Research Article
Austin J Anal Pharm Chem. 2015; 2(5): 1050.
Three Spectrophotometric Methods for Simultaneous Determination of Ampicillin and Dicloxacillin in Presence of Their Major Impurity 6Aminopenicillanic Acid
Abdelrahman MM¹, Naguib IA¹, Elsayed MA¹* and Zaazaa HA²
¹Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, BeniSuef University, Alshaheed Shehata Ahmad Hegazy St, Egypt
²Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr ElAini St., Egypt
*Corresponding author: Mohamed A Elsayed, Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, BeniSuef University, Alshaheed Shehata Ahmad Hegazy St, Egypt.
Received: October 04, 2015; Accepted: October 14, 2015; Published: October 19, 2015
Abstract
Three simple, selective, accurate, precise and economic spectrophotometric methods were developed for simultaneous determination of ampicillin (AMP) and dicloxacillin (DX) in presence of their major impurity namely; 6aminopenicillanic acid (APA). Method A is derivative spectrophotometry, where DX was determined by first derivative D¹ spectrophotometric method at 207.4 nm, while AMP was determined by second derivative D2 spectrophotometric method at 229.6 nm. Method B is first derivative of ratio spectra spectrophotometry which allows determination of both AMP at 265.4 nm and DX at 244.2 nm without interference of each other and in presence of 6amino penicillanic acid (APA). Method C is the mean centering of ratio spectra spectrophotometric (MCR) method, which depends on measuring the mean centered values of ratio spectra of AMP, DX and APA at 223, 237 and 209 nm respectively. The accuracy and precision of the developed methods were determined while their selectivity was tested by their application for determination of the studied component in different synthetic prepared mixtures. Additionally, the developed methods were successfully used for determination of the studied drugs in their dosage form. Statistical comparison of the suggested methods with the reported HPLC one using F and student’s t tests showed no significant difference regarding both accuracy and precision. The specificity of the developed methods was investigated by analyzing different laboratory prepared mixtures of the AMP and DX.
Keywords: Ampicillin; Dicloxacillin; 6Aminopenicillanic acid; First derivative spectrophotometry; Second derivative spectrophotometric; Derivative ratio spectrophotometry; Mean centring of ratio spectra spectrophotometry
Introduction
Ampicillin shown in (Figure1), is known chemically as (2S, 5R, 6R)6([(2R)2amino2phenylacetyl] amino)3, 3 dimethyl 7oxo4thia1azabicyclo [3.2.0] heptane2carboxylic acid. Dicloxacillin shown in (Figure 1), is known chemically as (2S, 5R, 6R)6{[3(2, 6dichlorophenyl)5methyl oxazole4carbonyl] amino}3, 3dimethyl7oxo4thia1azabicyclo [3.2.0] heptane2 carboxylic acid. Both of them are used individually and in fixed dose combination as antibacterial agents [1, 2]. 6Aminopenicillanic acid shown in (Figure 1), is known chemically as (2S, 5R, 6R)6Amino3, 3dimethyl7oxo4 thia1azabicyclo [3.2.0] heptane2 carboxylic acid.
Figure 1: Chemical structures of Ampicillin (AMP), Dicloxacillin (DX) and 6Aminopenicillanic acid (APA).
Ampicillin is betalactam antibiotic that was used extensively to treat bacterial infections since 1961. It is considered part of the amino penicillin family and is nearly equivalent to amoxicillin in terms of spectrum and level of activity.
Ampicillin is a potent antibiotic with relatively shorttermed stability in aqueous solutions [3, 4]. It is used clinically to treat a broad range of bacterial infections [57]. With injection; ampicillin is distributed rapidly and widely, resulting in a high concentration of the drug in bile [8]. From bile, it is excreted into the gut and is known to cause disruption of the normal intestinal micro flora by diminishing the main flora and increasing the presence of yeast as well as inducing a high risk of Clostridium difficile colitis [9].
Dicloxacillin (INN) is a narrow spectrum antibiotic of the penicillin class. It is used to treat infections caused by susceptible grampositive bacteria. It is very similar to flucloxacillin and these two agents are considered interchangeable, also dicloxacillin is more acidstable than many other penicillins and can be given orally, in addition to parenteral routes. However, like methicillin, it is less potent than benzyl penicillin against nonβlactamaseproducing grampositive bacteria [10].
6Aminopenicillanic acid has a beta lactam nucleus and it is the major impurity for both of ampicillin and dicloxacillin [11].
Literature survey revealed the analytical methods for determination of ampicillin and dicloxacillin which include high performance liquid chromatography (HPLC) method for determination of ampicillin in its pharmaceutical formulation [12]. Another HPLC methods used for determination of ampicillin and dicloxacillin with other penicillins [1315], several spectrophotometric methods were reported for the assay of ampicillin and dicloxacillin but none of them can determine AMP and DX in the prescience of APA [1618].
The aim of the presented work is to develop and conduct a comparative study on three well established spectrophotometric methods for resolving the spectral interference problem of AMP and DX mixtures in presence of 19 % from their impurity APA without preliminary separation. The new methods are simple, accurate, precise, stability indicating and do not require any sophisticated apparatus or computer programs. The mean centering of ratio spectra spectrophotometric (MCR) method can determine the concentrations of AMP, DX and APA mixtures without preliminary separation.
Experimental
Instruments
A double beam UVvisible spectrophotometer (SHIMADZU, Japan) model UV1601 PC with quartz cell of 1 cm path length,connected to IBM compatible computer. The software was UVPC personal spectroscopy software version 3.7. The spectral band width was 2 nm and wavelengthscanning speed 2800 nm/min.
