Three Spectrophotometric Methods for Simultaneous Determination of Ampicillin and Dicloxacillin in Presence of Their Major Impurity 6-Aminopenicillanic Acid

    

    

    Figure 2:  Zero-order absorption spectra of 28 μg mL-1 of AMP (……), 18 μg
mL-1 of DX (―) and 15 μg mL-1 of APA(---) using 0.05 mol L-1 HCl as a solvent.
    
    

Laboratory prepared mixtures: Mixtures containing different ratios of AMP, DX and APA were prepared using their respective working solutions in 0.05 mol L^-1 HCl.

Construction of calibration curves: Aliquots equivalent to 3 –28 μg of AMP, 5-20 μg of DX and 5-30 μg of APA were accurately transferred from their working standard solutions (0.1 mg mL^-1) into two separate series of 10 mL volumetric flasks then completed to volume with 0.05 mol L^-1 HCl. The spectra of the prepared standard solutions were scanned from 200-400 nm.

First derivative D¹ spectrophotometric method: The D¹ spectra of DX solutions in the range of 5 – 20 μg mL^-1 were recorded using Δλ = 4 and scaling factor =10, then the peak amplitude was measured at 207.4 nm.

The D2 spectra of AMP solutions in the range of 3 – 28 μg mL^-1 were recorded using Δλ = 4 and scaling factor =100, then the peak amplitude was measured at 229.6 nm.

First derivative of ratio spectra spectrophotometric method: The DD¹ spectra of AMP solutions in the range of 3-28 μg mL^-1 were recorded using 5μg mL^-1 of DX product as a divisor, Δλ = 4 and scaling factor =10, then peak amplitude was measured at 265.4 nm.

The DD¹ spectra of DX solutions in the range of 5-20μg mL^-1 were recorded using 5 μg mL^-1 of AMP as a divisor, Δλ = 4 and scaling factor =10, then peak amplitude was measured at 244.2 nm.

The calibration curves were constructed relating the peak amplitudes against the corresponding drug concentrations and the regression equations were calculated in the presence of APA.

Mean centering of ratio spectra (MCR) method: Aliquots of AMP equivalent to 5-35 μg mL^-1 were accurately transferred from its standard working solution (0.1 mg mL^-1) into set of 10 ml measuring flasks and the volume was adjusted using 0.05 mol L^-1 HCl. The absorption spectra of the prepared solutions were recorded in the range of 200-255 nm and divided by the spectrum of 20 μg mL^-1 of DX and 15μg mL^-1 of APA then the obtained ratio spectra mean centered.

By the same way the spectra of different concentrations of standard DX 5-30 μg mL^-1 were recorded. The stored spectra were divided by the standard spectrum of 25 μg mL^-1 of AMP and 15 μg mL^-1 of APA to obtain the ratio spectra then mean centering of these ratio spectra was carried out.

Finally the spectra of different concentrations of standard APA 3-28 μg mL^-1 were recorded. The stored spectra were divided by the standard spectrum of 25 μg mL^-1 of AMP and 20 μg mL^-1 of DX to obtain the ratio spectra then mean centering of these ratio spectra was carried out.

Calibration curves for AMP, DX and APA were constructed by plotting the amplitudes of their respective mean centered ratio spectra from 200 to 255 nm (peak to peak) for both drugs and APA against their corresponding concentrations.

Analysis of laboratory prepared mixtures of AMP and DX in presence of APA

In two series of 10 mL measuring flasks, aliquots of AMP, DX and APA were transferred accurately from their corresponding working solutions (0.1 mg mL^-1) to prepare mixtures containing APA from 4-9 % of the pure AMP and DX in the mixture. The volume was completed with 0.05 mol L^-1 HCl. The spectra of the prepared solutions were recorded from 200-400 nm and stored in the computer. Procedures under calibration for each of the proposed methods were followed. The concentrations of the AMP and DX were calculated using the specified regression equations.

