Zolmitriptan (ZMT), 4(S)-4-[3-(2-dimethyl aminoethyl)-1H- 5-indolyl-methyl]-1, 3-oxazolan-2 one (Figure 1) belongs to a group of medicines known as Serotonin 5-HT1D Receptor Agonists. It works by stimulating serotonin receptors in the brain. Serotonin is a natural substance in the brain that, among other things, causes blood vessels in the brain to narrow. It is used to treat severe migraine headaches¹.

The assay of ZMT in pure and dosage forms, as far as we know, is not official in any pharmacopoeia, and therefore, requires much more investigation.

The different analytical methods that have been reported for its determination include HPLC with mass spectrometry detection^2-7, with coulometric detection⁸, electrospray ionization mass spectrometry⁹, tandem mass spectrometry^10-11, fluorescence detection^12,13 in pharmaceutical preparations and biological fluids and spectrophotometric and extractive methods^14-22, Comparative Study in the Presence of its Degradation products²³, LC-MS-MS^24-25, Titrimetric Assay²⁵. In the present work, spectroscopic analytical study for the analysis of zolmitriptan in pure and its Syrian pharmaceutical dosage forms through complex formation with Sulphonphthalein dyes in dichloromethane and chloroform mediums have been applied.

Fig. 1 Zolmitriptan structure.

2. EXPERIMENTAL:

2.1. Apparatus:

Measurements were made on a Jasco V-650 mode spectrophotometer UV-VIS (Japan Spectroscopic Co. Ltd., Tokyo) with a scanning speed of 400nm/min and a bandwidth of 2.0nm, equipped with 10mm matched quartz cells. All absorption spectra were made for electronic spectral measurements between (190-1100nm). UV-Visible spectrophotometer with 1.00cm quartz cells. Ultrasonic processor model powersonic 405 was used to sonicate the sample solutions. The diluter pipette model DIP-1 (Shimadzu), having 100μL sample syringe and five continuously adjustable pipettes covering a volume range from 20 to 5000μL (model PIPTMAN P), were used for preparation of the experimental solutions.

2.2. Reagents and solutions:

Pharmaceutical grade zolmitriptan (ZMT 99.2%) was received from Univision Pharmaceutical Co. Ltd. (China). A stock solutions of ZMT (100μg.mL^-1) were prepared by dissolving the appropriate weight of ZMT in 20 mL with chloroform and the volume was diluted to the mark in a 100mL calibrated flask with dichloromethane for method A and in chloroform for method B. Working standard solutions were prepared from suitable dilution of the standard stock solution. All solutions are stable for a period of 3.0 days when refrigerated (4°C).

Working standards were prepared daily by added different volumes of stock solutions to 3mL of reagent TB (1.10^-4M) and 2ml of reagent PhR (3. 10^-4 M) diluting to 10mL with dichloromethane and chloroform respectively.

The concentrations of ZMT were used for the analysis of ZMT by the spectrophotometric methods (A and B). The methods were based on the complex formation between Sulphonphthalein dyes (TB, PhR) and ZMT in dichloromethane medium. The colored product was quantified spectrophotometrically using absorption bands at 398nm for complex of (ZMT–TB) and at 418 nm for (ZMT–PhR).

Thymol Blue AR, TB(1.10^-4M) Prepared by dissolving 46.7mg of TB dye (Sd fine, Chem limited, India) in10mL dichloromethane and made up to mark with dichloromethane in a 100mL calibrated flask and diluted 5ml of the previous solution to the calibrated flask 50ml to the mark.

Phenol Red, PhR (3.10^-4M) Prepared by dissolving 10.6mg of PhR dye (BDH, England company) in 10mL chloroform to dissolve and made up to mark with chloroform in a 100mL calibrated flask.

Zomitan (Pharmasyr Company, Damascus, Syria) containing 5 mg/tab. and 2.5mg/tab. from local medical stores.

All reagents and solvents were of analytical grade.

2.3. Spectrophotometric procedure:

Increasing volumes of ZMT working standard solution were transferred into series of 10mL volumetric flasks that contain 3mL of TB reagent (1.10^-4M) for method A and 2ml of PhR (3.10^-4M) for method B. Volumes were made up to mark with dichloromethane for method A and with chloroform for method B. Solutions were mixed gently and allowed to stand at room temperature only for 20 minutes for method B, and mixed before the spectra was recorded at 398 nm for method (A) and 418 nm for method (B) against reagent blank that had been treated similarly.

2.4. Determination of ZMT/Dye stoichiometric relationship:

The composition ratio of drug ZMT to dyes (TB, PhR) of the colored complex was determined using the molar ratio and continuous variation methods^27,28.

