Accepted Manuscript Highly Sensitive Colorimetric Determination of Amoxicillin in Pharmaceutical Formulations Based on Induced Aggregation of Gold Nanoparticles Morteza Akhond, Ghodratollah Absalan, Hamid Ershadifar PII: DOI: Reference:
S1386-1425(15)00092-X http://dx.doi.org/10.1016/j.saa.2015.01.071 SAA 13239
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
12 May 2014 19 January 2015 28 January 2015
Please cite this article as: M. Akhond, G. Absalan, H. Ershadifar, Highly Sensitive Colorimetric Determination of Amoxicillin in Pharmaceutical Formulations Based on Induced Aggregation of Gold Nanoparticles, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.01.071
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Highly
Sensitive
Colorimetric
Determination
of
Amoxicillin
in
Pharmaceutical Formulations Based on Induced Aggregation of Gold Nanoparticles Morteza Akhond, Ghodratollah Absalan*, Hamid Ershadifar Professor Massoumi Laboratory, Department of Chemistry, College of Sciences, Shiraz University, Shiraz, 71454, Iran Corresponding author: E–mail address: [email protected]; [email protected] Tel.: +98 (71) 3613-7137; Fax: +98 71 36460788
Abstract A novel, simple and highly sensitive colorimetric method is developed for determination of Amoxicillin (AMX). The system is based on aggregation of citrate-capped gold nanoparticles (AuNP) in acetate buffer (pH =4.5) in the presence of the degradation product of Amoxicillin (DPAMX). It was found that the color of gold nanoparticles changed from red to purple and the intensity of surface plasmon resonance (SPR) peak of AuNPs decreased. A new absorption band was appeared in the wavelength range of 600-700 nm upon addition of DPAMX. The absorbance ratio at the wavelength of 660 and 525 nm (A660/A525) was chosen as the analytical signal indirectly related to AMX concentration. The linearity of the calibration graph was found over the concentration range of 0.3 to 4.5 µM AMX with a correlation coefficient of 0.9967. Under the optimum experimental conditions, the detection limit was found to be 0.15 µM. The applicability of the method was successfully demonstrated by analysis of AMX in pharmaceutical formulations including capsules and oral suspensions.
1
Keywords: Amoxicillin; Gold
nanoparticles; Pharmaceutical formulations,
Colorimetric method
1. Introduction Amoxicillin (AMX), D-α-amino-p-hydroxybenzylpenicillin trihydrate, Fig. 1, is one of the most frequently used β-lactam antibiotics that has been employed to treat and reduce the spread of bacterial infections in human, with the benefits of maintaining animal welfare [1, 2]. Because of this, development of new procedures for AMX with sensitive and rapid measurement abilities, for the quality controlling purposes and for clinical monitoring, is important. One of the most frequently and successfully used methods for determination of AMX is HPLC [3-7]. This method suffers from some disadvantages such as requiring large amount of highly pure organic solvents, long equilibration times, and derivatizing treatment. Other analytical methods reported for determination of AMX in pharmaceutical formulations includes capillary electrophoresis [8], chemiluminescence [9] and voltammetry [10, 11]. The main disadvantages of these methods are complicated procedures and expensive laboratory equipments. By development of nanotechnology, preparation and characterization of nano-size materials provided new research area in different scientific approaches including chemistry, physics, biology, materials science, medicine, and catalysis. In this regard, novel optical properties of noble metal nanoparticles which originating from their surface plasmon resonances made them applicable in a wide variety of investigations in the field of analytical chemistry [12]. Since the plasmon frequency is sensitive to the refractive index nature of an interface with the local medium, any local changes to the environment of these nanoparticles could lead to sharp colorimetric changes of their dispersions. 2
Among the noble metal nanoparticles, gold nanoparticles are being extensively studied for biological and medical applications [13, 14]. As early as 1951, Turkevich introduced the citrate reduction of HAuCl4 to gold colloids in water [15], which was improved by Frens to tune particle sizes by changing reagent ratios [16]. Later, many attempts were made to synthesize and stabilize gold nanoparticles [17-19], that finally were successfully used for determination of traces of pharmaceutical and biological compounds [20-27]. Amoxicillin contains sulfur atoms that convert to thiol groups upon alkaline or acidic degradation which can induce aggregation of citrate-stabilized AuNPs (Fig. 1). In this paper, a simple and sensitive colorimetric detection system for AMX based on citrate-stabilized AuNPs aggregates in the presence of degradation product of Amoxicillin (DPAMX) is reported. The absorption ratio (A660/A525) was followed as analytical signal by UV-Vis spectrometer. The experimental parameters such as solution pH and buffer concentration were studied.
2. Experimental
2.1 Reagents and solutions Standard reference Amoxicillin trihydrate, AMX capsules (250 and 500 mg per capsule) and AMX oral suspension sample (125 mg per 5 mL) was purchased from Farabi Co., Iran. Tetrachloroauric(III) acid trihydrate, HAuCl4.3H2O (99.5%), and trisodium citrate dihydrate, Na3C6H5O7.2H2O, were obtained from Merck Chemical Company. Other reagents were of the highest purity available from Merck and were used without further purification. Aliquot of 0.1 g L−1 HAuCl4 stock solution was prepared by dissolving 0.25 g HAuCl4.3H2O in 250 mL of deionized water and was stored at 4 °C. The stock solution of AMX (5 mM) was prepared by dissolving appropriate amount of AMX 3
in deionized water-ethanol (5:2, v/v) diluted to 10 mL and was stored at 4 °C. The working standard solutions were prepared by suitable dilution of the stock solution with deionized water.
2.2 Synthesis of gold nanoparticles The citrate-capped AuNPs with different sizes (13, 25 and 44 nm) were prepared by means of the chemical reduction of HAuCl4 in the aqueous phase based on the well-documented Frens’ method [16]. According to this procedure, all glasswares should be placed in 3:1 HCl/HNO3 solutions and then rinsed with triply-distilled water for three times. An aqueous solution of HAuCl4.3H2O (0.01%, 50 mL) was refluxed under vigorous stirring in a round-bottom flask. A solution of Na3C6H5O7.2H2O (1 %, 0.75 mL for synthesis of 25 nm AuNPs) was added to HAuCl4 solution and after observing the color changing from pale yellow to red, the solution was heated up in boiling point for 30 min. After stopping heating, the solution was cooled down to room temperature while stirring and the resulted nanoparticles were stored at 4 ºC.
