Spectrophotometric determination of amoxicillin by

Analytica Chimica Acta 554 (2005) 184–189

Spectrophotometric determination of amoxicillin by reaction with N,N-dimethyl-p-phenylenediamine and potassium hexacyanoferrate(III) Mouayed Q. Al-Abachi, Hind Haddi, Anas M. Al-Abachi ∗ Chemistry Department, College of Science, Baghdad University, Al-Jaderia, Baghdad, Iraq Received 22 May 2005; received in revised form 3 August 2005; accepted 15 August 2005 Available online 19 September 2005

Abstract A batch and flow injection analysis (FIA) spectrophotometric methods have been developed for the determination of amoxicillin (AMX) in aqueous solution and in pharmaceutical preparations. The methods are based on the reaction of AMX with N,N-dimethyl-p-phenylenediamine in the presence of potassium hexacyanoferrate(III) in alkaline medium. The water soluble blue colour product was measured at λmax 660 nm. Linearity was observed from 20 to 400 and 100 to 700 ␮g AMX in a final volume of 10 ml (i.e. 2–40 and 10–700 ␮g ml−1 AMX) with detection limits of 0.637 and 4.90 ␮g ml−1 AMX by batch and FIA procedure respectively. The effect of chemical and physical parameters have been carefully considered and the proposed procedures were successfully applied to the determination of AMX in pharmaceutical formulations. © 2005 Elsevier B.V. All rights reserved. Keywords: Spectrophotometric; Amoxicillin; Potassium hexacyanoferrate(III); Flow injection analysis

1. Introduction Amoxicillin (AMX),6-(p-hydroxy-␣-amino phenyl acetamido) penicillanic acid, is the only phenolic penicillin, and it is used as an antibacterial drug [1]. The B.p. recommended a liquid chromatography (LC) method for the determination of AMX in raw material and a spectrophotometric method using imidazol-mercury reagent for its determination in capsules and injections [2]. The therapeutic importance of AMX required the development of sensitive and rapid method for industrial quality control and clinical monitoring. A review of the literature revealed that many methods have been described for its determination in pharmaceutical formulation and biological fluids. They include titrimetry [3], LC [4,5], spectrophotometry [6,7], polarography [8] and fluorometry [9]. Oxidative coupling organic reactions seems to be one of the most popular FIA spectrophotometric methods for the determination of several drugs such as sulphonamide [10], paracetamol

∗

Corresponding author. Tel.: +96417758996. E-mail address: anas [email protected] (A.M. Al-Abachi).

0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.08.030

[11], phenylephrine HCL [12], methyl dopa [13], folic acid [14] and catecholamine drugs [15]. In the present paper, an automated procedures are proposed for the spectrophotometric determination of AMX by reaction with N,N-dimethyl-p-phenyenediamine (DMPD) in the presence of potassium hexacyanoferrate(III) in alkaline medium. The reaction can be carried out in batch and in FIA and in this paper the two approaches are compared. The reaction product has been spectrophotometrically measured at 660 nm. 1.1. Reaction mechanism of the method Amoxicillin forms a blue-coloured product (λmax of 660 nm with a molar absorption coefficient of 9537 l mole−1 cm−1 ) with DMPD in the presence of potassium hexacyanoferrate(III) in alkaline medium. The absorption spectra of the coloured product is given in Fig. 1. Under the reaction conditions, DMPD, upon oxidation with potassium hexacyanoferrate(III) loses two electrons and one proton, forming an electrophilic intermediate(II), which is an active coupling species. The intermediate of DMPD undergoes electrophilic substitution with the phenolic moieties of AMX to form a coloured product(III) according to Scheme 1.

M.Q. Al-Abachi et al. / Analytica Chimica Acta 554 (2005) 184–189

185

Scheme 1. Proposed mechanism of the reaction between AMX and DMPD.

Channel A was used to transport DMPD, channel B to transport potassium hexacyanoferrate(III) and channel C to transport ammonium hydroxide solution. The sample was injected into the stream of the mixture of DMPD with potassium hexacyanoferrate(III) solution, through the injection valve. Solutions were propelled by peristaltic pump with individual flow rate of 1.5 ml min−1 . The absorbance was measured at 660 nm. 2.2. Reagent and materials Fig. 1. Absorption spectra of A (50 ␮g ml−1 ) of AMX treated as described under procedure and measured against reagent blank and B the reagent blank measured against distilled water.

