Validated Simple and Affordable Visible Spectrophotometric Methods Development for the Assay of Almotriptan malate in Bulk and Pharmaceutical Preparations

 

U. Viplava Prasad¹, M. Syam Bab¹*, B. Kalyana Ramu²

¹Department of Organic Chemistry and Analysis of Foods Drugs and water Laboratories, AU College of Science and Technology, Andhra University, Visakhapatnam -530003 Andhra Pradesh (India)

²Department of Chemistry, Maharajah’s College (Aided and Autonomous), Vizianagaram-535002 (AP) India.

ABSTRACT:

Two simple, sensitive and affordable visible spectrophotometric methods (M₁ and M₂) have been developed for the estimation of Almotriptan malate (AM) in bulk and dosage forms. Method M₁ involves Internal salt formation of aconitic anhydride, dehydration product of citric acid [CIA] with acetic anhydride [Ac₂O] to form colored chromogen with an absorption maximum of 560 nm and the method M₂ is based on the formation of green colored coordination complex by the drug with cobalt thiocyanate which is quantitatively extractable into nitro benzene with an absorption maximum of 620 nm. Beer’s law obeyed in the concentration range of 8-24µg/ml for method M₁ and 20-60 µg/ml for method M₂.  Axert tablets were analyzed and the results are statistically compared with those obtained by the reference UV method and validated by recovery studies. The results are found satisfactory and reproducible. These methods are applied successfully for the estimation of the almotriptan malate in the presence of other ingredients that are usually present in dosage forms. These methods offer the advantages of rapidity, simplicity and sensitivity and normal cost and can be easily applied to resource-poor settings without the need for expensive instrumentation and reagents.    

 

 

 

INTRODUCTION:

Almotriptan malate (AM) (Fig.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 butanedioate¹ (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.

 

In literature, several analytical methods such as HPLC^2-3, HPTLC⁴, HPLC-MS/MS⁵, LC-ESI-MS/MS⁶, UV spectrometric^7-8 and fluorometric and coloricmetric⁹ have been reported for the determination of AM in biological fluids (considerable more) and formulations (less). Even though there is one visible spectrophotometric method using TCNQ reported for the determination of the drug they are tedious and less specificity. Nevertheless, there still exists a need for development of sensitive accurate and flexible visible spectrophotometric methods for the determination of AM in pharmaceutical preparations. The authors have made some attempts in this direction and succeeded in developing two methods based on the reaction between the drug and citric acid-acetic anhydride reagent ¹⁰ (M₁) or drug and cobalt thiocyanate ¹¹ (M₂). These methods can be extended for the routine assay of AM formulations.

 

Fig. 1: Chemical structure of Almotriptan malate

 

MATERIALS AND METHODS :

Apparatus and chemicals:

A Milton Roy UV/Visible spectrophotometer model-1201 with 10mm matched quartz cells was used for all spectral measurements. Systronics model-362 pH meter was used for all the pH measurements.  All the chemicals used were of analytical grade. AXERT tablets procured from Ortho Mc Nell Pharmaceuticals, USA. Citric acid monohydrate (Prepared by dissolving 1.2 grams of (1.2%,  6.245X10^-2M) Citric acid in 5 ml methanol initially followed by dilution up to 100ml with acetic anhydride) and Acetic anhydride (SD Fine chemicals), CTC (2.50x10^-1M, solution prepared by dissolving 7.25 g of cobalt nitrate and 3.8 g of ammonium thiocyanate in 100ml distilled water), Citrate buffer pH(2.0) (prepared by mixing 306ml of 0.1M tri sodium citrate with 694ml of 0.1M HCl and pH was adjusted to 2.0) were prepared . 

 

Preparation of Standard and sample drug stock solution:

 An accurately weighed quantity of AM (pure or tablet powder) equivalent to 100mg was mixed with 5ml of 10% Na₂CO₃ solution and  transferred into 125ml separating funnel. The freebase released was extracted with 3x15ml portion of chloroform and the combined chloroform layer was brought up to 100ml with the same solvent to get 1mg/ml AM drug stock solution in free base form. This free base stock solution was further diluted step wise with the same solvent to get the working standard solution concentrations [M₁-200 µg/ml, M₂-500 µg/ml].

 

Determination of wavelength maximum (λ _max)

Method M₁:

The 3.0 ml of working standard solution of AM (200µg/ml) (free base form) in chloroform was taken in 25ml standard flask and gently evaporated in a boiling water bath to dryness. To this, 10ml of citric acid- Acetic anhydride reagent was added and the tubes were immersed in a boiling water bath for 30 minutes then the tubes were cooled to room temperature and made up to the mark with acetic anhydride and sonicated for 1 min. to get a concentration of 24µg/ml. In order to investigate the wavelength maximum, the above standard stock solution was scanned in the range of 400-660nm by UV-Visible spectrophotometer. From the spectra (Fig.2), it was concluded that 560nm is the most appropriate wavelength for analyzing AM with suitable sensitivity.

 

Fig. 2: Absorption spectra of AM-CIA/AC₂O

 

Method M₂:

The 3.0 ml of working standard solution of AM (500µg/ml) (free base form) in chloroform was taken in 125 ml separating funnel. Then 2.0ml of buffer solution (pH 2.0) and 5.0ml CTC solution were added. The total volume of aqueous phase in each separating funnel was adjusted to 15.0ml with distilled water. To separating funnel 10.0ml of nitrobenzene was added and contents were shaken for 2 minutes, to get a concentration of 60µg/ml. The two phases were allowed to separate In order to investigate the wavelength maximum, the parrot green colored nitro benzene solution was scanned in the range of 400-700nm by UV-Visible spectrophotometer. From the UV spectra (Fig.3), it was concluded that 620nm is the most appropriate wavelength for analyzing AM with suitable sensitivity.