2.3 Instruments The UV-Vis absorption spectra were recorded against the solvent blank at room temperature, using Ultrospec 4000 spectrophotometer (Pharmacia Biotech.). The pH measurements were made with a Metrohm 780 pH meter using a combined glass electrode. The TEM micrographs of the colloidal dispersions were obtained by a transmission electron microscope (Zeiss, model ELMCO) operated at an accelerating voltage of 80 kV. Statistical analysis of TEM data has been done with image processing program (ImageJ software). The Dynamic Light Scattering
4
(DLS) experiment was conducted with HORIBA L-550 at a fixed-scattering angle of 90° and at a constant temperature of 25 °C.
2.4 Degradation of AMX samples To prepare the pharmaceutical sample solutions, contents of five AMX capsules were thoroughly mixed and 21.0 mg of the mixture was transferred into a 10-mL volumetric flask and diluted to the mark with distilled water-ethanol (5:2, v/v). Also, 2 mL of an oral AMX suspension was transferred into a 10-mL volumetric flask and was sonicated till complete dissolution. In order to degrade AMX, aliquot of 2.0 mL of either its standard or pharmaceutical sample solutions along with 3.0 mL of deionized water and 1.0 mL of 12 M HCl were mixed in a glass test tube. The resulting solution was heated in a water bath (98 ºC) for about 20 min. After cooling to room temperature, the solution was adjusted to pH 7.0 with 1.0 M NaOH and then diluted to 10 mL with deionized water in a volumetric flask.
3. Results and discussion 3.1 Characterization of AuNPs A previously reported procedure was used for synthesize of citrate-stabilized gold nanoparticles with different sizes [25]. The mean diameter and size distribution of AuNPs were obtained by both TEM micrographs and DLS. Fig. 2A shows the TEM image of AuNPs. Using the statistical analysis, the mean particle diameter was calculated and found to be 25±3 nm while DLS data indicated to a mean particle diameter of 32 nm (Fig. 2B). It should be mentioned that in DLS the diameter is hydrodynamic diameter while in TEM it refers to metal nanoparticles core [28].
5
The concentration of AuNPs (CAu) was calculated to be 5.25×10−10 mol L−1 (31.6×1013 particles per liter) by using the following equations [29]: Np =
C Au =
2 D π 3 a
n Np
3
(1) (2)
where Np is the number of Au atoms in each nanoparticle; ɑ refers to the edge of a unit cell with a value of 4.0786 Å; D refers to the diameter of the AuNPs with a value of 25±3 nm according to TEM image; and n refers to the final concentration (2.54×10−4 mol L−1) of HAuCl4 precursor.
3.2 Gold nanoparticle aggregation induced by degradation product of AMX Both nanoparticle and colloidal forms of gold that have been widely used for most biological studies are citrate-based particles. These particles are stabilized by the citrate layer through electrostatic interactions [30]. It has been noticed that the thiol group of a biomolecule exchanges with the citrate molecule capped over the gold particle [31]. Thereby, the presence of any other functional groups in these molecules that may interact with each other may lead to the aggregation of the gold particles. The UV-Vis spectroscopy has been used to characterize the absorbance of the gold particle suspension because surface plasmon excitation frequencies depend on the refractive index of the surrounding medium [32]. Figure 3 shows that the intensity of the plasmon absorption band of AuNPs at 525.0 nm decreased and a new peak appeared in longer wavelength (600-700 nm) upon addition of degradation product of AMX (i.e. DPAMX). The appearance of the new absorption band at longer wavelengths could be explained due to the dipole coupling between the plasmons of the neighboring aggregated particles. While the
6
increased refractive index causes a moderate red shift in the surface plasmon resonance (SPR) of the individual nanoparticles, the collective SPR of the coupled (aggregated) nanoparticles is highly sensitive to the refractive index of the surrounding environment [33]. Moreover, the color changed from red to purple, when DPAMX was added, but there was no color or spectral change upon addition of AMX itself. As it is seen in Fig. 1, AMX does not have any free −SH functional group to bind to gold nanoparticle surface. However, the hydrolysis in acidic media that followed by heating, yields penicillamine which contains −SH functional group. The citrate molecules can be easily replaced on the surfaces of gold particles via chemisorption-type interaction in a ligand exchange process [34]. Generally, interparticles H-bonding [30], electrostatic attraction between molecules adsorbed on different particles [35] and diminishing of electrostatic repulsion between particles at high ionic strength [36] can resulted in aggregation of particles such as citrate-capped AuNPs. The AuNPs can also aggregate in the presence of low concentration of multivalent metal ions if they could coordinate with ligand (linker) present at the nanoparticles shell [37]. The hydrophobic and steric forces can be ruled out here because they should be taken into account, respectively, when hydrocarbons or polymers are involved as the stabilizing agents [30]. However, the interactions become often very complex when the kinetic aspects of ligand-exchange process come into play. Hepel et al. [38] reported that the ligand-exchange process is controlled by breakage of H-bonds in citrate layer (at the edge of nanoparticle) and
formation of intermolecular H-bonds with
incoming ligand (i.e. DPAMX in this work). The process could also affected by pH of the solution and hence by the charge of both initial protecting (stabilizing) agent and the incoming ligand.
