spectrophotometric determination of amikacin ...

‫‪Theia'a N. Al-Sabha‬‬

‫‪SPECTROPHOTOMETRIC DETERMINATION OF‬‬ ‫‪AMIKACIN SULPHATE VIA CHARGE TRANSFER‬‬ ‫‪COMPLEX FORMATION REACTION USING‬‬ ‫‪TETRACYANOETHY`LENE AND 2,3-DICHLORO-5,6‬‬‫‪DICYANO-1,4-BENZOQUINONE REAGENTS‬‬ ‫*‪Theia'a N. Al-Sabha‬‬ ‫‪Chemistry Department, College of Education, Mosul University‬‬ ‫‪Mosul, Iraq‬‬

‫اﻟﺨﻼﺻـﺔ‪:‬‬ ‫ﺗﻢ ‪ -‬ﻓﻲ هﺬﻩ اﻟﻮرﻗﺔ ‪ -‬وﺻﻒ ﻃﺮﻳﻘﺔ ﻃﻴﻔﻴﺔ ﺗﻌﺘﻤﺪ ﻋﻠﻰ ﺗﻔﺎﻋﻞ ﺗﻜﻮﻳﻦ ﻣﻌﻘﺪات اﻧﺘﻘﺎل اﻟﺸﺤﻨﺔ ﻟﺘﻘﺪﻳﺮ آﺒﺮﻳﺘﺎت اﻷﻣﻴﻜﺎﺳﻴﻦ ﺑﻮﺻﻔﻪ ﻣﺎﻧﺤﺎ ﻟﻺﻟﻜﺘﺮوﻧ ﺎت ﻧ ﻮع‬ ‫‪ ، n‬وذﻟﻚ ﻣﻦ ﺧﻼل ﺗﻔﺎﻋﻠﻪ ﻣﻊ آﻞ ﻣﻦ اﻟﻜﺎﺷﻔﻴﻦ رﺑﺎﻋﻲ ﺳﻴﺎﻧﻮأﺛﻴﻠﻴﻦ و ‪ -3،2‬ﺛﻨ ﺎﺋﻲ آﻠ ﻮرو‪ -6،5-‬ﺛﻨ ﺎﺋﻲ ﺳ ﻴﺎﻧﻮ‪ -4،1-‬ﺑ ﺎراﺑﻨﺰوآﻮﻳﻨﻮن ﺑﻮﺻ ﻔﻬﻤﺎ ﻣ ﺴﺘﻘﺒﻼت‬ ‫ﻟﻺﻟﻜﺘﺮوﻧﺎت ﻧﻮع ‪ π‬ﻹﻋﻄﺎء ﻣﻌﻘﺪات ﻣﻠﻮﻧﺔ ﻓ ﻲ اﻟﻤﺤﻠ ﻮل اﻟﻤ ﺎﺋﻲ ﺗﻤﺘﻠ ﻚ أﻗ ﺼﻰ اﻣﺘ ﺼﺎص ﻋﻨ ﺪ اﻷﻃ ﻮال اﻟﻤﻮﺟﻴ ﺔ ‪ 330‬و ‪ 340‬ﻧ ﺎﻧﻮﻣﻴﺘﺮ ﻋﻠ ﻰ اﻟﺘ ﻮاﻟﻲ‪ .‬وﻗ ﺪ‬ ‫أﻣﻜﻦ ﺗﻄﺒﻴﻖ ﻗﺎﻧﻮن ﺑﻴﺮ ﻟﻠﺘﺮاآﻴﺰ ‪ 6.8 - 0.4‬و ‪ 20.0 - 0.8‬ﻣﺎﻳﻜﺮوﻏﺮام‪/‬ﻣﻠﻠﺘﺮ ﺑﺎﻣﺘﺼﺎﺻﻴﺎت ﻣﻮﻻرﻳﺔ ‪ 410× 9.49‬و ‪ 410× 1.91‬ﻟﺘﺮ‪.‬ﻣﻮل‪1-‬ﺳﻢ‪ . 1-‬وﻗﺪ‬ ‫آﺎﻧﺖ ﺣﺪود اﻟﻜﺸﻒ ‪ 0.06‬و ‪ 0.18‬ﻣﺎﻳﻜﺮوﻏﺮام \ﻣﻠﻠﺘﺮ ودﻗﺔ )ﻣﻌﺪل ﻧﺴﺒﺔ اﻻﺳﺘﺮﺟﺎع( ‪ % 96.54‬و ‪ % 99.47‬ﺑﺎﺳﺘﺨﺪام اﻟﻜﺎﺷ ﻔﻴﻦ أﻋ ﻼﻩ ﻋﻠ ﻰ اﻟﺘ ﻮاﻟﻲ‪،‬‬ ‫آﻤﺎ وﺟﺪ اﻻﻧﺤﺮاف اﻟﻤﻌﻴﺎري اﻟﻨﺴﺒﻲ ≥ ‪ 4‬ﺑﺎﺳﺘﺨﺪام آﻼ اﻟﻜﺎﺷﻔﻴﻦ‪ .‬وﺗﻢ ﺗﻄﺒﻴﻖ اﻟﻄﺮﻳﻘﺔ ﺑﻨﺠ ﺎح ﻓ ﻲ ﺗﻘ ﺪﻳﺮ اﻷﻣﻴﻜﺎﺳ ﻴﻦ ﻓ ﻲ ﻣﺴﺘﺤ ﻀﺮﻩ اﻟ ﺼﻴﺪﻻﻧﻲ ﻋﻠ ﻰ ﺷ ﻜﻞ‬ ‫ﺣﻘﻦ‪.‬‬