2.5. Procedure for pharmaceutical samples:

Ten individual tablets were weighed and pulverized carefully. An accurately weighed amount of the powder equivalent to 2.5mg of ZMT was transferred into 25mL volumetric flask and dissolved in 20mL of dichloromethane for method (A) and in chloroform for method (B). The content of the flask was sonicated for 20min then diluted to volume with same solvent. A portion of this solution was centrifuged at 5000 rpm for 10 minutes. Then suitable volume 2mL of the filtrate solution was then transferred into10mL volumetric flask, and added a suitable volume 3mL of TB, 2ml PhR for the complete forming of complexes in methods A and B, respectively and diluted with dichloromethane for method (A) and in chloroform for method (B)for up to mark. Then the analysis of ZMT by the spectrophotometric method directly for method (A) and after 20 minuet till the completely forming for method (B).

3. RESULTS AND DISCUSSION:

3.1. Optimization of reaction conditions:

3.1.1. Effect of reaction time and stability:

The optimum reaction time for the development of color at ambient temperature (25±2C˚)was studied and it was found that a the complex forming directly after addition the dye for method A and 20 min standing time was sufficient for the complete formation method B. The formed color was stable for more than 24 h in methods A and B. Figure 1 shows the constant absorbance readings were obtained between (0-40 min) for method A and method B.

3.1.3. Effect of dye concentration:

The influence of the concentration of TB and PhR on the intensity of the color developed at the selected wavelength and constant drug concentration was studied. As shown in Figure 3the constant absorbance readings were obtaind between (0.5-5.0mL) of (1.10^-4M) of TB in method A and (0.5-5.0mL) of (3.10^-4M) PhR in method B. Hence, 3mL of TB and 2ml PhR were the suitable volume for the complete forming of complexes in methods A and B, respectively.

3.1.4. Stoichiometric ratio:

^{Molar ratio method: The
stoichiometry of}(ZMT^:Dye)^{complex by molar ratio method according to following equation: Amax= f([}ZMT^]/[Dye^{], confirms that the ratio of complex}ZMT^:TB and ZMT:PhR ^{are equal to 1:1for both methods (Figure 4).}

Job’s method :

In order to establish the stoichiometry of ZMT and dyes (TB, PhR) complex by Job’s method of continuous variations was applied. In both cases, the plot reached a maximum value at a mole fraction of 0.5 which indicated the formation of 1:1 (ZMT:Dye) complex (Figure 5) between ZMT and TB or PhR.

3.2. Validation of the proposed method:

3.2.1 Linearity:

Under the optimum experimental conditions, standard calibration curve was constructed at twelve concentration for method A and eleven concentrations for method B levels (n=5). The correlation coefficient was 0.9998 for method A, and 0.9998 for method B indicating very good linearity, over the concentration range of 0.125 – 9.0g/mL for method A and 1.25-40g/mL for method B (figure 6). The intercept, slope, limit of detection (LOD), and limit of quantitation (LOQ) are summarized in Table 1. LOD and LOQ values were calculated as 3.3S_b/m and 10S_b/m, respectively. Where molar absorptivity of regression and Sandell sensitivity Table 1.

3.2.2. Precision:

The repeatability of proposed methods were estimated by measuring five replicate samples of each concentration of zolmitriptan prepared in one laboratory on the same day. The precision expressed as the relative standard deviation (RSD%) ranged from 0.29% to 3.97% (method A) ,and from 0.02% to 1.81% (method B) for the smallest concentration, indicating good precision Table 2.

Table 1. Statistics and analytical parameters of ZMT determination by TB method (A) and by PhR method (B).

Parameter	Result(A)	Result (B)
λ_max (nm)	398	418
Linear range (μg/mL)	0. 125–9.0	1.25 – 40.0
Slope	0.2005	0.0460
Molar absorptivity (Ɛ), L / moL.cm	63642	12552
Intercept	0.0679	0.0127
Sandell sensitivity, μg/ cm²	0.029	0.072
Correlation coefficient	0.9998	0.9998
Limit of detection (g/mL)	0.08	0.73
Limit of quantification (g/mL)	0.24	2.20

Table 2. Precision for determination of ZMT in pure form using proposed methods(A, B)

Method	Taken ZMT (g/mL)	aFound ZMT (g/mL)	SD (μg/mL)	RSD%	Recovery %
TB (A)	0.125	0.12±0.006	0.005	3.97	99.67
	0.500	0.50±0.014	0.011	2.22	100.35
	1.000	0.97±0.018	0.015	1.51	97.13
	2.000	1.98±0.026	0.021	1.07	99.06
	4.000	4.07±0.018	0.015	0.36	101.63
	6.000	5.92±0.024	0.019	0.32	98.58
	9.000	9.04±0.032	0.026	0.29	100.42
PhR (B)	1.25	1.23±0.028	0.022	1.81	98.68
	2.50	2.49±0.036	0.029	1.17	99.69
	5.00	4.82±0.049	0.039	0.82	96.44
	10.00	9.71±0.072	0.058	0.59	97.12
	15.00	15.26±0.047	0.038	0.25	101.76
	30.00	30.09±0.015	0.012	0.04	100.31
	40.00	39.78±0.010	0.008	0.02	99.45

^aAverage of five determination ± Confidence limit

Figure 6: Calibration plot of ZMT complexs with TB and PhR.

