Spectrophotometric Determination of Amodiaquine

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z azotanem(III) sodu, a następnie z β-naftolem, w wyniku której powstaje barwny produkt. Maksima absorpcji występuj¹ odpowiednio przy 505 nm i 470 nm, ...

Chem. Anal. (Warsaw), 53, 305 (2008)

Spectrophotometric Determination of Amodiaquine and Sulfadoxine in Pharmaceutical Preparations by Muhammad Tayyab Ansari1, Tariq Mahmood Ansari2, Asad Raza2*, Muhammad Ashraf2 and Muhammad Yar2 1

Department of Pharmacy, Bahauddin Zakariya University, Multan, 60800, Pakistan 2 Department of Chemistry, Bahauddin Zakariya University, Multan, 60800, Pakistan


Amodiaquine; Sulfadoxine; Spectrophotometric determinations; Dosage forms

A new rapid and sensitive spectrophotometric method was developed for the determination of amodiaquine and sulfadoxine in both pure and dosage forms. The proposed method is based upon the reaction of amino group with sodium nitrite followed by β-naphthol that produces a colored compound. These species exhibit the maximum absorption at 505 nm for amodiaquine and at 470 nm for sulfadoxine with molar absorptivity of 6.83 × 104 and 3.74 × 104 L mol–1 cm–1 respectively. The range of linearity for the both drugs is 4–60 µg mL–1. The optimum reaction conditions and other analytical parameters have also been investigated. The influence of substrates commonly employed as excipients with these drugs have been studied. The proposed method is applicable for the determination of these drugs in pharmaceutical formulations. The results demonstrated that the method is accurate, reproducible and comparable to the official method. Opracowano now¹ szybk¹ i czu³¹ spektrofotometryczn¹ metodê oznaczania amodiachiny i sulfadioksyny w czystej postaci i w lekach. Metoda polega na reakcji grupy aminowej z azotanem(III) sodu, a nastêpnie z β-naftolem, w wyniku której powstaje barwny produkt. Maksima absorpcji wystêpuj¹ odpowiednio przy 505 nm i 470 nm, a wspó³czynniki absorpcji wynosz¹ odpowiednio: 6,83 × 104 i 3,74 × 104 L mol–1 cm–1 dla amodiachiny i sulfadioksyny. W przypadku obu leków zakres stosowalnoœci prawa Beera wynosi od 4 do 60 µg mL–1. Zbadano optymalne warunki oznaczenia i wp³yw substancji mog¹cych wystêpowaæ w lekach. Metoda mo¿e byæ stosowana do oznaczania leków w preparatach farmaceutycznych. Otrzymane wyniki wskazuj¹, ¿e metoda jest dok³adna, odtwarzalna i porównywalna z oficjalnie stosowanymi metodami.

* Corresponding author. E-mail: [email protected]


M.T. Ansari, T.M. Ansari, A. Raza, M. Ashraf and M. Yar

Counterfeiting of pharmaceuticals and proliferation of substandard drugs is one of the fastest growing economic crimes threatening both the developed and developing world alike. The United States Food and Drug Administration estimates that counterfeits make up more than 10% of the global medicines market and are present in both industrialized and developing countries. It is estimated that up to 25% of the medicines consumed in poor countries are counterfeit or substandard. The World Health Organization (WHO) figures suggest that developing countries account for around 60% of all reported cases of counterfeit and substandard drugs [1]. A recent WHO survey of the equality of antimalarial preparations in seven African countries revealed that up to 90% of sulphadoxine/pyrimethamine tablets were substandard. 33% of amodiaquine samples were found to be substandard in Africa [2]. The WHO estimates that around 200,000 annual malaria deaths could be avoided if medicines were of high enough quality to actually treat the illness [3]. For quantitative estimation of antimalarial drugs, methodology, time, cost and instrumentation is substantial while elimination of counterfeits demand rapid and low cost field testing methods. A few reports are available on comparative rapid analytical methods e.g. color reaction followed by semi-quantitative thin layer chromatography for chloroquine, amodiaquine, quinine, mefloquine and proguanil etc. [4]. Amodiaquine Hydrochloride, chemically 4-[(7-chloro-4-quinolyl)amino]-2-[(diethylamino)methyl] phenol dihydrochloride dehydrate, is a 4-aminoquinoline derivative similar to chloroquine in structure and activity. It has been used as both an antimalarial and an anti-inflammatory agent. Amodiaquine is an antimalarial with high schizonticidal activity. Amodiaquine is at least as effective as chloroquine, and is effective against some chloroquine-resistant strains. It was formerly used as a prophylactic agent against malaria but lost its importance due to the risk of development of agranulocytosis and hepatic disorders. For those reasons, the usefulness of amodiaquine for treatment of malaria remained limited. However, recently, amodiaquine received renewed interest since its safety and efficacy have been demonstrated in several clinical trials [5–7]. It is effective against the erythrocytic stages of all four species (falciparum, vivax, ovale, malariae) of plasmodium [8]. Sulfadoxine is 4-amino-N-(5,6-dimethoxypyrimidine-4-yl)benzene sulfonamide. A long acting sulfonamide that is used, usually in combination with other drugs, for respiratory, urinary tract, and malarial infections. Sulfadoxine has microbial activity similar to that of sulfadiazide. Its principal use, however, is in the prophylaxis or suppression of malaria caused by chloroquine- resistant p. falciparum [9]. A survey of literature reveals that there are very few methods available for determination of amodiaquine including HPLC [10, 11], spectrophotometric [12–14], conductometric [15] and PVC membrane ion-selective electrode [16]. Similarly a few methods have been developed for the determination of Sulfadoxine including deriva-