Application to pharmaceutical formulation (Cloxapen^® capsule)

The content of fourteen capsules of Cloxapen^® were powdered and mixed well. Accurately weighed amount of the powdered capsules equivalent to 100 mg of AMP and DX was transferred to 100 ml volumetric flask and 75 ml of 0.05 mol L^-1 HCl was added. The prepared solution was sonicated for 15 minutes, cooled and the volume was completed to obtain 1 mg mL^-1 stock solution and then the solution was filtered. Appropriate dilutions of the prepared sample were made to prepare its working solution (0.1 mg mL^-1) and the procedure under linearity was followed.

Results and Discussion

First derivative D¹ spectrophotometric method used for determination of DX and D2 spectrophotometric method used for determination of AMP in presence of APA

(Figure 2) shows the zero order absorption spectra of AMP, DX and APA which overlap seriously that direct determination of AMP and DX is not possible .The problem of overlapped spectra of AMP and DX is solved using first derivative (D¹) spectrophotometry to determine DX and second derivative (D2) spectrophotometry to determine DX in presence of APA.

(Figure 3) shows the first derivative spectra of AMP, DX and APA. It is obvious that the overlap observed in the zero order absorption spectra was eliminated at 207.4 nm for DX, which lies at the zero crossing of DX and APA. Different factors affecting resolution including type of solvent, Δλ and scaling factor were studied to optimize resolution of drugs. The best resolution was obtained using Δλ= 4nm, scaling factor=10 and 0.05 mol L^-1 HCl as solvent.

Figure 3: First derivative absorption spectra of 10 μg mL^-1 of Dx (―), 5 μg mL^-1 of AMP (-. -. - ) and 5 μg mL^-1 of APA (……) using 0.05 mol L^-1 HCl as a solvent.

    

    

    Figure 3:  First derivative absorption spectra of 10 μg mL-1 of Dx (―), 5 μg
mL-1 of AMP (-. -. - ) and 5 μg mL-1 of APA (……) using 0.05 mol L-1 HCl as
a solvent.
    
    

Close

(Figure 4) shows the second derivative spectra of AMP, DX and APA. It is obvious that the overlap observed in the zero order absorption spectra is eliminated at 229.6 nm for AMP which lies at the zero crossing of DX and APA. Different factors affecting resolution including type of solvent, Δλ and scaling factor were studied to optimize resolution of drugs. The best resolution was obtained using Δλ= 4nm, scaling factor=100 and 0.05 mol L^-1 HCl as solvent.

Figure 4: Second derivative absorption spectra of 28 μg mL^-1 of AMP (…..), 20 μg mL^-1 of DX (- - - -) and 10 μg mL^-1 of APA (-. -. - ) using 0.05 mol L^-1 HCl as a solvent.

    

    

    Figure 4:  Second derivative absorption spectra of 28 μg mL-1 of AMP (…..),
20 μg mL-1 of DX (- - - -) and 10 μg mL-1 of APA (-. -. - ) using 0.05 mol
L-1 HCl as a solvent.
    
    

Close

Linear correlations were obtained between peak amplitudes at 207.4 nm for D¹ spectra of DX in the concentration range 5–20 μg mL- 1, while linear correlations were obtained between peak amplitudes at 229.6 nm for D2 spectra of AMP in the concentration range 3–28 μg mL^-1 and the regression equation were computed as follows

P.A₁= 0.0260C₁ - 0.0027 r₁= 0.9997 at 207.4 nm for D¹ method of DX

P.A₂= 0.0049C₂ + 0.0003 r₂= 0.9998 at 229.6 nm for D2 method of AMP

Where P.A₁ and P.A₂, are the peak amplitudes of DX using D¹ and AMP using D2 methods, C₁ and C₂ are the concentration of DX in μg mL^-1 and AMP in μg mL^-1 respectively, and r₁, r₂ are the correlation coefficients.

Results described in (Table 1) showed that D¹ method and D2 method are selective, valid and applicable for the determination of DX and AMP in presence of APA in different laboratory prepared mixtures.