7
3.3 Optimization of the reaction conditions To monitor the aggregation of gold nanoparticles, the change in A660/A525value was monitored as it is directly related to the aggregation state of AuNPs and less sensitive to fluctuations of sampling and detection conditions. The A660 and A525 refer to absorbance at the wavelength of 660 and 525 nm, respectively. To investigate the effect of size on the aggregation behavior of citratestabilized AuNPs, in the presence of DPAMX, three sets of nanoparticles with different sizes (13, 25, and 44 nm) have been utilized. After addition of DPAMX to nanoparticles, different sets showed distinctly different absorption profiles (Fig. 4). The absorption peak ratio (A660/A525) for each profile, as the analytical signal, was calculated in order to find the particle size that could provide an efficient aggregation process in the presence of DPAMX. Since, the absorption peak ratio corresponding to the aggregation state of 25 nm AuNPs was the highest; this size of AuNPs was selected for further analysis. Such a behavior has been also reported for induced aggregation of citrate-stabilized gold nanoparticles that was explained by Maxwell-Garnett theory [39]. It should be mentioned that in the absence of DPAMX, depending on the size of the AuNPs, the color of the solution was observed to vary from red to pinkish-red indicating that the absorption peak must show a red shift when the particle size increases (Fig. 4). This was is in agreement with the Mie theory [39]. Among different buffer solutions including phosphate, citrate, acetate, and potassium hydrogen phthalate which were tested for choosing the best buffer type, acetate buffer was selected for further investigation as it improved the sensitivity of the measurement. In order to evaluate the effect of solution pH on aggregation of AuNPs, HCl and NaOH were used for pH adjustment in the range of 2.0 to 9.0 pH unit. As it is
8
shown in Fig. 5d, A660/A525 increases with increasing pH, reaches to a maximum value at pH 4.5 and then decreases. The pKa values for citric acid are reported to be 3.2, 4.8, and 6.4 [33], while for penicillamine the pKa values are not reported but its chemical structure is similar to cysteine. So, it is assumed that penicillamine behaves almost similar to cysteine, i.e., it must be in cationic form at low pH; zwitterionic at moderate pH; and anionic at high pH values. It should be mentioned that at low pH values the aggregation of citrate-stabilized AuNPs is observed even before addition of DPAMX that could be due to lack of electrostatic repulsion and interparticle Hbonding, as indicated for pH 2.0 in Fig. 5a. In the pH range of 3.0-7.0, citrate is mostly in anionic form and penicillamine presents as zwitterionic species and it can diffuse toward the surface of AuNPs where citrate ions are expected to be replaced by penicillamine through the thiol group. At higher pH values, diffusion of the anionic penicillamine toward the negatively-charged AuNPs is prohibited because of electrostatic repulsion so the ligand exchange process expected to be prohibited [40]. The effect of ionic strength has been evaluated by changing the buffer concentration in a series of AuNPs solutions just before injecting DPAMX and recording the spectra. As shown in Fig. 6, the absorbance ratio (A660/A525) increased by increasing the buffer concentration and reached a maximum value at 100 mM buffer solution. It should be mentioned that at such low concentration of the analyte that used, citrate molecules could not be completely replaced by penicillamine, so a mixed ligand shell is expected to surround AuNPs [41]. Under the applied pH, citrate molecules are negatively charged and penicillamine molecules are in their zwitterionic form. By increasing ionic strength, the charges on these molecules (including AuNPs) screened and hence diffusion of penicillamine toward the surface of AuNPs is improved. Consequently, the inter9
particle distance reduced which facilitate the formation of H-bonds as well as van der Waals attraction forces between nanoparticles. At such a high ionic strength, the electrostatic force between adjacent AuNPs is strongly screened and consequently the H-bonding could be responsible for the aggregation process. For further study, a buffer concentration of 100 mM was chosen.
3.4 Analytical parameters Under the optimal experimental conditions (pH=4.5 and buffer concentration of 100 mM), the analytical figures of merit were obtained for determination of AMX. Fig. 7 shows the effect of successive addition of DPAMX on the spectroscopic properties of the gold nanoparticle. As can be seen, two points must be taken into consideration; blue shift in the second peak (the peak due to aggregation process), and increasing in the background absorption. These observations prove the dependencies of the maximal wavelength and scattering intensity on the shape and size of gold nanoparticles aggregates [42]. Plotting A660/A525 vs. concentration in Fig. 7 indicates to a mathematical relationship of the form A660/A525 =0.196(±0.004)×CAMX+0.270(±0.010) which is linear in the range of 0.30-4.5 µM AMX with a regression coefficient of 0.9967. The theoretical limit of detection was found to be 0.15 µM evaluated by statistical treatment 3σb/m, where σb is equivalent to the standard deviation of intercept and m is the slope of the calibration curve [43]. The relative standard deviation (RSD) for five replicate measuring of 2.5 µM AMX was 2.1%. Table 1 compares the analytical figures of merits of some reported spectrophotometric methods for amoxicillin determination with the present work. In terms of lowest detection concentration and working range, the proposed method offers better results. Some chromatographic methods give better results but they require expensive instrument and high consumption of organic solvents. 10
3.5 Interference study of coexisting compounds with AMX In order to apply the proposed method to the determination of AMX in real samples, a study of the discrimination against possible interferants was carried out. The analytical signal for a fixed concentration of 2.5 µM AMX was compared with the analytical signal values obtained in the presence of variable concentrations of each possible interferant. The compounds taken into consideration were lactose, starch, glucose, fructose, sucrose, maltose and sorbitol (Fig. 8). The value of the relative response is defined as Si /Sa ×100, where Si is the signal recorded for solutions containing both AMX and possible interferant and Sa is the signal recorded for AMX solutions. Considering acceptable deviation of 5%, as a criterion for no interfering effect, the relative response obtained for most of these chemicals was found to be negligible. The results showed that 50-fold excess of all these compounds did not have any interference in the determination of 2.5µM AMX. The only interfering species was sorbitol. 3.6 Determination of AMX in pharmaceutical products The applicability of the method to the real samples was evaluated by determining the labeled amount of AMX in pharmaceutical formulations. The AMX in pharmaceutical formulation is marketed mainly as capsules (250 mg and 500 mg) and oral suspension. The ingredients other than amoxicillin trihydrate are commonly sugars and starch. The analytical Figures of merit of the procedure were evaluated by comparing AMX contents obtained by the officially available methods [51]. The results are summarized in Table 2. The results for AMX determination showed a satisfactory agreement with those specified by the respective manufacturers.
4. Conclusions 11
An exceptionally simple, rapid and highly sensitive method for measurement of AMX without any complicated protocols or expensive instrumentation except a spectrophotometer which is commonly available in all laboratories have been developed. The proposed sensor exhibited a low detection limit of 0.15 µM and a good linear working range from 0.30 to 4.5 µM AMX as well as a good precision evaluated as RSD=2.1%. It was found that the presence of some possible coexisting components in pharmaceutical formulations does not have any significant effect on determination of Amoxicillin; this makes feasible and accurate determination of AMX in real samples including capsules and oral suspension.
Acknowledgements The authors wish to acknowledge the support of this work by Shiraz University Research Council.
