INTRODUCTION:

Almotriptan malate (AM) (Figure 1) is a selective and potent serotonin 5-hydroxy trytamine1B/1D (5-HT 1B/1D) receptor agonist. It is chemically designated as 1[[[3-[2-(Di methyl amine) ethyl]-1H-indol-5-yl] methyl] sulfonyl] pyrrolidine ± - hydroxy butanedioate [1] (1:1). Its empirical formula is C₁₇H₂₅N₃O₂S.C₄H₆O₅ representing molecular weight of 469.56. It is a white to slightly yellow crystalline powder that is soluble in water and sparingly soluble in methanol. Almotriptan is available in market as conventional tablets (AXERT).The drug is absorbed well orally, with an absolute bioavailability of around 70%. The drug is used to treat severe migraine headaches and vascular headaches; acute treatment of migraine attacks with or without aura. The drug binds with high affinity to 5-HT 1D, 5-HT 1B and 5-HT 1F receptors. Because of the particular distribution of the 5-HT 1B/1D receptors, almotriptan basically constricts the human meningeal arteries; therefore it has a limited effect on arteries supplying blood to the brain and little effect on cardiac and pulmonary vessels. Ameliorate migraine through selective constriction of certain intracranial blood vessels, inhibition of neuro peptide release and reduced transmission in trigeminal pain pathway

Figure 1: Chemical structure of Almotriptan malate

In literature, several analytical methods such as HPLC [2-5], HPTLC [6], HPLC-MS/MS [7], LC-ESI-MS/MS [8], UV Spectrometric [9-10], Fluorometric and Coloricmetric [11], visible spectrophotometric [12-16] have been reported for the determination of AM in biological fluids and formulations. For routine analysis, simple, rapid and cost effective visible spectrophotometric methods are useful and preferred in small scale pharmaceutical industries. Nevertheless, there still exists a need for development of sensitive accurate and flexible visible spectrophotometric method for the determination of AM in pharmaceutical preparations and quality control analysis. So the authors have made some attempts in this direction and succeeded in developing a method based on the redox reaction and colored charge-transfer complex formation between the drug and NBS-PMAP-SA reagent [17] under specified experimental conditions.

The proposed method for AM determination has many advantages over other analytical methods due to its rapidity, normal cost and environmental safety. Unlike HPLC, HPTLC procedures, the instrument is simple and is not costly. Economically, all the analytical reagents are inexpensive and available in any analytical laboratory. The method can be extended for the routine quality control analysis of pharmaceutical products containing AM.

MATERIALS AND METHODS (EXPERIMENTAL):

Apparatus and chemicals:

A Milton Roy UV/Visible spectrophotometer model-1201 with 10mm matched quartz cells was used for all spectral measurements. A Systronics digital pH meter mode-361 was used for pH measurements. All the chemicals used were of analytical grade. AXERT tablets procured from Ortho Mc Nell Pharmaceuticals, USA.

NBS solution (BDH, 0.01%, 5.618x10^-4M): Prepared by dissolving 10 mg NBS in100ml distilled water and standardized iodometricallly, PMAP (metol) solution(Wilson labs, 0.3%,8.75x10^-3M): prepared by dissolving 300mg of P-N-methyl amino phenol sulphate in 100ml of distilled water, SA solution (Sd-fine,0.2%,1.16x10^-2M): prepared by dissolving 200mg Sulfanilamide in 10ml of 0.1M HCl followed by dilution to 100ml with distilled water, AcOH solution(Qualigens, 0.5%, 8.75x10^-1M) : Prepared by diluting 5.0ml of glacial acetic acid to 100ml with distilled water.

Preparation of standard drug stock solution:

An accurately weighed quantity of 50mg pure AM drug was dissolved in little amount of water and made to 50ml with glacial acetic acid. The prepared stock solution was stored at 4⁰ C protected from light. This stock standard solution was further diluted stepwise with distilled water to obtain working standard solution and a series of standards were freshly prepared during the analysis day.

