Research article Received: 19 November 2014,
Revised: 27 April 2015,
Accepted: 28 April 2015
Published online in Wiley Online Library: 23 June 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2952
Sensitive determination of enoxacin in pharmaceutical formulations by its quench effect on the fluorescence of glutathionecapped CdTe quantum dots Qiong Yang,a Xuanping Tanb and Jidong Yangb,c* ABSTRACT: A sensitive and simple method for the determination of enoxacin (ENX) was developed based on the fluorescence quenching effect of ENX for glutathione (GSH)-capped CdTe quantum dots (QDs). Under optimum conditions, a good linear relationship was obtained from 4.333 × 109 molL1 to 1.4 × 105 molL1 with a correlation coefficient (R) of 0.9987, and the detection limit (3σ/K) was 1.313 × 109 molL1. The corresponding mechanism has been proposed on the basis of electron transfer supported by ultraviolet–visible (UV) light absorption, fluorescence spectroscopy, and the measurement of fluorescence lifetime. The method has been applied to the determination of ENX in pharmaceutical formulations (enoxacin gluconate injections and commercial tablets) with satisfactory results. The proposed method manifested several advantages such as high sensitivity, short analysis time, low cost and ease of operation. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: CdTe quantum dots; enoxacin; fluorescence; pharmaceutical formulation
Introduction
Luminescence 2016; 31: 241–246
* Correspondence to: Jidong Yang, School of Chemistry and Chemical Engineering, Southwest University, Chongqing, Beibei 400715, China. E-mail: [email protected] a
School of Chemistry and Chemical Engineering, Yangtze Normal University, Chongqing, Fuling 408100, China
b
School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
c
School of Chemistry and Environmental Engineering, Chongqing Three Gorges University, Chongqing, Wanzhou 404000, China Abbreviations: BR, Britton-Robinson; BSA, bovine serum albumin; HPLC, highperformance liquid chromatography; PBS, phosphate-buffered saline; QD, quantum dots; TEM, Transmission electron microscopy; TGA, thioglycolic acid.
Copyright © 2015 John Wiley & Sons, Ltd.
241
Enoxacin (ENX) belongs to the third generation of multiple fluorinated antibacterial quinolone derivatives widely used in the treatment of systemic infections including urinary tract, respiratory, gastrointestinal and skin infections. The mechanism of action of ENX is based primarily on the inhibition of bacterial cell DNA gyrase and prohibiting DNA replication. Nowadays it is used extensively in clinical treatment and with higher potential as a broad antibacterial spectrum lower side effects (1). However, public and scientific concerns about the potential risk of quinolones to human health have been increasing due to their latent allergic hypersensitivity reactions or toxic effects on the articular cartilages causing arthralgia or juvenile arthropathies (2,3). To better exploit and safely utilize ENX in clinical disease diagnosis and treatment, it is necessary to develop a simple, rapid, high sensitivity, low-cost and convenient method for quantitative determination of ENX in pharmaceutical formulation. To date, for ENX detection, methods such as spectrophotometry (4,5), high-performance liquid chromatography (HPLC) (6,7), chemical luminescence (8), capillary electrophoresis (9), nuclear magnetic resonance spectroscopy (10) and so on have developed. However, many of these methods are not readily adaptable to routine analysis because they are time-consuming and require relatively expensive and complicated instruments. Based on this problem, a room-temperature phosphorescence assay for ENX with Mn-doped ZnS Dots as probe has been reported (3). Compared with those methods mentioned above, this assay is simple but only limited to the doped quantum dots (QDs), and therefore led indirectly to increases in detection costs and difficulty. Given all these factors, it is therefore necessary to develop a more low-cost, practical and convenient method for ENX detection.
Semiconductor QDs, one of the most useful type of nanoparticle, have attracted extensive application studies in many fields due to their several distinct properties compared with other luminescent materials, such as greater brightness, better stability with respect to photobleaching, broad excitation wavelength range, narrower symmetrical emission bands and size-tunable photoluminescence (11–13). All of these properties therefore promise that QDs will be ideal fluorescence indicators for chemical and biological assays. Recently, QDs have been exploited increasingly as fluorescent probes to determine drug molecules based on fluorescence quenching or enhancement. For example, Shen et al. (14) used CdTe QDs as luminescent probes for ellagic acid determination based on the quenching of CdTe QDs fluorescence by ellagic acid. Wang et al. (15) have developed a new assay for melamine based on the measurement of enhanced fluorescence intensity signal resulting from the interaction of thioglycolic
Q. Yang et al. acid-capped CdS QDs with melamine and Liang et al. (16) developed a fluorescence method for spironolactone determination with CdSe Dots as luminescent probes. To our knowledge, the use of glutathione (GSH)-capped CdTe QDs as fluorescence probes for the quantitative determination of ENX has not been reported so far. In this article, a facile, rapid, low-cost, convenient and sensitive assay for ENX has been proposed for the first time based on the decrease in fluorescence intensity of CdTe QDs. The corresponding interaction mechanism between CdTe QDs and ENX was investigated by ultraviolet–visible (UV-vis) light absorption, fluorescence spectroscopy and the measurement of fluorescence lifetime. It was found that the fluorescence intensity of CdTe QDs was quenched at 558 nm in the presence of ENX. In addition, interaction information regarding the mechanism of the reaction, such as suitable reaction conditions, affecting factors and the influence of coexisting substances, was studied.
Synthesis of CdTe QDs GSH-capped CdTe QDs were synthesized according to a previously described method (17). Under N2 atmosphere and magnetic stirring, tellurium powder (0.0383 g) was reacted with excess sodium borohydride in deionized water to produce the colorless solution of sodium hydrogen telluride (NaHTe). CdCl2.2.5H2O (0.1028 g) and GSH (0.1844 g) were dissolved in 150 mL deionized water. Under magnetic stirring, the pH of the mixture was adjusted to 10.2 by the dropwise addition of NaOH solution (1 mol × L1). The solution was deaerated by Ar bubbling for about 40 min. Under stirring, H2Te gas generated by the reaction of NaHTe solution with diluted H2SO4 (0.5 mol × L1) and was passed through an oxygen-free Cd2+ solution together with a slow nitrogen flow. Then the resulting solution mixture was heated to 373 K and refluxed under nitrogen for about 1 h. Salmon pink coloured CdTe solution was obtained. The concentration of GSH-capped CdTe QDs was 3 × 103 molL1 (determined by the Cd2+ concentration).
Experimental Reagents and apparatus
General procedure for ENX detection
All chemicals used were of at least analytical grade. The following reagents were purchased: tellurium power (99.99%, Sinopharm Chemical Reagent Co., Shanghai, China), NaBH4 (98%, Tianjin Huanwei Fine Chemical Co., Tianjin, China), CdCl2 · 2.5H2O (99.95%, Shanghai Chemicals Reagent Co., Shanghai, China), l-glutathione (GSH, 98%, Aladdin Reagent Co., Shanghai, China), and enoxacin (ENX, 99%, Aladdin Reagent Co., Shanghai, China). Phosphate-buffered saline (PBS) solutions of different pH were prepared by mixing 1/30 mol · L1 KH2PO4 with 1/30 mol · L1 Na2HPO4 in different proportions. All solutions were prepared using deionized water as solvent. A Hitachi F-4500 spectrofluorophotometer (Hitachi Company, Tokyo, Japan) was used to record the fluorescence spectra. A U-3010 spectrophotometer (Tianmei Corporation, Shanghai, China) was applied to record the absorption spectra. Transmission electron microscopy (TEM) images of CdTe QDs were acquired on a ZEISS LIBRA 200 FE transmission electron microscope (ZEISS, Berlin, Germany). A FL-TCSPC Fluorolog-3 fluorescence spectrometer (Horiba Jobin Yvon Inc., France) was used to measure the fluorescence lifetime of the CdTe QDs–ENX system at room temperature (λex/λem = 350 nm/558 nm). A PHS-3C pH meter (Leici, Shanghai, China) was used to adjust the pH values of the aqueous solutions.