Spectrophotometric determination of amodiaquine and sulfadoxine


tive spectroscopy, multi-wavelength spectroscopy [17, 18], refractometric [19] and spectrophotometric methods [20–22]. Most of the spectrophotometric methods reported suffer from the disadvantages of narrow range of determination, long duration for the completion of reaction, use of non-aqueous system, need of heating or extraction, stability of the colored product formed etc. The objective of this study was to develop a sensitive spectrophotometric method to rapidly assess the quality of commercially available tablets containing amodiaquine or sulfadoxine.

EXPERIMENTAL Chemicals and reagents All chemicals used were of analytical reagent grade without further purification. Prepared solutions were: 0.1 mol L–1 sodium nitrite (Fluka), 2 mol L–1 sodium hydroxide (Fluka) and 2 mol L–1 hydrochloric acid (Riedel). 0.1 mol L–1 of β-naphthol (Merck) was prepared in 2 mol L–1 sodium hydroxide. Deionised water was used throughout this work. Equipment UV–VIS spectrophotometer (Model 6305, Jenway, UK) with 1 cm matched quartz cells was used for all absorbance measurements. Standard solutions Stock standard solutions of amodiaquine (Parke–Davis, Pakistan) and sulfadoxine (Roche (Pvt.) Ltd Karachi, Pakistan) were prepared by dissolving powdered drugs (0.1 mg mL–1) in 2 mol L–1 HCl solution. Working solutions were prepared by diluting the stock standard solutions. Analytical procedure Standards. An aliquot of sample containing 4–60 µg mL–1 of amodiaquine or sulfadoxine was transferred to a series of 25 mL measuring flasks. 1 mL of 0.1 mol L–1 sodium nitrite solution was added and kept the mixture for two minutes to form diazonium salt. Then 1 mL of 0.1 mol L–1 β-naphthol solution was added and shaken for two minutes until the complete color development was attained. The contents were diluted up to 25 mL with 2 mol L–1 HCl solution and absorbance was measured at 505 nm for amodiaquine and 470 nm for sulfadoxine respectively against the corresponding blank reagents after 10 min at room temperature (25°C). The calibration graph was drawn and regression equation was applied. Procedure for the assay of the drugs in commercial samples. An accurately weighed amount of commercial sample (powdered contents of 20 tablets, equivalent to 20 µg mL–1 of the respective drug) was dissolved in 2 mol L–1 HCl solution in a 100 mL calibrated flask, diluted the solution up to the mark with 2 mol L–1 HCl and filtered. Appropriate aliquots of the drug solutions were taken and determined the drug content using the same procedure as mentioned above.


M.T. Ansari, T.M. Ansari, A. Raza, M. Ashraf and M. Yar

RESULTS AND DISCUSSION Different analytical parameters i.e. wavelength of maximum absorption (λmax), pH, effect of temperature, color stability studies and measuring range were investigated. Spectral characteristics Amodiaquine and sulfadoxine were diazotized and coupled with β-naphthol in acidic medium to form the colored products of λmax equal to 505 nm and 470 nm respectively. These wavelengths were used for all the absorbance measurements. The corresponding reagent blanks showed negligible absorbance at these wavelengths practically. The unknown concentration of the drug was calculated by knowing the absorbance at the λmax using the regression equation. pH and effect of temperature The pH of the system was found to be 2 that can be attributed to 2 mol L–1 HCl solutions, which was used as a solvent. The diazotization of the studied drugs was completed in 2 min at room temperature (25°C). When effect of temperature was studied on reaction of drugs (amodiaquine and sulfadoxine) with sodium nitrite and β-naphthol, a high color intensity was produced within 5 min at 20–50°C while it took longer times for complete color development at other temperatures. Color stability of the system When color stability of the system was investigated, it was found that the developed color remained constant for one hour at 25–45°C. After this time, the color intensity decreased slowly. One hour duration is sufficient for an analytical chemist to carry out absorbance measurements. Optical characteristics and statistical data for the regression equation of the proposed method are shown in Table 1.

