The Use of Charge-Transfer Complexation in The Spectrophotometric Determination of Amlodipine Besylate

A simple and sensitive analytical method has been developed for the spectrophotometric assay of amlodipine besylate (ADB) in pure forms and tablets have been described. The method is based on the formation of a charge-transfer complex between the drug and tetrachloquinone (TCQ). This complex exhibit intense absorption bands in the electronic spectrum. The molecular ratio of the reactant in the complex was established and the experimental conditions leading to maximum charge-transfer band was also studied. The reaction proceeds quantitatively at pH 9 and 55°C for 10 min, the absorbance was measured at 346 nm. The method was applied to commercially available tablets and the results were statistically compared wrth those obtained by UV spectrophotometric method, using Newman-Keuls tests. In our method, Beer's Law limits to 5-25 @ml. The optimum experimental parameters for colour production with reagent were studied and incorporated into procedure.

It has been determined in human plasma by I-IPLC [6] and GC [7,8,9].Only two spectrophotometric methods have been reported so far for the determination of ADB [lo, 1 11.But the charge-transfer complexes with amlodipine had not been occurred in these methods which reported.
Charge-transfer complex forming reactions used in the determination of electro-donating basic compounds through interaction with &acceptors [12-181 or IT-acceptors [19-2 I], offered sensitive, accurate and easy to carry out analytical methods.This paper reports a simple and sensitive spectrophotometric method for the ADB in tablets.The method involves the formation of a charge-transfer complex (CTC) between the drug and chloranil (TCQ).

Apparatus
Double beam spectrophotometer ( Schimadzu 160 A ) with I -cm quartz cuvettes was used.
Tacussel TS 70 N-1 was used for the pH measurements.

Chemicals
Amlodipine Besylate was kindly provided by Pfizer, ~stanbul, Turkey and was used without prior purification, TCQ and other chemicals were purchased from Merck.All chemicals used were of analytical reagent grade quality.

Reagent Solution
TCQ, saturated solution in ethanol.Freshly prepared.
The Use of Charge-Transfer Complexation in the Spectrophotometric Determination ... 237

Stock Solution of Drug
Prepared by weighing accurately 25 mg of the drug into a 100-ml volumetric flask, dissolving in absolute ethano1:water (1: 10) mixture and diluting to volume with distilled water.

Assay procedure for dosage forms 2.3.1 Tablets
Twenty tablets of ADB were weighed and pulverized to a fine powder.An aliquot equivalent to about 25 mg of ADB is transferred in 50 ml flasks.A suspension with 10 mL of ethanol and 10,O ml water is shaken for 2 min and filtered to a 100 ml volumetric flask.The first flask is rinsed w f i small portions of water which are transferred through the same filter paper to the volumetric flask to obtain a solution of 100 ml.

Recommended Procedure For Pure Forms Calibration Curves
0,2-1,O ml standard solution has been transferred to test tubes.1,O ml buffer solution and 1,O ml of chloranil solution are added to each tube.The tubes were maintained at 55°C in a water hath for 10 min.The absorbance of the resulting solution was measured at 346 nm against a reagent blank.
In this work, it has been studied that the TCQ with amlodipin at pH= 7,8,9,10 and 11 reads or not.
Coloured complex formation has been observed only at pH=9 and 10 with the studied effective substance concentration.At the both pH condition reasonable relative standart deviation results were obtained.Since maximum absorbtton values were obtained at pH=9, the experiments were decided to be carried out at that pH (Table 1).
Table 1 The pH effect to formation of CTC Coloured complex formation occurs at room temperature therefore the equilibrium time of the reaction was investigated.In order to determine the equilibrium time, the colour insenties after 10-20-30-40 minutes were compared to each other (Table 2).To determine the effect of heat on the reaction rate, the the colour intensities of series at 55°C 5-10-15 min and at 65°C 5 min, were also compared (Table 2).The maximum absorbance were obtained by heating for 10 min at 55°C (A= 0, 5 14).The experiment at room temperature showed very close absorbance to the maximum values (A= 0,506).The followed experiments gave the results in very short time therefore the optimum conditions were determined by heating 10 rnin at 5S°C.The determined conditions were applied to the serial which forms coloured complex in 30 min at room temperature.
In order to determine the buffer amount to keep pH constant, buffer solution of 0.5, 1.0, 1.5, 2.0, 2.5 ml were added respectively and the results were compared.It has been obsorved that the series with 0.5 and 2.5 rnl of buffer solution do not show colour reaction (Table 3).
Table 3 The effect of added buffer solution (pH=9) to colour formation In order to determine the reagent amount required for optimum colour formation, saturated chloranil solution of 0.5,1.0,1.5, 2.0,2.5 ml were added and the results were compared (Table 4).

