International Journal of Drug Development and Research

Key words

Anti migraine, Assay, FC reagent, o- phenanthroline,Spectrophotometer, Tablets.

Introduction

Almotriptan malate (AM) (Fig.1) is a selective andpotent serotonin 5-hydroxy trytamine1B/1D (5-HT 1B/1D) receptor agonist. It is chemically designatedas 1[[[3-[2-(Di methyl amine) ethyl]-1H-indol-5-yl]methyl] sulfonyl] pyrrolidine ± - hydroxybutanedioate [1] (1:1). Its empirical formula isC17H25N3O2S.C4H6O5 representing molecular weightof 469.56. It is a white to slightly yellow crystallinepowder that is soluble in water and sparingly solublein methanol. Almotriptan is available in market asconventional tablets (AXERT).The drug is absorbedwell orally, with an absolute bioavailability of around70%. The drug is used to treat severe migraineheadaches and vascular headaches; acute treatmentof migraine attacks with or without aura. The drugbinds with high affinity to 5-HT 1D, 5-HT 1B and 5-HT 1F receptors. Because of the particulardistribution of the 5-HT 1B/1D receptors,almotriptan basically constricts the humanmeningeal arteries; therefore it has a limited effect onarteries supplying blood to the brain and little effecton cardiac and pulmonary vessels. Amelioratemigraine through selective constriction of certainintracranial blood vessels, inhibition of neuro peptiderelease and reduced transmission in trigeminal painpathway.

In literature, several analytical methods such asHPLC [2-3], HPTLC [4], HPLC-MS/MS [5], LC-ESIMS/MS [6], UV Spectrometric [7-8] andFluorometric and Colorimetric [9] have beenreported for the determination of AM in biologicalfluids (considerable more) and formulations (less).Even though there is one visible spectrophotometricmethod using TCNQ reported for the determinationof the drug they are tedious and less specificity andthe functional groups present in the drug not fullyexploited. For routine analysis, simple, rapid and cost effective visible spectrophotometric methods arerequired and preferred. Nevertheless, there stillexists a need for development of sensitive accurateand flexible visible spectrophotometric methods forthe determination of AM in pharmaceuticalpreparations and quality control analysis. So theauthors have made some attempts in this directionand succeeded in developing two methods based onthe reaction between the drug and FC reagent [10-11](M1) or Fe(III)-1,10-phenanthroline [12] (M2) underspecified experimental conditions.

The proposed methods for AM determination havemany advantages over other analytical methods dueto its rapidity, normal cost and environmental safety.Unlike HPLC, HPTLC procedures, the instrument issimple and is not costly. Economically, all theanalytical reagents are inexpensive and available inany analytical laboratory. These methods can beextended for the routine quality control analysis ofpharmaceutical products containing AM.

MATERIALS & METHODS (EXPERIMENTAL)

Apparatus and chemicals

A Milton Roy UV/Visible spectrophotometer model-1201 with 10mm matched quartz cells was used forall spectral measurements. A Systronics digital pHmeter mode-361 was used for pH measurements. Allthe chemicals used were of analytical grade. AXERTtablets procured from Ortho Mc NellPharmaceuticals, USA. Fe (III) solution (Wilson labs,0.05%, 3.32x10-3M prepared by dissolving 50mganhydrous ferric chloride in 100ml of distilled water),PHEN solution (Merck, 0.2%, 1.10x10-2M preparedby dissolving 200g of o-phenanthroline in 100ml ofdistilled water with warming), o-phosphoric acid(Qualigens, 2.0x10-2M prepared by diluting 1.27ml ofo-phosphoric acid to 100ml with distilled water. Tenml of this stock solution was diluted to 100ml withdistilled water) were prepared for method M2 andcommercial available FC reagent (Loba, 2N), 10%Na2CO3 (BDH, 9.43x10-1M) solution was used formethod M1.

Preparation of Standard stock solution: Thestandard stock solution (1mg/ml) of AM wasprepared by dissolving 100mg of AM in 100 mldistilled water. The working standard solutions ofAM were obtained by appropriately diluting thestandard stock solution with the same solvent (M1-100 μg/ml & M2- 50 μg/ml). The prepared stocksolution was stored at 4°C protected from light.From this stock solution, a series of standards werefreshly prepared during the analysis day.

Preparation of Sample solution: About 20tablets were weighed to get the average tablet weightand pulverized. The powder equivalent to 100mg ofAM was weighed, dispersed in 25ml of Isopropylalcohol, sonicated for 15 minutes and filtered throughWhatman filter paper No 41.The filtrate wasevaporated to dryness and the residue was dissolvedas under standard solution preparation.

Determination of wavelength maximum (:max)

Method M1: 3.0ml of Standard AM solution wastransferred into 25ml calibrated tube. To this 2.5mlof FC (2N) reagent was added. After 3 minutes 7ml of10% Na2CO3 was added. The solutions were mixedand kept at room temperature for 30minutes forcomplete color development and diluted to the markwith distilled water. In order to investigate thewavelength maximum, the above colored solutionwas scanned in the range of 400-660 nm UV-Visiblespectrophotometers against a reagent blank. Fromthe absorption spectra (Fig.2), it was concluded that770nm is the most appropriate wavelength foranalyzing AM with suitable sensitivity.

