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Surface plasmon resonance based selective and sensitive colorimetric determination of azithromycin using unmodified silver nanoparticles in pharmaceuticals and human plasma
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 170 (2017) 97–103
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Surface plasmon resonance based selective and sensitive colorimetric determination of azithromycin using unmodified silver nanoparticles in pharmaceuticals and human plasma Vijay D. Chavada a, Nejal M. Bhatt a, Mallika Sanyal b, Pranav S. Shrivastav a,⁎ a b
Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad 380009, Gujarat, India Department of Chemistry, St. Xavier's College, Navrangpura, Ahmedabad 380009, Gujarat, India
a r t i c l e
i n f o
Article history: Received 17 May 2016 Received in revised form 25 June 2016 Accepted 6 July 2016 Available online 8 July 2016 Keywords: Surface plasmon resonance Azithromycin Silver nanoparticles Colorimetry Pharmaceuticals Human plasma
a b s t r a c t In this article we report a novel method for colorimetric sensing and selective determination of a non-chromophoric drug-azithromycin, which lacks native absorbance in the UV–Visible region using unmodified silver nanoparticles (AgNPs). The citrate-capped AgNps dispersed in water afforded a bright yellow colour owing to the electrostatic repulsion between the particles due to the presence of negatively charged surface and showed surface plasmon resonance (SPR) band at 394 nm. Addition of positively charged azithromycin at a concentration as low as 0.2 μM induced rapid aggregation of AgNPs by neutralizing the negative charge on the particle surface. This phenomenon resulted in the colour change from bright yellow to purple which could be easily observed by the naked eye. This provided a simple platform for rapid determination of azithromycin based on colorimetric measurements. The factors affecting the colorimetric response like pH, volume of AgNPs suspension and incubation time were suitably optimized. The validated method was found to work efficiently in the established concentration range of 0.2–100.0 μM using two different calibration models. The selectivity of the method was also evaluated by analysis of nanoparticles-aggregation response upon addition of several anions, cations and some commonly prescribed antibiotics. The method was successfully applied for the analysis of azithromycin in pharmaceuticals and spiked human plasma samples with good accuracy and precision. The simplicity, efficiency and cost-effectiveness of the method hold tremendous potential for the analysis of such non-chromophoric pharmaceuticals. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Azithromycin (AZT) is an azalide, a subclass of macrolide antibiotics and is one of the world's best-selling antibiotics [1]. AZT is derived from erythromycin yet it shows higher activity against gram-negative organisms preserving good activity against gram-positive microorganisms. Due to its dibasic nature, AZT possesses greater oral bioavailability and improved acid stability compared to erythromycin. It shows excellent pharmacokinetic properties which includes extensive distribution within tissues and high drug concentrations within cells. AZT prevents bacterial growth by blocking bacterial protein synthesis. This action stops the growth of the bacteria and relieves symptoms of the bacterial infection, which include inflammation and pain. Hence, AZT is useful for the treatment of upper and lower respiratory tract infections, skin infections, intestinal infections and sexually transmitted infections [2,3]. The most widely used simple and economical methods for the quantification of AZT are based on spectrophotometric measurements. These methods involve formation of complex between AZT and the reagent ⁎ Corresponding author. E-mail address: [email protected] (P.S. Shrivastav).
http://dx.doi.org/10.1016/j.saa.2016.07.011 1386-1425/© 2016 Elsevier B.V. All rights reserved.
by charge transfer and\or ion-pair interactions [4]. Further no direct spectrophotometric measurement is reported for AZT, apparently due to its non-aromatic structure. Other detection and/or quantitative methods are largely based on chromatographic techniques [5–9]. Owing to the unique optical sensing properties of noble metal nanoparticles, particularly those of silver (AgNPs) and gold (AuNPs), they find widespread use in almost every field of chemistry [10–13]. Among these AgNPs has a significant advantage over AuNPs due to their high extinction coefficients and low cost, which make them more favourable compared to AuNPs [14,15]. Basically, the synthesized AgNPs remain in a dispersed state as a result of electrical repulsion among the negatively charged citrate molecules coated on the particle surface, and thus exhibit a bright yellow colour. The colour and optical properties of unmodified AgNPs can be controlled by triggering the surface charges upon reacting with some target molecules. This generally results in the loss of surface charge, aggregation of the AgNPs and thereby changes in colour. The same strategy has been extensively utilized for the colorimetric sensing of various metal ions [16,17] as well as drug molecules [18,19] and bio-molecules [20]. This fundamental strategy was adopted for the present work by explicitly balancing the surface charge of the AgNPs in presence of a target drug to obtain a response
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Fig. 1. TEM images and DLS histograms of AgNPs in the (a) absence and (b) presence of azithromycin.
which is visible to the naked eye. Till now, there are no reports on the use of metal nanoparticles for the colorimetric sensing of azithromycin. Therefore, the main objective of the present work was to fabricate a visual colorimetric sensor for highly facile, selective, sensitive and cost-effective determination of azithromycin based on selective aggregation reaction between azithromycin and unmodified AgNPs. 2. Experimental 2.1. Chemicals and materials Reference standard of azithromycin dihydrate (99.72%) and other drugs used for interference study, erythromycin dihydrate (99.23%), clarithromycin (99.35%), roxithromycin (98.97%) and doxycycline (99.82%) were procured from Clearsynth Laboratories Pvt. Ltd., (Mumbai,
India). HPLC grade methanol, dichloromethane, ethyl acetate and analytical reagent grade sodium hydroxide, hydrochloric acid, trisodium citrate, NaBH4 were purchased from E. Merck (Mumbai, India). Analytical grade metal salts were obtained from CDH Pvt. Ltd. (New Delhi, India). Water used in the study was prepared from Milli-Q water purification system from Millipore (Bangalore, India). Twenty tablets of Azintas® (Intas Pharmaceuticals Pvt. Ltd., India) and Azipro® (Cipla Ltd., India) claimed to possess 250 mg of AZT, were purchased from a local pharmacy store. Blank human plasma was obtained from Supratech Micropath (Ahmedabad, India) and was stored at − 20 ⁰C until use. 2.2. Instrumentation A Jasco V-570 double beam spectrophotometer (Kyoto, Japan) with a matched pair of 10 mm quartz cells were used for spectral
Fig. 2. UV absorption spectra of AgNPs with increasing concentration of azithromycin (0–100 μM).
