HPLC and TLC-Densitometric Methods for the Determination of some Antimigraine Drugs in Bulk Powder and in Pharmaceutical Preparations

Abd El-Aziz B. Abd El-Aleem¹, Shaban M. Khalile², Amr M. Badawy¹ and Omneya K. El-Naggar²*

¹Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo Egypt

²National Organization for Drug Control and Research (NODCAR), Giza, Egypt.

ABSTRACT:

Two simple and accurate chromatographic methods were developed for the determination of zolmitriptan, and sumatriptan in raw material and in tablets. The first method uses isocratic high performance liquid chromatographic (HPLC) method. Analysis was performed on Agilent Zorbax C18 column using a mobile phase consisting of phosphate buffer pH3: acetonitrile: methanol (2:1:1, v/v/v) with a flow rate of 0.75 ml/ min and UV detection at 253 nm. The second method uses thin-layer chromatographic (TLC) separation of sumatriptan, zolmitriptan and eletriptan from their impurities followed by densitometric measurements of drug spots at 254 nm. The separation was carried out on silica gel 60 F254 using chloroform: ethylacetate: methanol: ammonia (72:10:18:2, v/v/v/v) as mobile phase. The methods were validated according to ICH guidelines and the acceptance criteria for linearity, accuracy, precision, specificity and system suitability were met in all cases. The methods were linear in the range of 10-40 µg/ml and 10-50 µg/ml for zol. and sum. respectively by HPLC method and in range of 1-20 µg/spot, 1-20 µg/spot and 1-10 µg/spot for ele., sum. and zol. respectively by TLC method. The proposed methods were successfully applied for the determination of zol., sum. and ele. in bulk and tablets forms. The results were compared statistically at 95% confidence level with reported methods. There was no significant difference between the mean percentage recoveries and precision of the methods.

KEYWORDS: Zolmitriptan, sumatriptan, eletriptan, HPLC, TLC-densitometry.

INTRODUCTION:

Sumatriptan is a selective serotonin agonist that acts at 5-HT₁ receptors and produces vasoconstriction of cranial arteries. Drugs like sumatriptan, which are commonly known as triptans are believed to act mainly at 5-HT_1B and 5-HT_1D subtype receptors and are therefore sometimes referred to as 5HT_1B/1D-receptor antagonists. Sumatriptan is used for the acute treatment of migraine attacks and of cluster headache. It should not be used for prophylaxis.

Zolmitriptan is a selective serotonin (5-HT₁) agonist with actions and uses similar to those of sumatriptan. It is used for the acute treatment of migraine attacks. Zolmitriptan should not be used for prophylaxis.

Eletriptan hydrobromide is a selective serotonin (5-HT₁) agonist with actions and uses similar to those of sumatriptan. It is used for acute treatment of the headache phase of migraine attacks. It should not be used for prophylaxis.⁽¹⁾

Various methods were used in determination of sumatriptan in its different forms, such as spectrophotometry⁽²⁾, HPLC⁽³⁾ and electrocatalytic determination⁽⁴⁾, for zolmitriptan the spectrophotometric method⁽⁵⁾, HPLC⁽⁶⁾ and elecrochemical assay⁽⁷⁾, for eletriptan the spectrophotometric method⁽⁸⁾, UPLC⁽⁹⁾and RP-HPLC⁽¹⁰⁾.

Fig (1): Chemical structure of sumatriptan.

Fig (2): Chemical structure of zolmitriptan.

Fig (3): Chemical structure of eletriptan.

MATERIALS AND METHODS:

Instrument:

· Shimadzu-Dual wavelength lamp flying CS9301 densitometer with PC. With Hamilton syringe 10 µl capacity.

· Florescent TLC plates (20 × 20 cm) with 0.25 mm thickness silica gel F254, (E. Merck).

· The HPLC system comprised an Agilent pump with different flow rates (model 1260 series, Agilent, USA), equipped with a variable wavelength detector and a 20 µl volume injection loop. A Zorbax SB-C18 (4.6 x 100 mm, 3.5µm) column was used as stationary phase. The samples were injected with a 50 µl Hamilton analytical syringe.

Reagents and Chemicals:

All chemicals and solvents used in this investigation were of analytical reagent grade (A.R.) used as such without any further purification.

Sumatriptan (Sum.), zolmitriptan (Zol.) and eletriptan (Ele.) were of sufficient purity and passed the British Pharmacopeia (B.P.) requirements.