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Captions and legends for figures Fig. 1. The schematic representation of degradation of AMX in acidic medium and its induced aggregation on citrate-capped gold nanoparticles. Fig. 2.(A) The TEM image and (B) Hydrodynamic diameter
distributions
of
AuNPs. Fig. 3. (A) The UV-Vis absorption spectra of citrate-capped AuNPs, (a) in the absence of AMX or its degradation product, (b) in the presence of 2.5 µM AMX, and (c) in the presence of 2.5 µM degradation product. All solutions contained 0.1 M acetate buffer (pH 4.5). The values were acquired at 1 min after addition of the analytes. (B) The sample number corresponds to: citrate-capped AuNPs, (1) in the absence of AMX or its degradation product, (2) in the presence of 2.5 µM AMX, (3) in the presence of 0.1 µM of degradation product of AMX, (4) in the presence of 0.8 µM of the degradation product, and (5) in the presence of 2.5 µM of the degradation product of AMX. Fig. 4. UV–Vis absorption spectra of citrate -capped AuNPs with different sizes as indicated in absence (—) and presence (…) of 2.5 µM of degradation product AMX. All of the solutions contained 0.1 M acetate buffer (pH 4.5) Fig. 5. UV–Vis absorption spectra of citrate -capped AuNPs in absence (—) and presence (…) of 2.5 µM of degradation product AMX in three pH values, (a) pH=2.0, (b) pH=5.0, (c) pH=8.0. (d) Plot of A660/A525 vs. pH. Fig. 6. Effect of buffer concentration on aggregation of citrate-capped AuNPs in presence of 2.5 µM of degradation product AMX. (pH 4.5). The values were acquired at 1 min after addition of the analyte. Fig. 7.(A) Absorption spectral changes of a citrate-capped AuNPs solution containing, 0.10 M acetate buffer (pH=4.5), upon the addition of analyte 19
concentration with the range of 0.0 to 6.0 µM. (B) A plot of A660/A525 nm versus the concentrations of analyte in the range of 0.30 to 4.5 µM. Fig. 8. Recovery percent for 2.5 µM of degradation product of AMX in the presence of 50 µM of interferant species calculated as described above.
20
Table 1 The most important reported researches on determination of AMX Method
Detection limit(µM)
Linear range (µM)
[Refs.]
Bienzymatic UV-sectrophotometry
0.77
0.0-100
[44]
Spectrophotometric determination
-
3.6-184.8
[45]
HPLC with UV detection
240
480-9600
[3]
HPLC with UV detection
-
240-3600
[46]
HPLC with UV detection
1.5
1.5-48
[47]
0.012
120-1200
[48]
0.2
2-55
[9]
LC with fluorescence detection
0.144
0.24-96
[49]
HPLC with UV detection
0.01
0.36-1440
[50]
Capillary Electrophoresis
0.67
24-168
[8]
Colorimetric determination
0.15
0.3- 4.5
This work
LC-MS/MS Chemiluminescence determination
21
Table 2 Determination of AMX in capsules and oral suspension (n=5) Sample Capsule 1 Capsule 2 Oral suspension
Labeled (mg)
Found ± SD (mg)
Relative error (%)
250
262±5
4.8
500
526±11
5.3
2500
2593±51
3.7
22
Amoxicillin H 2N
HO
O
Penicillamine
H
H N
S H+
N O
OH O
HS
O
H2N
H N
OH + NH 2
HO
S
Dispersed
O
+ CO2
O
COO- + H3N NH3+ -OOC
S
Aggregated
Figure 1
23
(A)
40nm
250 nm
Figure 2
(B)
Figure 3
24
Figure 4
25
Figure 5
26
Figure 6
Figure 7
27
Figure 8
28
Amoxicillin H 2N
HO
O
Penicillamine
H
H N
S H+
N O
OH O
HS
O
H2N
H N
OH + NH 2
O
HO
S
Dispersed
COONH3+
+
H3N
-OOC
Aggregated
29
O
S
+ CO2
Highlights • The proposed method for determination of Amoxicillin is exceptionally simple, rapid, and sensitive. •
No complicated protocols or expensive instrumentation is required.
• The method has advantages of a higher sensitivity, a broader linear dynamic range and particularly a lower detection limit. • Other components usually present in pharmaceutical formulations have no significant affect on the analysis of Amoxicillin in real samples.
30
S1386-1425(15)00092-X http://dx.doi.org/10.1016/j.saa.2015.01.071 SAA 13239
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
12 May 2014 19 January 2015 28 January 2015
Please cite this article as: M. Akhond, G. Absalan, H. Ershadifar, Highly Sensitive Colorimetric Determination of Amoxicillin in Pharmaceutical Formulations Based on Induced Aggregation of Gold Nanoparticles, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.01.071
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highly
Sensitive
Colorimetric
Determination
of
Amoxicillin
in
Pharmaceutical Formulations Based on Induced Aggregation of Gold Nanoparticles Morteza Akhond, Ghodratollah Absalan*, Hamid Ershadifar Professor Massoumi Laboratory, Department of Chemistry, College of Sciences, Shiraz University, Shiraz, 71454, Iran Corresponding author: E–mail address: [email protected]; [email protected] Tel.: +98 (71) 3613-7137; Fax: +98 71 36460788
Abstract A novel, simple and highly sensitive colorimetric method is developed for determination of Amoxicillin (AMX). The system is based on aggregation of citrate-capped gold nanoparticles (AuNP) in acetate buffer (pH =4.5) in the presence of the degradation product of Amoxicillin (DPAMX). It was found that the color of gold nanoparticles changed from red to purple and the intensity of surface plasmon resonance (SPR) peak of AuNPs decreased. A new absorption band was appeared in the wavelength range of 600-700 nm upon addition of DPAMX. The absorbance ratio at the wavelength of 660 and 525 nm (A660/A525) was chosen as the analytical signal indirectly related to AMX concentration. The linearity of the calibration graph was found over the concentration range of 0.3 to 4.5 µM AMX with a correlation coefficient of 0.9967. Under the optimum experimental conditions, the detection limit was found to be 0.15 µM. The applicability of the method was successfully demonstrated by analysis of AMX in pharmaceutical formulations including capsules and oral suspensions.