Determination of wavelength maximum (λ _max):

The 5.0 ml of working standard solution of AM (100µg/ml) was transferred into 25 ml calibrated tube containing 0.5 ml of 8.75x10^-1M AcOH and 2.5ml of (5.618x10^-4M) NBS solution. The volume was made up to 10ml with distilled water and kept aside for 15 minutes at room temperature. Then 1.0 ml of 8.71x10^-3M PMAP solution and after 2 minutes 2.0ml of (1.16x10^-2M) SA was added and mixed thoroughly. The volume was made up to 25ml with distilled water. In order to investigate the wavelength maximum, the above solution was scanned in the range of 350-750nm by UV-Visible spectrophotometer. From the spectra (Figure 2), it was concluded that 520nm is the most appropriate wavelength for analyzing AM with suitable sensitivity.

Figure 2: Absorption spectra of AM-NBS-PMAP-SA system

Analysis of Bulk samples:

Aliquots of the standard AM solution ((1.0-5.0ml, 100µg/ml) were transferred into a series of 25ml calibrated tubes containing 0.5ml of 8.75x10^-1M AcOH and 2.5ml of (5.618x10^-4M) NBS solution. The volume was made up to 10ml with distilled water and kept aside for 15 minutes. Then 1.0 ml of 8.71x10^-3M PMAP solution and after 2 minutes 2.0ml of (1.16x10^-2M) SA was added and mixed thoroughly. The volume was made up to 25ml with distilled water. The absorbances were measured against distilled water at 520 nm during the stability of 10-30 min. A blank experiment was also performed omitting drug solution. The decrease in absorbance corresponding to AM drug content was obtained by subtracting the absorbance of the test solution from that of blank solution. The amount of drug in a sample was calculated from its calibration curve (Figure 3).

Figure 3: Beer’s Law plot of AM-NBS-PMAP-SA system

Analysis of Tablets:

Twenty tablets were weighed accurately and powdered and an amount of the tablet powder equivalent to 10mg of AM drug was treated with 8.0ml of glacial acetic acid (warmed on a boiling water-bath for 5 min with occasional shaking). The solution was cooled to room temperature, filtered and made up to 10ml with glacial acetic acid to give a solution of 1 mg/ml. This stock standard solution was further diluted stepwise with distilled water to obtain working standard solution and was analyzed as described in the analysis of bulk samples.

RESULTS AND DISCUSSION:

The optimum conditions for the development of the method was established by varying the parameters one at a time and keeping the others fixed and observing the effect produced on the absorbance of the colored species. The method involves two stages, namely oxidation with excess NBS and the determination of unreacted NBS using PMAP-SA reagent. Oxidation of AM drug with 1.5-3.0ml of NBS solution gave maximum and reproducible absorbance values. The effect of time and temperature of oxidation on the absorbance of the colored species was studied by conducting the oxidation at different temperatures for different time intervals. Oxidation times ranging from 20-30 min at room temperature (28±5^◦C) gave constant and reproducible absorbance values. Prolonging the oxidation time beyond 30 min and increasing the temperature gave erratic results. Maintaining the pH of the solution at 2.9±0.2 was found to be the best for attaining the maximum sensitivity. This was achieved by the addition of 0.5 ml of 3.5x10^-1M acetic acid. Use of 1.0 -2.0 ml of PMAP solution and 1.0 -2.5 ml of SA solution afforded the maximum absorbance values. A waiting period of 1-3 min was necessary between the additions of PMAP SA solutions for the generation of p-N-methyl benzoquinone monoimine (PMBQMI) by the action of NBS on PMAP. Prolonging the waiting period beyond 3 min resulted in low absorbance values, probably owing to the partial hydrolysis of the PMBQMI formed in situ to the quinone state. Among the water miscible solvents examined, water was found to be the best for final dilution of the solution. Maximum color intensity was attained within 10 min after the final dilution and remained stable for the next 20 min. The optical characteristics such as Beer’s law limit, Sandell‘s sensitivity, molar absorptivity, percent relative standard deviation (calculated from the six measurements containing 3/4^th of the amount of the upper Beer’s law limits ) were calculated and the results are summarized in Table-1. Regression characteristics like standard deviation of slope (S_b), standard deviation of intercept (S_a), standard error of estimation (S_e) and % range of error (0.05 and 0.01 confidence limits) were calculated and are shown in Table 1.