Table 4
The effect of reagent amount to colour formation The series with 0.5 and 2.5 ml of reagent showed no colour reaction.It has been thought that addition of 0.5 ml of reagent gives unsufficient reagent concentration to form complex.It could not be explained not ot form complex by addmg 2.5 ml of reagent.The relative standart deviations of the series added 1.0, 1.5 and 2.0 ml of reagent found very close to each other.Comparison of the absorbance values of the series showed that addxtion of 1.0 ml of reagent was enough to form complex.
Having determined the optimum conditions, the series obtained at room temperature for 30 rnin and at 55°C for 10 min were also investigated in forms of colour stability (Table 5).The Use of Charge-Transfer Complexation in the Spectrophotometric Determination ... 241 The experiment carried out at room temperature showed no colour change in first 30 rnin but in second 36 min 1.9% increase has been observed in the colour intensity.The colour of the serial obtained by heating at 55°C has been constant for 60 min.Consequently under the both conditions colour has been within the acceptable error limits for 60 min.Depending on this results, it has been decided that the chloranil method can be applied on larger sample series.
Following the stability tests, calibration curves have been plotted by using 5-25 pglml amlodipine added standard solution at room temperature and 55"C, respectively (Table 6).The equations obtained by least square method are as follows: For 20°C, 30 min y=3.8. 1 O -~X -5 .8 ~1 o -~ ( ~0 .9 9 9 9 ) For 55"C, 10min y=3.8.10~~x+2.3~10"( ~0 .9 9 9 2 ) These show that the method has given correct results for both conditions with 5-25 M m l amlodipine.It has been decided that plotting calibration curves for each experiment was necessary due to the colour reactions were effected by condition of media.
Molar absorbtivitiy of colour has been calculated as follow (Table 7).The method has been applied to the samples containing 10 mg amlodipine and the results (Table 8) were compared using Newman-Keuls test [24] as statisties by spectrophotometric method supplied by Pfizer company (Table 9).The Use of Charge-Transfer Complexation in the Spectrophotometric Determination ... 243 Table 9 Comparative studies for amlodipine dosage forms * * Meaningful difference for p = 0.0 1 .
There has been no significant difference at pH= 0.05 level between the results of serial forming coloured complex by heating at 55OC and the results of serial using W method. Depending on this, it has been decided that this method can be applied safely on determination of amlodipine by forming wloured charge-transfer complex at 55OC.The serial studied at room temperature showed significant difference at p=0.01 level with both those at 55°C and those used UV method.In this case it has been decided that the colour forming at room temperature will not use to determination of arnlodipine.

Chemistry of Coloured Species:
In this method, the formation of charge-transfer complex is based on the basic nature of the drug (ADB), which under specified experimental conditions forms a charge-transfer complex with the tetrachloroquinone (TCQ).The stoichiometric balance between ADB and TCQ was investigated using Job's Continous Varitation method [25].The results obtained showed that 1:2 complexes were formed between ADB and TCQ.The structural representation of the complex is shown in Scheme 1. Fig. 1 shows the W-VIS absorbtion spectra of ADB-TCQ charge-transfer complexes.

Conclusions
Amlodipine possesses a primary amine group on the side-chain attached to the dihyropyridine ring.
Colorimetric assay method based on the reaction of the amine group with chloranil was developed.
Chloranil gives with amlodipine a colored charge-transfer complexe, if heated 55°C for 10 rnin.

The
Use of Charge-Transfer Complexation in the Spectrophotometric Determination ... 239

A. G 6 1
G u et al.:

Table 2
The effect of heat and heating time to colour formation