1
Keywords: Amoxicillin; Gold
nanoparticles; Pharmaceutical formulations,
Colorimetric method
1. Introduction Amoxicillin (AMX), D-α-amino-p-hydroxybenzylpenicillin trihydrate, Fig. 1, is one of the most frequently used β-lactam antibiotics that has been employed to treat and reduce the spread of bacterial infections in human, with the benefits of maintaining animal welfare [1, 2]. Because of this, development of new procedures for AMX with sensitive and rapid measurement abilities, for the quality controlling purposes and for clinical monitoring, is important. One of the most frequently and successfully used methods for determination of AMX is HPLC [3-7]. This method suffers from some disadvantages such as requiring large amount of highly pure organic solvents, long equilibration times, and derivatizing treatment. Other analytical methods reported for determination of AMX in pharmaceutical formulations includes capillary electrophoresis [8], chemiluminescence [9] and voltammetry [10, 11]. The main disadvantages of these methods are complicated procedures and expensive laboratory equipments. By development of nanotechnology, preparation and characterization of nano-size materials provided new research area in different scientific approaches including chemistry, physics, biology, materials science, medicine, and catalysis. In this regard, novel optical properties of noble metal nanoparticles which originating from their surface plasmon resonances made them applicable in a wide variety of investigations in the field of analytical chemistry [12]. Since the plasmon frequency is sensitive to the refractive index nature of an interface with the local medium, any local changes to the environment of these nanoparticles could lead to sharp colorimetric changes of their dispersions. 2
Among the noble metal nanoparticles, gold nanoparticles are being extensively studied for biological and medical applications [13, 14]. As early as 1951, Turkevich introduced the citrate reduction of HAuCl4 to gold colloids in water [15], which was improved by Frens to tune particle sizes by changing reagent ratios [16]. Later, many attempts were made to synthesize and stabilize gold nanoparticles [17-19], that finally were successfully used for determination of traces of pharmaceutical and biological compounds [20-27]. Amoxicillin contains sulfur atoms that convert to thiol groups upon alkaline or acidic degradation which can induce aggregation of citrate-stabilized AuNPs (Fig. 1). In this paper, a simple and sensitive colorimetric detection system for AMX based on citrate-stabilized AuNPs aggregates in the presence of degradation product of Amoxicillin (DPAMX) is reported. The absorption ratio (A660/A525) was followed as analytical signal by UV-Vis spectrometer. The experimental parameters such as solution pH and buffer concentration were studied.
2. Experimental
2.1 Reagents and solutions Standard reference Amoxicillin trihydrate, AMX capsules (250 and 500 mg per capsule) and AMX oral suspension sample (125 mg per 5 mL) was purchased from Farabi Co., Iran. Tetrachloroauric(III) acid trihydrate, HAuCl4.3H2O (99.5%), and trisodium citrate dihydrate, Na3C6H5O7.2H2O, were obtained from Merck Chemical Company. Other reagents were of the highest purity available from Merck and were used without further purification. Aliquot of 0.1 g L−1 HAuCl4 stock solution was prepared by dissolving 0.25 g HAuCl4.3H2O in 250 mL of deionized water and was stored at 4 °C. The stock solution of AMX (5 mM) was prepared by dissolving appropriate amount of AMX 3
in deionized water-ethanol (5:2, v/v) diluted to 10 mL and was stored at 4 °C. The working standard solutions were prepared by suitable dilution of the stock solution with deionized water.
2.2 Synthesis of gold nanoparticles The citrate-capped AuNPs with different sizes (13, 25 and 44 nm) were prepared by means of the chemical reduction of HAuCl4 in the aqueous phase based on the well-documented Frens’ method [16]. According to this procedure, all glasswares should be placed in 3:1 HCl/HNO3 solutions and then rinsed with triply-distilled water for three times. An aqueous solution of HAuCl4.3H2O (0.01%, 50 mL) was refluxed under vigorous stirring in a round-bottom flask. A solution of Na3C6H5O7.2H2O (1 %, 0.75 mL for synthesis of 25 nm AuNPs) was added to HAuCl4 solution and after observing the color changing from pale yellow to red, the solution was heated up in boiling point for 30 min. After stopping heating, the solution was cooled down to room temperature while stirring and the resulted nanoparticles were stored at 4 ºC.
2.3 Instruments The UV-Vis absorption spectra were recorded against the solvent blank at room temperature, using Ultrospec 4000 spectrophotometer (Pharmacia Biotech.). The pH measurements were made with a Metrohm 780 pH meter using a combined glass electrode. The TEM micrographs of the colloidal dispersions were obtained by a transmission electron microscope (Zeiss, model ELMCO) operated at an accelerating voltage of 80 kV. Statistical analysis of TEM data has been done with image processing program (ImageJ software). The Dynamic Light Scattering
4
(DLS) experiment was conducted with HORIBA L-550 at a fixed-scattering angle of 90° and at a constant temperature of 25 °C.
2.4 Degradation of AMX samples To prepare the pharmaceutical sample solutions, contents of five AMX capsules were thoroughly mixed and 21.0 mg of the mixture was transferred into a 10-mL volumetric flask and diluted to the mark with distilled water-ethanol (5:2, v/v). Also, 2 mL of an oral AMX suspension was transferred into a 10-mL volumetric flask and was sonicated till complete dissolution. In order to degrade AMX, aliquot of 2.0 mL of either its standard or pharmaceutical sample solutions along with 3.0 mL of deionized water and 1.0 mL of 12 M HCl were mixed in a glass test tube. The resulting solution was heated in a water bath (98 ºC) for about 20 min. After cooling to room temperature, the solution was adjusted to pH 7.0 with 1.0 M NaOH and then diluted to 10 mL with deionized water in a volumetric flask.
3. Results and discussion 3.1 Characterization of AuNPs A previously reported procedure was used for synthesize of citrate-stabilized gold nanoparticles with different sizes [25]. The mean diameter and size distribution of AuNPs were obtained by both TEM micrographs and DLS. Fig. 2A shows the TEM image of AuNPs. Using the statistical analysis, the mean particle diameter was calculated and found to be 25±3 nm while DLS data indicated to a mean particle diameter of 32 nm (Fig. 2B). It should be mentioned that in DLS the diameter is hydrodynamic diameter while in TEM it refers to metal nanoparticles core [28].
5
The concentration of AuNPs (CAu) was calculated to be 5.25×10−10 mol L−1 (31.6×1013 particles per liter) by using the following equations [29]: Np =
C Au =
2 D π 3 a
n Np
3
(1) (2)
where Np is the number of Au atoms in each nanoparticle; ɑ refers to the edge of a unit cell with a value of 4.0786 Å; D refers to the diameter of the AuNPs with a value of 25±3 nm according to TEM image; and n refers to the final concentration (2.54×10−4 mol L−1) of HAuCl4 precursor.