In order to determine ENX, 1 mL above synthesized CdTe QDs, 1 mL PBS (pH 7.4) and an appropriate amount of ENX were added into a 10 mL volumetric flask in turn, then diluted with deionized water to the mark and mixed thoroughly with gentle shaking. After incubation for 10 min, the fluorescence, absorption spectra and fluorescence lifetime of the solution were examined.
Results and discussion Characterization of CdTe QDs Fig. 1(A) shows the absorption (curve 1) and fluorescence (curve 2) spectra of these as-prepared CdTe QDs at room temperature. It can be seen that the line width of the fluorescence spectrum is narrow (with the full width at half-maximum about 40 nm), showing that the as-prepared CdTe QDs are nearly monodisperse and homogeneous (16). In order to verify our finding, the morphologies and diameters of the as-prepared CdTe QDs were investigated by TEM (Fig. 1B). The result showed that these nanoparticles were close to spherical and were uniform with an average diameter of 3–4 nm (Fig. 1B).
4
242
Figure 1. (A) Uv–vis light absorption (curve 1) and fluorescence (curve 2) spectra of the as-prepared CdTe QDs in PBS solution at pH 7.4 (CdTe QDs, 3 × 10 image of the as-prepared CdTe QDs in PBS solution at pH 7.4.
wileyonlinelibrary.com/journal/luminescence
Copyright © 2015 John Wiley & Sons, Ltd.
1
mol · L ). (B) TEM
Luminescence 2016; 31: 241–246
Quantum dots based fluorescence probe for the determination of enoxacin Factors affecting ENX quenched fluorescence of CdTe QDs It was found that the fluorescence intensity of CdTe QDs was greatly quenched in the presence of ENX, and that the fluorescence quenching was related to for example solution pH, QD concentration, and reaction time. Generally, the pH of the solution had a great effect on the fluorescence intensity of the QDs (18). In this study, three buffer solutions, Tris–HCl, PBS and Britton-Robinson (BR) buffer were used to investigate the effect of the CdTe QDsENX solution system on fluorescence intensity. The corresponding results showed that PBS was the most suitable one to use. Therefore, PBS was selected to control the pH of the solution. As shown in Fig. 2, the effect of pH on CdTe QD fluorescence intensity in the presence of ENX was studied. Maximal fluorescence quenching intensity (F0 – F) of CdTe QDs by ENX was obtained at pH 7.4 (F0 and F represent the fluorescence intensities of CdTe QDs before and after the addition of ENX, respectively). Considering that the CdTe QD fluorescence intensity was strongly affected by pH, QDs were more stable and exhibited better optical properties in basic solution than in acidic solution. We propose that ENX interacts with QDs through electrostatic interaction. If the pH of the system is higher than 8, it will hinder ENX protonization, if the pH is lower that 8, it is not a suitable environment for QD optical properties. Thus, in order to obtain a lower detection limit, the pH of the solution used in the experiment was set at 7.4. At the same time, the effect of PBS concentration on the fluorescence intensity of the CdTe QDs–ENX solution system was also discussed. The results showed that the optimal dosage of PBS was 1 mL. The influence of CdTe QD concentration on ENX decreased fluorescence (F0 – F) was also studied (F0 and F represent the fluorescence intensity of CdTe QDs before and after the addition of ENX, respectively). The results are shown in Fig. 3. It can be seen that the suitable concentration of CdTe QDs was 3 × 104 mol × L1. If CdTe QD concentration was higher than this value, the extent of CdTe QD fluorescence quenching decreased, possibly reducing the sensitivity. Meanwhile, if the CdTe QD concentration was lower than the above value, the detection range of the method would reduce. Considering these factors, CdTe QDs at a 3 × 104 mol · L1 concentration were adopted. Maximal fluorescence quenching intensity was observed after a 10 min interaction between ENX and CdTe QDs; fluorescence intensity was stable for 100 min at room temperature. When the
Figure 3. Effects of CdTe QDs concentration on the fluorescence intensity of the so6 1 lution system (ENX, 3 × 10 mol · L ; PBS, 1 mL, pH 7.4).
sample was stored at 0 °C, it was stable for 24 h. This finding demonstrated the good stability of the detection system. In our experiment, fluorescence intensity was measured after 10 min of adding ENX to CdTe QDs.
Calibration and sensitivity Under optimum conditions, CdTe QD emission spectra in the absence and presence of ENX were recorded and are shown in Fig. S1(A). It was found that the CdTe QD fluorescence intensity was quenched efficiently by ENX centered around 558 nm (excitation 350 nm), a finding that was commonly attributed to suppression of electron-hole recombination. At the same time, no emission peak shift was found even at relatively high ENX concentrations (1.4 × 105 molL1) and a quenching efficiency of 73.95% was observed at this time. On this basis, the possibility of developing a sensitive method for ENX was evaluated. To evaluate the sensitivity of CdTe QDs for ENX detection, the fluorescence intensity of CdTe QDs at 558 nm was monitored as a function of ENX concentration over a certain range. As shown in Fig. S1(B), a linear calibration plot of quenched fluorescence intensity (ΔF = F0 – F) against ENX concentration was observed in the range 4.333 × 109 molL1 to 1.4 × 105 molL1 with a correlation coefficient (R) of 0.9987 and a linear regression equation of ΔF = 1408. 5C + 51.1 (where C is the ENX concentration in molL1). A detection limit of 1.313 × 109 molL1 for ENX was determined using 3σ/S, where σ is the standard deviation of 11 replicate measurements of fluorescence intensity of the blank samples and S is the slope of the calibration plot. Thus, it is quite obvious that this approach has good sensitivity for detecting ENX in aqueous solution.
Selectivity of this method
Luminescence 2016; 31: 241–246
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
243
Figure 2. Effects of pH on the decrease of fluorescence intensity of CdTe QDs after 4 1 the addition of ENX in PBS buffer solution at pH 7.4 (CdTe QDs, 3 × 10 mol × L ; 6 1 ENX, 3 × 10 mol · L ; PBS, 1 mL).
To explore the selectivity of the method using CdTe QDs as probe for ENX detection in aqueous solution, the influences of foreign substances such as relevant metal ions, inorganic anions, and biomolecules on the determination of 3 × 106 mol · L1 ENX were investigated (Table 1). If the coexisting substances caused a relative error of less than ±5% on the fluorescence intensity change of the CdTe QDs, they were considered to have no interference with the detection of ENX. It was found that ions (Na+, NH4+, K+,
Q. Yang et al. Table 1. Effect of coexistence materials for the CdTe QDs–ENX system in PBS at pH 7.4. (CENX = 3 × 106 mol · L1) Coexistence materials
Concentration (μg · mL1)
Relative error (%)
Coexistence materials
Concentration (μg · mL1)
Relative error (%)
200 200 200 1.32 1 0.30 0.22 200 200 200 200 200
–2.50 –0.43 –3.22 +1.21 +2.22 +3.10 –3.31 +0.11 +1.10 –1.40 –1.04 –2.84
CO32– HCO3 Leucine Threonine Sarcosine Glycine Histidine Glucose Uracil Thymine BSA ATP
200 200 200 220 200 200 200 500 30 20 20 200
+2.74 –3.55 +2.76 +3.42 –3.21 –2.13 +3.70 +3.98 –2.24 –4.97 +4.22 –1.83
K+ Na+ NH4+ Mg2+ Zn2+ Ca2+ Al3+ Cl Br NO3 NO2 SO42–
ATP, disodium adenosine triphosphate; BSA, bovine serum albumin.
