Different spectrophotometric methods applied for the ...

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 161 (2016) 64–69

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Different spectrophotometric methods applied for the analysis of binary mixture of flucloxacillin and amoxicillin: A comparative study Khalid A.M. Attia a, Mohammed W.I. Nassar a, Mohamed B. El-Zeiny b, Ahmed Serag a,⁎ a b

Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, 11751, Nasr City, Cairo, Egypt Analytical Chemistry Department, Faculty of Pharmacy, Modern University for Technology and Information (MTI), 12582, Al Hadaba Al Wosta, Cairo, Egypt

a r t i c l e

i n f o

Article history: Received 13 January 2016 Received in revised form 25 February 2016 Accepted 27 February 2016 Available online 02 March 2016 Keywords: Flucloxacillin Amoxicillin Ratio subtraction Absorbance subtraction Amplitude modulation

a b s t r a c t Three different spectrophotometric methods were applied for the quantitative analysis of flucloxacillin and amoxicillin in their binary mixture, namely, ratio subtraction, absorbance subtraction and amplitude modulation. A comparative study was done listing the advantages and the disadvantages of each method. All the methods were validated according to the ICH guidelines and the obtained accuracy, precision and repeatability were found to be within the acceptable limits. The selectivity of the proposed methods was tested using laboratory prepared mixtures and assessed by applying the standard addition technique. So, they can be used for the routine analysis of flucloxacillin and amoxicillin in their binary mixtures. © 2016 Elsevier B.V. All rights reserved.

1. Introduction

2. Experimental

Flucloxacillin (FLX), (6R)-6-[3-(2-chloro-6-fluorophenyl)-5methylisoxazole-carboxamido] penicillanic acid Fig. 1(a), is a bactericidal agent used primarily for the treatment of infections due to staphylococci resistant to benzyl penicillin [1–3]. There are several methods available in the literature for the quantification of FLX including spectrophotometry [4–8], high-performance liquid chromatography [9–13], polarography [14] and potentiometry [15]. Amoxicillin (AMX), (6R)-6-[α-D-(4-hydroxyphenyl) glycylamino] penicillanic acid Fig. 1(b), is one of the most frequently used β-lactam antibiotics in the world. It is employed to treat humans and animals [1–3]. Various analytical methods have been reported for the determination of AMX based on spectrophotometry [4,16–24], capillary electrophoresis [25,26], high-performance liquid chromatography [27–29] and electrochemical techniques [30–33]. Few methods were reported for determination of both components including high-performance liquid chromatography [34–37], spectrophotometry [38] and chemometric assisted techniques [39]. In the present work, a comparative study was done between three methods namely ratio subtraction [40,41] for the determination of FLX in the presence of AMX, absorbance subtraction [42–44] and amplitude modulation [42–44] for the determination of FLX and AMX listing the advantages and the disadvantages of these methods.

2.1. Materials and reagents

⁎ Corresponding author. E-mail address: [email protected] (A. Serag).

http://dx.doi.org/10.1016/j.saa.2016.02.028 1386-1425/© 2016 Elsevier B.V. All rights reserved.

A. Flucloxacillin (99.73%) and amoxicillin (99.84%); kindly supplied by EIPICO pharmaceutical Company, Egypt; B. Flumox® Capsules dosage form; labeled to contain 250 mg of each of FLX and AMX batch number 1405340, manufactured by EIPICO Pharmaceuticals Company; C. Sodium hydroxide; El-NASR Pharmaceutical Chemicals Co., Egypt; D. Distilled water.

2.2. Instruments SHIMADZU dual beam UV–visible spectrophotometer (Kyoto/ Japan), model UV-1800 PC connected to a compatible IBM and an HP1020 laser jet printer. The bundled software, UV-Probe personal spectroscopy software version 2.43 (SHIMADZU) was used. The spectral band was 2 nm and scanning speed is 2800 nm/min with 0.2 nm interval. 2.3. Standard solutions 2.3.1. Preparation of AMX working standard solution AMX standard working solutions; 100 μg/mL in 0.1 N sodium hydroxide.