2. Experimental 2.1. Apparatus All spectral and absorbance measurements were carried out on a Shimadzu UV–vis 260 digital double beam recording spectrophotometer. A flow cell with 50 ␮l internal volume and 1 cm bath length was used for the absorbance measurements. A three-channel manifold (Fig. 2) was employed for the FIA spectrophotometric determination of AMX drug. A peristaltic pump (Ismatec, Labortechnik – Analytik, CH – 8152, Glatbrugg – Zurich – Switzerland) was used to transport the carries solutions. (Rheodyne, Altex 210, Supelco – USA) injection valve was employed to provide appropriate injection volumes of standard solutions and samples. Flexible vinyl tubing of 0.5 mm internal diameter was used for the peristaltic pump. Reaction coil (RC) was of Teflon with internal diameter of 0.5 mm.

Analytical reagent grade chemicals and distilled water were used throughout. Pure amoxicillin drug sample was kindly provided from state company for Drug Industries and Medical Appliance, SDI, Samara, Iraq. Dosage forms were obtained from commercial sources. DMPD (BDH) 10 mM aqueous solution was prepared daily. Potassium hexacyanoferrate(III), (Fluka) 20 mM solution. More dilute solutions were prepared by suitable dilutions with distilled water.

Fig. 2. Manifold employed for FIA-Spectrophotometric determination of amoxicillin by reaction with DMPD and potassium hexacyanoferrate(III), where: IV, injection valve; Rc, reaction coil; S, sample; P, peristaltic pump; FC, flow cell; D, detector; W, waste.


186

Table 1 Analytical features of the procedures developed for the determination of AMX

3. Results and discussion

Parameter

Batch procedure

FIA procedure

Regression equation Linear range (␮g ml−1 ) Correlation coefficient Limit of detection (s/n = 3) (␮g ml−1 ) Reproducibility (%) for 10 ␮g ml−1 Recovery (%) for 10 ␮g ml−1 Through-put (h−1 )

Y = 0.0201X + 0.0794 2–40 0.9991 0.637

Y = 0.002X + 0.0006 10–700 0.9992 4.90

The factors affecting on the sensitivity and stability of the coloured product resulting from the oxidative coupling reaction of AMX with DMPD and potassium hexacyanoferrate(III) in alkaline medium were carefully studied. The blue dye product was only formed in alkaline medium, therefore, the effect of different alkaline solutions were studied such as sodium acetate, sodium carbonate, sodium hydroxide and ammonium hydroxide. Maximum sensitivity and stability were obtained only when the reaction was carried out in the presence of ammonium hydroxide solution.

a

0.46

0.56a

99.93

100.50a

6

120

For 50 ␮g ml−1 AMX.

3.1. Batch spectrophotometric determination 2.3. Preparation of standard solutions A 1 mg ml−1 stock solution of AMX was prepared in distilled water. Serial dilutions with distilled water were made to cover the working range (Table 1). 2.4. Procedure 2.4.1. General batch procedure An aliquol of sample containing 20–400 ␮g of AMX was transfered into a series of 10 ml standard flasks. A volume of 0.8 ml of 5 mM DMPD solution, 0.8 ml of 10 mM of potassium hexacyanoferrate(III) and 0.5 ml of 50 mM ammonium hydroxide solution were added. The contents of the flasks were diluted to the mark with distilled water, mixed well and left for 15 min. The absorbance was measured at 660 nm (at room temperature 25 ◦ C) against reagent blank containing all materials except AMX. A calibration graph was drawn and the regression equation calculated. For the optimization of conditions and in all subsequent experiments, a solution of 300 ␮g was used and the final volume was 10 ml. 2.4.2. General FIA procedure Working solutions of AMX in the range cited in Table 1 were prepared from stock solutions. A 100 ␮l portion of AMX was injected into the stream of the mixture of 5 mM DMPD and 10 mM potassium hexacyanoferrate(III) solution and was then combined with a stream of 20 mM ammonium hydroxide with a flow rate of 1.5 ml min−1 in each channel (Fig. 2). The resulting absorbance of the blue dye was measured at 660 nm and a calibration graph was prepared over the range cited in Table 1. Optimization of conditions were carried out on 50 ␮g ml−1 of AMX. 2.4.3. Procedure for capsules and injections An accurately weighed amount of 10 powdered capsules or mixed content of 10 vials equivalent to 100 mg of the pure drug, was transfered into a 100 ml calibrated flask and completed to the mark with distilled water. The flask with its contents was shaked well and filtered. A sample of 400 ␮g of AMX in a final volume of 10 ml was taken and the measurement was carried out as described earlier under general procedure.