‫__________________‬ ‫‪*Corresponding Author:‬‬ ‫‪E-mail: [email protected]‬‬

‫_____________________________________________________________________________________________‬ ‫‪Paper Received January 11, 2009; Paper Revised October 29, 2009; Paper Accepted December 9, 2009‬‬

‫‪27‬‬

‫‪The Arabian Journal for Science and Engineering, Volume 35, Number 2A‬‬

‫‪July 2010‬‬

Theia'a N. Al-Sabha

ABSTRACT A spectrophotometric method based on the complex formation reaction has been described for the determination of amikacin sulphate as n-donor with terecyanoethylene (TCNE) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) reagents as π-acceptors to give a colored complex species in aqueous solution which can absorb maximally at 330 and 340 nm, respectively. Beer's law is obeyed in the concentration ranges 0.4–6.8 and 0.8–20.0 µg/ml with high apparent molar absorptivities of 9.49×104 and 1.91×104 and limits of detections are 0.06 and 0.18 µg/ml, whereas the average recoveries are 96.54% and 99.47% when using TCNE and DDQ, respectively. The relative standard deviation is ≤4. Application of the proposed method to commercial pharmaceutical injuctions is presented. Key words: TCNE, DDQ, charge-transfer complex, amikacin sulphate, aqueous solution

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The Arabian Journal for Science and Engineering, Volume 35, Number 2A

July 2010

Theia'a N. Al-Sabha

SPECTROPHOTOMETRIC DETERMINATION OF AMIKACIN SULPHATE VIA CHARGE TRANSFER COMPLEX FORMATION REACTION USING TETRACYANOETHYLENE AND 2,3-DICHLORO-5,6-DICYANO-1,4BENZOQUINONE REAGENTS

1. INTRODUCTION Amikacin sulphate {6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-4-O-(6-amino-6-deoxy-α-D-glucopyranosyl)N1-[(2S)-4-amino-2-hydroxybutanoyl]-2-deoxy-D-streptamine} (Illustration 1) [1] is a broad-spectrum and potent aminoglycoside with limited clinical use owing to a high dose requirement and renal and audio-vestibular apparatus toxicity [2].