Sum. was supplied by SMS Pharmaceuticals Ltd, India and zol. was supplied by Western Pharmaceutical Industries, China. Ele. was supplied by Pfizer, U.S.A.

Pharmaceutical dosage forms of Sum., Zol. and Ele. are supplied by local market companies. GlaxoSmithKline for sum. (Imigran^®), AstraZeneca for Zol. (Zomig^®), Pfizer for Ele. (Relpax^®).

Solutions:

Stock standard solution:

For TLC:

Solutions were prepared by transferring accurately weighed 100 mg of Ele., Zol., Sum. into 50 ml volumetric flasks. 20 ml of the suitable solvents were added to dissolve by shaking and the volume was completed to the mark with the used solvent. In case of Zol. the solvent is ethanol, for sum. the solvent is water and for Ele. the solvent is acetonitrile.

For HPLC:

An accurate weight (200 mg) of each of Zol. and Sum. was transferred into 50 ml volumetric flask. Twenty five ml aliquot of the mobile phase was added for each flask and the flasks were shaken for 10 min. The solutions were completed to mark using the same solvent. One ml aliquot of each solution was transferred into 10 ml volumetric flask and the volume was completed with the mobile phase to form 400 µg/ml of the intact standard solutions.

Preparation of internal standard solution: An accurate weight (200 mg) of hydrochlorothiazide (HCZ) was transferred into 100 ml volumetric flask. Twenty five ml aliquot of the mobile phase was added and the flask was shaken for 10 min. The solution was completed to mark using the same solvent. Five ml aliquot was transferred into 10 ml volumetric flask and the volume was completed with the mobile phase to form 1mg/ml of the internal standard solution.

General analytical procedure:

Bulk sample:

For TLC:

In 10 ml measuring flasks aliquots containing 1-20 mg/ml of Sum., Zol., Ele. working solutions were transferred. The volumes of the solutions were completed to the mark with ethanol in case of Zol., with water in case of sum. and with acetonitrile in case of Ele.

The calibration curve was constructed relating the area under peak to the corresponding concentration of each drug spot and regression equation was computed.

For HPLC:

Aliquot portions (0.25- 4ml) of Zol. and Sum. intact standard solutions equivalent to (0.1- 1.6mg) were transferred into two series of 10 ml volumetric flasks. One ml aliquot of HCZ internal standard solution was added to each flask and the volumes were completed to mark using the mobile phase. Twenty µl of each solution was injected to HPLC system.

The peak area ratio of the intact drug / internal standard was plotted versus the concentration (µg/ml) for constructing the calibration curves.

Assay of pharmaceutical dosage forms:

For TLC:

Weigh twenty tablets and thoroughly grind well to fine powder, extract an accurate weighed portions of the obtained powder equivalent to 100 mg of Zol. in 50 ml ethanol, of Ele. in 50 ml acetonitrile and of sum. in 50 ml water. Shake for about 15 minutes, filter the solutions in 100 ml measuring flasks, wash the residues several time by small portions of the used solvents and dilute to the mark with the solvents. The procedure of the proposed method was applied under the specified conditions mentioned above. The concentration of each drug was calculated from its corresponding regression equation.

For HPLC:

Accurate weights of the powdered Zomig and Imigran tablets equivalent to 100 mg of Zol. and Sum. respectively were transferred into two 150 ml conical flasks. Forty ml aliquot of the mobile phase was added to each flask. The flasks were shaken for 15 min. the solutions were filtered. The filter papers and the residues were washed three times each with 5 ml mobile phase. The combined filtrates and washings were collected into 100 ml volumetric flasks and the volumes were completed with the same solvent. Ten ml aliquot of each solution was transferred into 25 ml volumetric flask and the volume was completed to mark with the mobile phase to form a solution of 400 µg/ml of pharmaceutical dosage form.

RESULTS AND DISCUSSION:

TLC-densitometry:

Several developing systems were tested to show which will perform good separation of the drugs Ele., Sum. and Zol. Chloroform/ ethylacetate/ methanol/ ammonia (72:10:18:2 v/v/v/v) was the best solvent system.

Results are computerized by chromatogram and area under peak. Selection of the wavelength was tested and the best wavelength was 254nm. After developing the spots, the plates are removed and dried. The spots of the drugs can be seen, detected and identified by UV lamp 254 nm to determine the R_f of each drug.