K.A.M. Attia et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 161 (2016) 64–69

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two drugs at 235.6 nm (isoabsorptive point) and AMX only at 291 nm (λ2) were recorded, then the absorbance factor of AMX at 235.6 nm and 291 nm [=A235.6/A291] was calculated. 3.1.3. Amplitude modulation method Different aliquots of FLX and AMX standard solutions ranging from 50–400 μg were transferred to a two separated sets of 10-mL volumetric flasks and completed to volume with 0.1 N sodium hydroxide. The absorption spectra (from 200 to 400 nm) of these solutions were recorded using 0.1 N sodium hydroxide as a blank, and then divided by the normalized absorption spectrum of AMX solution (1 μg/mL). The amplitude of ratio spectra for FLX and AMX at isoabsorptive point 235.6 nm and 290 were recorded respectively. 3.2. Application to laboratory prepared mixtures

Fig. 1. Chemical structure of (a) flucloxacillin and (b) amoxicillin.

2.3.2. Preparation of FLX working standard solution FLX standard working solutions; 100 μg/mL in 0.1 N sodium hydroxide. 3. Procedure 3.1. Linearity and construction of calibration curves 3.1.1. Ratio subtraction method Different aliquots of FLX standard solution (100 μg/mL) ranging from 50–400 μg were transferred to 10-mL volumetric flasks and completed to volume with 0.1 N sodium hydroxide. The absorption spectra (from 200 to 400 nm) of these solutions were recorded using 0.1 N sodium hydroxide as a blank. The absorbance was measured at 223 nm. 3.1.2. Absorbance subtraction method Different aliquots of FLX and AMX standard solutions ranging from 50–400 μg were transferred to a two separated sets of 10-mL volumetric flasks and completed to volume with 0.1 N sodium hydroxide. The absorption spectra (from 200 to 400 nm) of these solutions were recorded using 0.1 N sodium hydroxide as a blank. The absorbances of

Accurate aliquots of FLX and AMX were transferred from their working solutions into a series of 10-mL volumetric flasks to prepare mixtures containing 1:1 ratios of both drugs. The volumes were completed with the solvent. The spectra of the prepared series from 200 to 400 nm were recorded and stored. The concentrations of FLX and AMX were calculated as described under linearity for each of the proposed methods. 3.3. Application to pharmaceutical preparation Content of eight Flumox® capsules were weighed and finely powdered. A portion of powder equivalent to 10 mg of both components was weighed, transferred into 100-mL volumetric flask and dissolved in 75 mL of 0.1 N sodium hydroxide. The solution was shaken vigorously for 15 min then sonicated for 30 min. The volume was completed to 100 mL with 0.1 N sodium hydroxide and filtered. Necessary dilutions of the filtrate were made with 0.1 N sodium hydroxide to obtain different concentrations of FLX and AMX samples as stated under linearity. To assess the accuracy of the proposed methods, standard addition technique was applied. 4. Results and discussion The zero-order absorption spectr of FLX and AMX, show a certain degree of overlapping with isoabsorptive point at 235.6 nm and AMX is more extended in plateau region in which FLX has no absorbance as shown in Fig. 2. This overlap does not permit direct determination of FLX and AMX in their binary mixture. To overcome this problem, many manipulations have been done allowing the determination of

Fig. 2. Absorption spectra of FLX (10 μg/mL), AMX (10 μg/mL) and their mixture (5 μg/mL) of each.