MWt = 782 Illustration 1: Chemical formula of amikacin sulphate

British and United States Pharmacopoeias [1,3] describe the liquid chromatography method for the assay of amikacin. The different analytical techniques that have been reported for the determination of this drug in biological fluids and pharmaceutical formulations include chromatography [4–7], chemiluminescence [8], voltammetry [9], and fluorimetry [10]. However, spectrophotometric techniques continue to be the most preferred method for routine analytical work due to their simplicity and reasonable sensitivity, along with significant economical advantages. Few spectrophotometric methods have been reported in the literature for the determination of amikacin, probably due to the complexity of the amikacin molecule. These methods involve the use of a purified enzyme from R-factor E.coli which acetylates amikacin with the production of coenzyme A, the latter in turn being reacted with a sulfhydryl reagent to produce stoichiometric amounts of a sensitive chromophore that is measured at 412 nm [11]; nitrosation of the primary amino groups present in amikacin sulphate drug is followed by reaction with cyanoacetamide in ammonia medium at 100 °C for 30 min. The reaction product exhibit absorption maximum at 270 nm [12], ninhydrin at certain pH and heating conditions formed a purple colored complex with amikacin sulphate measured at 568 nm [13]. Two spectrophotometric methods have been reported for the determination of amikacin depending on the Hantzsch reaction by forming dihydrolutidine derivatives [14,15], and the Rimini test with disodium pentacyanonitrosylferrate(II) reagents is used for the determination of amikacin [16]. Charge transfer complex formation reactions have also been used for the determination of amikacin and other antibiotics as n-donor with chloranil as π-acceptor [17,18]. On the other hand, it is well known tetracyanoethylene (TCNE) and 2,3-dichloro5,6-dicyano-1,4-benzoquinone (DDQ) π-electron acceptors often form charge transfer (CT) complexes with various donors which provides the possibility of determination of drugs by spectrophotometric methods. This work describes a simple spectrophotometric method for the determination of amikacin sulphate, in its pure form and pharmaceutical formulation as injections, by exploiting its electron donating property. The method is based on the charge transfer complexation reaction of amikacin with TCNE and DDQ.

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2. EXPERIMENTAL Apparatus All absorption measurements were made on a computerized Shimadzu UV/VIS-1650 double-beam spectrophotometer and matched 1-cm optical silica cells. Reagents All reagents used were of analytical grade and obtained from the Fluka and BDH companies. TCNE solution (1x10-2M): This solution is prepared freshly by dissolving 0.129g of tetracyanoethylene (recrystilized by chlorobenzene) in absolute ethanol and diluted to the mark in a 100 ml-volumetric flask with absolute ethanol. DDQ solution (1x10-3M): This solution is prepared by dissolving 0.0127g of 2,3-dichloro-5,6-dicyano-pbenzoquinone in absolute ethanol and diluted to the mark in 100-ml volumetric flask with absolute ethanol. This solution was freshly prepared. Borate buffer solution 0.05M ( pH9) [19]: This solution is prepared by dissolving 4.7675 g of borax in distilled water and is completed into a 250 ml. Sodium carbonate solution (5x10-3M): This solution is prepared by dissolving 0.0528g of sodium carbonate with distilled water and diluted to the mark in a 100 ml-volumetric flask in distilled water. Standard solutions of amikacin sulphate (100µg/ml): This solution is prepared by dissolving 0.01g of pure form, provided from SDI, and diluted to the mark with distilled water in a 100 ml-volumetric flask. This solution was further diluted with water to the required concentration for working solution. Recommended procedures TCNE Aliquots of 100 µg/ml amikacin sulphate solution (0.2 – 1.5ml) were pipetted into a series of 25-ml standard volumetric flasks. One ml of 5×10-3 M sodium bicarbonate and 1.2 ml of 10-2 M TCNE solutions were added to each flask and the content was diluted to volume with distilled water. The absorbance of the colored product was measured at 330 nm within the stability period of 2–40 min at room temperature (20°C) against the reagent blank prepared simultaneously. DDQ Aliquots of 200 µg/ml amikacin sulphate solution (0.1–3.0 ml) were pipetted into a series of 25-ml standard volumetric flasks. A 0.5 ml of borate buffer solution of pH9 and 1.5 ml of 10-3 M DDQ were added to each flask and left for 30 min in water bath adjusted at 40°C±1. Then the content was diluted to volume with distilled water. The absorbance of the colored complex was measured at 340 nm against the reagent blank prepared simultaneously. Analysis of amikacin sulphate injections: An accurately measured volume of the mixed content of five ampoules, equivalent to 100 mg of amikacin sulphate, was diluted to 100 ml with distilled water. An aliquot of this solution containing 10–170 and 20–500 µg of amikacin was treated in the same way as the standards in the recommended procedures using TCNE and DDQ reagents, respectively. The concentration of the drugs per ampoul was determined using their respective calibration graphs constructed for pure drugs. 3. RESULTS AND DISCUSSION The spectrum of TCNE disolved in the ethanol and diluted with distilled water exhibits two absorption bands at 296 nm and 396 nm. The addition of amikacin dissolved in distilled water to this solution causes a new characteristic band at 330 nm in the presence of a base (Figure 1). Therefore, the observation of this band may be assigned to the formation of a charge transfer complex between amikacin sulphate and TCNE. The amino group present in amikacin sulphate and TCNE reagent in this complex behave as n-donor and π-acceptor, respectively [20]. This compound is believed to be an intermediate molecular association complex, which is followed instantly by conversion to the N-tricyanovinylamine as final product [21].