Cl, Br, NO3, NO2, CO32–, HCO3, SO42–) and biomolecules ( glucose, common amino acids, ATP) posed no interference on the determination. Whereas Al3+, Ca2+, Zn2+ and Mg2+ could be allowed at lower concentration levels without significant interference. These data demonstrated that the proposed method had high selectivity and might be applied to the detection of ENX in biological samples with satisfactory results. The mechanism of reaction The fluorescence quenching behavior is customarily classified as either dynamic or static. Dynamic and static quenching can be distinguished by their differing dependence on temperature. Quenching constants increase with temperature increase for dynamic quenching, whereas the reverse effect is observed in the case of static quenching. To the best of our knowledge, the quenching behavior of ENX on the fluorescence of CdTe QDs can be described by the well known Stern–Volmer equation (19): F0
F
¼ 1 þ K SV ½Q
(1)
where F0 and F are the fluorescence intensities of CdTe QDs in the absence and presence of a quencher (ENX), respectively; [Q] is the concentration of the quencher and KSV is the Stern–Volmer quenching constant, which defines the quenching efficiency of the quencher. Hence, eqn (1) was applied to determine the KSV of the CdTe QDs–ENX solution system at three different temperatures (292 K, 301 K and 310 K) by linear regression of a plot of F0/F against [Q] (as shown in Fig. S2). The corresponding values of KSV are listed in Table 2. It can be seen that the quenching
constant (KSV) increased with rise in temperature, which indicated that the probable quenching mechanism of the CdTe QDs–ENX solution system was initiated by dynamic quenching rather than by static quenching. One additional method to distinguish static and dynamic quenching is by careful examination of the absorption spectra of the fluorophore. Dynamic quenching only affects the excited states of the fluorophores, and thus no changes in absorption spectra are expected. In contrast, ground-state complex formation will frequently result in perturbation of the absorption spectrum of the fluorophore (20,21). To reconfirm that the probable quenching mechanism of CdTe QD fluorescence caused by ENX is initiated by dynamic quenching, UV–vis absorption spectra of CdTe QDs-ENX solution system were studied and the results are presented in Fig. 4. In the CdTe QD spectrum (curve 1), there was strong absorption in the UV region, whereas absorption in the visible region was relatively weak. In the ENX spectrum (curve 2) with distilled water as the reference, there were two strong absorption peaks at 261 and 334 nm, respectively. Curve 4 is the ENX absorption spectrum with CdTe QDs as the reference. There was no indication of a spectral change when comparing curve 2 with curve 4, implying that curve 3 is a linear combination of the spectra of each component (curves 1 and 2). Namely, the quenching type was dynamic (22). As we know, the measurement of fluorescence lifetime is the most definitive method to distinguish static and dynamic quenching. The lifetime of a fluorescence molecule in the excited state does not change in the presence of quencher when static quenching takes place. Conversely, fluorescence lifetime is shorter if dynamic quenching occurs (23).
Table 2. Stern–Volmer quenching constants for the interaction of CdTe QDs with ENX in PBS (pH 7.4) at three different temperatures pH
Temperature (K)
Stern–Volmer linear equation
R
SD
7.4
292 301 310
F0/F = 0.9694 + 1308 × 105 [Q] F0/F = 0.9604 + 1.736 × 105 [Q] F0/F = 0.9837 + 2.086 × 105 [Q]
0.9982 0.9988 0.9991
0.03312 0.03591 0.03812
244
R, correlation coefficient; SD, standard deviation for the KSV values.
wileyonlinelibrary.com/journal/luminescence
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2016; 31: 241–246
Quantum dots based fluorescence probe for the determination of enoxacin lated to electron transfer from CdTe QDs to ENX (24). Upon photoexcitation of a QD, the electrons from the valence band are excited to the conduction band. The excited electron and the oppositely charged ’hole’ attract one another. When the excited electron recombines with the hole, a photon is emitted in the form of fluorescence (25). Introduction of ENX (which serves as efficient electron acceptor for the conduction band electron from CdTe QDs) to the CdTe QD solution prevented electronhole recombination at the CdTe QD interface, and caused the fluorescence quenching (26).
Analytical applications Figure 4. UV–vis absorption spectra of (1) CdTe QDs, (2) ENX (with distilled water as the reference), (3) mixture solution system (CdTe QDs and ENX), and (4) ENX (with 4 1 6 1 CdTe QDs as the reference) (CdTe QDs, 3 × 10 mol × L ; ENX, 3 × 10 mol · L ; PBS, 1 mL, pH 7.4).
As shown in Fig. 5, CdTe QD fluorescence lifetime shortened when ENX concentration increased (0, 5 × 106 mol · L1 and 10 × 106 mol · L1), which illustrated that the fluorescence quenching process was dynamic. The decrease in fluorescence lifetime of CdTe QDs in the presence of ENX was directly re-
4
1
Figure 5. Fluorescence decay of CdTe QDs (3 × 10 mol × L ) in the absence 6 1 6 1 (black) and presence (red: 5 × 10 mol × L and green: 10 × 10 mol × L ) of ENX. (PBS, 1 mL, pH 7.4).
To confirm the feasibility of the proposed method for real sample determination, the present method was applied to determine ENX in enoxacin gluconate injections (Wuhan Jianuo Pharmaceutical Group Co. Ltd, China) and commercial tablets (Zhejiang Haizheng Pharmaceutical Co. Ltd, China). The results are shown in Table 3 and recoveries of the spiked samples were generally satisfactory. The accuracy of the developed method was evaluated by analyzing the samples with the reference procedures (an HPLC method (6) for the ENX assay was adopted). The results obtained by the developed system were in good agreement with those by the HPLC method. In order to further investigated the feasibility of the fluorescent probe, the present sensor was also applied to determine ENX in fresh serum samples from healthy human in accordance with the procedure reported in the references (27). A 2 mL aliquot of fresh serum sample (healthy human) and 2 mL trichloroacetic acid were mixed thoroughly and centrifuged at 5000 rpm for 5 min; the supernatant fluid was diluted to 100 mL and 1 mL of this solution was pipetted into a 10 mL volumetric flask, and then 1 mL QDs, 1 mL PBS buffer solution and appropriate amounts of ENX were added for determination of ENX concentration. The standard addition method was used to determine five parallel samples for each concentration and the results are listed in Table 4. It can be seen that the fluorescence quenching method had good repeatability for the determination of ENX in fresh healthy human serum. Therefore, the proposed fluorescent sensor can be applied to the rapid monitoring of ENX in real samples and fresh serum samples from healthy humans.