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Fig. 3. FT-IR spectra of (a) citrate capped AgNPs, (b) pure azithromycin, and (c) AgNPs in the presence of azithromycin.
measurements. The parameters set for measurement were, scan range: 200–800 nm, bandwidth: 1 nm and scan speed: 400 nm/min. The recorded spectral data were processed with Jasco Spectra Manager© software version 1.53.01. A Sartorius GD503 analytical balance (Bradford, MA, USA), with a readability of 0.0001 g, was used for weighing through entire studies. Prime micropipette used for accurate and precise transfer of solutions with a varying volume capacity of 0.1 to 1.0 mL was obtained from Biosystem Company (Mumbai, India). A EQ-614A digital pH meter (Equiptronics Pvt. Ltd., Gujarat, India) was used to measure the pH values of the solutions. The mean particle size and size distribution were acquired using a dynamic light scattering (Microtrac-NanotracTM 10.5.2, USA) and a Transmission Electron Microscope (H-7500, Hitachi, Japan). FT-IR spectra in the spectral range of 4000–400 cm−1 were recorded using a Bruker Tensor–27® IR spectrophotometer.
Fig. 5. Effect of pH on the colorimetric response of AgNPs based sensor using (a) calibration model 1 and (b) calibration model 2.
dark. For pH-adjustment, spectral measurement and colorimetric sensing purpose, the prepared AgNPs was used after 5 times dilution with double-distilled water. The synthesized AgNPs could effectively be used for a minimum period of two months since no detectable change in the UV spectra was observed, indicating good stability of the nanosuspension.
2.3. Synthesis of unmodified silver nanoparticles (AgNPs) 2.4. Colorimetric determination of azithromycin Unmodified silver nanoparticles were prepared by liquid phase reduction of metal salt precursor (silver nitrate) according to the reported Creighton method [21] with a slight modification. Briefly, 50 mL solution of AgNO3 with a concentration of 1.0 × 10−3 M was first prepared in the ice bath. After cooling the solution for 5 min, 1 mL 1.0% trisodium citrate was introduced, followed by immediate addition of 0.6 mL 1.0 × 10−2 M NaBH4. The solution was stirred continuously for about 45 min, and the prepared AgNps suspension was aged for 24 h in
Typically, 1.0 mL of prepared AZT solution (concentration ranging from 0.2–100 μM) was successively added to 2.0 mL of diluted and pH-adjusted 0.36 nM AgNps suspension, in a 5 mL calibrated tube. The contents were thoroughly mixed and allowed to react for exactly 5 min. Thereafter the mixture was transferred to 10 mm quartz cell for spectrophotometric measurement. From the spectral results, two calibration curves were constructed.
Fig. 4. Suggested mechanism for colorimetric sensing of azithromycin upon aggregation of silver nanoparticles.
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3. Results and discussion 3.1. Characterization of the synthesized AgNPs The particle morphology was investigated in using dynamic light scattering (DLS) spectra of the prepared AgNPs before and after the addition of AZT, under the optimized conditions. From the histograms and TEM images in Fig. 1, it is clearly evident that the synthesized citrate capped AgNPs were mono dispersed and the distribution of particle size was centralized around 12 nm, which was in good agreement with the TEM images of the same. After the addition of AZT, the interparticle distance for AgNPs was reduced due to the formation of a larger assembly, and this aggregation reaction was confirmed from the particle size distribution as indicated by DLS and TEM results. Owing to the uniform electronic repulsion, the synthesized AgNPs exhibited bright yellow colour and correspondingly a single peak at 394 nm (Fig. 2). The concentration of AgNP solution after five times dilution was determined to be 0.36 nM according to the Beer's law. The extinction coefficient (e = 6.14 × 109 /M.cm) was calculated using the equation lne = 1.4418 lnD + 18.955, where D is the mean diameter of the nanoparticles, i.e. 12 nm [23]. 3.2. Colorimetric sensing mechanism The as-prepared AgNPs remain in a regular dispersed state as a consequence of electronic repulsion between the surface bound negatively charged citrate ions. This uniform repulsion prevents the particles to come closer and aggregate. The optical properties are largely dependent on the size, shape, and most importantly their inter-particle distance. However some positively charged molecules or electrolytes can
Fig. 6. Effect of incubation time on the colorimetric response for varying concentration of azithromycin using (a) calibration model 1 and (b) calibration model 2.
2.5. Preparation of sample from tablets Twenty tablets of both the generics were separately weighed and ground to fine powder. Thereafter, an amount of powder equivalent to 50 mg of AZT was immersed in minimum amount of methanol. To dissolve the drug completely dissolved, the mixture was ultrasonicated for 30 min and kept for 1 hour. The mixture was then centrifuged and the supernatant containing the extracted drug was collected and the residues were washed 2 times with minimum amount of methanol and centrifuged again. Finally, the supernatants were collected in a standard flask and were diluted to 100 mL with methanol. The working sample solution was prepared by aqueous dilution of the extracted sample. In order to explore the potential of the proposed colorimetric method, standard addition method was applied to detect AZT.