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b

Absorbance

a

Wavelength, nm Figure 1: Absorption spectra of: (a) amikacin sulphate (8µg/ml) with TCNE vs reagent blank; (b) reagent blank vs distilled water

DDQ is π-acceptor, which reacts instantaneously with basic nitrogenous compounds to form charge transfer complexes of the n-π type. The absorption spectrum of DDQ reagent in ethanol and diluted with distilled water shows a characteristic band peaking at 305 nm. The addition of amikacin sulphate to this solution causes an immediate change in the absorption spectrum, with a new characteristic band peaking at 340 nm. This band may be attributed to the formation of a charge transfer complex between amikacin sulphate and DDQ (Figure 2). 0.60

b

Absorbance

0.40

a

0.20

0.00 260

300

350

Wavelength, nm

400

450

Figure 2: Absorption spectra of: (a) amikacin sulphate(12µg/ml) with DDQ vs reagent blank; (b) reagent blank vs distilled water

3.1. Study of the Optimum Reaction Conditions The spectrophotometric properties of the colored species formed with TCNE and DDQ, as well as the different parameters affecting the color development, were extensively studied. The optimum conditions for the assay procedures have been established by studying the nature of the solvent, the reactions as a function of the concentration of reagent, the effect of surfactants, the temperature, and the stability of the colored species.

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3.2. Effect of the Solvent In our work [22,23], it was found that TCNE and DDQ reagents reacted with most of the primary, secondary, and tertiary aliphatic and aromatic amines in the medium of methylene chloride, acetonitrile, acetone, ethanol, methanol, and water, and produced various colors, except when using water as a solvent for amines and ethanol for TCNE or DDQ with dilution by water leading to the production of colored complexes with aliphatic and aromatic primary amines in the presence of a base, whereas other amines were either unreacted or gave a low response. Therefore, this system of solvents is recommended in this method for the selective determination of drugs containing primary amino groups such as amikacin. 3.3. Effect of pH and Buffer Solutions on the Absorbance The effect of pH on the absorption of amikacin–TCNE product and amikacin–DDQ complex was studied using different concentrations of HCl and NaOH of pH ranging from 2–12. TCNE: It was found that the product amikacin–TCNE formed in the final pH of 4.6 in the presence of sodium hydroxide. Different buffers of pH 4.6 were prepared to examine the sensitivity of the amikacin–TCNE product. A negative effect on the color intensity of the product was observed. Therefore, different bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and ammonium hydroxide of 5×10-3 M concentration were examined in order to obtain high sensitivity. lt was found that 1.0 ml of sodium bicarbonate gave maximum color intensity, and beyond this amount, the absorbance would be decreased. Therefore, 1ml of 5 x 10-3M was chosen as the optimum concentration of sodium bicarbonate, (Figure 3).