Table 3. Results for the determination of ENX in enoxacin gluconate injections and commercial tablets Sample 3
Enoxacin gluconate injections (10 Commercial tablets ( g/tablet)
mg/L)
Found
Average
Mark concentration
HPLC
6.915, 6.929, 6.967, 7.001, 6.957 0.0958, 0.1002, 0.1005, 0.0998, 0.09991
6.954 0.09924
7.0 0.1
6.988 0.101
Table 4. Results for the determination of ENX in fresh human serum samples, n = 5 Sample
Found (n = 5, mol · L1)
Added (105 mol · L1)
Found (105 mol · L1)
Recovery (n = 5, %)
RSD (n = 5, %)
Serum 1 Serum 2 Serum 3
ND ND ND
4 6 8
4.046 5.990 7.982
101.2 99.83 99.78
2.1 3.6 2.8
Luminescence 2016; 31: 241–246
245
ND, not detected.
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
Q. Yang et al.
Conclusions We studied the interaction between CdTe QDs and ENX in PBS at pH 7.4, and a simple, time-saving, low-cost, high sensitive and specific quantitative method based on the fluorescence quenching of CdTe QDs was developed for the detection of ENX. The quenching mechanism of CdTe QDs by ENX was dynamic quenching, arising from electron transfer from CdTe QDs to ENX. In addition, interferences caused by common metal ions, amino acids and other several common compounds to ENX detection were also investigated. The proposed method was successfully applied to determine ENX in enoxacin gluconate injections and commercial tablets. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21175015) and all authors here express their deep thanks.
References 1. Tong CL, Xiang GH. Sensitive determination of enoxacin by its enhancement effect on the fluorescence of terbium(III)-sodium dodecylbenzene sulfonate and its luminescence mechanism. J Lumin 2007;126:575–80. 2. Botsoglou NA, Fletouris DJ. Analysis of residual antibacterials in food of animal origin. In: Nollet LML editor. Handbook of food analysis. Marcel Dekker: Ghent, Belgium, 2004:931–1037. 3. He Y, Wang HF, Yan XP. Exploring Mn-doped ZnS quantum dots for the room-temperature phosphorescence detection of enoxacin in biological fluids. Anal Chem 2008;80:3832–37. 4. Süslü I, Tamer A. Spectrophotometric determination of enoxacin as ion-pairs with bromophenol blue and bromocresol purple in bulk and pharmaceutical dosage form. J Pharm Biomed Anal 2002;29: 545–54. 5. Hopkala H, Kowalczuk D. Application of derivative UV spectrophotometry for the determination of enoxacin and nalidixic acid in tablets. Pharmazie 2000;55:432–5. 6. Samanidou VF, Christodoulou EA, Papadoyannis IN. Determination of fluoroquinolones in edible animal tissue samples by high performance liquid chromatography after solid phase extraction. J Sep Sci 2005;28:555–65. 7. Samanidou VF, Demetriou CE, Papadoyannis IN. Direct determination of four fluoroquinolones, enoxacin, norfloxacin, ofloxacin, and ciprofloxacin, in pharmaceuticals and blood serum by HPLC. Anal Bioanal Chem 2003;375:623. 8. Yi L, Zhao HC, Chen S, Jin LP, Zheng DD, Wu ZL. Flow-injection analysis of two fluoquinolones by the sensitizing effect of terbium(III) on chemiluminescence of the potassium permanganate–sodium sulfite system. Talanta 2003;61:403–9. 9. Tuncel M, Dogrukol-Ak D, Senturk Z, Ozkan SA, Aboul-Enein H. Capillary electrophoretic behaviour and determination of enoxacin in pharmaceutical preparations and human serum. J Liquid Chromatogr Relat Technol 2001;24:2455–67. 10. Hu M, Hu CQ, Liu WY. Determination of drug reference substance 1 content by H-nuclear magnetic resonance spectroscopy. Chin J Anal Chem 2004;32:451–5.
11. Shen YZ, Liu SP, Wang J, Li D, Wang XD, He YQ. A sensitive assay of chelerythrine using a fluorescence quenching approach with glutathione capped CdTe/CdS quantum dots as a probe. Anal Methods 2013;5:5309–16. 12. Yan H, Wang HF. Turn-on room temperature phosphorescence assay of heparin with tunable sensitivity and detection window based on target-induced self-assembly of polyethyleneimine capped Mndoped ZnS quantum dots. Anal Chem 2011;83:8589–95. 13. Shen YZ, Liu SP, Kong L, Li D, He YQ. Sensitive detection of polymyxin B sulfate at the nanogram level using enhanced resonance scattering signals and decreased fluorescence signals with thioglycolic acid capped CdTe/CdS quantum dots as probe. Sens Actuat B 2013;188: 555–63. 14. Shen YZ, Liu SP, Wang J, Li D, He YQ. Determination of ellagic acid by fluorescence quenching method with glutathione capped CdTe quantum dots as the probe. Anal Methods 2013;5:3228–34. 15. Wang GL, Jiao HJ, Zhu XY, Dong YM, Li ZJ. Enhanced fluorescence sensing of melamine based on thioglycolic acid-capped CdS quantum dots. Talanta 2012;93:398–403. 16. Liang JG, Huang S, Zeng DY, He ZK, Ji XH, Ai XP, et al. CdSe quantum dots as luminescent probes for spironolactone determination. Talanta 2006;69:126–30. 17. Shen YZ, Liu SP, Yang JD, Wang LL, Tan XP, He YQ. A novel and sensitive 3+ turn-on fluorescent biosensor for the DNA detection using Sm -modulated glutathione-capped CdTe quantum dots. Sens Actuat B 2014;199:389–97. 18. Skoog DA, Holler FJ, Nieman TA. Principles of instrumental analysis 7th ed. Saunders College: Philadelphia, 1998:601–8. 19. Hu YJ, Yang W, Yu OY, Juan Z, Yi L. Characterize the interaction between naringenin and bovine serum albumin using spectroscopic approach. J Lumin 2010;130:1394–99. 20. Mi R, Li PQ, Hu YJ, Fan XY, Li HY, Yu XC, et al. Biophysical studies on the interactions of jatrorrhizine with bovine serum albumin by spectroscopic and molecular modeling methods. Mol Biol Rep 2013;40:4397–404. 21. Lakowicz JR. Principles of fluorescence spectroscopy3rd ed. Springer: New York, 2006. 22. Shen YZ, Liu SP, Liu ZQ, He YQ. Sensitive detection of luteolin using thioglycolic acid–capped cadmium telluride/cadmium sulphide quantum dots as the fluorescent probe. Spect Lett 2013;46:483–92. 23. Gan XJ, Liu SP, Liu ZF, Hu XL. Determination of tetracaine hydrochloride by fluorescence quenching method with some aromatic amino acids as probes. J Fluoresc 2012;22:129–35. 24. Asha JM, Renganathan R. Investigations on the photoinduced interaction of water soluble thioglycolic acid (TGA) capped CdTe quantum dots with certain porphyrins. J Coll Inter Sci 2010;344:596–602. 25. Zhao D, Chan WH, He ZK, Qiu T. Quantum dot ruthenium complex dyads: recognition of double-strand DNA through dual-color fluorescence detection. Anal Chem 2009;81:3537–43. 26. Shen YZ, Liu SP, He YQ. Fluorescence quenching investigation on the interaction of glutathione-CdTe/CdS quantum dots with sanguinarine and its analytical application. Luminescence 2014;29:176–82. 27. Gan XJ, Liu SP, Liu ZF, Hu XL, Cui ZP, Wang YQ. Determination of carbazochrome by fluorescence quenching method. Spectrochim Acta A 2012;97:161.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
246 wileyonlinelibrary.com/journal/luminescence
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2016; 31: 241–246
Revised: 27 April 2015,
Accepted: 28 April 2015
Published online in Wiley Online Library: 23 June 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2952
Sensitive determination of enoxacin in pharmaceutical formulations by its quench effect on the fluorescence of glutathionecapped CdTe quantum dots Qiong Yang,a Xuanping Tanb and Jidong Yangb,c* ABSTRACT: A sensitive and simple method for the determination of enoxacin (ENX) was developed based on the fluorescence quenching effect of ENX for glutathione (GSH)-capped CdTe quantum dots (QDs). Under optimum conditions, a good linear relationship was obtained from 4.333 × 109 molL1 to 1.4 × 105 molL1 with a correlation coefficient (R) of 0.9987, and the detection limit (3σ/K) was 1.313 × 109 molL1. The corresponding mechanism has been proposed on the basis of electron transfer supported by ultraviolet–visible (UV) light absorption, fluorescence spectroscopy, and the measurement of fluorescence lifetime. The method has been applied to the determination of ENX in pharmaceutical formulations (enoxacin gluconate injections and commercial tablets) with satisfactory results. The proposed method manifested several advantages such as high sensitivity, short analysis time, low cost and ease of operation. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: CdTe quantum dots; enoxacin; fluorescence; pharmaceutical formulation
Introduction
Luminescence 2016; 31: 241–246
* Correspondence to: Jidong Yang, School of Chemistry and Chemical Engineering, Southwest University, Chongqing, Beibei 400715, China. E-mail: [email protected] a
School of Chemistry and Chemical Engineering, Yangtze Normal University, Chongqing, Fuling 408100, China
b
School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
c
School of Chemistry and Environmental Engineering, Chongqing Three Gorges University, Chongqing, Wanzhou 404000, China Abbreviations: BR, Britton-Robinson; BSA, bovine serum albumin; HPLC, highperformance liquid chromatography; PBS, phosphate-buffered saline; QD, quantum dots; TEM, Transmission electron microscopy; TGA, thioglycolic acid.
