statistical analysis proves that the methods suitable for the analysis of Clomiphene Citrate as bulk drug and in tablet formulation without any interference from the excipients. Hence, these proposed methods can be used for the routine analysis of Clomiphene Citrate in API and their tablet dosage forms.

6. ACKNOWLEDGMENTS:

The authors are grateful to the principal of Rajarshi Shahu College of Pharmacy and Research, Pune, for providing the facilities for working and also to the laboratory assistance.

7. REFERENCES:

1. Proteau PJ. Steroid hormones and therapeutically related compounds. In Wilson and Gisvold’s. Textbook of Organic medicinal and pharmaceutical chemistry, Edited by Beale JM and Block JH. Lippincott Williams and Wilkins, Philadelphia. 2006: 12th ed: pp. 835.

2. Adashi EY. Clomiphene citrate: mechanism(s) and site(s) of action—a hypothesis revisited. Fertility and Sterility. 1984; 42: 331–344.

3. Kousta E, White DM and Franks S. Modern use of clomiphene citrate in induction of ovulation. Human Reproduction Update. 1997; 3(4): 359–365.

4. Vaghasiya N, Antala H and Mahajan T. Development and validation of first order spectrophotometric method for estimation of clomiphene citrate in bulk drug and formulation. International Journal of Research in Pharmaceutical and Nano Sciences. 2013; 2(1): 107- 114.

5. Hewala II. First derivative spectrophotometric and colorimetric determination of non-steroidal antiestrogens. Analytical Letters. 1993; 26(4): 625-640.

6. Rao Mallikarjuna GPV, Devi AP, Krisna Prasad KMM and Sastry CSP. A note on the estimation of Clomiphene citrate and Pimozide in bulk forms and pharmaceutical Formulations by Precipitation Reagents. Indian Drugs. 2002; 39: 395.

7. Rao Malikarjuna GPV, Aruna Devi P, Krishna Prasad KMM and Sastry CSP, DDQ (2,3-dichloro-5,6-dicyano-p-benzoquinone) as a chromogenic reagent for the determination of clindamycin hydrochloride, clomiphene citrate and mefloquine hydrochloride. Journal of Indian Chemical Society. 2002; 79: 848.

8. Vaddempudi V, Puranik SB, Kumar GVS and Sridhar KA. Development and validation of zero order spectrophotometric method for estimation of clomiphene citrate in bulk and tablet dosage form. International Journal of Pharm Tech Research. 2013; 5(1): 27-30.

9. Kim SB, Ozawa T, Umezawa Y. A screening method for estrogens using an array-type DNA glass slide. Analytical Sciences. 2003; 19: 499-504.

10. Peter JH, Harman, Graeme LB and George P. High performance liquid chromatographic determination of clomiphene using post-column on-line photolysis and fluorescence detection. Journal of Chromatography. 1981; 225(1): 131-138.

11. Urmos I, Benko SM and Klebovich I. Simple and rapid determination of clomiphene cis and trans isomers in human plasma by high-performance liquid chromatography using online post column photochemical derivatization and fluorescence detection. Journal of Chromatography B. 1993; 617: 168-172.

12. Hage DS and Sengupta A. Studies of protein binding to nonpolar solutes by using zonal elution and high-performance affinity chromatography: interactions of cis- and trans-clomiphene with human serum albumin in the presence of beta-cyclodextrin. Analytical Chemistry. 1998; 70(21): 4602-4609.

13. Sengupta A and Hage DS. Characterization of minor site probes for human serum albumin by high-performance affinity chromatography. Analytical Chemistry. 1999; 71(17): 3821-3827.

14. Bempong DK and Honigberg IL. Multivariate analysis of capillary electrophoresis separation conditions for Z-E isomers of clomiphene. Journal of Pharmaceutical and Biomedical Analysis.1996; 15(2): 233-239.

15. Hays PA. Proton nuclear magnetic resonance spectroscopy (NMR) methods for determining the purity of reference drug standards and illicit forensic drug seizures. Journal of Forensic Scienec. 2005; 50(6): 1342-1360.