Absorbance

0.4 0.3 0.2 0.1 0

0.5

0

1

1.5

2

2.5

NaHCO3 ,NaHCO ml 3 Figure 3: Effect of NaHCO3 amount on the absorption of amikacin sulphate –TCNE product

DDQ: It was found that the amikacin – DDQ complex formed in the final pH of 9 by addition of sodium hydroxide. Therefore, a fixed amount (0.5 ml) of different buffers of pH9 such as carbonate, borate, phosphate, and ammonium buffers were prepared to investigate the sensitivity of the complex. The Borate buffer solution increased the sensitivity of this complex. However, the optimum amount of borate buffer solution of pH9 for the drug was studied and it was found that 0.5 ml is the optimum amount. Beyond this amount, the absorbance would be decreased. Therefore, 0.5 ml of pH9 was recommended in the subsequent experiments (Figure 4).

Absorbance

0.3 0.2 0.1 0 0

0.5

1

1.5

2

2.5

pH9, ml

Figure 4: Effect of borate buffer (pH9) amount on the absorption of amikacin sulphate-DDQ complex

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However, the addition of HCl lead to the destruction of the amikacin sulphate complexes with TCNE or DDQ probably due to liberation of HCN. 3.4. Effect of Reagent Concentration The effects of changing the volume of 1×10-2 M TCNE and 1×10-3 M DDQ were studied over the range of 0.2– 2.0 ml in a solution containing 4 and 8 µg/ml amikacin sulphate, respectively. The results, as shown in Figure 5, revealed the fact that 1.2 ml of TCNE and 1.5 ml of DDQ were required to achieve the maximum intensity of the color in final dilution with water. Therefore, these volumes were used as optimum values and maintained throughout the experiments in this method.

0.5

Absorbance

0.4 0.3 0.2 0.1 0 0

0.5

1

1.5

2

2.5

Volume (ml) of reagent Figure 5: Effect of TCNE (♦) and DDQ (■) concentration on the absorption of 4 and 8 µg/ml amikacin sulphate

3.5. Effect of Reaction Time and Temperature The reaction time was determined by following the color development at room temperature and in a thermostatically controlled water-bath at different temperatures. The absorbance was measured against the reagent blank and treated similarly. It was observed that the absorbance reached its maximum after 10 min at room temperature (20°C) after addition of the TCNE reagent solution and remained constant for 30 minutes. Fading was observed thereafter. By addition of the DDQ reagent, the maximum color intensity of the reaction mixture was attained after 30 minutes at 40°C and remain constant for 20 min at this temperature. Fading was observed thereafter, which may be attributed to the destruction of the complex (Figure 6). These temperatures and reaction time were chosen for color development in the proposed method.

0.5 R.T (20°C)

Absorbance

0.4 0.3

40°C

0.2 0.1 0 0

10

20

30

40

50

60

70

Time, min. Figure 6: Effect of the time and temperature on the absorbance of 2 µg/ml and 8µg/ml amikacin sulphate sulphate with TCNE (■) and DDQ (▲), respectively

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3.6. Effect of Order of Addition In order to obtain the high color intensity, the order of the addition of reagents should be followed as given in the recommended procedures; otherwise, a loss in color intensity was observed. 4. QUANTIFICATION AND ANALYTICAL DATA The results for the determination of amikacin sulphate by both reagents are summarized in Table 1, which shows the sensitivity, recovery, and reproducibility of the proposed method. These are reasonably precise and accurate. The calibration graph is linear in the range of 0.4–6.8 µg/ml for the TCNE reagent and 0.8–20 µg/ml for the DDQ reagent. The apparent molar absorptivities are 9.49×104 l.mol-1cm-1 and 1.91×104 l.mol-1cm-1 using the above reagents, respectively, which indicate that the method is sensitive and the sensitivity of the TCNE reagent is about five times more than that of the DDQ reagent. Also, Table 1 illustrates regression equations, correlation coefficients (R2), and Sandell’s sensitivity for the proposed method. The reproducibility of the proposed method was checked by estimating three different concentration levels within the Beer's law limit in six replicates. The average recoveries were 96.54 % using TCNE and 99.47% using DDQ, revealing good accuracy for the proposed method. The relative standard deviation can be considered to be very satisfactory. The limits of detection (LOD) and limits of quantitation (LOQ) were determined using the formula LOD or LOQ= к S.D.a/b, where к = 3 for LOD and 10 for LOQ, S.D.a is the standard deviation, and b is the slope. Based on the six replicate measurements, as cited in Table 3, LOD is well below the lower limit of the Beer’s law range, and LOQ is approximately 3 times greater than LOD when using both reagents. Table 1. Quantitative Parameters of the Proposed Method