Copyright © 2015 John Wiley & Sons, Ltd.
241
Enoxacin (ENX) belongs to the third generation of multiple fluorinated antibacterial quinolone derivatives widely used in the treatment of systemic infections including urinary tract, respiratory, gastrointestinal and skin infections. The mechanism of action of ENX is based primarily on the inhibition of bacterial cell DNA gyrase and prohibiting DNA replication. Nowadays it is used extensively in clinical treatment and with higher potential as a broad antibacterial spectrum lower side effects (1). However, public and scientific concerns about the potential risk of quinolones to human health have been increasing due to their latent allergic hypersensitivity reactions or toxic effects on the articular cartilages causing arthralgia or juvenile arthropathies (2,3). To better exploit and safely utilize ENX in clinical disease diagnosis and treatment, it is necessary to develop a simple, rapid, high sensitivity, low-cost and convenient method for quantitative determination of ENX in pharmaceutical formulation. To date, for ENX detection, methods such as spectrophotometry (4,5), high-performance liquid chromatography (HPLC) (6,7), chemical luminescence (8), capillary electrophoresis (9), nuclear magnetic resonance spectroscopy (10) and so on have developed. However, many of these methods are not readily adaptable to routine analysis because they are time-consuming and require relatively expensive and complicated instruments. Based on this problem, a room-temperature phosphorescence assay for ENX with Mn-doped ZnS Dots as probe has been reported (3). Compared with those methods mentioned above, this assay is simple but only limited to the doped quantum dots (QDs), and therefore led indirectly to increases in detection costs and difficulty. Given all these factors, it is therefore necessary to develop a more low-cost, practical and convenient method for ENX detection.
Semiconductor QDs, one of the most useful type of nanoparticle, have attracted extensive application studies in many fields due to their several distinct properties compared with other luminescent materials, such as greater brightness, better stability with respect to photobleaching, broad excitation wavelength range, narrower symmetrical emission bands and size-tunable photoluminescence (11–13). All of these properties therefore promise that QDs will be ideal fluorescence indicators for chemical and biological assays. Recently, QDs have been exploited increasingly as fluorescent probes to determine drug molecules based on fluorescence quenching or enhancement. For example, Shen et al. (14) used CdTe QDs as luminescent probes for ellagic acid determination based on the quenching of CdTe QDs fluorescence by ellagic acid. Wang et al. (15) have developed a new assay for melamine based on the measurement of enhanced fluorescence intensity signal resulting from the interaction of thioglycolic
Q. Yang et al. acid-capped CdS QDs with melamine and Liang et al. (16) developed a fluorescence method for spironolactone determination with CdSe Dots as luminescent probes. To our knowledge, the use of glutathione (GSH)-capped CdTe QDs as fluorescence probes for the quantitative determination of ENX has not been reported so far. In this article, a facile, rapid, low-cost, convenient and sensitive assay for ENX has been proposed for the first time based on the decrease in fluorescence intensity of CdTe QDs. The corresponding interaction mechanism between CdTe QDs and ENX was investigated by ultraviolet–visible (UV-vis) light absorption, fluorescence spectroscopy and the measurement of fluorescence lifetime. It was found that the fluorescence intensity of CdTe QDs was quenched at 558 nm in the presence of ENX. In addition, interaction information regarding the mechanism of the reaction, such as suitable reaction conditions, affecting factors and the influence of coexisting substances, was studied.
Synthesis of CdTe QDs GSH-capped CdTe QDs were synthesized according to a previously described method (17). Under N2 atmosphere and magnetic stirring, tellurium powder (0.0383 g) was reacted with excess sodium borohydride in deionized water to produce the colorless solution of sodium hydrogen telluride (NaHTe). CdCl2.2.5H2O (0.1028 g) and GSH (0.1844 g) were dissolved in 150 mL deionized water. Under magnetic stirring, the pH of the mixture was adjusted to 10.2 by the dropwise addition of NaOH solution (1 mol × L1). The solution was deaerated by Ar bubbling for about 40 min. Under stirring, H2Te gas generated by the reaction of NaHTe solution with diluted H2SO4 (0.5 mol × L1) and was passed through an oxygen-free Cd2+ solution together with a slow nitrogen flow. Then the resulting solution mixture was heated to 373 K and refluxed under nitrogen for about 1 h. Salmon pink coloured CdTe solution was obtained. The concentration of GSH-capped CdTe QDs was 3 × 103 molL1 (determined by the Cd2+ concentration).
Experimental Reagents and apparatus
General procedure for ENX detection
All chemicals used were of at least analytical grade. The following reagents were purchased: tellurium power (99.99%, Sinopharm Chemical Reagent Co., Shanghai, China), NaBH4 (98%, Tianjin Huanwei Fine Chemical Co., Tianjin, China), CdCl2 · 2.5H2O (99.95%, Shanghai Chemicals Reagent Co., Shanghai, China), l-glutathione (GSH, 98%, Aladdin Reagent Co., Shanghai, China), and enoxacin (ENX, 99%, Aladdin Reagent Co., Shanghai, China). Phosphate-buffered saline (PBS) solutions of different pH were prepared by mixing 1/30 mol · L1 KH2PO4 with 1/30 mol · L1 Na2HPO4 in different proportions. All solutions were prepared using deionized water as solvent. A Hitachi F-4500 spectrofluorophotometer (Hitachi Company, Tokyo, Japan) was used to record the fluorescence spectra. A U-3010 spectrophotometer (Tianmei Corporation, Shanghai, China) was applied to record the absorption spectra. Transmission electron microscopy (TEM) images of CdTe QDs were acquired on a ZEISS LIBRA 200 FE transmission electron microscope (ZEISS, Berlin, Germany). A FL-TCSPC Fluorolog-3 fluorescence spectrometer (Horiba Jobin Yvon Inc., France) was used to measure the fluorescence lifetime of the CdTe QDs–ENX system at room temperature (λex/λem = 350 nm/558 nm). A PHS-3C pH meter (Leici, Shanghai, China) was used to adjust the pH values of the aqueous solutions.