Table 1: Linear regression and analytical parameters of the proposed methods for determination of AMP and DX.

  

    
 
    
Derivative    Method
    
Ratio    Derivative Method
    
Mean    Centre Method
  
  

    
Parameters
    
AMP
    
DX
    
AMP
    
DX
    
AMP
    
DX
    
APA
  
  

    
Linearity range
    
3-28
      
µg ml-1
    
5-20
      
µg ml-1
    
3-28
      
µg ml-1
    
5-20
      
µg ml-1
    
5-35
      
µg ml-1
    
5-30
      
µg ml-1
    
3-28
      
µg ml-1
  
  

    
Slope 
    
  0.005
    
0.026
    
0.022
    
0.013
    
0.035
    
0.062
    
0.318
  
  

    
Intercept
    
0.001
    
-0.003
    
0.001
    
-0.001
    
-0.038
    
0.480
    
-0.386
  
  

    
Correlation coefficient
    
0.9998
    
0.9996
    
0.9999
    
0.9997
    
0.9998
    
0.9999
    
0.9999
  
  

    
Precision Repeatability(RSD%)a*
    
0.954
    
1.061 
    
0.943 
    
1.023 
    
  1.021
    
1.052
    
0.989
  
  

    
Intermediate
      
Precision(RSD%)b* 
    
  1.353 
    
  1.152
    
1.724
    
1.162
    
1.326
    
1.134
    
1.298
  
  

    
Accuracy
      
(Mean ± SD)
    
100.96 % ± 0.903
    
99.28 % ± 1.090
    
99.44 % ± 0.919
    
99.88 % ± 0.918
    
100.23    % ± 0.945
    
99.91 % ± 0.807
    
99.62 % ± 0.96 
  
  

    
LOD**
    
0.80
    
1.30
    
0.91
    
1.38
    
1.33
    
  1.28 
    
0.87
  
  

    
LOQ**
    
2.48
    
4.03
    
2.79
    
  4.28 
    
4.12
    
  3.96 
    
2.67 
  
  

    
*(RSD %) a* and (RSD %) b*;    the intra- and inter-day relative standard deviation of three concentrations    for each spectrophotometric method.
      
**Limit of detection and quantitation are determined    via calculations (LOD=3.3xSD of the response/slope, LOQ=10xSD of the    response/slope).
      
 
  

Table 1:  Linear regression and analytical parameters of the proposed methods for determination of AMP and DX.

Close

First derivative of ratio spectra spectrophotometric method

(Figure 5) shows the first derivative of ratio spectra of AMP, DX and APA. As seen, the trough at 265.4 nm for AMP which lies at the zero crossing of DX and APA can be adopted for determination of AMP in presence of DX and APA. Different factors affecting resolution including divisor concentration, type of solvent, Δλ and scaling factors were studied to optimize resolution of drugs. The best resolution was obtained using 5 μg mL^-1 of DX as a divisor, Δλ=4 nm, scaling factor=10 and 0.05 M HCl as solvent in terms of signal to noise ratio, sensitivity and selectivity. (Figure 6) shows the first derivative of ratio spectra of AMP, DX and APA. As seen, the trough at 244.2 nm for DX which lies at the zero crossing of AMP and APA can be adopted for determination of DX in presence of AMP and APA. Different factors affecting resolution including divisor concentration, type of solvent, Δλ and scaling factors were studied to optimize resolution of drugs. The best resolution was obtained using 5μg mL^-1 of AMP as a divisor, Δλ=4nm, scaling factor=10 and 0.05 mol L^-1 HCl as solvent in terms of signal to noise ratio, sensitivity and selectivity.

Figure 5: First derivative of ratio spectra of 28 μg mL^-1 of AMP (- - - -),10 μg mL^-1 of DX(……) and 5 μg mL^-1 of APA (―) using 5 μg mL^-1 of DX as a divisor and 0.05 mol L^-1 HCl as a solvent.