2.6. Preparation of plasma samples To further access the applicability of the assay, the method was used to determine the drug content in a plasma sample. Following the procedure of Xu et al. [22], briefly 0.2 mL blank human plasma was spiked with 0.2 mL of AZT solution (equivalent to 50, 75, and 100 μM), along with 80 μL of 1.0 M sodium hydroxide solution. The contents were mixed well and extracted with 2.0 mL of dichloromethane: ethyl acetate (20:80, v/v) solution. After vortexing for about 5 min, the sample was centrifuged at 14,000 rpm for 10 min. The organic layer was separated and evaporated to dryness under nitrogen atmosphere at 40 °C. The dried residue was reconstituted with 1.0 mL methanol, and then analyzed as described in Section 2.4.
Fig. 7. Linear relationship between the colorimetric response and azithromycin concentration using (A) calibration model 1, and (B) calibration model 2.
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Table 1 Analytical features of the developed method.
Curve Linear range (μM) LOD (μM) LOQ (μM) Slope Intercept Correlation coefficient (r2)
Calibration model 1
Calibration model 2
A394 vs. Concentration 0.2–20 μM 0.053 0.160 −0.0459 2.1649 0.9888
A500/A394 vs. log(C) 6.0–100 μM 1.084 1.274 0.3983 2.1325 0.9823
efficiently interact with the surface charge and neutralize it. The charge neutralization process leads to a decrease in the inter-particle distance resulting in a visible change in their surface plasmon resonance (SPR) properties. Consequently, the original SPR band is reduced with the emergence of a new SPR band at longer wavelength that can be measured using a simple spectrophotometer [19]. In the present investigation, citrate capped AgNPs were synthesized which showed a single SPR band centralized at 394 nm. Since the particles are surrounded with a uniform negative charge, the addition of positively charged AZT at pH 4.4 introduced a new SPR band at around 500 nm. This results due to increased inter-particle Van der Waal's forces. Apparently, the presence of sterically large structure is unfavourable for the functional groups of AZT to directly interact with the citrate ions on the silver atoms via a ligand exchange mechanism. However at the experimental pH values, AZT has two positively charged N-atoms and five –OH groups which can react with the surface bound citrate ions. This proposed interaction between the surface charge and AZT molecules resulted in the aggregation of the silver nanosuspension. As a result the original SPR band at 394 nm was systematically reduced in presence of increasing concentration of AZR with corresponding increase in the intensity of the band at 500 nm. The proposed results were also supported by DLS histograms of the as-prepared AgNPs and the aggregated AgNPs upon addition of AZT. The FT-IR spectra of citrate capped AgNPs, AZT and AZT bound with AgNPs is shown in Fig. 3a–c. The spectra of citrate capped AgNPs showed presence of sharp –COO– symmetric stretching band at 1385 cm−1. Upon addition of AZT, this peak displays a high-frequency shift (red shift) to 1392 cm−1, while the characteristic C\\N stretching band at 1054 cm−1 in the FT-IR spectra of pure AZT exhibits slightly visible differentiating shift to 1063 cm−1 due to the interaction with the citrate anions on the AgNPs surface. This band displacement primarily suggests some specific interaction, presumably ionic in nature between the drug and the citrate capped silver nanoparticles. The sensing mechanism is briefly represented in Fig. 4, which provides a simple platform for the sensitive determination of AZT based on colour change from monodispersed to aggregated state of AgNPs. 3.3. Factors affecting the colorimetric response Analytical conditions such as the pH of nanosuspension (3.0–6.7), volume of AgNPs suspension (1.0–3.0 mL), concentration of AZT (0.2– 100.0 μM) and incubation time (2.5–20 min) were examined in detail. It is well known that the pH of the medium largely affects the working
Fig. 8. Colorimetric response of the developed sensor towards azithromycin in presence of some antibiotics and inorganic interferants using (a) calibration model 1 and (b) calibration model 2.
range and sensitivity of nanoparticles based colorimetric sensing. In the present work, colorimetric determination was tested by varying the pH of silver nanosuspension. The pH of 0.36 nM AgNPs solution was adjusted in the range of 3.0–6.7 using 0.1 M HCl and 0.1 M NaOH solutions. From the results shown in Fig. 5, it is quite apparent that at pH below 3.5 the blank solution itself produces an observable colour response (self-aggregation), due to the neutralization of citrate ions as its pKa value is ~3.2. On the contrary at higher pH (N5.2) the response was too less to observe with a naked eye, and this condition might possibly lead to a higher calibration range. More precisely, AZT remains positively charged at pH values quite lower than the pKa value of the drug, so
Table 2 Results for intra-batch and inter-batch accuracy and precision. Calibration model
A394 vs. Concentration
A500/A394 vs. log of concentration
Standard added (μM)
2.5 5.0 7.5 10 15 20s
Intra-batch (n = 5; single batch) Found value (Mean ±
Inter-batch (n = 15; 5 from each batch) Precision (% RSD)
Found value (Mean ±
SD)
Accuracy (%)
SD)
Accuracy (%)
Precision (% RSD)
2.52 ± 0.05 5.07 ± 0.10 7.44 ± 0.17 10.11 ± 0.19 14.92 ± 0.24 19.73 ± 0.35
100.98 101.38 99.20 101.07 99.49 98.67
2.11 2.06 2.29 1.83 1.64 1.77
2.46 ± 0.06 5.06 ± 0.11 7.43 ± 0.17 9.87 ± 0.16 14.97 ± 0.33 20.09 ± 0.20
98.49 101.24 99.02 98.70 99.78 100.43
2.66 2.07 2.31 1.64 2.23 0.99
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Table 3 Results for real sample analysis (n = 3). Sample
Amount claimed (mg)
Amount found (mg, mean ± SD)
Accuracy (%)
Amount added (μM)
Amount recovered (μM, ± SD)
Recovery (%)
RSD (%)
Plasmaa
–
–
–
Azintas® tabletb
250
253.7 ± 5.58
101.49
Azipro® tabletb
250
247.9 ± 2.32
99.16
0.20 0.50 1.00 10.0 15.0 20.0 10.0 15.0 20.0
0.19 ± 0.01 0.49 ± 0.01 1.01 ± 0.02 9.94 ± 0.14 15.16 ± 0.26 19.75 ± 0.26 10.10 ± 0.15 14.91 ± 0.27 19.76 ± 0.39
99.60 99.16 101.34 99.42 101.08 98.74 101.04 99.43 98.79
2.61 2.19 1.85 1.45 1.71 1.32 1.45 1.81 2.01
a b
Using calibration model 1. Using calibration model 2.