In order to determine ENX, 1 mL above synthesized CdTe QDs, 1 mL PBS (pH 7.4) and an appropriate amount of ENX were added into a 10 mL volumetric flask in turn, then diluted with deionized water to the mark and mixed thoroughly with gentle shaking. After incubation for 10 min, the fluorescence, absorption spectra and fluorescence lifetime of the solution were examined.
Results and discussion Characterization of CdTe QDs Fig. 1(A) shows the absorption (curve 1) and fluorescence (curve 2) spectra of these as-prepared CdTe QDs at room temperature. It can be seen that the line width of the fluorescence spectrum is narrow (with the full width at half-maximum about 40 nm), showing that the as-prepared CdTe QDs are nearly monodisperse and homogeneous (16). In order to verify our finding, the morphologies and diameters of the as-prepared CdTe QDs were investigated by TEM (Fig. 1B). The result showed that these nanoparticles were close to spherical and were uniform with an average diameter of 3–4 nm (Fig. 1B).
4
242
Figure 1. (A) Uv–vis light absorption (curve 1) and fluorescence (curve 2) spectra of the as-prepared CdTe QDs in PBS solution at pH 7.4 (CdTe QDs, 3 × 10 image of the as-prepared CdTe QDs in PBS solution at pH 7.4.
wileyonlinelibrary.com/journal/luminescence
Copyright © 2015 John Wiley & Sons, Ltd.
1
mol · L ). (B) TEM
Luminescence 2016; 31: 241–246
Quantum dots based fluorescence probe for the determination of enoxacin Factors affecting ENX quenched fluorescence of CdTe QDs It was found that the fluorescence intensity of CdTe QDs was greatly quenched in the presence of ENX, and that the fluorescence quenching was related to for example solution pH, QD concentration, and reaction time. Generally, the pH of the solution had a great effect on the fluorescence intensity of the QDs (18). In this study, three buffer solutions, Tris–HCl, PBS and Britton-Robinson (BR) buffer were used to investigate the effect of the CdTe QDsENX solution system on fluorescence intensity. The corresponding results showed that PBS was the most suitable one to use. Therefore, PBS was selected to control the pH of the solution. As shown in Fig. 2, the effect of pH on CdTe QD fluorescence intensity in the presence of ENX was studied. Maximal fluorescence quenching intensity (F0 – F) of CdTe QDs by ENX was obtained at pH 7.4 (F0 and F represent the fluorescence intensities of CdTe QDs before and after the addition of ENX, respectively). Considering that the CdTe QD fluorescence intensity was strongly affected by pH, QDs were more stable and exhibited better optical properties in basic solution than in acidic solution. We propose that ENX interacts with QDs through electrostatic interaction. If the pH of the system is higher than 8, it will hinder ENX protonization, if the pH is lower that 8, it is not a suitable environment for QD optical properties. Thus, in order to obtain a lower detection limit, the pH of the solution used in the experiment was set at 7.4. At the same time, the effect of PBS concentration on the fluorescence intensity of the CdTe QDs–ENX solution system was also discussed. The results showed that the optimal dosage of PBS was 1 mL. The influence of CdTe QD concentration on ENX decreased fluorescence (F0 – F) was also studied (F0 and F represent the fluorescence intensity of CdTe QDs before and after the addition of ENX, respectively). The results are shown in Fig. 3. It can be seen that the suitable concentration of CdTe QDs was 3 × 104 mol × L1. If CdTe QD concentration was higher than this value, the extent of CdTe QD fluorescence quenching decreased, possibly reducing the sensitivity. Meanwhile, if the CdTe QD concentration was lower than the above value, the detection range of the method would reduce. Considering these factors, CdTe QDs at a 3 × 104 mol · L1 concentration were adopted. Maximal fluorescence quenching intensity was observed after a 10 min interaction between ENX and CdTe QDs; fluorescence intensity was stable for 100 min at room temperature. When the
Figure 3. Effects of CdTe QDs concentration on the fluorescence intensity of the so6 1 lution system (ENX, 3 × 10 mol · L ; PBS, 1 mL, pH 7.4).
sample was stored at 0 °C, it was stable for 24 h. This finding demonstrated the good stability of the detection system. In our experiment, fluorescence intensity was measured after 10 min of adding ENX to CdTe QDs.
Calibration and sensitivity Under optimum conditions, CdTe QD emission spectra in the absence and presence of ENX were recorded and are shown in Fig. S1(A). It was found that the CdTe QD fluorescence intensity was quenched efficiently by ENX centered around 558 nm (excitation 350 nm), a finding that was commonly attributed to suppression of electron-hole recombination. At the same time, no emission peak shift was found even at relatively high ENX concentrations (1.4 × 105 molL1) and a quenching efficiency of 73.95% was observed at this time. On this basis, the possibility of developing a sensitive method for ENX was evaluated. To evaluate the sensitivity of CdTe QDs for ENX detection, the fluorescence intensity of CdTe QDs at 558 nm was monitored as a function of ENX concentration over a certain range. As shown in Fig. S1(B), a linear calibration plot of quenched fluorescence intensity (ΔF = F0 – F) against ENX concentration was observed in the range 4.333 × 109 molL1 to 1.4 × 105 molL1 with a correlation coefficient (R) of 0.9987 and a linear regression equation of ΔF = 1408. 5C + 51.1 (where C is the ENX concentration in molL1). A detection limit of 1.313 × 109 molL1 for ENX was determined using 3σ/S, where σ is the standard deviation of 11 replicate measurements of fluorescence intensity of the blank samples and S is the slope of the calibration plot. Thus, it is quite obvious that this approach has good sensitivity for detecting ENX in aqueous solution.
Selectivity of this method
Luminescence 2016; 31: 241–246
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
243
Figure 2. Effects of pH on the decrease of fluorescence intensity of CdTe QDs after 4 1 the addition of ENX in PBS buffer solution at pH 7.4 (CdTe QDs, 3 × 10 mol × L ; 6 1 ENX, 3 × 10 mol · L ; PBS, 1 mL).
To explore the selectivity of the method using CdTe QDs as probe for ENX detection in aqueous solution, the influences of foreign substances such as relevant metal ions, inorganic anions, and biomolecules on the determination of 3 × 106 mol · L1 ENX were investigated (Table 1). If the coexisting substances caused a relative error of less than ±5% on the fluorescence intensity change of the CdTe QDs, they were considered to have no interference with the detection of ENX. It was found that ions (Na+, NH4+, K+,
Q. Yang et al. Table 1. Effect of coexistence materials for the CdTe QDs–ENX system in PBS at pH 7.4. (CENX = 3 × 106 mol · L1) Coexistence materials
Concentration (μg · mL1)
Relative error (%)
Coexistence materials
Concentration (μg · mL1)
Relative error (%)
200 200 200 1.32 1 0.30 0.22 200 200 200 200 200
–2.50 –0.43 –3.22 +1.21 +2.22 +3.10 –3.31 +0.11 +1.10 –1.40 –1.04 –2.84
CO32– HCO3 Leucine Threonine Sarcosine Glycine Histidine Glucose Uracil Thymine BSA ATP
200 200 200 220 200 200 200 500 30 20 20 200
+2.74 –3.55 +2.76 +3.42 –3.21 –2.13 +3.70 +3.98 –2.24 –4.97 +4.22 –1.83
K+ Na+ NH4+ Mg2+ Zn2+ Ca2+ Al3+ Cl Br NO3 NO2 SO42–
ATP, disodium adenosine triphosphate; BSA, bovine serum albumin.