3.2 Gold nanoparticle aggregation induced by degradation product of AMX Both nanoparticle and colloidal forms of gold that have been widely used for most biological studies are citrate-based particles. These particles are stabilized by the citrate layer through electrostatic interactions [30]. It has been noticed that the thiol group of a biomolecule exchanges with the citrate molecule capped over the gold particle [31]. Thereby, the presence of any other functional groups in these molecules that may interact with each other may lead to the aggregation of the gold particles. The UV-Vis spectroscopy has been used to characterize the absorbance of the gold particle suspension because surface plasmon excitation frequencies depend on the refractive index of the surrounding medium [32]. Figure 3 shows that the intensity of the plasmon absorption band of AuNPs at 525.0 nm decreased and a new peak appeared in longer wavelength (600-700 nm) upon addition of degradation product of AMX (i.e. DPAMX). The appearance of the new absorption band at longer wavelengths could be explained due to the dipole coupling between the plasmons of the neighboring aggregated particles. While the
6
increased refractive index causes a moderate red shift in the surface plasmon resonance (SPR) of the individual nanoparticles, the collective SPR of the coupled (aggregated) nanoparticles is highly sensitive to the refractive index of the surrounding environment [33]. Moreover, the color changed from red to purple, when DPAMX was added, but there was no color or spectral change upon addition of AMX itself. As it is seen in Fig. 1, AMX does not have any free −SH functional group to bind to gold nanoparticle surface. However, the hydrolysis in acidic media that followed by heating, yields penicillamine which contains −SH functional group. The citrate molecules can be easily replaced on the surfaces of gold particles via chemisorption-type interaction in a ligand exchange process [34]. Generally, interparticles H-bonding [30], electrostatic attraction between molecules adsorbed on different particles [35] and diminishing of electrostatic repulsion between particles at high ionic strength [36] can resulted in aggregation of particles such as citrate-capped AuNPs. The AuNPs can also aggregate in the presence of low concentration of multivalent metal ions if they could coordinate with ligand (linker) present at the nanoparticles shell [37]. The hydrophobic and steric forces can be ruled out here because they should be taken into account, respectively, when hydrocarbons or polymers are involved as the stabilizing agents [30]. However, the interactions become often very complex when the kinetic aspects of ligand-exchange process come into play. Hepel et al. [38] reported that the ligand-exchange process is controlled by breakage of H-bonds in citrate layer (at the edge of nanoparticle) and
formation of intermolecular H-bonds with
incoming ligand (i.e. DPAMX in this work). The process could also affected by pH of the solution and hence by the charge of both initial protecting (stabilizing) agent and the incoming ligand.
7
3.3 Optimization of the reaction conditions To monitor the aggregation of gold nanoparticles, the change in A660/A525value was monitored as it is directly related to the aggregation state of AuNPs and less sensitive to fluctuations of sampling and detection conditions. The A660 and A525 refer to absorbance at the wavelength of 660 and 525 nm, respectively. To investigate the effect of size on the aggregation behavior of citratestabilized AuNPs, in the presence of DPAMX, three sets of nanoparticles with different sizes (13, 25, and 44 nm) have been utilized. After addition of DPAMX to nanoparticles, different sets showed distinctly different absorption profiles (Fig. 4). The absorption peak ratio (A660/A525) for each profile, as the analytical signal, was calculated in order to find the particle size that could provide an efficient aggregation process in the presence of DPAMX. Since, the absorption peak ratio corresponding to the aggregation state of 25 nm AuNPs was the highest; this size of AuNPs was selected for further analysis. Such a behavior has been also reported for induced aggregation of citrate-stabilized gold nanoparticles that was explained by Maxwell-Garnett theory [39]. It should be mentioned that in the absence of DPAMX, depending on the size of the AuNPs, the color of the solution was observed to vary from red to pinkish-red indicating that the absorption peak must show a red shift when the particle size increases (Fig. 4). This was is in agreement with the Mie theory [39]. Among different buffer solutions including phosphate, citrate, acetate, and potassium hydrogen phthalate which were tested for choosing the best buffer type, acetate buffer was selected for further investigation as it improved the sensitivity of the measurement. In order to evaluate the effect of solution pH on aggregation of AuNPs, HCl and NaOH were used for pH adjustment in the range of 2.0 to 9.0 pH unit. As it is
8
shown in Fig. 5d, A660/A525 increases with increasing pH, reaches to a maximum value at pH 4.5 and then decreases. The pKa values for citric acid are reported to be 3.2, 4.8, and 6.4 [33], while for penicillamine the pKa values are not reported but its chemical structure is similar to cysteine. So, it is assumed that penicillamine behaves almost similar to cysteine, i.e., it must be in cationic form at low pH; zwitterionic at moderate pH; and anionic at high pH values. It should be mentioned that at low pH values the aggregation of citrate-stabilized AuNPs is observed even before addition of DPAMX that could be due to lack of electrostatic repulsion and interparticle Hbonding, as indicated for pH 2.0 in Fig. 5a. In the pH range of 3.0-7.0, citrate is mostly in anionic form and penicillamine presents as zwitterionic species and it can diffuse toward the surface of AuNPs where citrate ions are expected to be replaced by penicillamine through the thiol group. At higher pH values, diffusion of the anionic penicillamine toward the negatively-charged AuNPs is prohibited because of electrostatic repulsion so the ligand exchange process expected to be prohibited [40]. The effect of ionic strength has been evaluated by changing the buffer concentration in a series of AuNPs solutions just before injecting DPAMX and recording the spectra. As shown in Fig. 6, the absorbance ratio (A660/A525) increased by increasing the buffer concentration and reached a maximum value at 100 mM buffer solution. It should be mentioned that at such low concentration of the analyte that used, citrate molecules could not be completely replaced by penicillamine, so a mixed ligand shell is expected to surround AuNPs [41]. Under the applied pH, citrate molecules are negatively charged and penicillamine molecules are in their zwitterionic form. By increasing ionic strength, the charges on these molecules (including AuNPs) screened and hence diffusion of penicillamine toward the surface of AuNPs is improved. Consequently, the inter9
particle distance reduced which facilitate the formation of H-bonds as well as van der Waals attraction forces between nanoparticles. At such a high ionic strength, the electrostatic force between adjacent AuNPs is strongly screened and consequently the H-bonding could be responsible for the aggregation process. For further study, a buffer concentration of 100 mM was chosen.