that it possesses maximum interaction with the NPs surface charge and aggregation effect. Finally, pH 4.4 was selected as the optimum pH which offered optimum sensitivity, minimum blank response and observable colorimetric response for AZT. The volume of AgNPs suspension was also adjusted for adequate response [19], i.e. the colorimetric behavior was evaluated using different amounts of AgNPs (within the range of 1.0–3.0 mL) for various concentrations of AZT. It was found that the diluted and pH adjusted suspension was unable to produce a desirable optical changes at relatively higher amount, and lower amount deviated the linearity at higher concentration of AZT. It was concluded that higher AgNPs volume suffered from lower sensitivity; on the other hand lower volume restricted the linear range. Finally, 2.0 mL was selected as the optimum volume. The AgNPs aggregation kinetics was studied at different time intervals of 2.5 min after mixing the contents (Fig. 6). At higher concentration of AZT the colorimetric response reached a limiting value at around 5 min, while the reaction was much slower at lower concentration of AZT and was found to be completed within 7.5 min. Hence, all the colorimetric assays were carried out using 7.5 min of incubation time.
response under the defined conditions remained significantly unaltered. It was also demonstrated from the selectivity data that some common monovalent, bivalent and trivalent metal ions (Na+, K+, NH+ 4 , 2− Mg2 +, Ca2 + and Fe3 +) and anions (Cl−, NO− 3 and SO4 ) even up to 50 times higher concentration than that of AZT, did not interfere with the colorimetric aggregation reaction of AgNPs with AZT (Fig. 8). 3.5. Analysis of real samples In order to establish the practical applicability of the colorimetric assessment of AZT in real samples, the method was tested for the analysis of pharmaceutical formulations and also in human plasma. The mean recoveries of AZT ranged from 98.74–101.08% for pharmaceutical samples, and 99.16–101.34% in spiked blank plasma samples (Table 3). The precision (%RSD) of the method ranged from 1.32 to 2.61% for triplicate analysis of the samples. The accuracy and precision of the results obtained signifies the analytical merit of the method, and can have wide applications in drug testing laboratories. 4. Conclusions
3.4. Analytical features and validation of the method Using the above selected experimental conditions, several analyses were carried out to establish the sensitivity of the developed sensor. From the results obtained, it was found that as the concentration of AZT was increased from 0.2 to 100 μM, the colour of the solution gradually changed from yellow to red-brown and then to purple depending upon the AZT concentration. At much higher concentration, i.e. above 200 μM, the solution turned to almost clear liquid because of excessive aggregation of AgNPs. In order to establish a calibration range two models were adopted [18,24]. The first model used the absolute change in characteristic SPR band at 394 nm as the analytical signal and was directly correlated with the concentration of AZT. This approach was found to be linear for 0.2–20.0 μM concentration range with a correlation coefficient of 0.9888 (Fig. 7a). On the other hand, the second model was based on the correlation between the natural logarithm of analyte concentration and the ratio of absorbance values at two different wavelengths, i.e. 500 nm and 394 nm. The calibration plot was linear over the concentration range of 6.0–100 μM with correlation coefficient of 0.9823 (Fig. 7b). The summary of analytical performance of the developed method is shown in Table 1. The accuracy and precision of the method was evaluated for the analysis of AZT at three different concentration levels (Table 2). The accuracy values ranged from 98.49 to 101.43%, while the intra- and inter– batch precision values (% RSD) were ≤ 2.66% for both the calibration models, indicating good reproducibility of the method. In order to assess the selectivity of the developed nanosensor for AZT, spectral measurements were carried out in presence of several common antibiotics like erythromycin, clarithromycin, roxithromycin and doxycycline at 10 times higher concentration in the same manner. In terms of the colorimetric absorbance ratio (A500/A394), it could be said the analytical
A simple, rapid, and sensitive colorimetric method has been developed for determination of a non-chromophoric antibiotic drug, AZT using unmodified silver nanoparticles. Basically, the synthesized silver nanoparticles possess regular shell of citrate ions on the particle surface, which helps to stabilize the AgNPs by counteracting the effects of Van der Waals' force between AgNPs. The nanoparticles upon reacting with AZT displayed a distinct colour change from yellow to purple and thus afforded quick colorimetric determination. The parameters affecting colorimetric response were suitably optimized, under which the method could be used from 0.2–100.0 μM AZT concentration range without interference of some common antibiotic drugs and selected cations and anions. The method was applied for the analysis of AZT from pharmaceutical dosage and plasma samples, and the resultant recovery demonstrated its applicability for routine analysis of AZT. Above all, the developed colorimetric analytical method could be transferred to any pharmaceutical laboratory without the need for expensive instrumentation. Acknowledgements The authors gratefully acknowledge Department of Chemistry, Gujarat University for providing infrastructure and instrumental facility to carry out this work. One of the authors, Ms. Nejal M. Bhatt is also thankful to UGC F 4-1/2009 (BSR)/7-74/2007 for providing financial assistance through UGC BSR fellowship, New Delhi. References [1] WIPO case study report, Azythromycin: A world best-selling antibiotic–Pliva, 2009 http://www.wipo.int/ipadvantage/en/details.jsp?id=906 (Accessed on April 2016). [2] M.J. Parnham, V.E. Haber, E.J. Giamarellos-Bourboulis, G. Perletti, G.M. Verleden, R. Vos, Pharmacol. Ther. 143 (2014) 225–245.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Surface plasmon resonance based selective and sensitive colorimetric determination of azithromycin using unmodified silver nanoparticles in pharmaceuticals and human plasma Vijay D. Chavada a, Nejal M. Bhatt a, Mallika Sanyal b, Pranav S. Shrivastav a,⁎ a b
Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad 380009, Gujarat, India Department of Chemistry, St. Xavier's College, Navrangpura, Ahmedabad 380009, Gujarat, India