Spectrophotometric determination of amodiaquine and sulfadoxine

Table 1.


Optical characteristics and statistical data for the regression equation of the proposed method




λmax (nm)



Beer’s law verification range (µg mL–1)



Molar absorptivity (L mol–1 cm–1)

6.8 × 104

3.7 × 104

Sandell’s sensitivity (µg mL–1 per 0.001 A)

5.2 × 10–3

8.3 × 10–3

Slope (b)

2.4 × 10–3

2.1 × 10–3

Intercept (a)

3.8 × 10–3

4.1 × 10–3

Correlation coefficient (r)



Limit of detection (µg mL–1)



Regression equation (Y)*

* Y = a + bC, where C is the concentration of analyte (µg mL–1) and Y is absorbance.

Quantification and reaction sequence Under the given experimental conditions, a linear calibration graph was obtained and Beer’s law was verified in the concentration range 4–60 µg mL–1 for both investigated drugs. The development of spectrophotometric method for the determination of amodiaquine and sulfadoxine is based on the formation of nitrosonium salt. It was noted that secondary aromatic amine in amodiaquine and primary aromatic amine in sulfadoxine were responsible to react with sodium nitrite to form nitrosonium ion which thereafter reacts with color producing reagent i.e. β-naphthol to produce color. Literature indicate that this color reaction has never been exploited as basis for spectrophotometric determination of these drugs. Interferences The problem of interference did not arise in the analysis of commercially available products. When the effects of additives associated with amodiaquine and sulfadoxine in their formulations were investigated using our developed methods, no interference from common excipients and other substances was observed. In addition, the percentage recovery of the drugs was substantial i.e. ranged from 98–99% (Tab. 2). Other amines such as morphine, aniline, etc. showed positive reactions under the same diazotization reaction conditions.


Table 2.


M.T. Ansari, T.M. Ansari, A. Raza, M. Ashraf and M. Yar

Recovery of amodiaquine and sulfadoxine from various excipients by the proposed method

Average of five determinations.

Applications The applicability of method for the assay of pharmaceutical preparations was examined by applying the proposed procedure to commercially available tablet formulations of amodiaquine and sulfadoxine. The results have been found to be comparable with the reference method (Tab. 3). Table 3.


Assay of amodiaquine and sulfadoxine in various dosage forms by proposed and reference method [23]

Average of five determinations.

Spectrophotometric determination of amodiaquine and sulfadoxine


The reproducibility of the method was checked by five replicate determinations at the 30 µg mL–1 level of amodiaquine and sulfadoxine. The assay of available tablets by our method was highly reproducible which was cross checked by the reference method [23]. Relative standard deviation was found to be in the range 0.5 and 1.6%. The proposed method is applicable only for the determination of amodiaquine and sulfadoxine drugs in pure and dosage forms for quality control purposes. It can not be used for the determination of these drugs in the clinical samples i.e. blood and urine because of presence of various metabolites containing amino groups in these samples. The robustness of the method is that it does not involve extraction. Different analysts determined these drugs in different laboratories using the proposed method. They found that the results of recovery and reproducibility justified the ruggedness of the proposed method. The comparison among proposed and existing spectrophotometric methods shows that the developed method is more sensitive and involve the reagents which are easily available as well as the chemistry of these reagents is already well established (Tab. 4). Table 4.

Comparison of UV–VIS spectrophotometric methods for the determination of amodiaquine and sulfadoxine


M.T. Ansari, T.M. Ansari, A. Raza, M. Ashraf and M. Yar

CONCLUSION The present methodology is found to be economical and more sensitive than the few reported spectrophotometric methods. The statistical parameters and the percentage recovery data clearly indicate the reproducibility and accuracy of the method. The analysis of real samples containing amodiaquine and sulfadoxine showed no interference from common excipients and additives. Hence, these methods could be considered for the determination of amodiaquine and sulfadoxine in pharmaceutical preparations. Acknowledgement Authors are thankful to the Department of Chemistry and the Department of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan and the Bio Fine Pharmaceutical (Pvt.) Ltd. Multan, Pakistan for providing the necessary research facilities to carry out this work.

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Spectrophotometric determination of amodiaquine and sulfadoxine


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Received October 2007 Revised January 2008 Accepted February 2008