Table 3. precision and accuracy results

Method	Taken ZMT μg/ml	Intra-dayn=5			Inter day n=5
Method	Taken ZMT μg/ml	^aFound ZMT±CL μg/ml	^bRSD%	Recovery%	^aFound ZMT±CL μg/ml	^bRSD%	Recovery%
TB (A)	2	2.04±0.015	0.58	101.83	2.02±0.032	1.26	101.13
	4	4.03±0.015	0.31	100.66	4.02±0.027	0.55	100.59
	6	5.95±0.021	0.29	99.14	5.95±0.021	0.28	99.15
	8	7.96±0.027	0.27	99.53	7.99±0.038	0.39	99.85
PhR (B)	5	5.00±0.041	0.66	100.08	5.01±0.024	0.38	100.14
	10	9.83±0.023	0.19	98.33	9.89±0.010	0.08	98.87
	20	20.27±0.036	0.14	101.33	20.22±0.030	0.12	101.10
	30	30.01±0.021	0.06	100.04	30.01±0.021	0.06	100.04

^aMean ± Confidence limit, ^b Mean of five determination

Table 4. determination of ZMT in tablets

Brand name	Label claim	^aAverage ZMT found ± SD ^b(Recovery%)			t- ( F- ) ^ctest
		Reference method^[8]	Proposed method		t- ( F- ) ^ctest
		Reference method^[8]	Method A	Method B	Method A	Method B
Zomitan	2.5 mg/tab	2.48±0.122 (99.20)	2.46±0.015 (98.48)	2.51±0.059 (100.46)	0.47(0.45)	0.90(0.13)
Zomitan	5 mg/tab	5.02±0.106 (100.40)	5.08±0.019 (101.56)	5.07±0.095 (101.49)	0.46(0.79)	0.79(1.18)

^aAverage and standard deviation of five determinations for the proposed method

^b Recoveries were calculated considering the labeled amount reported by the manufacturer.

^c the tabulated t value at 95% confidence limit for 4 degrees of freedom (n =5) is 2.776 and the tabulated F value at 95% confidence limit for 4 degrees of freedom for the proposed methods is 6.25.

3.2.3. Intraday and inter day results:

The accuracy and precision of the methods were evaluated by performing five replicate analysis in pure drug solution at four different concentration levels (within the working range). Percentage relative standard deviation (RSD%) as precision and percentage relative error (RE%) as accuracy of the proposed spectrophotometric (A,B) methods were calculated. The relative standard deviation (RSD) values were less than 2% in all cases, indicating good repeatability of the suggested methods. The percentage relative error calculated using the following equation:

RE % = [(found – taken) / taken]×100

The intra-day and inter-day precision and accuracy results show that the proposed methods have good repeatability and reproducibility Table 3.

3.2.4. Accuracy:

Accuracy is judged by comparing the results obtained from the presently proposed method, that has been applied on commercial tablets, with those obtained from a reference method such as HPLC. The resulted values were statistically compared with each other Table 4, using t- and F-tests. With respect to t- and F-tests, no significant differences were found between the calculated values of both the proposed and the reported methods at 95% confidence level.

3.3. Application to Tablets:

The proposed methods were applied to the determination of ZMT in tablets. The results in Table 5 showed that the methods are successful for the determination of ZMT and that the excipients in the dosage forms do not interfere. A statistical comparison of the results for determination of ZMT from the same batch of material by the proposed and reference method is shown in Table 5. The results agreed well with the label claim and also are in agreement with the results obtained by the reference method. Statistical analysis of the results using Student’s t-test for accuracy and F-test for precision revealed no significant difference between the proposed and reference method at the 95% confidence level with respect to accuracy and precision Table 5.

4. CONCLUSION:

The proposed methods application of complex formation for the determination of ZMT in pharmaceutical formulations. Compared with the existing visible spectrophotometric methods, the methods are rapid, simple, accuracy, cost-effective, extractive free and more sensitive as can be seen from the molar absorptivity values. Moreover, the proposed methods are free from tedious experimental steps such as heating or extraction steps.

The most attractive feature of these methods is its relative freedom from interference by the usual tablet diluents and excipients in amounts far in excess of the normal occurrence in pharmaceutical formulations. The statistical parameters and the recovery data reveal good accuracy and precision of the methods. Hence, offering economic and acceptable method for the routine determination of zolmitriptan in its formulations.

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Received on 10.02.2020 Modified on 16.04.2020

Research J. Pharm. and Tech. 2021;14(3):1215-1220.

DOI: 10.5958/0974-360X.2021.00216.X