a r t i c l e
i n f o
Article history: Received 17 May 2016 Received in revised form 25 June 2016 Accepted 6 July 2016 Available online 8 July 2016 Keywords: Surface plasmon resonance Azithromycin Silver nanoparticles Colorimetry Pharmaceuticals Human plasma
a b s t r a c t In this article we report a novel method for colorimetric sensing and selective determination of a non-chromophoric drug-azithromycin, which lacks native absorbance in the UV–Visible region using unmodified silver nanoparticles (AgNPs). The citrate-capped AgNps dispersed in water afforded a bright yellow colour owing to the electrostatic repulsion between the particles due to the presence of negatively charged surface and showed surface plasmon resonance (SPR) band at 394 nm. Addition of positively charged azithromycin at a concentration as low as 0.2 μM induced rapid aggregation of AgNPs by neutralizing the negative charge on the particle surface. This phenomenon resulted in the colour change from bright yellow to purple which could be easily observed by the naked eye. This provided a simple platform for rapid determination of azithromycin based on colorimetric measurements. The factors affecting the colorimetric response like pH, volume of AgNPs suspension and incubation time were suitably optimized. The validated method was found to work efficiently in the established concentration range of 0.2–100.0 μM using two different calibration models. The selectivity of the method was also evaluated by analysis of nanoparticles-aggregation response upon addition of several anions, cations and some commonly prescribed antibiotics. The method was successfully applied for the analysis of azithromycin in pharmaceuticals and spiked human plasma samples with good accuracy and precision. The simplicity, efficiency and cost-effectiveness of the method hold tremendous potential for the analysis of such non-chromophoric pharmaceuticals. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Azithromycin (AZT) is an azalide, a subclass of macrolide antibiotics and is one of the world's best-selling antibiotics [1]. AZT is derived from erythromycin yet it shows higher activity against gram-negative organisms preserving good activity against gram-positive microorganisms. Due to its dibasic nature, AZT possesses greater oral bioavailability and improved acid stability compared to erythromycin. It shows excellent pharmacokinetic properties which includes extensive distribution within tissues and high drug concentrations within cells. AZT prevents bacterial growth by blocking bacterial protein synthesis. This action stops the growth of the bacteria and relieves symptoms of the bacterial infection, which include inflammation and pain. Hence, AZT is useful for the treatment of upper and lower respiratory tract infections, skin infections, intestinal infections and sexually transmitted infections [2,3]. The most widely used simple and economical methods for the quantification of AZT are based on spectrophotometric measurements. These methods involve formation of complex between AZT and the reagent ⁎ Corresponding author. E-mail address: [email protected] (P.S. Shrivastav).
http://dx.doi.org/10.1016/j.saa.2016.07.011 1386-1425/© 2016 Elsevier B.V. All rights reserved.
by charge transfer and\or ion-pair interactions [4]. Further no direct spectrophotometric measurement is reported for AZT, apparently due to its non-aromatic structure. Other detection and/or quantitative methods are largely based on chromatographic techniques [5–9]. Owing to the unique optical sensing properties of noble metal nanoparticles, particularly those of silver (AgNPs) and gold (AuNPs), they find widespread use in almost every field of chemistry [10–13]. Among these AgNPs has a significant advantage over AuNPs due to their high extinction coefficients and low cost, which make them more favourable compared to AuNPs [14,15]. Basically, the synthesized AgNPs remain in a dispersed state as a result of electrical repulsion among the negatively charged citrate molecules coated on the particle surface, and thus exhibit a bright yellow colour. The colour and optical properties of unmodified AgNPs can be controlled by triggering the surface charges upon reacting with some target molecules. This generally results in the loss of surface charge, aggregation of the AgNPs and thereby changes in colour. The same strategy has been extensively utilized for the colorimetric sensing of various metal ions [16,17] as well as drug molecules [18,19] and bio-molecules [20]. This fundamental strategy was adopted for the present work by explicitly balancing the surface charge of the AgNPs in presence of a target drug to obtain a response
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Fig. 1. TEM images and DLS histograms of AgNPs in the (a) absence and (b) presence of azithromycin.
which is visible to the naked eye. Till now, there are no reports on the use of metal nanoparticles for the colorimetric sensing of azithromycin. Therefore, the main objective of the present work was to fabricate a visual colorimetric sensor for highly facile, selective, sensitive and cost-effective determination of azithromycin based on selective aggregation reaction between azithromycin and unmodified AgNPs. 2. Experimental 2.1. Chemicals and materials Reference standard of azithromycin dihydrate (99.72%) and other drugs used for interference study, erythromycin dihydrate (99.23%), clarithromycin (99.35%), roxithromycin (98.97%) and doxycycline (99.82%) were procured from Clearsynth Laboratories Pvt. Ltd., (Mumbai,
India). HPLC grade methanol, dichloromethane, ethyl acetate and analytical reagent grade sodium hydroxide, hydrochloric acid, trisodium citrate, NaBH4 were purchased from E. Merck (Mumbai, India). Analytical grade metal salts were obtained from CDH Pvt. Ltd. (New Delhi, India). Water used in the study was prepared from Milli-Q water purification system from Millipore (Bangalore, India). Twenty tablets of Azintas® (Intas Pharmaceuticals Pvt. Ltd., India) and Azipro® (Cipla Ltd., India) claimed to possess 250 mg of AZT, were purchased from a local pharmacy store. Blank human plasma was obtained from Supratech Micropath (Ahmedabad, India) and was stored at − 20 ⁰C until use. 2.2. Instrumentation A Jasco V-570 double beam spectrophotometer (Kyoto, Japan) with a matched pair of 10 mm quartz cells were used for spectral
Fig. 2. UV absorption spectra of AgNPs with increasing concentration of azithromycin (0–100 μM).