Parameters Beer's law limits (µg/ml) Molar absorptivity (l.mol-1 cm-1) Limit of detection (µg/ml) Limit of quantification (µg/ml) Sandell's sensitivity (µg/cm2) Regression equation (Y)# Slope, a Intercept, b Correlation coefficient (R2) RSD# # Average recovery % # ##

Values of using TCNE DDQ 0.4-6.8 0.8-20.0 9.49×104 1.91×104 0.06 0.18 0.19 0.63 0.0123 0.0581 0.1213 -0.0267 0.9952 ≤4.00 96.54

0.0244 0.0485 0.9988 ≤2.88 99.47

Y = aX+ b, where X is the concentration of amikacin sulphate in µg/ml Average of six determinations

Interference The potential interference by the excipients in the dosage form was also studied. Samples were prepared by mixing known amounts of amikacin sulphate (0.1 mg in the case of TCNE and 0.2 mg in the case of DDQ) with various amounts of the common excipients such as acacia, glucose, succrose, starch, magnesium stearate, and talc in a final volume of 25 ml. The results cited in Table 2 revealed that no interference was observed from any of these excipients with the proposed method.

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Table 2. Analysis of Amikacin Sulphate in the Presence of Common Excipients by the Proposed Method Ingredient

Amount added (mg)

Recoverya (%) ± RSD TCNE

Acacia Glucose Succrose Stach MSb Talc

a

0.25 1.25 2.50 0.25 1.25 2.50 0.25 1.25 2.50 0.25 2.00 2.50 0.25 2.00 2.50 0.25 1.25 2.50

101.2 ± 1.20 103.9 ± 0.10 105.3 ± 1.30 100.0 ± 0.09 103.5 ± 1.19 107.1 ± 2.82 100.5 ± 0.78 103.5 ± 1.49 107.0 ± 4.78 100.2 ± 0.69 104.7 ± 0.51 106.3 ± 3.84 100.4 ± 1.36 103.9 ± 0.32 107.1 ± 2.11 100.3 ± 1.07 100.4 ± 0.55 103.6 ± 2.91

DDQ 98.7 ± 3.90 99.7 ± 1.30 100.4 ± 1.38 101.5 ± 1.89 99.0 ± 3.11 97.2 ± 1.50 99.9 ± 0.52 100.1 ± 0.15 102.8 ± 2.13 101.4 ± 4.60 104.1 ± 2.61 105.3 ± 3.94 100.3 ± 1.54 103.9 ± 3.76 98.5 ± 4.11 100.2 ± 0.64 98.2 ± 0.33 97.4 ± 1.04

Average for three determinations Magnisium stearate

b

Application The applicability of the proposed method for the determination of amikacin sulphate in commercial dosage form as injections (0.1g/2ml), the only available pharmaceutical formulation, was examined by analyzing the marketed product (Germany medicines GMBH). The method was established by performing five replicate analyses on solutions containing three different amounts (within the Beer's law limit) of the drug and calculating the percentage recovery. The precision was ascertained by calculating the relative standard deviation (RSD) for five determinations of each level. The recovery percent, standard deviation (SD), and RSD % are given in Table 3. The comparision of the actual difference between the mean and the true value (x-µ) with the largest difference that could be expected as a result of indeterminate error (± ts/√n) was made in the last two columns of Table 3. It is clear from the results that at all three levels (concentrations) studied, the values of (x-µ) are less than (± ts/√n) indicating that no significant difference exists between the mean and true values. Moreover, to check the validity of the proposed method, the standard addition procedure was applied by adding pure amikacin sulphate to the previously analyzed injection. Figure 7 and Table 3 show the results of analysis of the commercial ampoules and the recovery of the studied drug.

