Two Simple Spectrophotometric Methods for the Simultaneous Determination of Amoxicillin Trihydrates and Flucloxacillin Sodium

 

Warid Khayata, Duaa AL Zakri

Department of Analytical and food Chemistry, Faculty of Pharmacy/ University of Aleppo, Syria

 

ABSTRACT:

Simple, specific, accurate and precise spectrophotometric methods were developed and validated for simultaneous estimation of amoxicillin Trihydrates and flucloxacillin Sodium in their pure form and pharmaceutical dosage form. Two spectrophotometric methods were applied: derivative ratio spectra and absorbance ratio method.

The linearity range for the spectrophotometric methods was found to be( 1-40μg/mL)  and( 0.7-40μg/mL) for amoxicillin and flucloxacillin respectively in derivative ratio method and to be (1.5-65μg/mL) for bothamoxicillin and flucloxacillin in absorbance ratio method.

Both methods were simple, sensitive, ecofriendly and don’t need a special program, so they could be easily applied as alternative methods to LC methods in quality control laboratories lacking the required facilities for those techniques.

 

 

 

1. INTRODUCTION:

Amoxicillin trihydrate (AMX)(Fig.1) is chemically a (2S, 5R, 6R) [[( 2R )- 2 - amino-2 (4 hydoxyphenyl ) acetyl ] amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptanes-2-carboxyic acid, and it belongs to class of antibiotics¹. Amoxicillin trihydrate is official in BP and Eur pharmacopoeia^2,3.AMX was determined alone and in combination with other drugs for pharmaceutical formulation and biological fluidsby spectrophotometry^4-9 and HPLC methods^10-11.Flucloxacillin sodium (FLX)(Fig.1) is chemically (2S,5R,6R)-6-[[[3-(2-chloro-6- flurophenyl)-5 methoxylisoxazol-4-yl] carbonyl]amino]- 3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptanes-2-carboxylate and it belongs to class of antibiotics having bactericidal action¹.Flucloxacillin sodium is official in BP and Eur Pharmacopoeia^2,3.

 

FLX was determined alone by spectrophotometry methods^12-13.Simultaneous determination of AMX and FLX has been reported in the literature by HPLC methods^14-16and spectrophotometric methods^17-18. Our aim was to develop more sensitive spectrophotometric methods than previous ones for the simultaneous determination of this binary mixture.

 

Amoxicillin trihydrates C₁₆H₁₉N₃O₅S,3H₂O               M.Wt 419.4

 

 

Flucloxacillin sodiumC₁₉H₁₆CIFN₃NaO₅S,H₂O           M.Wt 493.9

Fig.1:The chemical structures of Amoxicillin trihydrates and Flucloxacillin sodium.

2. MATERIALS AND METHODS:

2.1. Instruments:

Spectrophotometric measurements were made in Jasco company (Japan) model V650. UV-Visible spectrophotometer with 1.00 cm quartz cells.

 

2.2. Chemicals and solvents:

All chemicals used were of analytical grade: Double distilled Water, Ethanol (panreac).

 

2.3. Samples:

2.3.1. Pure samples:

Amoxicillin(99.88%), flucloxacillin(99.84%) were supplied by Parabolic Drugs Ltd.

 

2.3.2.Pharmaceutical formulation

Amoxipen capsules are labeled to contain 250 mg AMX and FLX in capsule, It is manufactured by Barakat for Pharmaceutical Industries, (Alepp, Syria).

 

2.4.Standard solutions:

2.4.1.For derivative ratio method:

2.4.1.1. Stock standard solutions of AMX and FLX (1mg/ mL):

The solution was prepared by dissolving 100mg of AMX and FLX into two separate 100mL double distilled water.

 

2.4.1.2. Working standard solutions of AMX and FLX (100µg/ mL):

AMX and FLX working solutions were prepared by diluting10mL from their respective stock standard solutions 1mg/mL into two separate 100mL volumetric flasks and the volume was completed with double distilled water.

 

2.4.1.3. Laboratory prepared mixtures:

Different aliquots of AMX and FLX were separately transferred from their working standard solutions (100 µg /mL) into a set of 10mL volumetric flasks then the volume was completed to the mark with double distilled water to produce laboratory prepared mixtures containing different ratios of the cited drugs.

 

2.4.2. For absorbance ratio method:

2.4.2.1.Stock standard solutions of AMX and FLX

 

·         amoxicillin(0.4mg/ mL):

The solution was prepared by dissolving 100mg of AMX in 250mL ethanol 96%.

 

·         flucloxacillin(1mg/ mL):

The solution was prepared by dissolving 100mg of AMX in 100mL ethanol 96%.

 

2.4.2.2. Working standard solutions of AMX and FLX (100µg /mL):

AMX and FLX working solutions were prepared by diluting 25ml and 10mL respective from their  stock standard solutions 0.4mg/mL and 1mg/ mL (for AMX and FLX, respectively) into two separate 100mL volumetric flasks and the volume was completed with ethanol 96%.