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Fig. 3. FT-IR spectra of (a) citrate capped AgNPs, (b) pure azithromycin, and (c) AgNPs in the presence of azithromycin.
measurements. The parameters set for measurement were, scan range: 200–800 nm, bandwidth: 1 nm and scan speed: 400 nm/min. The recorded spectral data were processed with Jasco Spectra Manager© software version 1.53.01. A Sartorius GD503 analytical balance (Bradford, MA, USA), with a readability of 0.0001 g, was used for weighing through entire studies. Prime micropipette used for accurate and precise transfer of solutions with a varying volume capacity of 0.1 to 1.0 mL was obtained from Biosystem Company (Mumbai, India). A EQ-614A digital pH meter (Equiptronics Pvt. Ltd., Gujarat, India) was used to measure the pH values of the solutions. The mean particle size and size distribution were acquired using a dynamic light scattering (Microtrac-NanotracTM 10.5.2, USA) and a Transmission Electron Microscope (H-7500, Hitachi, Japan). FT-IR spectra in the spectral range of 4000–400 cm−1 were recorded using a Bruker Tensor–27® IR spectrophotometer.
Fig. 5. Effect of pH on the colorimetric response of AgNPs based sensor using (a) calibration model 1 and (b) calibration model 2.
dark. For pH-adjustment, spectral measurement and colorimetric sensing purpose, the prepared AgNPs was used after 5 times dilution with double-distilled water. The synthesized AgNPs could effectively be used for a minimum period of two months since no detectable change in the UV spectra was observed, indicating good stability of the nanosuspension.
2.3. Synthesis of unmodified silver nanoparticles (AgNPs) 2.4. Colorimetric determination of azithromycin Unmodified silver nanoparticles were prepared by liquid phase reduction of metal salt precursor (silver nitrate) according to the reported Creighton method [21] with a slight modification. Briefly, 50 mL solution of AgNO3 with a concentration of 1.0 × 10−3 M was first prepared in the ice bath. After cooling the solution for 5 min, 1 mL 1.0% trisodium citrate was introduced, followed by immediate addition of 0.6 mL 1.0 × 10−2 M NaBH4. The solution was stirred continuously for about 45 min, and the prepared AgNps suspension was aged for 24 h in
Typically, 1.0 mL of prepared AZT solution (concentration ranging from 0.2–100 μM) was successively added to 2.0 mL of diluted and pH-adjusted 0.36 nM AgNps suspension, in a 5 mL calibrated tube. The contents were thoroughly mixed and allowed to react for exactly 5 min. Thereafter the mixture was transferred to 10 mm quartz cell for spectrophotometric measurement. From the spectral results, two calibration curves were constructed.
Fig. 4. Suggested mechanism for colorimetric sensing of azithromycin upon aggregation of silver nanoparticles.
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3. Results and discussion 3.1. Characterization of the synthesized AgNPs The particle morphology was investigated in using dynamic light scattering (DLS) spectra of the prepared AgNPs before and after the addition of AZT, under the optimized conditions. From the histograms and TEM images in Fig. 1, it is clearly evident that the synthesized citrate capped AgNPs were mono dispersed and the distribution of particle size was centralized around 12 nm, which was in good agreement with the TEM images of the same. After the addition of AZT, the interparticle distance for AgNPs was reduced due to the formation of a larger assembly, and this aggregation reaction was confirmed from the particle size distribution as indicated by DLS and TEM results. Owing to the uniform electronic repulsion, the synthesized AgNPs exhibited bright yellow colour and correspondingly a single peak at 394 nm (Fig. 2). The concentration of AgNP solution after five times dilution was determined to be 0.36 nM according to the Beer's law. The extinction coefficient (e = 6.14 × 109 /M.cm) was calculated using the equation lne = 1.4418 lnD + 18.955, where D is the mean diameter of the nanoparticles, i.e. 12 nm [23]. 3.2. Colorimetric sensing mechanism The as-prepared AgNPs remain in a regular dispersed state as a consequence of electronic repulsion between the surface bound negatively charged citrate ions. This uniform repulsion prevents the particles to come closer and aggregate. The optical properties are largely dependent on the size, shape, and most importantly their inter-particle distance. However some positively charged molecules or electrolytes can
Fig. 6. Effect of incubation time on the colorimetric response for varying concentration of azithromycin using (a) calibration model 1 and (b) calibration model 2.
2.5. Preparation of sample from tablets Twenty tablets of both the generics were separately weighed and ground to fine powder. Thereafter, an amount of powder equivalent to 50 mg of AZT was immersed in minimum amount of methanol. To dissolve the drug completely dissolved, the mixture was ultrasonicated for 30 min and kept for 1 hour. The mixture was then centrifuged and the supernatant containing the extracted drug was collected and the residues were washed 2 times with minimum amount of methanol and centrifuged again. Finally, the supernatants were collected in a standard flask and were diluted to 100 mL with methanol. The working sample solution was prepared by aqueous dilution of the extracted sample. In order to explore the potential of the proposed colorimetric method, standard addition method was applied to detect AZT.