The best experimental conditions for the determination of AMX were established for DMPD (from 0.05 to 1 mM), potassium hexacyanoferrate(III) (from 0.1 to 2.5 mM) and ammonium hydroxide (from 0.5 to 5 mM) by altering one variable at a time and measuring the absorbance at 660 nm. The obtained results show that 0.4 mM DMPD, 0.8 mM of potassium hexacyanoferrate(III) and 2.5 mM of ammonium hydroxide are the concentrations that can give a higher absorption intensity and stability of the dye product at 660 nm for 300 ␮g of AMX in a final volume of 10 ml (i.e. 30 ␮g ml−1 ). The development of the colour of AMX from a mixture containing 30 ␮g ml−1 in 0.4 mM DMPD, 0.8 mM potassium hexacyanoferrate(III) and 2.5 mM ammonium hydroxide gave evidence that the colour develops immediately and remains stable for more than 120 min. The effect of temperature on the colour intensity of the dye was studied. In practice, high absorbance was obtained when the colour was developed at room temperature (25 ◦ C) than when the calibrated flasks were placed in an ice-bath at (0 ◦ C) or in a water bath at (60 ◦ C). The stoichiometry of the reaction was investigated by changing the molar ratio of the reagents [16]. The results obtained (Fig. 3) show a 1:1 AMX to DMPD product was formed at 660 nm. The formation of the dye may probably occur as given in Scheme 1. The stability constant of the dye product was 2.46 × 104 l mol−1 . In order to assess the possible analytical applications of the proposed methods. The effect of some common excipients

Fig. 3. Study of the mole ratio of the reaction between AMX and DMPD.


frequently found with AMX drugs in pharmaceutical formulations, such as sucrose, glucose, fructose, lactose, starch, talc and magnesium stearate was studied by analyzing synthetic sample solutions containing 30 ␮g ml−1 of AMX and excess amounts (10-fold excess) of each excipient, none of these substances interfered seriously. The regression equation obtained, from a series of AMX standards, and the analytical figures of merits of this procedure are summarized in Table 1 in which are also summarized the main performance of the flow procedure developed for AMX determination in order to make an effective comparison between the two approaches.

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Fig. 5. Effect of the concentration of potassium hexacyanoferrate(III) in (M).

3.2. FIA spectrophotometric determination The batch method for the determination of AMX was adopted as a basis to develop FIA procedure. The manifold used for the determination of AMX was so designed to provide different reaction conditions for magnifying the absorbance signal generated by the reaction of AMX drug with DMPD and potassium hexacyanoferrate(III) in ammonium hydroxide medium. Maximum absorbance intensity was obtained when the sample was injected into a stream of mixed DMPD with potassium hexacyanoferrate(III) and was then combined with the stream of ammonium hydroxide (Fig. 2). The influence of different chemical and physical FIA parameters on the absorbance intensity of the coloured product were optimized as follows.

Fig. 6. Effect of the concentration of ammonium hydroxide in (M).

3.2.1. Optimization of reagents concentration The effects of various concentrations of DMPD were investigated. A concentration of 5 mM gave the highest absorbance and was chosen for further use. The results are shown in Fig. 4. It was observed that the reaction between AMX and DMPD depends on the oxidation process with potassium hexacyanoferrate(III) in alkaline medium. The effects of various concentration of potassium hexacyanoferrate(III) were similarly studied. A concentration of (10 mM) gave the best results and minimum blank value as shown in Fig. 5 and was considered as optimum value. Ammonium hydroxide was found necessary for developing the coloured product and increase its stability. The effect of ammonium hydroxide was studied in the concentration range of 10 mM–0.2 M and a greatest absorbance intensity was obtained with 20 mM (Fig. 6).

3.2.2. Optimization of manifold parameters The variables studied under the optimized reagent concentrations were the flow rate, the injected sample volume and the reaction coil length. The effect of flow rate on the sensitivity of the coloured reaction product was investigated in the range of 1–6 ml min−1 . The results obtained showed that a total flow rate of 4.5 ml min−1 (1.5 ml min−1 in each line) gave the highest absorbance as shown in Fig. 7 and was used in all subsequent experiments. The volume of the injected sample was varied between 50 and 300 ␮l using different length of sample loop. The results obtained showed that injected sample of 100 ␮l gave the best absorbance. Coil length is an essential parameter that affected on the sensitivity of the coloured reaction product and was investigated in the range of 25–150 cm The result obtained showed that a coil length of 75 cm gave the highest absorbance as shown in Fig. 8 and was used in all subsequent experiments.

Fig. 4. Effect of the concentration of DMPD on the coloured reaction product.

Fig. 7. Effect of the total flow rate (ml min−1 ).