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Table 3. Results of Assay of Amikacin Sulphate in Injection (0.1g/2ml) by the Proposed Method and Standard Addition Procedure Drug Drug Average Drug R.S.D. amount amount Recovery* ± ts amount Procedure applied (%)±S.D. % x-µ taken found found √n (g/2ml) (µg/ml) (µg/ml) 0.8 0.81 101.20±0.74 3.72 0.01 0.85 0.0996 TCNE 2.0 2.00 100.00±0.05 2.50 0.0 0.06 4.8 4.76 99.30±0.13 2.70 0.04 0.15 5.0 5.08 101.60±0.79 1.80 0.08 0.91 0.0995 DDQ 15.0 14.81 99.98±0.29 1.96 0.19 0.33 20.0 19.37 96.85±0.48 2.42 0.36 0.55 0.5 0.51 102.00±0.02 3.89 0.01 0.03 0.0992 Standard TCNE 1.0 0.98 98.00±0.04 3.82 0.02 0.05 Addition 2.0 2.04 97.50±0.04 2.16 0.04 0.05 2.5 2.58 103.20±0.07 2.66 0.08 0.10 DDQ 0.1011 5.0 5.02 100.40±0.18 3.52 0.02 0.25 10.0 9.97 99.70±0.44 4.36 0.03 0.61 * Mean for five determinations (four determinations for standard addition procedure). X = mean value; µ = true value; t = 2.571 for n = 5 and 2.766 for n = 4 at 95%confedence level. S = standard deviation; R.S.D. = relative standard deviation.

□ 0.5µg/ml ∆ 1.0µg/ml ◊ 2.0µg/ml

1

(a)

0.9

(b)

0.5

0.8 Absorbance

0.7 Absorbance

0.6

■ 2.5µg/ml ▲5.0µg/ml ◆ 10.0µg/ml

0.6 0.5

0.4

0.3

0.4 0.2

0.3 0.2

0.1

0.1 0 -2.5 -1.5 -0.5 0.5 1.5 2.5 3.5 4.5 5.5 Concentration , µg/ml

0 -12 -10 -8 -6 -4 -2 0

2

4

6

8 10 12 14 16 18 20

Concentration., µg/ml

Figure 7: Standard addition procedure for amikacin sulphate in its injection formulation using (a)TCNE and (b) DDQ

Stoichiometric Relationship Job’s method of continuous variation and mole ratio method [24] were used for determining the molar ratio of amikacin sulphate to each of the analytical reagents employed in the method. These ratios were 1:1 amikacin: TCNE (Figure 8a), which may be attributed to the fact that only one amino group present in the drug is responsible for the product formation. However, these ratios were found to be 1:4 amikacin : DDQ (Figure 8b), which indicated that the four amino groups present in the drug are possible for the charge-transfer complex formation.

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0.6

(a) 0.6

0.5 Absorbance

Absorbance

0.5 0.4 0.3 0.2

0.4 0.3 0.2 0.1

0.1

0

0 0

0.2

0.4

0.6

0.8

0

1

1

[Am ikacin]/[Amikacin]+[TCNE]

3

0 .8

(b)

0 .7 0 .6

0.3 Absorbance

Absorbance

0.4

2

[TCNE] / [Amikacin]

0.2 0.1

0 .5 0 .4 0 .3 0 .2 0 .1

0

0

0

0.2

0.4

0.6

0.8

[Amikacin] / [Amikacin]+[DDQ]

1

0

2

4

6

8

[DDQ] / [Amikacin]

Figure 8: Continuous variation mole ratio plots for reaction of amikacin with (a) TCNE (1×10-3 M) and (b) DDQ (1.022×10-3 M)