    

    

    Figure 5:  First derivative of ratio spectra of 28 μg mL-1 of AMP (- - - -),10 μg
mL-1 of DX(……) and 5 μg mL-1 of APA (―) using 5 μg mL-1 of DX as a divisor
and 0.05 mol L-1 HCl as a solvent.
    
    

Close

Figure 6: First derivative of ratio spectra of 10 μg mL^-1 of AMP (―),5 μg mL^-1 of DX(……) and 5 μg mL^-1 of APA (- - - -) using 5 μg mL^-1 of AMP as a divisor and 0.05 mol L^-1 HCl as a solvent.

    

    

    Figure 6:  First derivative of ratio spectra of 10 μg mL-1 of AMP (―),5 μg mL-1
of DX(……) and 5 μg mL-1 of APA (- - - -) using 5 μg mL-1 of AMP as a divisor
and 0.05 mol L-1 HCl as a solvent.
    
    

Close

Linear correlations were obtained between peak amplitudes at 265.4nm for DD¹ spectra of AMP in the concentration range 3–28 μg mL^-1 and peak amplitudes at 244.2 nm for DD¹ spectra of DX in concentration range of 5-20 μg mL^-1 from which the regression equations were computed as follows

P.A₁= 0.0218 C₁+ 0.0002 r₁= 0.9999 at 265.4 nm for DD¹ method of AMP

P.A₂= 0.0130 C₂- 0.0009 r₂= 0.9998 at 244.2 nm for DD¹ method of DX

Where P.A₁ and P.A₂, are the peak amplitudes of AMP using DD¹ and DX using DD¹ methods, C₁ and C₂ are the concentration of AMP in μg mL^-1 and DX in μg mL^-1 respectively, and r₁, r₂ are the correlation coefficients. Results described in (Table 1) showed that DD¹ method is selective, valid and applicable for the determination of AMP and DX in presence of each other and in presence of APA in different laboratory prepared mixtures.

Mean centering of ratio spectra (MCR) method

As shown in (Figure 1) the absorption spectra of AMP, DX and APA in 0.05 mol L^-1 HCl overlapped in the wavelength region of 200- 350 nm. To construct the calibration curves of the proposed method the absorption spectra of the standard solutions of the AMP with different concentrations were recorded in the wavelength range of 200-290 nm and divided by the standard spectrum of DX (20 μg mL^-1) and APA (5 μg mL^-1) and mean centering of the obtained ratio spectra were carried out and the concentrations of AMP were determined by measuring the amplitudes of the mean centered ratio spectra from 200 to 255 nm (peak to peak) as shown in (Figure 7). By the same way different standard solutions of the DX with different concentrations scanned then recorded spectra were divided by the standard spectrum of AMP (25 μg mL^-1) and APA (5 μg mL^-1) and the ratio spectra were obtained then mean centered. The values from 200 to 255 nm (peak to peak) in the obtained mean centered ratio spectra were used for determination of DX as shown in (Figure 8).

Figure 7: Mean centered ratio spectra of AMP (3-28μg mL^-1) using 20 μg mL^-1 of DX and 5μg mL^-1of APA as a divisor and 0.05 mol L^-1 HCl as a solvent.

    

    

    Figure 7:  Mean centered ratio spectra of AMP (3-28μg mL-1) using 20 μg mL-1
of DX and 5μg mL-1of APA as a divisor and 0.05 mol L-1 HCl as a solvent.
    
    

Close

Figure 8: Mean centered ratio spectra of DX (5-20μg mL^-1) using 25μg mL^-1 of AMP and 5μg mL^-1of APA as a divisor and 0.05 mol L^-1 HCl as a solvent.

    

    

    Figure 8:  Mean centered ratio spectra of DX (5-20μg mL-1) using 25μg mL-1
of AMP and 5μg mL-1of APA as a divisor and 0.05 mol L-1 HCl as a solvent.
    