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Fig. 3. Ratio spectra of laboratory prepared mixtures of FLX and AMX (5–40 μg/mL) using 30 μg/mL of AMX as a divisor.

one or both of the compounds without previous separation as will be shown.

point (235.6 nm) in their mixture according to the following equations. Absorbance of AMX in the mixture at λiso ¼

4.1. Ratio subtraction method In this method, careful choice of the divisor concentration was of great importance; therefore several trials were done using different values of divisor concentrations. The absorption spectra of binary mixtures of FLX and AMX were divided by the absorption spectrum of AMX (30 μg/mL) as a divisor to get the ratio spectra, as shown in Fig. 3, then subtraction of the absorbance values in plateau region at 300 nm (the constant), as shown in Fig. 4, followed by multiplication of the obtained spectra by the spectrum of the divisor, as shown in Fig. 5. The peak amplitude at 223 nm in the final spectra is proportional to the concentrations of FLX without interference from AMX.

4.2. Absorbance subtraction method 235:6 was used In this method; the absorbance factor of pure AMX ½abs abs291

for calculating the absorbance values of FLX and AMX at isoabsorptive

abs235:6 absλ291 ðFLXþAMXÞ abs291

Absorbance of FLX in the mixture at λiso abs235:6 ¼absλ235:6ðFLXþAMXÞ ‐ absλ291ðFLXþAMXÞ abs291 Where, abs λ (FLX + AMX) is the absorbance of the binary mixture and abs235:6 abs291

is the absorbance factor of pure AMX at 235.6 nm to 291 nm and it

was calculated and found to be 3.032. The calculated absorbance values corresponding to FLX and AMX can be separately used to identify each of their concentrations using the corresponding regression equations at isoabsorptive point (235.6 nm). 4.3. Amplitude modulation method In this method; by dividing the spectra of the binary mixture by the normalized AMX divisor spectrum, we obtain the ratio spectra as shown in Fig. 6. At the isoabsorptive point of ratio spectra the amplitude value

Fig. 4. Ratio spectra of laboratory prepared mixtures of FLX and AMX (5–40 μg/mL) using 30 μg/mL of AMX as a divisor after subtraction of the constant.


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Fig. 5. The zero-order absorption spectra of FLX (5–40 μg/mL) obtained by the proposed ratio subtraction method for the analysis of laboratory prepared mixtures with AMX (5–40 μg/mL) after multiplication by the divisor.

Fig. 6. Ratio spectra of laboratory prepared mixtures of FLX and AMX (5–40 μg/mL) using normalized spectrum (1 μg/mL) of AMX as a divisor.

was modulated to concentration. The amplitude value of the constant can be determined at the plateau region at 290 nm which is equivalent to the recorded concentration of AMX in the mixture. At the isoabsorptive point (λiso) at 235.6 nm, the amplitude of the ratio spectra at this point will be equal to the sum of the amplitudes of FLX and AMX. After the subtracting the recorded amplitude at 235.6 nm from the previously obtained constant at 290 nm, we get the

corresponding recorded amplitude of FLX, which is equivalent to recorded concentration of FLX in the mixture. To eliminate any error due to signal to noise ratio, the actual concentration of FLX and AMX could be calculated by using their corresponding regression equations at isoabsorptive point 235.6 nm and 290 nm, respectively. These equations can be obtained by plotting the amplitudes versus the actual concentrations of both drugs at their selected wavelengths.

Table 1 Assay validation sheet of the proposed methods. Parameter

Ratio subtraction

Absorbance subtraction

Accuracy (mean ± RSD)a

FLX 100.46 ± 1.567

FLX 99.66 ± 1.644

AMX 100.29 ± 1.154

FLX 99.41 ± 1.439

AMX 99.99 ± 1.018

Precision Repeatability (RSD)b Intermediate precision (RSD)c

0.636 1.431

0.929 0.851

0.993 1.356

0.953 1.188

0.616 1.101

0.0183 0.0018 0.9997

(5–40 μg/ml) (5–40 μg/ml) 0.0194 −0.0090 0.9995

0.9884 0.0657 0.9997

0.9918 0.0395 0.9999

Linearity Range Slope Intercept Regression coefficient (r2) a b c

0.0319 −0.0451 0.9996

Amplitude modulation

Average of three determinations for three concentrations (10, 20, and 30 μg/m) for FLX and AMX repeated three times. The intraday (n = 3), average of three concentrations (10, 20, and 30 μg/m) for FLX and AMX repeated three times within the day. The interday (n = 3), average of three concentrations (10, 20, and 30 μg/m) for FLX and AMX repeated three times in three days.