2.5ml of Standard AM solution wastransferred into 25ml calibrated tube. Then 1.5ml ofFecl3 3.32x10-3M solution and 2.0ml of 1,10PHEN(1.10x10-2M) solution were added successively andtotal volume in tube was made to 10.0ml withdistilled water and kept in boiling water bath for 30 minutes. After cooling to room temperature, 2.0ml ofo-phosphoric acid was added and the total volumemade up to 25ml with distilled water. In order toinvestigate the wavelength maximum, the abovecolored solution was scanned in the range of 400-660 nm UV-Visible spectrophotometers against areagent blank. From the absorption spectra (Fig.3), itwas concluded that 510nm is the most appropriatewavelength for analyzing AM with suitablesensitivity.

Preparation of calibration curve

Method M1: Aliquots of Standard AM solution (1.0-3.0ml, 100μg/ml) were transferred into a series of25ml calibrated tubes. To each tube 2.5ml of FC (2N)reagent was added. After 3 minutes 7.0ml of 9.43x10-1M Na2CO3 was added. The solutions were mixed andkept at room temperature for 30minutes forcomplete color development and then diluted to themark with distilled water. The absorbance was measured at 770nm against a reagent blank preparedsimultaneously. The amount of drug was computedfrom its calibration graph (Fig.4).

Method M2:

Aliquots of Standard AM solution (0.5-2.5ml, 50μg/ml) were transferred into a series of 25mlcalibrated tubes. Then 1.5ml of Fecl3 (3.32x10-3M)solution and 2.0ml of 1,10-PHEN (1.10x10-2M)solution were added successively and total volume intube was made to 10.0ml with distilled water andkept in boiling water bath for 30minutes. Aftercooling to room temperature, 2.0ml of o-phosphoricacid was added and the total volume made up to25ml with distilled water. The absorbances of thecolored complex solution were measured after 5minutes before 60minutes at 510nm against thereagent blank prepared similarly. The content of thedrug computed from the appropriate calibrationgraph (fig.5).

RESULTS AND DISCUSSION

Optimum operating conditions used in the procedurewere established by adopting variation of onevariable at a time (OVAT) method. The effect ofvarious parameters such as time, volume andstrength of reagents, the order of addition of reagentsand solvent for final dilution of the colored specieswere studied. In method M1, Na2CO3 preferredamong other bases like NaOH or Pyridine as theywere found to be inferior. Distilled water was foundto be best solvent for final dilution. Other watermiscible solvents like methanol, ethanol, propan-2-oland acetonitrile have no additional advantage inincreasing the intensity of the color in both methods.The optical characteristics such as Beer’s law limit,Sandell‘s sensitivity, molar absorptivity, percentrelative standard deviation, (calculated from the sixmeasurements containing 3/4th of the amount of theupper Beer’s law limits), Regression characteristicslike standard deviation of slope (Sb), standarddeviation of intercept (Sa), standard error ofestimation (Se) and % range of error (0.05 and 0.01confidence limits) were calculated and the results aresummarized in Table-1.

Commercial formulations containing AM weresuccessfully analyzed by the proposed methods. Thevalues obtained by the proposed and referencemethods for formulations were compared statisticallyby the t-and F-test and found not to differsignificantly. As an additional demonstration of accuracy, recovery experiments were performed byadding a fixed amount of the drug to the pre analyzedformulations at three different concentration levels.MS Excel Software-2007 used for calculations andgraphs. These results are summarized in Table-2.

Chemistry of color species

Method M1: The color formation by Folin- Ciocalteureagent with AM may be explained basing on theanalogy with the reports of earlier workers. Themixed acids in the FC preparation are the finalchromogen and involve the following chemicalspecies.

3H2O.P2O5.13WO3.5MoO3.10H2O

And

3H2O.P2O5.14WO3.4MoO3.10H2O

AM probably effects a reduction of the 1,2 or 3oxygen atoms from tungstate and /or molybdate inFC preparation (phosphomolybdo tungstate), therebyproducing one or more several reduced species whichhave characteristic intense blue color.

Method M2: AM drug exhibits reducing propertydue to the presence of functional moieties vulnerableto oxidation selectively with oxidizing agents such asFe (III) under controlled experimental conditions.When treated with known excess of oxidant, AMundergoes oxidation, giving products of oxidation (inclusive of reduced form of oxidant, Fe II from FeIII) besides unreacted oxidant. It is possible toestimate the drug content colorimetrically, which isequivalent to either reacted oxidant or reduced formof oxidant formed. The reduced form of Fe III (i.e. FeII) has a tendency to give a colored complex ontreatment with 1, 10-PTL.

AM + Fe (III)→ oxidation products + Fe (II) + un reacted Fe (III)

Fe (III) + o-phosphoric acid →Complex (un reactive)

Scheme for Method M2

Conclusion

The proposed methods applicable for the assay ofdrug, the advantage of wider range under Beer’s lawlimits, validated as per ICH guide lines and possessreasonable precision, accuracy, and simple, sensitive.

These methods can be extended for the routine assayof AM formulations.

Conflict of Interest

NIL

Source of Support

NONE

Acknowledgements

The authors (MS Bab & BKR) are thanks to theUniversity Grants Commission, New Delhi forproviding financial assistance under teacher fellowship and also thanks to University authorities forproviding necessary facilities in this work.

Tables at a glance

Table 1 Table 2

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

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