 

Fig. 3: Absorption spectra of AM-CTC

 

Preparation of calibration curve: 

Method M₁:

Aliquots of standard AM drug solution [1.0-3.0ml;200µg/ml in free base form] in chloroform were taken into a series of 25ml graduated tubes and gently evaporated in a boiling water bath to dryness. To this, 10ml of citric acid- Acetic anhydride reagent was added and the tubes were immersed in a boiling water bath for 30 minutes then the tubes were cooled to room temperature and made up to the mark with acetic anhydride. The absorbance of the colored solutions was measured after 15minutes at 560 nm against the reagent blank (within the stability period of 15-60min.The amount of AM was computed from its calibration graph (Fig-4 showing Beer’s law plot).

 

Fig.4: Beer’s Law plot of AM-CIA/Ac₂O

 

Method M₂:

Aliquots of standard AM solution (1.0ml - 3.0ml, 500µg/ml in free base form) were delivered into a series of 125ml separating funnels. Then 2.0ml of buffer solution (pH 2.0) and 5.0ml CTC solution were added. The total volume of aqueous phase in each separating funnel was adjusted to 15.0ml with distilled water. To each separating funnel 10.0ml of nitrobenzene was added and contents were shaken for 2 minutes. The two phases were allowed to separate and absorbance of nitrobenzene layer was measured at 620nm against a similar reagent blank .The colored product was stable for 1 hour. The amount of AM in the sample solution was computed from its calibration graph (Fig-5 showing Beer’s law plot).

 

Fig.5: Beer’s Law plot of AM-CTC

RESULTS AND DISCUSSION:

In developing these methods, systematic studies of the effects of various parameters were undertaken by varying one parameter at a time and controlling all others fixed (OVAT method). The effect of various parameters such as time, volume and strength of reagents, pH buffer solution and order of addition of reagents, stability period and solvent for final dilution of the colored species were studied and the optimum conditions were established. Among the various water immiscible organic solvents (C₆H₆, CHCl₃, dichloro methane, nitro benzene, chloro benzene and CCl₄) tested for the extraction of colored coordinate complex into organic layer, nitrobenzene was preferred for selective extraction of colored complex from organic phase in method M₂. Different solvents like acetic anhydride, acetic acid, methanol, ethanol and isopropanol were also used as diluents but acetic anhydride was found to be ideal for final dilution in method M₁. The ratio of organic to aqueous phase was found to be 1:1.5 by slope ratio method for method M₂. 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. 

 

Commercial formulations containing AM were successfully analyzed by the proposed methods. 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.

 

Table - 1 Optical characteristics, precision and accuracy of the proposed methods

Parameters	Method A	Method B
λ max(nm)	560	620
Beer’s law limit (µg/ml)	8- 24	20-60
Sandell’s sensitivity (µg/cm2/0.001 abs. unit)	0.0025	0.00569395
Molar absorptivity (Litre/mole/cm)	187824	82466.475
Regression equation        (Y) *= a +b x
Intercept (a)	-0.089	-0.083
Slope(b)	0.022	0.09
%RSD	1.399	0.95
% Range of errors (95% Confidence  limits) 0.05 significance level 0.01 significance level	1.469	0.997
	2.30	1.56

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

 

 

Table-2 Analysis of AM in pharmaceutical formulations

Method	*Formulations	Labeled Amount (mg)	Found by Proposed Methods			Found by Reference Method  ± SD	#% Recovery by Proposed Method ± SD
			**Amount found ± SD	t	F
A	Tablet-1	6.25	6.21±0.027	0.361	1.522	6.21±0.034	99.30±0.44
	Tablet-2	12.5	12.54±0.093	0.70	2.64	12.44±0.15	100.30±0.74
B	Tablet-1	6.25	6.23±0.044	1.25	1.69	6.21±0.034	99.69 ± 0.703
	Tablet-2	12.5	12.43  ± 0.229	2.09	2.31	12.44±0.15	99.97 ± 0.94

* Tablet- 1 and Tablet-2: 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).

 

 

Recovery experiments indicated the absence of interference from the commonly encountered pharmaceutical excipients present in formulations. The proposed methods are found to be simple, sensitive and accurate and can be used for the routine quality control analysis of AM in bulk and dosage forms.  

 

Chemistry of colored species

In method M₂ the green color species formation is the coordination complex of the drug (electron donor) and the central metal of cobalt thiocyanate, which is extractable into nitro benzene from aqueous solution and in method M₁ red-violet color internal salt of aconitic anhydride is formed when AM was treated with CTC or CIA/Ac₂O reagents. The formations of colored species are due to the presence of the tertiary amino group in it. It is based on the analogy of tertiary amine as given in scheme (Fig-6).

 

 

 

 

Fig. 6: Probable Schemes for methods M₁ and M_2.

CONCLUSION: 

The reagents utilized in the proposed methods are normal cost, readily available and the procedures do not involve any critical reaction conditions or tedious sample preparation. The proposed visible spectrophotometric methods are validated as per ICH guide lines and  possess reasonable precision, accuracy, simple, sensitive and can be used as alternative methods to the reported ones for the routine determination of AM depending on the need and situation. 

 

ACKNOWLEDGEMENT:

The authors (MS Bab and BKR) are thanks to the University Grants Commission, New Delhi for providing financial assistance under teacher fellow ship and also thanks to University authorities for providing necessary facilities in this work.

 

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Received on 24.04.2012        Modified on 12.05.2012

Accepted on 24.05.2012        © AJRC All right reserved