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Table 2 Determination of FLX and AMX in laboratory prepared mixtures by the proposed methods.

FLX (μg/mL)

5 10 20 30 40

Ratio subtraction



FLX

FLX

AMX

FLX

AMX

99.06 98.78 101.57 98.94 98.29

100.79 98.44 98.93 98.93 99.59

100.11 100.06 99.25 98.46 100.02

98.96 99.64 99.16 98.63 99.19

99.77 99.53 98.15 100.93 100.96

AMX (μg/mL)

5 10 20 30 40

Table 3 Application of standard addition technique to the analysis of Flumox® capsule by applying the proposed methods. Pharmaceutical conc. μg/mL

Added standard μg/mL

Ratio subtraction



FLX

FLX

AMX

FLX

FLX

10

10 15 20

98.76 100.31 99.38

98.69 100.35 100.66

98.85 99.10 100.78

99.86 99.17 100.82

100.12 99.77 98.27

5. Validation of the methods Validation of the proposed methods were assessed as per the ICH guidelines [45] of accuracy, precision, repeatability, interday precision and linearity. It was found that good results were obtained as shown in Table 1. Table 2 shows the specificity; recovery percentages of the laboratory prepared mixtures of both components. The validity of the proposed procedures is further assessed by applying the standard addition technique showing no interference from excipients. The obtained results were shown in Table 3. The above discussion shows that some methods are simpler in their manipulation than the others. Ratio subtraction method has two major disadvantages. One of them is its capability to determine only one component in the presence of the other. The other disadvantage is that several trials are required for the divisor selection which is a tedious and non-convenient procedure. On the other hand, absorbance subtraction method can overcome these disadvantages and can measure both components simultaneously without any need of manipulation or division to obtain ratio spectra. However, its major disadvantage is that it requires two conditions; an isoabsorptive point and one component is more extended in plateau region in which the other has no absorbance. Amplitude modulation method has almost the same disadvantages as absorbance subtraction method but its major advantage is its simplicity since the amplitudes have been modulated to the concentrations. Table 4 Statistical comparison for the results obtained by the proposed methods and the reported methods for the analysis of FLX and AMX in Flumox® capsule. Value Mean

SD

N Variance

Method Ratio subtraction Absorbance subtraction

Amplitude modulation Reported method

FLX

99.68 1.094

5

1.197

FLX

100.08 1.396

5

2.792

AMX

99.68 1.164

5

1.335

FLX

100.24 1.453

5

2.111

AMX

100.10 1.619

5

2.621

FLX AMX

99.58 1.126 100.69 1.646

4 4

1.268 2.709

Student's F value t test 0.129 (2.365) 0.601 (2.365) 1.043 (2.365) 0.768 (2.365) 0.539 (2.365)

1.059 (6.591) 1.536 (9.117) 2.001 (6.591) 1.665 (9.117) 1.034 (6.591)

The values in the parenthesis are the corresponding theoretical values of t and F at (P = 0.05).

Table 5 One-way ANOVA testing for the different proposed methods used for the determination of AMX and FLX in Flumox®Capsules. Drug FLX AMX

Source

DF

Sum of squares

Mean square

F value

Between exp. Within exp. Between exp. Within exp.

3 15 2 11

0.986 24.178 2.295 24.046

0.329 1.612 1.147 2.186

0.204 (3.287) 0.525 (3.982)

The values between parentheses are the theoretical F values. The population means are not significantly different.

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