Cl, Br, NO3, NO2, CO32–, HCO3, SO42–) and biomolecules ( glucose, common amino acids, ATP) posed no interference on the determination. Whereas Al3+, Ca2+, Zn2+ and Mg2+ could be allowed at lower concentration levels without significant interference. These data demonstrated that the proposed method had high selectivity and might be applied to the detection of ENX in biological samples with satisfactory results. The mechanism of reaction The fluorescence quenching behavior is customarily classified as either dynamic or static. Dynamic and static quenching can be distinguished by their differing dependence on temperature. Quenching constants increase with temperature increase for dynamic quenching, whereas the reverse effect is observed in the case of static quenching. To the best of our knowledge, the quenching behavior of ENX on the fluorescence of CdTe QDs can be described by the well known Stern–Volmer equation (19): F0
F
¼ 1 þ K SV ½Q
(1)
where F0 and F are the fluorescence intensities of CdTe QDs in the absence and presence of a quencher (ENX), respectively; [Q] is the concentration of the quencher and KSV is the Stern–Volmer quenching constant, which defines the quenching efficiency of the quencher. Hence, eqn (1) was applied to determine the KSV of the CdTe QDs–ENX solution system at three different temperatures (292 K, 301 K and 310 K) by linear regression of a plot of F0/F against [Q] (as shown in Fig. S2). The corresponding values of KSV are listed in Table 2. It can be seen that the quenching
constant (KSV) increased with rise in temperature, which indicated that the probable quenching mechanism of the CdTe QDs–ENX solution system was initiated by dynamic quenching rather than by static quenching. One additional method to distinguish static and dynamic quenching is by careful examination of the absorption spectra of the fluorophore. Dynamic quenching only affects the excited states of the fluorophores, and thus no changes in absorption spectra are expected. In contrast, ground-state complex formation will frequently result in perturbation of the absorption spectrum of the fluorophore (20,21). To reconfirm that the probable quenching mechanism of CdTe QD fluorescence caused by ENX is initiated by dynamic quenching, UV–vis absorption spectra of CdTe QDs-ENX solution system were studied and the results are presented in Fig. 4. In the CdTe QD spectrum (curve 1), there was strong absorption in the UV region, whereas absorption in the visible region was relatively weak. In the ENX spectrum (curve 2) with distilled water as the reference, there were two strong absorption peaks at 261 and 334 nm, respectively. Curve 4 is the ENX absorption spectrum with CdTe QDs as the reference. There was no indication of a spectral change when comparing curve 2 with curve 4, implying that curve 3 is a linear combination of the spectra of each component (curves 1 and 2). Namely, the quenching type was dynamic (22). As we know, the measurement of fluorescence lifetime is the most definitive method to distinguish static and dynamic quenching. The lifetime of a fluorescence molecule in the excited state does not change in the presence of quencher when static quenching takes place. Conversely, fluorescence lifetime is shorter if dynamic quenching occurs (23).
Table 2. Stern–Volmer quenching constants for the interaction of CdTe QDs with ENX in PBS (pH 7.4) at three different temperatures pH
Temperature (K)
Stern–Volmer linear equation
R
SD
7.4
292 301 310
F0/F = 0.9694 + 1308 × 105 [Q] F0/F = 0.9604 + 1.736 × 105 [Q] F0/F = 0.9837 + 2.086 × 105 [Q]
0.9982 0.9988 0.9991
0.03312 0.03591 0.03812
244
R, correlation coefficient; SD, standard deviation for the KSV values.
wileyonlinelibrary.com/journal/luminescence
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2016; 31: 241–246
Quantum dots based fluorescence probe for the determination of enoxacin lated to electron transfer from CdTe QDs to ENX (24). Upon photoexcitation of a QD, the electrons from the valence band are excited to the conduction band. The excited electron and the oppositely charged ’hole’ attract one another. When the excited electron recombines with the hole, a photon is emitted in the form of fluorescence (25). Introduction of ENX (which serves as efficient electron acceptor for the conduction band electron from CdTe QDs) to the CdTe QD solution prevented electronhole recombination at the CdTe QD interface, and caused the fluorescence quenching (26).
Analytical applications Figure 4. UV–vis absorption spectra of (1) CdTe QDs, (2) ENX (with distilled water as the reference), (3) mixture solution system (CdTe QDs and ENX), and (4) ENX (with 4 1 6 1 CdTe QDs as the reference) (CdTe QDs, 3 × 10 mol × L ; ENX, 3 × 10 mol · L ; PBS, 1 mL, pH 7.4).
As shown in Fig. 5, CdTe QD fluorescence lifetime shortened when ENX concentration increased (0, 5 × 106 mol · L1 and 10 × 106 mol · L1), which illustrated that the fluorescence quenching process was dynamic. The decrease in fluorescence lifetime of CdTe QDs in the presence of ENX was directly re-
4
1
Figure 5. Fluorescence decay of CdTe QDs (3 × 10 mol × L ) in the absence 6 1 6 1 (black) and presence (red: 5 × 10 mol × L and green: 10 × 10 mol × L ) of ENX. (PBS, 1 mL, pH 7.4).
To confirm the feasibility of the proposed method for real sample determination, the present method was applied to determine ENX in enoxacin gluconate injections (Wuhan Jianuo Pharmaceutical Group Co. Ltd, China) and commercial tablets (Zhejiang Haizheng Pharmaceutical Co. Ltd, China). The results are shown in Table 3 and recoveries of the spiked samples were generally satisfactory. The accuracy of the developed method was evaluated by analyzing the samples with the reference procedures (an HPLC method (6) for the ENX assay was adopted). The results obtained by the developed system were in good agreement with those by the HPLC method. In order to further investigated the feasibility of the fluorescent probe, the present sensor was also applied to determine ENX in fresh serum samples from healthy human in accordance with the procedure reported in the references (27). A 2 mL aliquot of fresh serum sample (healthy human) and 2 mL trichloroacetic acid were mixed thoroughly and centrifuged at 5000 rpm for 5 min; the supernatant fluid was diluted to 100 mL and 1 mL of this solution was pipetted into a 10 mL volumetric flask, and then 1 mL QDs, 1 mL PBS buffer solution and appropriate amounts of ENX were added for determination of ENX concentration. The standard addition method was used to determine five parallel samples for each concentration and the results are listed in Table 4. It can be seen that the fluorescence quenching method had good repeatability for the determination of ENX in fresh healthy human serum. Therefore, the proposed fluorescent sensor can be applied to the rapid monitoring of ENX in real samples and fresh serum samples from healthy humans.