Analyses were performed on 20X20 cm TLC florescent aluminium sheet silica gel plates. The plate was activated in the oven at 105°C for 5 minutes and cool. 10 µl spot of each working drug solutions were applied to the plate using Hamilton microsyringe (10 µl). Spots were spaced 2 cm apart from each other and from the bottom edge of the plate. The plate was developed in chromatographic chamber previously saturated for one hour with mobile phase. Ascending chromatography was performed with chloroform/ethylacetate/methanol/ammonia (72:10:18:2) as developing system through distance 16 cm at room temperature. Plates were air dried then the spots were detected under UV lamp 254 nm. The spots were scanned using densitometer under the following conditions:

Photomode (reflection)

Scan mode (Zigzag)

Result output (chromatogram and area under peak)

Swing width (12 cm)

Wavelength (254 nm)

Fig (4) shows that the selected developing system give complete separation of the intact drugs with R_f values 0.45, 0.45 and 0.77 for Zol., sum. and Ele. respectively.

The linearity was confirmed by plotting the area under peak versus the corresponding concentration of each drug spot where a linear response was obtained fig (5, 6, 7) and regression equations were computed.

A= 0.5166 × C + 0.496 r²= 0.9982 for Ele.

A= 0.3504 × C + 0.8627 r²= 0.9998 for Sum.

A= 1.105 × C + 0.1416 r²= 0.9995 for Zol.

Where A is the AUP ×10^-3, C is the concentration in µg/spot and r² is the regression coefficient.

Fig (4): TLC chromatogram of (A) Ele.,(B) Sum. and (C) Zol. visualized under UV lamp at 254 nm, using chloroform: ethyl acetate: methanol: ammonia (7.2:1.8:1:0.2)

Fig (5): Linearity of the peak area to the corresponding concentration of eletriptan (1-20 µg/spot) at 254 nm

Fig (6): Linearity of the peak area to the corresponding concentrations of sumatriptan (1-20 µg/spot) at 254 nm

Fig (7): Linearity of the peak area to the corresponding concentrations of zolmitriptan (1-10 µg/spot) at 254 nm

High performance liquid chromatographic method:

A simple isocratic high-performance liquid chromatography method was developed for the determination of zolmitriptan and sumatriptan in pure form and in pharmaceutical formulations using a Zorbax SB-C18 (4.6 x 100 mm, 3.5µm) in the presence of hydrochlorothiazide as internal standard. The mobile phase consisted of phosphate buffer pH 3: methanol: acetonitrile (2:1:1 v/v/v). The mobile phase was chosen after several trials to reach the optimum stationary /mobile –phase matching. The average retention times under the conditions described were 1.34 minutes for both drugs and 1.614 minutes for HCZ. The chromatographic system in this work allowed complete baseline separation of sumatriptan and zolmitrirtan from HCZ [Fig. 8]. Calibration graphs were obtained by plotting the ratio of peak areas of drugs/peak area of internal standard versus concentrations of zolmitriptan and sumatriptan [Fig.9.10], Linearity ranges were found to be 10-40 μg/ml for zolmitriptan and 10-50 μg/ml for sumatriptan using the following regression equations:

R= 0.0187 C+ 0.08 r²= 0.9998 for Zol.

R= 0.0272 C+ 0.134 r²= 0.9998 for Sum.

Where R is the area under peak ratio, C is the corresponding concentration in µg/ml, and r² is the regression coefficient.

The robustness of the HPLC method was investigated by analysis of samples under a variety of experimental conditions. It was found that the method was robust when the column and the mobile phase ratio were varied. During these investigations, the retention times were modified, however the areas and peak symmetry were conserved.

Fig (8): HPLC chromatogram of sumatriptan at t_R= 1.339 min, in the presence of hydrochlorothiazide as internal standard at t_R= 1.615 min.

Fig (9): Calibration curve for the determination of zolmitriptan using HPLC method (R=0.0187C+0.08 r²=0.9998)

Fig (10): Calibration curve for the determination of sumatriptan using HPLC method (R=0.0272C+0.134 r²=0.9998)

Table (1): Assay validation sheet of the proposed TLC-densitometric method for the determination of eletriptan sumatriptan and zolmitriptan.