3.4 Analytical parameters Under the optimal experimental conditions (pH=4.5 and buffer concentration of 100 mM), the analytical figures of merit were obtained for determination of AMX. Fig. 7 shows the effect of successive addition of DPAMX on the spectroscopic properties of the gold nanoparticle. As can be seen, two points must be taken into consideration; blue shift in the second peak (the peak due to aggregation process), and increasing in the background absorption. These observations prove the dependencies of the maximal wavelength and scattering intensity on the shape and size of gold nanoparticles aggregates [42]. Plotting A660/A525 vs. concentration in Fig. 7 indicates to a mathematical relationship of the form A660/A525 =0.196(±0.004)×CAMX+0.270(±0.010) which is linear in the range of 0.30-4.5 µM AMX with a regression coefficient of 0.9967. The theoretical limit of detection was found to be 0.15 µM evaluated by statistical treatment 3σb/m, where σb is equivalent to the standard deviation of intercept and m is the slope of the calibration curve [43]. The relative standard deviation (RSD) for five replicate measuring of 2.5 µM AMX was 2.1%. Table 1 compares the analytical figures of merits of some reported spectrophotometric methods for amoxicillin determination with the present work. In terms of lowest detection concentration and working range, the proposed method offers better results. Some chromatographic methods give better results but they require expensive instrument and high consumption of organic solvents. 10
3.5 Interference study of coexisting compounds with AMX In order to apply the proposed method to the determination of AMX in real samples, a study of the discrimination against possible interferants was carried out. The analytical signal for a fixed concentration of 2.5 µM AMX was compared with the analytical signal values obtained in the presence of variable concentrations of each possible interferant. The compounds taken into consideration were lactose, starch, glucose, fructose, sucrose, maltose and sorbitol (Fig. 8). The value of the relative response is defined as Si /Sa ×100, where Si is the signal recorded for solutions containing both AMX and possible interferant and Sa is the signal recorded for AMX solutions. Considering acceptable deviation of 5%, as a criterion for no interfering effect, the relative response obtained for most of these chemicals was found to be negligible. The results showed that 50-fold excess of all these compounds did not have any interference in the determination of 2.5µM AMX. The only interfering species was sorbitol. 3.6 Determination of AMX in pharmaceutical products The applicability of the method to the real samples was evaluated by determining the labeled amount of AMX in pharmaceutical formulations. The AMX in pharmaceutical formulation is marketed mainly as capsules (250 mg and 500 mg) and oral suspension. The ingredients other than amoxicillin trihydrate are commonly sugars and starch. The analytical Figures of merit of the procedure were evaluated by comparing AMX contents obtained by the officially available methods [51]. The results are summarized in Table 2. The results for AMX determination showed a satisfactory agreement with those specified by the respective manufacturers.
4. Conclusions 11
An exceptionally simple, rapid and highly sensitive method for measurement of AMX without any complicated protocols or expensive instrumentation except a spectrophotometer which is commonly available in all laboratories have been developed. The proposed sensor exhibited a low detection limit of 0.15 µM and a good linear working range from 0.30 to 4.5 µM AMX as well as a good precision evaluated as RSD=2.1%. It was found that the presence of some possible coexisting components in pharmaceutical formulations does not have any significant effect on determination of Amoxicillin; this makes feasible and accurate determination of AMX in real samples including capsules and oral suspension.
Acknowledgements The authors wish to acknowledge the support of this work by Shiraz University Research Council.
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[32] S.K. Ghosh, T. Pal, Interparticle coupling effect on the surface plasmon resonance of gold nanoparticles: From theory to applications, Chem. Rev.107 (2007) 4797. [33] I. Ojea-Jimenez, and V. Puntes, Instability of cationic gold nanoparticle bioconjugates: the role of citrate ions, J. Am. Chem. Soc. 131(2009) 13320. [34] F. Belal, M. M. El-Kerdawy, S. M. El-Ashry, D.R. El-Wasseef, Kinetic spectrophotometric determination of ampicillin and amoxicillin in dosage forms, Il Farmaco 55 (2000) 680. [35] M. Stobiecka, M. Hepel Double-shell gold nanoparticle-based DNA-carriers with poly-L-lysine binding surface, Biomaterials 32 (2011) 3312. [36] I.M.S. Lim, D. Mott, W. Ip, P.N. Njoki, Y. Pan, S. Zhou, C.J. Zhong, Interparticle interactions of glutathione mediated assembly of gold nanoparticles, Langmuir 24 (2008) 8857. [37] M. Hepel, J. Dallas, M.D. Noble, Interactions and reactivity of Hg(II) on glutathione modified gold electrode studied by EQCN technique, J. Electroanal. Chem. 622 (2008) 173. [38] M. Stobiecka, M. Hepel, Rapid functionalization of metal nanoparticles by moderator-tunable ligand-exchange process for biosensor designs, Sensor. Actuat. B 149 (2010) 373. [39] S. Basu, S.K. Ghosh, S. Kundu, S. Panigrahi, S. Praharaj, S. Pande, S. Jana, T. Pal, Biomolecule induced nanoparticle aggregation: Effect of particle size on interparticle coupling, J. Colloid Interface Sci. 313 (2007) 724. [40] C. Lu, N. Zhang, J. Li, Q. Li, Colorimetric detection of cephradine in pharmaceutical formulations via fluorosurfactant-capped gold nanoparticles, Talanta 81 (2010) 698.
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[41] M. Stobiecka, J. Deeb, M. Hepel, Ligand-exchange effects in gold nanoparticle, assembly induced by oxidative stress biomarkers: Homocysteine and cysteine, Biophys. Chem. 146 (2010) 98. [42] N.G. Khlebtsov, L.A. Dykman, Optical properties and biomedical applications of plasmonic nanoparticles, J. Quant. Spectrosc. Radiat. Transfer 111 (2010) [43] J. Miller, J.C. Miller , Statistics and Chemometrics for Analytical Chemistry, 5th Ed., Prentice-Hall, England 2005, p.123. [44] T. Rojanarata, P. Opanasopit, T. Ngawhirunpat, C. Saehuan, S. Wiyakrutta, V. Meevootisom,
A simple, sensitive and
green bienzymatic UV-
spectrophotometric assay of amoxicillin formulations, Enzyme Microb. Technol. 46 (2010) 292. [45]
M.Q.