Table 3. Results for the determination of ENX in enoxacin gluconate injections and commercial tablets Sample 3
Enoxacin gluconate injections (10 Commercial tablets ( g/tablet)
mg/L)
Found
Average
Mark concentration
HPLC
6.915, 6.929, 6.967, 7.001, 6.957 0.0958, 0.1002, 0.1005, 0.0998, 0.09991
6.954 0.09924
7.0 0.1
6.988 0.101
Table 4. Results for the determination of ENX in fresh human serum samples, n = 5 Sample
Found (n = 5, mol · L1)
Added (105 mol · L1)
Found (105 mol · L1)
Recovery (n = 5, %)
RSD (n = 5, %)
Serum 1 Serum 2 Serum 3
ND ND ND
4 6 8
4.046 5.990 7.982
101.2 99.83 99.78
2.1 3.6 2.8
Luminescence 2016; 31: 241–246
245
ND, not detected.
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
Q. Yang et al.
Conclusions We studied the interaction between CdTe QDs and ENX in PBS at pH 7.4, and a simple, time-saving, low-cost, high sensitive and specific quantitative method based on the fluorescence quenching of CdTe QDs was developed for the detection of ENX. The quenching mechanism of CdTe QDs by ENX was dynamic quenching, arising from electron transfer from CdTe QDs to ENX. In addition, interferences caused by common metal ions, amino acids and other several common compounds to ENX detection were also investigated. The proposed method was successfully applied to determine ENX in enoxacin gluconate injections and commercial tablets. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21175015) and all authors here express their deep thanks.
References 1. Tong CL, Xiang GH. Sensitive determination of enoxacin by its enhancement effect on the fluorescence of terbium(III)-sodium dodecylbenzene sulfonate and its luminescence mechanism. J Lumin 2007;126:575–80. 2. Botsoglou NA, Fletouris DJ. Analysis of residual antibacterials in food of animal origin. In: Nollet LML editor. Handbook of food analysis. Marcel Dekker: Ghent, Belgium, 2004:931–1037. 3. He Y, Wang HF, Yan XP. Exploring Mn-doped ZnS quantum dots for the room-temperature phosphorescence detection of enoxacin in biological fluids. Anal Chem 2008;80:3832–37. 4. Süslü I, Tamer A. Spectrophotometric determination of enoxacin as ion-pairs with bromophenol blue and bromocresol purple in bulk and pharmaceutical dosage form. J Pharm Biomed Anal 2002;29: 545–54. 5. Hopkala H, Kowalczuk D. Application of derivative UV spectrophotometry for the determination of enoxacin and nalidixic acid in tablets. Pharmazie 2000;55:432–5. 6. Samanidou VF, Christodoulou EA, Papadoyannis IN. Determination of fluoroquinolones in edible animal tissue samples by high performance liquid chromatography after solid phase extraction. J Sep Sci 2005;28:555–65. 7. Samanidou VF, Demetriou CE, Papadoyannis IN. Direct determination of four fluoroquinolones, enoxacin, norfloxacin, ofloxacin, and ciprofloxacin, in pharmaceuticals and blood serum by HPLC. Anal Bioanal Chem 2003;375:623. 8. Yi L, Zhao HC, Chen S, Jin LP, Zheng DD, Wu ZL. Flow-injection analysis of two fluoquinolones by the sensitizing effect of terbium(III) on chemiluminescence of the potassium permanganate–sodium sulfite system. Talanta 2003;61:403–9. 9. Tuncel M, Dogrukol-Ak D, Senturk Z, Ozkan SA, Aboul-Enein H. Capillary electrophoretic behaviour and determination of enoxacin in pharmaceutical preparations and human serum. J Liquid Chromatogr Relat Technol 2001;24:2455–67. 10. Hu M, Hu CQ, Liu WY. Determination of drug reference substance 1 content by H-nuclear magnetic resonance spectroscopy. Chin J Anal Chem 2004;32:451–5.
11. Shen YZ, Liu SP, Wang J, Li D, Wang XD, He YQ. A sensitive assay of chelerythrine using a fluorescence quenching approach with glutathione capped CdTe/CdS quantum dots as a probe. Anal Methods 2013;5:5309–16. 12. Yan H, Wang HF. Turn-on room temperature phosphorescence assay of heparin with tunable sensitivity and detection window based on target-induced self-assembly of polyethyleneimine capped Mndoped ZnS quantum dots. Anal Chem 2011;83:8589–95. 13. Shen YZ, Liu SP, Kong L, Li D, He YQ. Sensitive detection of polymyxin B sulfate at the nanogram level using enhanced resonance scattering signals and decreased fluorescence signals with thioglycolic acid capped CdTe/CdS quantum dots as probe. Sens Actuat B 2013;188: 555–63. 14. Shen YZ, Liu SP, Wang J, Li D, He YQ. Determination of ellagic acid by fluorescence quenching method with glutathione capped CdTe quantum dots as the probe. Anal Methods 2013;5:3228–34. 15. Wang GL, Jiao HJ, Zhu XY, Dong YM, Li ZJ. Enhanced fluorescence sensing of melamine based on thioglycolic acid-capped CdS quantum dots. Talanta 2012;93:398–403. 16. Liang JG, Huang S, Zeng DY, He ZK, Ji XH, Ai XP, et al. CdSe quantum dots as luminescent probes for spironolactone determination. Talanta 2006;69:126–30. 17. Shen YZ, Liu SP, Yang JD, Wang LL, Tan XP, He YQ. A novel and sensitive 3+ turn-on fluorescent biosensor for the DNA detection using Sm -modulated glutathione-capped CdTe quantum dots. Sens Actuat B 2014;199:389–97. 18. Skoog DA, Holler FJ, Nieman TA. Principles of instrumental analysis 7th ed. Saunders College: Philadelphia, 1998:601–8. 19. Hu YJ, Yang W, Yu OY, Juan Z, Yi L. Characterize the interaction between naringenin and bovine serum albumin using spectroscopic approach. J Lumin 2010;130:1394–99. 20. Mi R, Li PQ, Hu YJ, Fan XY, Li HY, Yu XC, et al. Biophysical studies on the interactions of jatrorrhizine with bovine serum albumin by spectroscopic and molecular modeling methods. Mol Biol Rep 2013;40:4397–404. 21. Lakowicz JR. Principles of fluorescence spectroscopy3rd ed. Springer: New York, 2006. 22. Shen YZ, Liu SP, Liu ZQ, He YQ. Sensitive detection of luteolin using thioglycolic acid–capped cadmium telluride/cadmium sulphide quantum dots as the fluorescent probe. Spect Lett 2013;46:483–92. 23. Gan XJ, Liu SP, Liu ZF, Hu XL. Determination of tetracaine hydrochloride by fluorescence quenching method with some aromatic amino acids as probes. J Fluoresc 2012;22:129–35. 24. Asha JM, Renganathan R. Investigations on the photoinduced interaction of water soluble thioglycolic acid (TGA) capped CdTe quantum dots with certain porphyrins. J Coll Inter Sci 2010;344:596–602. 25. Zhao D, Chan WH, He ZK, Qiu T. Quantum dot ruthenium complex dyads: recognition of double-strand DNA through dual-color fluorescence detection. Anal Chem 2009;81:3537–43. 26. Shen YZ, Liu SP, He YQ. Fluorescence quenching investigation on the interaction of glutathione-CdTe/CdS quantum dots with sanguinarine and its analytical application. Luminescence 2014;29:176–82. 27. Gan XJ, Liu SP, Liu ZF, Hu XL, Cui ZP, Wang YQ. Determination of carbazochrome by fluorescence quenching method. Spectrochim Acta A 2012;97:161.
Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web site.
246 wileyonlinelibrary.com/journal/luminescence
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2016; 31: 241–246