    

Close

Finally different standard solutions of the APA with different concentrations were recorded and divided by the standard spectrum of AMP (25 μg mL^-1) and DX (20 μg mL^-1) and the ratio spectra were obtained which then mean centered. The values from 200 to 255 nm (peak to peak) in the obtained mean centered ratio spectra were used for determination of APA as shown in (Figure 9). The computed regression equation parameters for each of the studied drugs are given in (Table 1).

Figure 9: Mean centered ratio spectra of APA (5-30μg mL^-1) using 25μg mL^-1 of AMP and 20μg mL^-1 of DX as a divisor and 0.05 mol L^-1 HCl as a solvent.

    

    

    Figure 9:  Mean centered ratio spectra of APA (5-30μg mL-1) using 25μg mL-1
of AMP and 20μg mL-1 of DX as a divisor and 0.05 mol L-1 HCl as a solvent.
    
    

Close

P.A₁= 0.035 C₁- 0.038 r₁= 0.9998 at 223 nm for AMP

P.A₂= 0.062 C₂- 0.480 r₂= 0.9999 at 237 nm for DX

P.A₃= 0.318 C₃- 0.386 r3= 0.9999 at 209 nm for APA

Where A₁, A₂ and A₃ are the absorbance of AMP, DX and APA, respectively, C₁, C₂ and C₃ are the concentration of AMP, DX and APA in μg mL^-1, respectively, and r₁, r₂ and r3 are the corresponding correlation coefficients.

To optimize the developed MCR method different parameters were tested. The wavelength range used affects greatly the obtained mean centered ratio spectra, hence different ranges were tested and the good results were obtained when using range from 200-290 nm for AMP, DX and APA. Since the divisor concentration had great effect on the selectivity of the method different divisors were tried of DX and APA such as normalized spectra of 5, 15, 18 and 20 μg mL^-1 and 5, 10, 15 and 20 μg mL^-1 respectively (for AMP) and different divisors were tried of AMP and APA such as normalized spectra of 3, 5, 15 and 28 μg mL^-1 and 5, 10, 15 and 20 μg mL^-1 respectively (for DX). Finally different divisors were tried of AMP and DX such as normalized spectra of 3, 5, 15 and 28 μg mL^-1 and 5, 10, 15 and 20 μg mL^-1 respectively (for APA) .The best results regarding sensitivity and selectivity were obtained when using (25 μg mL^-1) of AMP, (20 μg mL- 1) of DX and (5 μg mL^-1) of APA as divisors.

The specificity of the proposed methods was assessed by analysis of different laboratory prepared mixtures containing different ratios of the suggested drugs, where satisfactory results were obtained in (Table 1). The developed spectrophotometric methods were also applied for determination of AMP and DX in Cloxapen^® capsules and the results obtained were acceptable. The validity of the methods was further assessed by applying the standard addition technique and confirmed the accuracy of the methods, (Table 2). The results obtained by applying the proposed methods for determination of tablets of AMP and DX in presence of APA were statistically compared to those obtained by applying a reported HPLC method [13] and the results showed no significant differences between the proposed methods and the reported one regarding accuracy and precision, (Table 2).

Table 2: Determination of the studied drugs in the pharmaceutical preparations by the proposed methods and statistical comparison with the reported HPLC method.

  

    
Parameters
    
Derivative    Method
    
Ratio    Derivative Method
    
Mean    Centre Method
    
Reported    HPLC Method13
  
  

    
 
    
AMP
    
DX
    
AMP
    
DX
    
AMP
    
DX
    
AMP
    
DX
  
  

    
Cloxapen® capsulesa
      
(B.N. 2941060404830)
    
99.325%±
      
0. 961
    
99.304%±
      
0.962
    
99.116%±
      
0.887
    
99.182%±
      
0.885
    
99.176%±
      
0.863
    
99.222%±
      
0.917
    
99.795%±
      
1.089
    
99.963%±
      
1.336
  
  

    
Standard additiona
    
100.541%
      
±1.025
    
101.012%
      
±1.012
    
100.108% 
      
  ±1.233
    
100.13%
      
±1.308
    
100.621%
      
±1.203
    
100.205% 
      
  ±1.001
    
 
      
-
    
 
      
-
  
  

    
F-test (5.050)b 
    
  1.284
    
1.929
    
1.507
    
2.279
    
1.592
    
2.122
    
-
    
-
  
  

    
Student's t-test(2.228)b 
    
  0.819
    
0.980
    
1.219
    
1.193
    
1.121
    
1.120
    
-
    
-
  
  

    
aAverage of six determination.
      
bThe values in the parenthesis are the corresponding    theoretical values at p= 0.05. 
      