Matlab^{®} version R2014a [19] was used for the proposed mean centering of ratio spectra (MCR) spectrophotometric method.
Material and reagents
Pure samples: Ampicillin was supplied by SIGMAALDRICH, Talat Harab, Cairo, Egypt. Its purity was found to be 99.24% according to company’s analysis certificate.
Dicloxacillin was supplied by SIGMAALDRICH, Talat Harab, Cairo, Egypt. Its purity was found to be 99.18% according to company’s analysis certificate.
6Aminopenicillanic acid was supplied by SIGMAALDRICH, Talat Harab, Cairo, Egypt. Its purity was found to be 99.48% according to company’s analysis certificate.
Pharmaceutical dosage form: Cloxapen^{®} capsules batch No. 2941060404830, are labeled to contain 250 mg Ampicillin and 250 mg Dicloxacillin. It was manufactured by Misr Co. Pharmaceutical Industries, El Matareya, Cairo, Egypt.
Chemicals and reagents
All chemicals and solvents used throughout this work were of analytical grade and were used without extra purification.
Methanol HPLC grade was purchased from SigmaAldrich Chemie GmbH, Germany.
Hydrochloric acid analytical grade was purchased from El Nasr Co. Pharmaceutical Industries, Cairo, Egypt.
Procedure
Standard stock and working solutions: AMP, DX and APA standard stock solutions were prepared in concentration of 1mg mL^{1} in 0.05 mol L^{1} HCl, while AMP, DX and APA standard working solutions were prepared in concentration of 0.1 mg mL^{1} by diluting 10 mLs of their respective stock standard solutions to100 mL in 0.05 mol L^{1} HCl.
Spectral characteristic of AMP, DX and APA: The zeroorder absorption spectra of 28 μg mL^{1} of AMP, 18 μg mL^{1} of DX and 15 μg mL^{1} of APA were recorded against 0.05 mol L^{1} HCl as a blank over the range of 200–400 nm as shown in (Figure 2).
Figure 2: Zeroorder absorption spectra of 28 μg mL1 of AMP (……), 18 μg mL1 of DX (―) and 15 μg mL1 of APA() using 0.05 mol L1 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, 520 μg of DX and 530 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 200400 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 328 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 520μg 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 535 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 200255 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 530 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 328 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 49 % 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 200400 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 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.
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.A_{1}= 0.0260C_{1}  0.0027 r_{1}= 0.9997 at 207.4 nm for D¹ method of DX
P.A_{2}= 0.0049C_{2} + 0.0003 r_{2}= 0.9998 at 229.6 nm for D2 method of AMP
Where P.A_{1} and P.A_{2}, are the peak amplitudes of DX using D¹ and AMP using D2 methods, C_{1} and C_{2} are the concentration of DX in μg mL^{1} and AMP in μg mL^{1} respectively, and r_{1}, r_{2} 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.
Derivative Method
Ratio Derivative Method
Mean Centre Method
Parameters
AMP
DX
AMP
DX
AMP
DX
APA
Linearity range
328
µg ml^{1}
520
µg ml^{1}
328
µg ml^{1}
520
µg ml^{1}
535
µg ml^{1}
530
µg ml^{1}
328
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 interday 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.
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 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.
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 520 μg mL^{1} from which the regression equations were computed as follows
P.A_{1}= 0.0218 C_{1}+ 0.0002 r_{1}= 0.9999 at 265.4 nm for DD¹ method of AMP
P.A_{2}= 0.0130 C_{2} 0.0009 r_{2}= 0.9998 at 244.2 nm for DD¹ method of DX
Where P.A_{1} and P.A_{2}, are the peak amplitudes of AMP using DD¹ and DX using DD¹ methods, C_{1} and C_{2} are the concentration of AMP in μg mL^{1} and DX in μg mL^{1} respectively, and r_{1}, r_{2} 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 200290 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 (328μg mL^{1}) using 20 μg mL^{1} of DX and 5μg mL^{1}of APA as a divisor and 0.05 mol L^{1} HCl as a solvent.
Figure 8: Mean centered ratio spectra of DX (520μg mL^{1}) using 25μg mL^{1} of AMP and 5μg mL^{1}of APA as a divisor and 0.05 mol L^{1} HCl as a solvent.
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 (530μg 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.
P.A_{1}= 0.035 C_{1} 0.038 r_{1}= 0.9998 at 223 nm for AMP
P.A_{2}= 0.062 C_{2} 0.480 r_{2}= 0.9999 at 237 nm for DX
P.A_{3}= 0.318 C_{3} 0.386 r3= 0.9999 at 209 nm for APA
Where A_{1}, A_{2} and A_{3} are the absorbance of AMP, DX and APA, respectively, C_{1}, C_{2} and C_{3} are the concentration of AMP, DX and APA in μg mL^{1}, respectively, and r_{1}, r_{2} 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 200290 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).
Parameters
Derivative Method
Ratio Derivative Method
Mean Centre Method
Reported HPLC Method^{13}
AMP
DX
AMP
DX
AMP
DX
AMP
DX
Cloxapen^{® }capsules^{a}
(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 addition^{a}
100.541%
±1.025
101.012%
±1.012
100.108%
±1.233
100.13%
±1.308
100.621%
±1.203
100.205%
±1.001


Ftest (5.050)^{b}
1.284
1.929
1.507
2.279
1.592
2.122


Student's ttest(2.228)^{b}
0.819
0.980
1.219
1.193
1.121
1.120


^{a}Average of six determination.
^{b}The 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.
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 328 μg mL^{1}, 520 μg mL^{1} and 530μg mL^{1}, respectively for derivative and ratio derivative methods and 535 μg mL^{1}, 530 μg mL^{1} and 328 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 intradaily 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 interdaily 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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