188

Table 2 Comparison of the proposed method with some spectrophotometric methods Reagent

Linear range (␮g ml−1 )

Molar absorptivity (l mol−1 cm−1 )

Remarks

Reference

3-Methyl benzo thiazolinone-hydrazone Pyrocatecol violet Potassium iodate

0.6–12 0.2–25 200–2000

1.27 × 104 9.23 × 103 Not reported

[17] [18] [19]

Diazotised sulphanilic acid

1–24

Not reported

DMPD

2–40

9.53 × 103

Reaction carried out at 25 ◦ C Reaction carried out at 60 ◦ C and pH 8.5 Reaction carried out at 80 ◦ C and the coloured product need extraction Reaction carried out at 0 ◦ C in borate buffer and in methanol medium Reaction carried out at room temperature (25 ◦ C)

[20] This work

Table 3 Application of the proposed and official methods to the determination of some AMX drug in dosage forms Drug form

Proposed methods Batch Recoverya

Amoline (injection 500 mg) Oubari-pharma-Syria Amoxicillin (injection 500 mg) Pan pharma-France Acamoxil (capsule 250 mg) ACAI-Iraq Amoxicillin (capsule 250 mg) Pharm-Inter-Belgica Amoxicillin (capsule 500 mg) Ajanta-pharm-Limited-India Amoxicillin (capsule 250 mg) SDI-Iraq a

Official method recovery

FIA (%)

100.93 101.80 99.73 100.25 100.38 97.88

R.S.D.a

(%)

0.48 0.68 2.06 0.40 0.50 0.46

Recoverya (%)

R.S.D.a (%)

99.43 100.98 98.31 97.47 99.71 101.96

1.52 0.51 0.42 0.86 1.07 1.09

102.50 101.00 102.00 99.00 98.00 99.00

For five determinations.

A standard calibration line, obtained for a series of AMX standards and the main analytical figures of merits of the developed procedure are indicated in Table 1. 3.3. Analytical application The developed methodology is very adequate for the determination of AMX in aqueous solution and in pharmaceutical preparation samples at a concentration level of traces (ppm) and without requiring any previous separation step, a temperature or a pH control (Table 2). Moreover, the proposed procedures are very economical when compared to other methods such as those based on the use of LC. In comparison of the batch with FIA procedure, the later is more convenient than the former method because of its speed (sample through-put of 120 injection h−1 ) and wider linear range of the calibration graph (Table 1).

Fig. 8. Effect of the length of the reaction coil in (cm).

The precision of the methods was evaluated by analyzing pure sample of AMX and a good recovery was obtained (Table 1). The proposed methods were applied successfully to the analysis of some capsules and injections containing AMX. The results in Table 3 are in accordance with those obtained by the official spectrophotometric method using imidazole-mercury reagent [2]. Finally, statistical analysis [21], F- and T-test, reveals that there is no significant difference in precision and accuracy between the proposed methods and the official spectrophotometric methods. References [1] A. Goodman, T. Rall, A. Nier, P. Taylor, The Pharmacology Bases of Therepeutics, McGraw-Hill, New York, 1996. [2] British Pharmacopaeia, H.M. Stationary Office, London, 1993. [3] F. Belal, A. El-Brashy, F. Ibrahim, J. Assoc. Off. Anal. Chem. 73 (1990) 896. [4] E.M. Abdel-Moety, M.A. Abounassif, E.A. Godkariem, N.A. Khattab, Talanta 40 (1993) 811. [5] T. Saesmaa, J. Chromatogr. 415 (1988) 450. [6] G.A. Saleh, Analyst (London) 121 (1996) 641. [7] H.F. Askal, G.A. Saleh, N.M. Omar, Analyst (London) 16 (1991) 387. [8] J.A. Squella, L.J. Nunez, Talanta 26 (1976) 1039. [9] F. Belal, A. El-Brashy, F. Ibrahim, Microchem. J. 39 (1989) 106. [10] M.Q. Al-Abachi, E.S. Salih, M.S. Salem, Fresenius J. Anal. Chem. 337 (1990) 408. [11] M.Q. Al-Abachi, H.S. Al-Ward, Natl. J. Chem. 4 (2001) 548. [12] M.Q. Al-Abachi, M.J. Hussan, M.A. Mustafa, Natl. J. Chem. 9 (2003) 79. [13] M.Q. Al-Abachi, Y.Y. Farid, M.J. Hamza, Natl. J. Chem. 8 (2002) 520. [14] M.Q. Al-Abachi, R.S. Al-Abaidi, Natl. J. Chem. 8 (2002) 527.

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