 
  

Table 2:  Determination of the studied drugs in the pharmaceutical preparations by the proposed methods and statistical comparison with the reported HPLC method.

Close

On the other hand the suggested MCR method provide higher selectivity and reproducibility compared to the reported one, where it can determine AMP, DX and APA concentrations in presence of each other.

Application of methods in assay of Capsule

The proposed UV methods were applied for determination of AMP and DX in its pharmaceutical formulation Cloxapen^® capsules. The results are shown in (Table 2) and compared to that of the reported method [13]. Standard addition technique was also successfully applied. The acceptable percentage recoveries values confirm the suitability of the proposed methods for the determination of these components in its pharmaceutical formulations.

Method Validation

Validation was done according to ICH recommendations [20].

Linearity

Linearity of methods was evaluated by analyzing 8 concentrations of AMP, DX and APA between 3-28 μg mL^-1, 5-20 μg mL^-1 and 5-30μg mL^-1, respectively for derivative and ratio derivative methods and 5-35 μg mL^-1, 5-30 μg mL^-1 and 3-28 μg mL^-1, respectively for mean center method. Each concentration was repeated three times. The assay was performed according to the experimental conditions previously mentioned.

Accuracy

Accuracy of results was checked by applying the proposed methods for determination of different blind samples of AMP, DX and APA. The concentrations were obtained from the corresponding regression equations and percentage recoveries of the proposed methods were calculated with mean percentage recovery, suggesting good accuracy as shown in (Table 1).

Range

The calibration range was established through considerations of the practical range necessary according to adherence to Beer’s law and the concentration of AMP and DX present in the pharmaceutical preparations to give accurate precise and linear results, (Table 1).

Precision

Repeatability: Three concentrations of AMP, DX and APA (5, 15, 20 μg mL^-1) were analyzed three times intra-daily using the proposed methods. Good relative standard deviation of three concentrations was obtained confirming the repeatability of the methods.

Reproducibility (intermediate precision): The previous procedures were repeated inter-daily on three different days for the analysis of the three chosen concentrations. Good results and acceptable relative standard deviation (RSD %) are illustrated in (Table 1).

Specificity

Specificity of the methods was achieved by the analysis of different laboratory prepared mixtures of AMP, DX and APA within the calibration range. Satisfactory results are shown in (Table 1).

Conclusion

The developed derivative spectrophotometry, derivative ratio spectrophotometry and mean centring of ratio spectra spectrophotometric methods were successfully applied for simultaneous determination of AMP and DX in presence of APA in their combined sample; however mean centring of ratio spectra spectrophotometric method was the only one that could determine AMP, DX and APA simultaneously. The developed methods were found to be rapid, simple, accurate and easy to understand and apply. On the other hand mean centring of ratio spectra spectrophotometric method had the advantages of being simple, accurate and rapid. When suggested methods were completely validated they showed satisfactory data for all the method validation parameters tested. Recovery studies indicated that practically there was no interference from the capsules additives, hence, these methods can be easily and conveniently adopted for routine quality control analysis of AMP and DX.

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Citation: Abdelrahman MM, Naguib IA, Elsayed MA and Zaazaa HA. Three Spectrophotometric Methods for Simultaneous Determination of Ampicillin and Dicloxacillin in Presence of Their Major Impurity 6-Aminopenicillanic Acid. Austin J Anal Pharm Chem. 2015; 2(5): 1050. ISSN : 2381-8913

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