Item	Eletriptan	Sumatriptan	Zolmitriptan
Linearity range (µg/spot) Regression equation*: Slope Intercept Regression coefficient (r^2) 95%confidence limit of slope 95%confidence limit of intercept LOD** LOQ***	1-20 0.5166 0.496 0.9982 0.4916-0.5417 0.2623-0.7298 0.81 2.46	1-20 0.3504 0.8627 0.9998 0.3451-0.3557 0.8131-0.9122 0.63 1.9	1-10 1.105 0.1416 0.9995 1.072-1.138 0.05357-0.3367 0.297 0.9

*A=a+bC, where A is area under peak x10³, a is the intercept, b is the slope and C is the concentration in µg/spot.

**LOD is limit of detection= 3.3 x σ /S where σ is the standard deviation of 5 replicate determinations under the same conditions as for the sample analysis in the absecnce of the analyte and S is the sensitivity ,namely the slope of the calibration graph.

***LOQ is the limit of quantification =10x σ /S.

Table (2): Assay validation sheet of the proposed HPLC method for the determination of zolmitriptan and sumatriptan.

Parameter	Zolmitriptan	Sumatriptan
Linearity Slope Intercept Correlation coefficient (r^2) Range 95% confidence limit of slope 95%confidence limit of intercept LOD* LOQ*	0.0187 0.08 0.9998 10 – 40 µg/ml 0.01795-0.01945 0.05959-0.1004 1.06 µg/ml 3.2 µg/ml	0.0272 0.134 0.9998 10 – 50 µg/ml 0.02647-0.02793 0.1096-0.1584 1.33 µg/ml 4.04 µg/ml

Table (3): Accuracy data for the analysis of ele., sum. and zol. in bulk powder by the proposed TLC-densitometric method.

Eletriptan			Sumatriptan			Zolmitriptan
Taken µg/spot	Found* µg/spot	Accuracy %	Taken µg/spot	Found* µg/spot	Accuracy %	Taken µg/spot	Found* µg/spot	Accuracy %
6 9 12	6.03 9.04 11.974	100.5 100.4 99.78	3 6 9	2.962 6.004 9.014	98.73 100.07 100.16	3 6 7	3.02 6 6.988	100.7 100 99.83
Mean		100.23	Mean		99.65	Mean		100.18
SD		0.39	SD		0.8	SD		0.46
RSD%		0.389	RSD%		0.803	RSD%		0.459

* Average of five determinations.

Table (4): Accuracy data for the analysis of zolmitriptan and sumatriptan in bulk powder by the proposed HPLC methods.

Zolmitriptan				Sumatriptan
Taken µg/ml	AUP drug/ AUP IS	Found µg/ml	Accuracy %	Taken µg/ml	AUP drug/ AUP IS	Found µg/ml	Accuracy %
10 20 30 40	0.27 0.45 0.64 0.83	10.03 19.9 30.6 41.1	100.3 99.5 102 100.2	10 20 30 40	0.41 0.67 0.95 1.23	10.12 20.2 29.7 40.9	101.2 101 99 102.25
Mean			100.5	Mean			100.86
SD			1.06	SD			1.36
RSD			1.054	RSD			1.348

Table (5): Determination of ele., sum. and zol. in commercial tablets by the proposed TLC-densitometric method and application of standard addition technique.

Product	TLC method (recovery%±SD)	Standard addition
		Taken µg/spot	Added µg/spot	Found µg/spot	Recovery %**
Eletriptan in Relpax^® tablets 40 mg / tablet	100.42±0.75	12	2 4 5	2.03 4.03 5.07	101.5 100.75 101.4
		Mean			101.22
		SD			0.41
		RSD%			0.405
Sumatriptan in Imigran^®tablets 50 mg / tablet	99.19±0.26	9	2 3 5	2.01 3.05 5.07	100.5 101.67 101.4
		Mean			101.9
		SD			0.61
		RSD%			0.599
Zolmitriptan in Zomig^® tablets 2.5 mg / tablet	100.61±0.92	2.5	1 2 3	1.01 2.03 3.05	101 101.5 101.67
		Mean			101.39
		SD			0.35
		RSD%			0.345

**Average of three determinations.

Table (6): Determination of zolmitriptan and sumatriptan in dosage forms by the proposed HPLC method and application of standard addition technique.