Suggested chemical reaction mechanism TCNE The nature of the reaction between drugs cotaining primary amino groups and TCNE reagent in aqueous solution is not clearly understood. Many methods have been used to describe the formation of π-π and n-π charge-transfer complexes between TCNE and organic molecules, including amines in organic medium [25–27]. Durgn [28] indicated that tetracyanoethylene reacts with primary and secondary aliphatic and aromatic amines in boiling tetrahydrofuran to give N-tricyanovinylamine, and Middleton et al. [21] observed that the pentacyanopropenide ion is formed in the reaction of TCNE with aqueous pyridine in the presence of sodium bicarbonate in a weak acidic medium with liberation of carbon dioxide and hydrogen cyanide. Ladilina et al. [29] reported that Ncyclohexyltricyanoacrylamidine was formed by the reaction of primary amino group present in cyclohexylamine and TCNE reagent in acetonitrile medium. In this work, it was observed that the reaction mixture of amikacin with TCNE in aqueous solution in the presence of sodium bicarbonate attained its maximum absorption after 10 min at room temperature (Figure 6) and was stable for about 30 min. In addition, it was found that only one primary amino group in amikacin was responsible for the product formation, as shown in Figure 8a. These results may be attributed to the formation of a shortlived intermediate of the charge-transfer complex between the starting compound and final reaction product of the N-tricyanovinylamine derivative, as shown in Illustration 2.

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Theia'a N. Al-Sabha

CN

H

C22H35NO13 (NH2)3

N

+

H

RT

C CN

Amikacin

CN C

CN

H

C22H35NO13 (NH2)3

N

C

H

CN

n → π CT complex

TCNE

H CN

CO2 + HCN + C22H35NO13 (NH2)3 N C C N-tricynovinylamine derivative

CN CN

HCO

-

3

H2 O

CN C

C22H35NO13 (NH2)3

CN

CN

H

CN

N

C

H

CN

CN C CN

Illustration 2: Probable mechanism of the product formation reaction between TCNE and the amikacin

DDQ DDQ is a strong electron π-acceptor having electron affinity 1.9 e.v [30], which reacts instantaneously with basic nitrogenous compounds to form charge transfer complexes of the n-π type. Most spectrophotometric methods with DDQ [31–32] are based on the charge transfer interaction of radical anion DDQ. – with the radical cation donors (D.+) formed in organic medium. The nature of the reaction between primary amines and DDQ in aqueous solution is not clearly understood. However, in the present work, it was observed that the complex was formed in aqueous medium in the ratio of 1:4 amikacin : DDQ [Figure 8b] after addition of the drug, including four primary amino groups, to DDQ reagent at pH9, and a new absorption band appeared at 340 nm, which is not shown by either of the components present at solution, which indicated that the four amino groups present in the drug are possible for the charge-transfer complex formation. Also, it was found that DDQ reagent has a maximum absorption band at 305 nm in the presence of pH9, which may be attributed to the formation of monochlorodicyanohydroxy-pbenzoquinone [33], which is considered a complexing agent for the drug. On this basis, a suggested chemical reaction has been proposed in Illustration 3.

O

O Cl

CN

Cl

CN

pH9

HO

CN

Cl

CN

O

O

Monochlorodicyanohydroxy-p-benzoquinone

DDQ

O

O C22H35NO13 (NH2)4 + Amikacin

H

CN

HO 4

C22H35NO13 Cl

CN

O

N:

HO

CN

Cl

CN

H CT-complex

O

4

Illustration 3: Probable mechanism of the charge-transfer complex formation reaction between DDQ and the amikacin

5. CONCLUSION The proposed method is simple, selective, sensitive, and economical compared to reported methods, and does not require any pretreatment of the drug or extraction procedure and has good accuracy and precision. The TCNE reagent was found to be more sensitive compared to the DDQ reagent for the assay of amikacin sulphate. The color is stable ≥20 min when using both reagents, which is sufficient time for the analyst to perform the analysis. The method is successfully applied for the determination of pure amikacin sulphate and in pharmaceutical injection form. However, the critical recommendations of using DDQ reagent might be based on the experimental conditions (e.g., reaction time and heating).

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