AXERT tablets containing AM were successfully analyzed by the proposed method. The values obtained by the proposed and reference methods for formulations were compared statistically by the t-and F-test and found not to differ significantly. As an additional demonstration of accuracy, recovery experiments were performed by adding a fixed amount of the drug to the pre analyzed formulations at three different concentration levels. These results are summarized in Table 2. The interference studies in the determination of AM in pharmaceutical formulation revealed that the normally existing excipients and additives like starch, talc, stearic acid, boric acid, gelatin, magnesium carbonate and sodium lauryl sulphate were found not to interfere even when present in excess (1-100 folds). However, preliminary clean up procedure with CHCl₃ is necessary prior to the estimation of AM in formulations if lactose is present.

TABLE 1: OPTICAL CHARACTERISTICS, PRECISION AND ACCURACY OF PROPOSED METHOD.

Parameter	Values
ג_max(nm)	520
Beer’s law limit(µg/ml)	4-20
Sandell’s sensitivity (µg/cm²/0.001 abs. unit	0.002742857
Molar absorptivity (Litre/mole/cm) Correlation Coefficient	171193.75 0.996
Regression equation (Y)*
Intercept (a)	0.003
Slope(b)	0.014
%RSD**	1.59
% Range of errors (95% Confidence limits)
0.05 significance level	1.67
0.01 significance level	2.62

*Y = a +b x, where Y is the absorbance and x is the concentration of AM in µg/ml

**calculated from six determinations

TABLE 2: ANALYSIS OF ALMOTRIPTAN MALATE IN TABLETS

Method	*Formulations	Labeled Amount (mg)	Found by Proposed Method			Found by Reference Method ± SD	#% Recovery by Proposed Method ± SD
			**Amount found ± SD	t	F
AM-NBS-PMAP-SA	Tablet-1	12.5	12.30±0.08	1.32	3.37	12.44±0.15	98.4±1.01

* Tablet- 1 AXERT tablets of Ortho Mc Nell Pharmaceuticals, USA

**Average ± Standard deviation of six determinations, the t- and f-values refer to comparison of the proposed method with UV reference method. Theoretical values at 95% confidence limits t =2.57 and F = 5.05.

# Recovery of 10mg added to the pre analyzed sample (average of three determinations).

Reference method (reported UV method) using methanol (λ _max=227nm).

Figure 4: probable sequence reactions of the proposed method

Chemistry of colored species:

This method based on the oxidation of AM drug by NBS to form oxidation products (probably a mixture but reproducible under proposed experimental conditions), excess NBS being determined with PMAP-SA reagent. The formation of charge-transfer complex involving two moles of PMBQMI and one mole of sulphanilamide may be represented as π- π* electron transfer and hydrogen bond formation between quinoneimine oxygen and amino hydrogen of sulphanilamide. The probable sequences of reactions in two steps based on analogy are presented in Figure 4.

CONCLUSION:

The reagents utilized in the proposed method are normal cost, readily available and the procedure does not involve any critical reaction conditions or tedious sample preparation. The proposed method possesses reasonable precision, accuracy and is simple, sensitive and can be used as alternative method to the reported ones for the routine determination of AM depending on the need and situation.

ACKNOWLEDGEMENTS:

The authors (BK Ramu & MS Bab) are thanks to the University Grants Commission, New Delhi for providing financial assistance under Teacher fellowship and also thanks to University authorities for providing facilities in this work.

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Received on 10.11.2018 Modified on 10.12.2018

Research J. Science and Tech. 2019; 11(1):77-81.

DOI: 10.5958/2349-2988.2019.00012.3