 

2.4.2.3. Laboratory prepared mixtures:

Different aliquots of AMX and FLX were separately transferred from their working standard solutions (100µg/ mL) into a set of 10mL volumetric flasks then the volume was completed to the mark with ethanol 96%to produce laboratory prepared mixtures containing different ratios of the cited drugs.

 

2. 5.Methods:

2.5.1first derivative ratio spectra method (¹DD)Linearity:

The absorption spectra of standard solutions of AMX (1-40µg/ml) and FLX (0.7-40µg/ml) were recorded and stored in the computer. The stored spectra of AMX and FLX were divided by the stored spectrum of FLX (5µg/ml) for determination of AMX and by the stored spectrum of AMX (30µg/ml) for the determination of FLX, respectively. Then, the first derivative of the above ratio spectra were obtained using the Savitzky-Golay method¹⁹ (first polynominal order, DELTA=7) for AMX and FLX.

 

The total amplitudes of the first derivative of the ratio spectra at the minimum at 226 nm and the maximum at 223 nm were plotted against the corresponding concentrations of AMX and FLX, respectively, and regression equations were computed.

 

Assay of Laboratory Prepared Mixtures: The same procedure mentioned under linearity was applied in order to determine AMX and FLX in the laboratory prepared mixtures. The concentrations of the two drugs were calculated from the computed regression equation.

 

2.5.2.QAnalysis Method (Absorbance Ratio) Linearity:

Standard solutions containing (1.5-65µg/ml) each of AMX and FLX were prepared separately using ethanol 96%. The absorption spectra of the prepared solutions were recorded in the range of 200-350 nm and the absorbance values at231.4 nm (max of AMX) and 227.9 nm (iso) were measured from which the absorptivity values for both drugs at the selected wavelengths were calculated and the average values were taken. The method employs Qvalues and the concentrations of the studied drugs in the prepared solutions were determined by using the following equations:

C_x=

 

C_y=

 

where Cxand Cyare the concentrations of AMX and FLX respectively; QM is the absorbance of sample at λ_231.4/absorbance of sample at λ_227.9; Qxis the absorptivity of AMX at λ _231.4/absorptivity of AMX at λ _227.9; Qyis the absorptivity of FLX at λ_231.4/absorptivity of FLX at λ_227.9; Axis the absorptivity of AMX at 227.9nm; Ay is the absorptivity of FLX at 227.9nm; and Ais the absorbance of the sample at 227.9nm.

 

Assay of Laboratory Prepared Mixtures: The absorbances of the laboratory prepared mixtures at λ 231.4 and λ 227.9 were recorded, absorptivities were calculated and substituted in the equations mentioned under linearity, in order to obtain the concentration of both drugs.

 

3. RESULTS AND DISCUSSION:

Derivative ratio method:

Fig.2 reveals severe overlap of the zero–order spectra of AMX and FLX in water. By applying ¹DD Method AMX concentration could be determined in binary mixture with FLX, without any interference. This method was based on obtaining the ratio spectra of studied drug by dividing its absorption spectra by a standard spectrum of the interfering compound then the first derivative of the ratio spectra was computed^20-22.The main parameters that affect the results are wavelength, the concentration of the standard solution used as a divisor and width of the boundaries over which the derivative is calculated (DELTA)are carefully tested.

 

The effect of divisor concentration is studied, different concentrations of divisor are used of AMX and FLX and the divisor concentrations 30µg/ml and 5µg/ml of AMX and FLX, respectively, are found the best regarding average recovery percent when they are used for the prediction of AMX and FLX concentrations, respectively, in bulk powder as well as in laboratory prepared mixtures (fig.3 and fig.4).

 

The absorption spectra of AMX are divided by the absorption spectrum of 5ug/ml FLX, and the absorption spectra of FLX are divided by the absorption spectrum of 30µg/ml AMX, for determination of AMX and FLX, respectively. These gave the best compromise in terms of sensitivity, repeatability and signal to noise ratio. The choice of wavelength for the measurement was carefully studied, the measuring at 226 and 225 nm For determination of AMX and FLX respect give the best linearity. Good linearity is obtained in the concentration range of 0.7-40µg/ml and 1-40µg/ml for AMX and FLX, respectively.

 

Fig.2: Zero-Order Absorption Spectra of 30µg/ml AMX(----). 30µg/ml FLX (____) and Mixture Containing 30µg/ml of each(^.-^.-^.-) in water.

 

Fig.3: The first derivative of ratio spectra of (1-40µg/ mL) AMX using the spectrum of 5µg/ml FLX as a divisor.

 

Fig.4: The first derivative of ratio spectra of (0.7-40µg/ mL) FLX using the spectrum of 30µg/ml AMX as a divisor.

 

Q-Analysis Method (Absorbance Ratio):

The principle of this method is that for the substance that obeys Beer–Lambert law at all wavelengths, the ratio of absorptivity (or absorbance) values at any two wavelengths are constant, independent of the concentration or path length. This ratio is referred as Q-ratio^23-24. One of the two selected wavelengths is an isosbestic point and the other is the wavelength of maximum absorption of one of the two components. From the overlain spectra of the two drugs and their mixture, (Fig. 5), absorbances were measured at 227.9 nm (isosbestic point) and at 231.4 nm (λmax of AMX) over the concentration range of (1.5-65 µg/ml). Absorptivity coefficients of each drug at both wavelengths were determined and the average values were taken.