Product	HPLC method	Standard addition
		Taken µg/ml	Added µg/ml	Found µg/ml	Recovery %**
Zolmitriptan in Zomig^® tablets 2.5 mg /tablet	101.2±0.988	10	10 15 20	10.02 15.06 20.4	100.2 100.4 102
		Mean			100.86
		SD			0.99
		RSD%			0.98
Sumatriptan in Imigran^® tablets 50 mg /tablet	99.29±0.266	10	10 15 20	10.12 15.04 20.11	101.2 100.27 100.55
		Mean			100.67
		SD			0.477
		RSD%			0.47

**Average of three determinations.

Table (7): Statistical analysis of the results obtained by applying the proposed TLC-densitometric and reported methods for the determination of ele., sum. and zol. in pharmaceutical formulations.

Drug	TLC method	Reported method^(8,11,12)	n	Student's t-test	F value
Eletriptan in Relpax^® tablet 40 mg / tablet (t-test = 2.78) (F-test = 6.39)	100.42±0.75	100.23±0.73	5	0.406	1.056
Sumatriptan in Imigran^® 50 mg / tablet (t-test = 2.78) (F-test = 6.39)	99.19±0.26	99.418±0.19	5	1.583	1.87
Zolmitriptan in Zomig^® 2.5 mg / tablet (t-test = 2.78) (F-test =6.39)	100.61±0.92	100.41± 0.522	5	0.423	3.1

The values in the parenthesis are the corresponding theoretical values of t and F at 95% confidence level.

Table (8): Statistical analysis of the results obtained by applying the proposed HPLC method and the reported methods ^(11,12) for the determination of zolmitriptan and sumatriptan.

Drug	HPLC method	Reported method^(11,12)	n	Student's t-test	F value
Zolmitriptan in Zomig^® tablets 2.5 mg/tablet	101.2±0.988	100.41±0.522	5	1.58	3.6
Sumatriptan in Imigran^® tablets 50 mg/tablet	99.29±0.266	99.418±0.19	5	0.875	2.29

Tabulated t-value at the 95% confidence level is 2.78.

CONCLUSION:

The suggested methods are found to be simple, accurate and selective with no significant difference of the precision compared with the reported methods of analysis. The proposed methods could be applied successfully, for routine analysis of eletriptan, zolmitriptan and sumatriptan in bulk powder and in their pharmaceutical formulations.

REFERENCES:

1- Martindale: The Complete Drug Reference 37 e.d.(2011).

2- M.R. Pourmand, M.S. Azar and M. Aghavalijamaat Pharmaceutical Chemistry Journal Volume 44, No. 10 (2011), p:585-589.

3- M.J. Nozal, J.L. Bernal, L. Toribio, M.T. Martin, F.J. Diez, Journal of Pharmaceutical and Biomedical Analysis vol. 30, Issue 2, 5 September (2002), p:285-291.

4- M. Amiri, Z. Pakdel, A. Bezaatpom, S. Shahrokhian, Bioelectrochemistry. Vol.81,Issue 2, June (2011), p:81-85.

5- S.K. Acharjya, M.E.B. Rao, B.V.V. Ravikumar, M.M. Annapurna, Journal of Advanced Scientific Research (2011), 2(3), p:42-47.

6- E.K.S. Vijayakumar, M.A. Samel, S.B. Bhatekai and S.M. Pakhale, Indian Journal of Pharmaceutical Sciences v.72(1); Jan-Feb (2010), p:119-122.

7- B. Uslu, D. Canbaz, An International Journal of Pharmaceutical Sciences, Vol 65,No.4, April (2010), pp.245-250(6).

8- P. Sunitha, C.H. Jhansirani, B. Kavitha, N. Sirisha and A. Pavani, International Journal of Pharmaceutical, Chemical and Biological Sciences, 2(4), (2012), p: 427-430.

9- N. Balaji, V.R. Sivaraman, Dr. P. Neeraja, IOSR Journal of Applied Chemistry, Jan-Feb (2013), vol.3, p: 20-28.

10- V. Venugopal, G. Ramu., A.B. Babu and C. Rambabu, International Journal of PharmTech Research, Oct-Dec (2012), Vol.4, No.4, p; 1504-1507.

11- S.K. Acharjya, M.E.B. Rao, B.V.V. Ravi Kumar, M.M. Annpurna, J adv Sci Res, ( 2011),2(3), p:42-47.

12- R.K. Nayak, S.K. Swain, S.K. Panda, K.C. Sahu, D. Mishra, S. Kumar, International Journal of Pharmaceutical and Biological Archives (2011);2(4), 1100-1105.

Received on 10.07.2013

Modified on 02.08.2013

Accepted on 12.08.2013

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(5): September-October, 2013, 288-294