Al-Abachi,
determination
of
H.
Haddi,
amoxicillin
A.M. by
Al-Abachi,
reaction
with
Spectrophotometric N,
N-dimethyl-p-
phenylenediamine and potassium hexacyanoferrate(III), Anal. Chim. Acta 554 (2005) 184. [46] H.J. Mascher, C. Kikuta, Determination of amoxicillin in human serum and plasma by high-performance liquid chromatography and on-line postcolumn derivatisation, J. Chromatogr. A 812 (1998) 221. [47] G. Hoizey, D. Lamiable, C. Frances, T. Trenque, M. Kaltenbach, J. Denis, H. Millart, Simultaneous determination of amoxicillin and clavulanic acid in human plasma by HPLC with UV detection, J. Pharm. Biomed. Anal. 30 (2002) 661. [48] A. Wen, T. Hang, S. Chen, Z. Wang, L. Ding, Y. Tian, M. Zhang, X. Xu, Simultaneous determination of amoxicillin and ambroxol in human plasma by LC–MS/MS: Validation and application to pharmacokinetic study, J. Pharm. Biomed. Anal. 48 (2008) 829.
17
[49] G. Delis, G. Batzias, G. Kounenis, M. Koutsoviti-Papadopoulou, Application and validation of a LC/fluorescence method for the determination of amoxicillin in sheep serum and tissue cage fluid, J. Pharm. Biomed. Anal. 49 (2009) 375. [50] H. Liu, H. Wang, V.B. Sunderland, An isocratic ion exchange HPLC method for the simultaneous determination of flucloxacillin and amoxicillin in a pharmaceutical formulation for injection, J. Pharm. Biomed. Anal. 37 (2005) 395. [51] A. Kar, Pharmaceutical drug analysis, 2nd ed., New Age International Publisher, New Delhi, India 2005, p. 307.
18
Captions and legends for figures Fig. 1. The schematic representation of degradation of AMX in acidic medium and its induced aggregation on citrate-capped gold nanoparticles. Fig. 2.(A) The TEM image and (B) Hydrodynamic diameter
distributions
of
AuNPs. Fig. 3. (A) The UV-Vis absorption spectra of citrate-capped AuNPs, (a) in the absence of AMX or its degradation product, (b) in the presence of 2.5 µM AMX, and (c) in the presence of 2.5 µM degradation product. All solutions contained 0.1 M acetate buffer (pH 4.5). The values were acquired at 1 min after addition of the analytes. (B) The sample number corresponds to: citrate-capped AuNPs, (1) in the absence of AMX or its degradation product, (2) in the presence of 2.5 µM AMX, (3) in the presence of 0.1 µM of degradation product of AMX, (4) in the presence of 0.8 µM of the degradation product, and (5) in the presence of 2.5 µM of the degradation product of AMX. Fig. 4. UV–Vis absorption spectra of citrate -capped AuNPs with different sizes as indicated in absence (—) and presence (…) of 2.5 µM of degradation product AMX. All of the solutions contained 0.1 M acetate buffer (pH 4.5) Fig. 5. UV–Vis absorption spectra of citrate -capped AuNPs in absence (—) and presence (…) of 2.5 µM of degradation product AMX in three pH values, (a) pH=2.0, (b) pH=5.0, (c) pH=8.0. (d) Plot of A660/A525 vs. pH. Fig. 6. Effect of buffer concentration on aggregation of citrate-capped AuNPs in presence of 2.5 µM of degradation product AMX. (pH 4.5). The values were acquired at 1 min after addition of the analyte. Fig. 7.(A) Absorption spectral changes of a citrate-capped AuNPs solution containing, 0.10 M acetate buffer (pH=4.5), upon the addition of analyte 19
concentration with the range of 0.0 to 6.0 µM. (B) A plot of A660/A525 nm versus the concentrations of analyte in the range of 0.30 to 4.5 µM. Fig. 8. Recovery percent for 2.5 µM of degradation product of AMX in the presence of 50 µM of interferant species calculated as described above.
20
Table 1 The most important reported researches on determination of AMX Method
Detection limit(µM)
Linear range (µM)
[Refs.]
Bienzymatic UV-sectrophotometry
0.77
0.0-100
[44]
Spectrophotometric determination
-
3.6-184.8
[45]
HPLC with UV detection
240
480-9600
[3]
HPLC with UV detection
-
240-3600
[46]
HPLC with UV detection
1.5
1.5-48
[47]
0.012
120-1200
[48]
0.2
2-55
[9]
LC with fluorescence detection
0.144
0.24-96
[49]
HPLC with UV detection
0.01
0.36-1440
[50]
Capillary Electrophoresis
0.67
24-168
[8]
Colorimetric determination
0.15
0.3- 4.5
This work
LC-MS/MS Chemiluminescence determination
21
Table 2 Determination of AMX in capsules and oral suspension (n=5) Sample Capsule 1 Capsule 2 Oral suspension
Labeled (mg)
Found ± SD (mg)
Relative error (%)
250
262±5
4.8
500
526±11
5.3
2500
2593±51
3.7
22
Amoxicillin H 2N
HO
O
Penicillamine
H
H N
S H+
N O
OH O
HS
O
H2N
H N
OH + NH 2
HO
S
Dispersed
O
+ CO2
O
COO- + H3N NH3+ -OOC
S
Aggregated
Figure 1
23
(A)
40nm
250 nm
Figure 2
(B)
Figure 3
24
Figure 4
25
Figure 5
26
Figure 6
Figure 7
27
Figure 8
28
Amoxicillin H 2N
HO
O
Penicillamine
H
H N
S H+
N O
OH O
HS
O
H2N
H N
OH + NH 2
O
HO
S
Dispersed
COONH3+
+
H3N
-OOC
Aggregated
29
O
S
+ CO2
Highlights • The proposed method for determination of Amoxicillin is exceptionally simple, rapid, and sensitive. •
No complicated protocols or expensive instrumentation is required.
• The method has advantages of a higher sensitivity, a broader linear dynamic range and particularly a lower detection limit. • Other components usually present in pharmaceutical formulations have no significant affect on the analysis of Amoxicillin in real samples.
30