2.6. Preparation of plasma samples To further access the applicability of the assay, the method was used to determine the drug content in a plasma sample. Following the procedure of Xu et al. [22], briefly 0.2 mL blank human plasma was spiked with 0.2 mL of AZT solution (equivalent to 50, 75, and 100 μM), along with 80 μL of 1.0 M sodium hydroxide solution. The contents were mixed well and extracted with 2.0 mL of dichloromethane: ethyl acetate (20:80, v/v) solution. After vortexing for about 5 min, the sample was centrifuged at 14,000 rpm for 10 min. The organic layer was separated and evaporated to dryness under nitrogen atmosphere at 40 °C. The dried residue was reconstituted with 1.0 mL methanol, and then analyzed as described in Section 2.4.
Fig. 7. Linear relationship between the colorimetric response and azithromycin concentration using (A) calibration model 1, and (B) calibration model 2.
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Table 1 Analytical features of the developed method.
Curve Linear range (μM) LOD (μM) LOQ (μM) Slope Intercept Correlation coefficient (r2)
Calibration model 1
Calibration model 2
A394 vs. Concentration 0.2–20 μM 0.053 0.160 −0.0459 2.1649 0.9888
A500/A394 vs. log(C) 6.0–100 μM 1.084 1.274 0.3983 2.1325 0.9823
efficiently interact with the surface charge and neutralize it. The charge neutralization process leads to a decrease in the inter-particle distance resulting in a visible change in their surface plasmon resonance (SPR) properties. Consequently, the original SPR band is reduced with the emergence of a new SPR band at longer wavelength that can be measured using a simple spectrophotometer [19]. In the present investigation, citrate capped AgNPs were synthesized which showed a single SPR band centralized at 394 nm. Since the particles are surrounded with a uniform negative charge, the addition of positively charged AZT at pH 4.4 introduced a new SPR band at around 500 nm. This results due to increased inter-particle Van der Waal's forces. Apparently, the presence of sterically large structure is unfavourable for the functional groups of AZT to directly interact with the citrate ions on the silver atoms via a ligand exchange mechanism. However at the experimental pH values, AZT has two positively charged N-atoms and five –OH groups which can react with the surface bound citrate ions. This proposed interaction between the surface charge and AZT molecules resulted in the aggregation of the silver nanosuspension. As a result the original SPR band at 394 nm was systematically reduced in presence of increasing concentration of AZR with corresponding increase in the intensity of the band at 500 nm. The proposed results were also supported by DLS histograms of the as-prepared AgNPs and the aggregated AgNPs upon addition of AZT. The FT-IR spectra of citrate capped AgNPs, AZT and AZT bound with AgNPs is shown in Fig. 3a–c. The spectra of citrate capped AgNPs showed presence of sharp –COO– symmetric stretching band at 1385 cm−1. Upon addition of AZT, this peak displays a high-frequency shift (red shift) to 1392 cm−1, while the characteristic C\\N stretching band at 1054 cm−1 in the FT-IR spectra of pure AZT exhibits slightly visible differentiating shift to 1063 cm−1 due to the interaction with the citrate anions on the AgNPs surface. This band displacement primarily suggests some specific interaction, presumably ionic in nature between the drug and the citrate capped silver nanoparticles. The sensing mechanism is briefly represented in Fig. 4, which provides a simple platform for the sensitive determination of AZT based on colour change from monodispersed to aggregated state of AgNPs. 3.3. Factors affecting the colorimetric response Analytical conditions such as the pH of nanosuspension (3.0–6.7), volume of AgNPs suspension (1.0–3.0 mL), concentration of AZT (0.2– 100.0 μM) and incubation time (2.5–20 min) were examined in detail. It is well known that the pH of the medium largely affects the working
Fig. 8. Colorimetric response of the developed sensor towards azithromycin in presence of some antibiotics and inorganic interferants using (a) calibration model 1 and (b) calibration model 2.
range and sensitivity of nanoparticles based colorimetric sensing. In the present work, colorimetric determination was tested by varying the pH of silver nanosuspension. The pH of 0.36 nM AgNPs solution was adjusted in the range of 3.0–6.7 using 0.1 M HCl and 0.1 M NaOH solutions. From the results shown in Fig. 5, it is quite apparent that at pH below 3.5 the blank solution itself produces an observable colour response (self-aggregation), due to the neutralization of citrate ions as its pKa value is ~3.2. On the contrary at higher pH (N5.2) the response was too less to observe with a naked eye, and this condition might possibly lead to a higher calibration range. More precisely, AZT remains positively charged at pH values quite lower than the pKa value of the drug, so
Table 2 Results for intra-batch and inter-batch accuracy and precision. Calibration model
A394 vs. Concentration
A500/A394 vs. log of concentration
Standard added (μM)
2.5 5.0 7.5 10 15 20s
Intra-batch (n = 5; single batch) Found value (Mean ±
Inter-batch (n = 15; 5 from each batch) Precision (% RSD)
Found value (Mean ±
SD)
Accuracy (%)
SD)
Accuracy (%)
Precision (% RSD)
2.52 ± 0.05 5.07 ± 0.10 7.44 ± 0.17 10.11 ± 0.19 14.92 ± 0.24 19.73 ± 0.35
100.98 101.38 99.20 101.07 99.49 98.67
2.11 2.06 2.29 1.83 1.64 1.77
2.46 ± 0.06 5.06 ± 0.11 7.43 ± 0.17 9.87 ± 0.16 14.97 ± 0.33 20.09 ± 0.20
98.49 101.24 99.02 98.70 99.78 100.43
2.66 2.07 2.31 1.64 2.23 0.99
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Table 3 Results for real sample analysis (n = 3). Sample
Amount claimed (mg)
Amount found (mg, mean ± SD)
Accuracy (%)
Amount added (μM)
Amount recovered (μM, ± SD)
Recovery (%)
RSD (%)
Plasmaa
–
–
–
Azintas® tabletb
250
253.7 ± 5.58
101.49
Azipro® tabletb
250
247.9 ± 2.32
99.16
0.20 0.50 1.00 10.0 15.0 20.0 10.0 15.0 20.0
0.19 ± 0.01 0.49 ± 0.01 1.01 ± 0.02 9.94 ± 0.14 15.16 ± 0.26 19.75 ± 0.26 10.10 ± 0.15 14.91 ± 0.27 19.76 ± 0.39
99.60 99.16 101.34 99.42 101.08 98.74 101.04 99.43 98.79
2.61 2.19 1.85 1.45 1.71 1.32 1.45 1.81 2.01
a b
Using calibration model 1. Using calibration model 2.