 

The concentration of each drug in laboratory prepared mixtures and tablet formulation was determined by substituting the absorbance ratio and the absorptivity coefficients in the equations mentioned under linearity in order to develop the following sets of equations: Q_AMX=1.04,Q_FLX=0.8,A_AMX=251, A_FLX=250.7

 

C _AMX=

 

C_FLX=

 

where C_AMX and C_FLX are the concentrations of AMX and FLX, respectively; QM is the absorbance of sample at λ_231.4/absorbance of sample at λ_227.9 and A is the absorbance of the sample at λ _227.9 .

 

3.1. Validation:

The methods were validated according to ICH guidelines²⁵for validation of analytical procedures.

 

Fig.5: Zero-Order Absorption Spectra of 30µg/ml AMX(------). 30µg/ml FLX (____) and Mixture Containing 30µg/ml of each(-^.-^.-^.) in ethanol 96%.

 

3.1.1. Linearity:

The linearity of the methods was evaluated by analyzing six concentrations of each drug and each concentration was repeated five times. Linear regression equations were obtained over the concentration ranges given in Table 1. The assays were performed according to the experimental conditions previously mentioned.

 

3.1.2. Range:

The calibration range was established through considerations of the practical range that obeys Beer–Lambert law and the concentrations of AMX and FLX present in the pharmaceutical preparation to give accurate, precise and linear results, Table 1.

 

Table 1. Assay Parameters and Methods’ Validation Obtained by Applying ¹DD and Q-analysis Spectrophotometric Methods for the Simultaneous Determination of AMX and FLX in Mixture.

* The intraday precision, average of four different concentrations repeated five times within day. The interday precision, average of five different concentrations repeated five times in five successive days.

 

Table 2. Determination of AMX and FLX in Laboratory Prepared Mixtures by the Proposed ¹DD and Q-analysis Spectrophotometric Methods.

* Average of five determinations

 

Table 3. Determination of AMX and FLX in Amoxipen capsules by ¹DD and Q-analysis Spectrophotometric Methods and Application of the Standard Addition Technique.

* Average of five determinations

 

Table 4. Statistical comparison of the results obtained by the proposed spectrophotometric methods and the reported methods for the determination of AMX and FLX in dosage form.

a. Mean Recoveries of five determinations for the proposed method. Recoveries were calculated considering the labeled amount reported by the manufacturer.

*the tabulated t value at 95% confidence limit for 4 degrees of freedom (n =5) is 2.776 and the tabulated F value at 95% confidence limit for 4 degrees of freedom for the proposed methods is 6.25.

**HPLC method¹⁴

 

3.1.3. Accuracy:

Accuracy was determined by comparing measured concentrations of AMX and FLX with the actual values. The accuracy of the results was checked by applying the proposed methods for the determination of laboratory prepared mixtures containing both drugs. The concentrations were obtained from the corresponding regression equations, results are shown in Table 2.

 

Accuracy of the methods was further assured by the use of the standard addition technique, which was performed by addition of known amounts of pure AMX and FLX to known concentrations of the pharmaceutical preparation, the resulting mixtures were assayed and the results obtained were compared with the expected results. The good recoveries, as revealed in Tables 2 and 3indicate good accuracy and that there is no interference from either the co-formulated drug or the frequently encountered tablet excipients. Statistical comparison between the results obtained by the proposed spectrophotometric methods and the reported method for the determination of AMX and FLX in pharmaceutical dosage form was also done. The values of calculated t and F tests are less than the tabulated ones, which reveals that there is no significant difference between the suggested methods with respect to accuracy and precision, Table 4.

 

3.1.4. Precision:

Repeatability: four different concentrations of AMX and FLX were analyzed five times, intradaily, using the proposed methods. The percentage relative standard deviation (%RSD) was calculated, Table 1.

 

Reproducibility (Intermediate Precision): The previous procedure was repeated, interdaily, five times on five different days for the analysis of the four chosen concentrations of AMX and FLX.

 

3.1.5 Limit of Detection and Limit of Quantification:

According to the ICH recommendations, the approach based on the SD of the intercepts and the slope was used for determining the limit of detection and limit of quantification, Table 1.

 

LOD=3.3×SD / slope      LOQ=10×SD / slope

 

4. CONCLUSION:

The proposed ¹DD and analysis spectrophotometric methods allowed the resolution of binary mixture of AMX and FLX with satisfactory results. Q analysis have the advantage of eliminating the derivative steps. The proposed methods are simple, rapid, sensitive, precise and could be easily applied in quality control laboratories for the simultaneous determination of the cited drugs in pure and dosage forms, without any preliminary separation step. Furthermore, they are economic in comparison to the more time consuming chromatographic techniques often used for the assay of formulations. The suggested methods were validated as per ICH guidelines.

 

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Accepted on 25.03.2017           © RJPT All right reserved