16. Ross MSF and Judelman H. Separation of E- and Z-isomers of clomiphene citrate by high performance liquid chromatography using methenamine as mobile phase modifier. Journal of Chromatography. 1984; 50(6): 172-174.

17. Crewe HK, Ghobadi C, Gregory A, Rostami-Hodjegan A and Lennard MS. Determination by liquid chromatography–mass spectrometry of clomiphene isomers in the plasma of patients undergoing treatment for the induction of ovulation. Journal of Chromatography B. 2007; 847(2): 296-299.

18. Godge RK, Damle MC, Pattan SR, Kendre PN, Lateef S N and Burange PJ. RP- HPLC Method for simultaneous estimation of pseudoephidrine sulphate and desloratidine from bulk and tablets. Asian Journal of Research in Chemistry. 2009; 2(2): 139-142.

19. Dedania Z, Dedania R, Karkhanis V, Vidya Sagar G, Baldania M and Sheth NR. RP-HPLC method for simultaneous estimation of omeprazole and ondansetron in combined dosage forms. Asian Journal of Research in Chemistry. 2009; 2(2): 108-111.

20. Phale MD and Hamrapurkar PD. A validated and simplified RP-HPLC of metoprolol succinate from bulk drugs. Asian Journal of Research in Chemistry. 2009; 2(2): 119-122.

21. Patel B , Dedania Z, Dedania R, Ramolia C, Vidya Sagar G and Mehta RS. Simultaneous estimation of lansoprazole and domperidone in combined dosage form by RP-HPLC. Asian Journal of Research in Chemistry. 2009; 2(2): 210-212.

22. ICH, Q2 (R1) validation of analytical procedures: text and methodology, Proceedings of the International Conference on Harmonization, November 1996.

23. Girase JC, Venkateshwarlu G and Banerjee SK. Development and validation of RP-HPLC method for simultaneous determination of ceftriaxone sodium and sulbactam sodium in bulk and pharmaceutical formulation. Asian Journal of Research in Chemistry. 2009; 2(3): 280-284.

24. Singh M, Kashkhedikar SG, Soni L , Garg A, Gandhi T and Patel A. Development of RP-HPLC method for estimation of carvedilol in tablet formulations. Research Journal of Pharmacy and Technology. 2008; 1(1): 18-22.

25. Pawar HI, Kothapalli L, Thomas A, Nanda RK and Mare S. Simultaneous RP-HPLC method for estimation of ezetimibe and fenofibrate in synthetic mixture. Research Journal of Pharmacy and Technology. 2008; 1(1): 25-28.

26. Chitlange SS, Imran M, Bagri K and Sakarkar DM. A stability-indicating reverse phase high performance liquid chromatography method for the simultaneous determination of ramipril and valsartan in pharmaceutical dosage form. Research Journal of Pharmacy and Technology. 2008; 1(3): 215-217.

27. Gupta A, Yadav JS, Rawat S and Gandhi M. Method Development and hydrolytic degradation study of doxofylline by RP-HPLC and LC-MS/MS. Asian Journal of Pharmaceutical Analysis. 2011; 1(1): 14-18.

28. Satyanarayana L, Naidu SV, Narasimha Rao M, Ayyanna C. and Kumar A. The estimation of raltigravir in tablet dosage form by RP-HPLC. Asian Journal of Pharmaceutical Analysis. 2011; 1(3): 56-58.

Received on 08.06.2019 Modified on 25.03.2020

Research J. Pharm. and Tech. 2021; 14(7):3483-3488.

DOI: 10.52711/0974-360X.2021.00604

Sr. No.	High Flow (1.2mL/min)			Low Flow (0.8mL/min)
Sr. No.	Area	Tailing Factor	Retention Time	Area	Tailing Factor	Retention Time
1	618956	0.92	3.22	717146	1.003	3.692
2	610867	0.91	3.23	717643	1.005	3.691
3	617658	0.92	3.21	718946	1.001	3.693
Mean	615828/	0.916	3.22	717611	1.003	3.692
S.D	4342.52			1173
% R.S.D	0.705			0.163