that it possesses maximum interaction with the NPs surface charge and aggregation effect. Finally, pH 4.4 was selected as the optimum pH which offered optimum sensitivity, minimum blank response and observable colorimetric response for AZT. The volume of AgNPs suspension was also adjusted for adequate response [19], i.e. the colorimetric behavior was evaluated using different amounts of AgNPs (within the range of 1.0–3.0 mL) for various concentrations of AZT. It was found that the diluted and pH adjusted suspension was unable to produce a desirable optical changes at relatively higher amount, and lower amount deviated the linearity at higher concentration of AZT. It was concluded that higher AgNPs volume suffered from lower sensitivity; on the other hand lower volume restricted the linear range. Finally, 2.0 mL was selected as the optimum volume. The AgNPs aggregation kinetics was studied at different time intervals of 2.5 min after mixing the contents (Fig. 6). At higher concentration of AZT the colorimetric response reached a limiting value at around 5 min, while the reaction was much slower at lower concentration of AZT and was found to be completed within 7.5 min. Hence, all the colorimetric assays were carried out using 7.5 min of incubation time.
response under the defined conditions remained significantly unaltered. It was also demonstrated from the selectivity data that some common monovalent, bivalent and trivalent metal ions (Na+, K+, NH+ 4 , 2− Mg2 +, Ca2 + and Fe3 +) and anions (Cl−, NO− 3 and SO4 ) even up to 50 times higher concentration than that of AZT, did not interfere with the colorimetric aggregation reaction of AgNPs with AZT (Fig. 8). 3.5. Analysis of real samples In order to establish the practical applicability of the colorimetric assessment of AZT in real samples, the method was tested for the analysis of pharmaceutical formulations and also in human plasma. The mean recoveries of AZT ranged from 98.74–101.08% for pharmaceutical samples, and 99.16–101.34% in spiked blank plasma samples (Table 3). The precision (%RSD) of the method ranged from 1.32 to 2.61% for triplicate analysis of the samples. The accuracy and precision of the results obtained signifies the analytical merit of the method, and can have wide applications in drug testing laboratories. 4. Conclusions
3.4. Analytical features and validation of the method Using the above selected experimental conditions, several analyses were carried out to establish the sensitivity of the developed sensor. From the results obtained, it was found that as the concentration of AZT was increased from 0.2 to 100 μM, the colour of the solution gradually changed from yellow to red-brown and then to purple depending upon the AZT concentration. At much higher concentration, i.e. above 200 μM, the solution turned to almost clear liquid because of excessive aggregation of AgNPs. In order to establish a calibration range two models were adopted [18,24]. The first model used the absolute change in characteristic SPR band at 394 nm as the analytical signal and was directly correlated with the concentration of AZT. This approach was found to be linear for 0.2–20.0 μM concentration range with a correlation coefficient of 0.9888 (Fig. 7a). On the other hand, the second model was based on the correlation between the natural logarithm of analyte concentration and the ratio of absorbance values at two different wavelengths, i.e. 500 nm and 394 nm. The calibration plot was linear over the concentration range of 6.0–100 μM with correlation coefficient of 0.9823 (Fig. 7b). The summary of analytical performance of the developed method is shown in Table 1. The accuracy and precision of the method was evaluated for the analysis of AZT at three different concentration levels (Table 2). The accuracy values ranged from 98.49 to 101.43%, while the intra- and inter– batch precision values (% RSD) were ≤ 2.66% for both the calibration models, indicating good reproducibility of the method. In order to assess the selectivity of the developed nanosensor for AZT, spectral measurements were carried out in presence of several common antibiotics like erythromycin, clarithromycin, roxithromycin and doxycycline at 10 times higher concentration in the same manner. In terms of the colorimetric absorbance ratio (A500/A394), it could be said the analytical
A simple, rapid, and sensitive colorimetric method has been developed for determination of a non-chromophoric antibiotic drug, AZT using unmodified silver nanoparticles. Basically, the synthesized silver nanoparticles possess regular shell of citrate ions on the particle surface, which helps to stabilize the AgNPs by counteracting the effects of Van der Waals' force between AgNPs. The nanoparticles upon reacting with AZT displayed a distinct colour change from yellow to purple and thus afforded quick colorimetric determination. The parameters affecting colorimetric response were suitably optimized, under which the method could be used from 0.2–100.0 μM AZT concentration range without interference of some common antibiotic drugs and selected cations and anions. The method was applied for the analysis of AZT from pharmaceutical dosage and plasma samples, and the resultant recovery demonstrated its applicability for routine analysis of AZT. Above all, the developed colorimetric analytical method could be transferred to any pharmaceutical laboratory without the need for expensive instrumentation. Acknowledgements The authors gratefully acknowledge Department of Chemistry, Gujarat University for providing infrastructure and instrumental facility to carry out this work. One of the authors, Ms. Nejal M. Bhatt is also thankful to UGC F 4-1/2009 (BSR)/7-74/2007 for providing financial assistance through UGC BSR fellowship, New Delhi. References [1] WIPO case study report, Azythromycin: A world best-selling antibiotic–Pliva, 2009 http://www.wipo.int/ipadvantage/en/details.jsp?id=906 (Accessed on April 2016). [2] M.J. Parnham, V.E. Haber, E.J. Giamarellos-Bourboulis, G. Perletti, G.M. Verleden, R. Vos, Pharmacol. Ther. 143 (2014) 225–245.
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