Research article Received: 17 February 2014,
Revised: 05 May 2014,
Accepted: 07 May 2014
Published online in Wiley Online Library: 27 June 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2714
A rapid and sensitive resonance Rayleigh scattering spectra method for the determination of quinolones in human urine and pharmaceutical preparation Man Qiao,a Yaqiong Wang,a Shaopu Liu,a Zhongfang Liu,a Jidong Yang,b Jinghui Zhua and Xiaoli Hua* ABSTRACT: A new method based on resonance Rayleigh scattering (RRS) was proposed for the determination of quinolones (QNS) at the nanogram level. In pH 3.3–4.4 Britton–Robinson buffer medium, quinolones such as ciprofloxacin, pipemidic acid (PIP), lomefloxacin (LOM), norfloxacin (NOR) and sarafloxacin (SAR) were protonated and reacted with methyl orange (MO) to form an ion-pair complex, which then further formed a six-membered ring chelate with Pd(II). As a result, new RRS spectra appeared and the RRS intensities were enhanced greatly. RRS spectral characteristics of the MO–QNS–Pd(II) systems, the optimum conditions for the reaction, and the influencing factors were investigated. Under optimum conditions, the scattering intensity (ΔI) increments were directly proportional to the concentration of QNS with in certain ranges. The method had high sensitivity, and the detection limits (3σ) ranged from 6.8 to 12.6 ng/mL. The proposed method had been successfully applied for the determination of QNS in pharmaceutical formulations and human urine samples. In addition, the mechanism of the reaction system was discussed based on IR, absorption and fluorescence spectral studies. The reasons for the enhancement of scattering spectra were discussed in terms of fluorescence-scattering resonance energy transfer, hydrophobicity and molecular size. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: quinolones; Pd(II); methyl orange; resonance Rayleigh scattering
Introduction
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* Correspondence to: X. Hu, Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China. E-mail: [email protected] a
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
b
College of Chemical and Environmental Engineering, Chongqing Three Gorges University, Wanzhou, Chongqing 404100, China
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Quinolones (QNS) are a large family of structurally related antibacterial agents that are widely used in the treatment of a variety of infections. The first quinolone, nalidixic acid, was accidentally discovered in 1962 (1). Since then, several modifications have been made to its chemical structure in an attempt to improve its antibacterial activity and pharmacological properties, leading to the development of four generations of quinolone compounds (2). Pipemidic acid (PIP) belongs to the first generation. PIP has moderate activity against aerobic Gram-negative bacteria and very little or no activity against aerobic Gram-positive bacteria, anaerobes or atypical pathogens. It is less used nowadays because of its poor oral bioavailability and limited distribution into systemic tissues (2,3). Ciprofloxacin (CIP), lomefloxacin (LOM) and norfloxacin (NOR) are second-generation quinolone derivatives which are obtained by the addition of a fluorine group at the 6-position of the basic quinolone structure. Because their activity against Gram-negative bacteria is remarkably expanded compared with first-generation QNS, they have been widely used in the treatment of urinary and respiratory tract infections, with good localized action on infected sites, and also in gastrointestinal diseases (4,5). Sarafloxacin (SAR) is a third-generation QNS and its activity against Gram-positive bacteria, anaerobes, Gram-negative bacteria and atypical pathogens have been largely improved (2). Because of their gradual enhanced activity against bacteria and pathogens, in recent decades, QNS have been frequently used as antimicrobials in veterinary medicine,
clinical medicine and food-producing animal husbandry. The wide applications have raised public health concerns because their residues may persist in edible animal tissues and result in the development of drug-resistant bacterial strains or allergies (6). Therefore, there is a need to establish a rapid and sensitive analytical method for the determination of trace amounts of QNS in pharmaceutical formulations and biological fluids to ensure human health. Various methods such as spectrophotometry (7), spectrofluorimetry (8,9), chemiluminescence (10,11), highperformance liquid chromatography (12,13) and electrochemical methods (14) have been used to determine QNS. Among these, Pascual-Reguera et al. (7) reported a solid-phase extraction UV spectrophotometric method for the determination of CIP with a detection limit of 12.0 ng/mL. The method improves the sensitivity of spectrophotometry, but is too complex to operate. Ulu et al. (9)
M. Qiao et al. present a highly sensitive spectrofluorimetric method for the determination of fluoroquinolone drugs based on the derivatization of fluoroquinolone with 4-chloro-7-nitrobenzofurazan, but the method is time consuming and requires a large amount of solvent. Chemiluminescence is a rapid and specific method, but its reproducibility and stability are relatively poor (10). Although the electrochemical method exhibits good sensitivity, it is not suitable for routine analysis. Nowadays, analysis of QNS is dominated by high-performance liquid chromatography, which offers high accuracy and efficiency, but requires too much time and costly instruments and skilled operators. Thus, it is important that a more sensitive, simple, economical and selective method is found for the determination of QNS. Because metal ions play a vital role in a vast number of biological processes, the interaction of metal ions with biologically active ligands, for example drugs, has attracted increasing attention. Many biologically active compounds act via chelation (15), and previous studies have shown that quinolone–metal complexes have stronger antibacterial activities than QNS themselves. Also, they can overcome cross-resistance between QNS and other antibiotic agents. For example, norfloxacin–Zn(II) and norfloxacin–Fe(III) complexes show increased activity over norfloxacin alone against Gram-negative Escherichia coli and Bacillus dysenteriae (16). Co(II), Ni(II), Mn(II) Cr(III), La(III) and UO2 (VI) complexes with sparfloxacin show remarkably higher antimicrobial activity than free quinolone ligands (16). Ciprofloxacin–Cu exhibits significant enhancement in the antitubercular activity (17). However, to the best of our knowledge, there are few reports of quinolone–Pd(II) complexes (18,19). Resonance Rayleigh scattering (RRS) spectroscopy belongs to the phenomenon of light scattering and can be obtained simply on a common low-cost fluorescence meter. When the wavelength of Rayleigh scattering is located at or close to the molecular absorption band, RRS is produced. For species that aggregate, enhancements in RRS of several orders of magnitude can be observed at wavelengths characteristic of these species (20). Owing to the various advantages of the RRS technique, such as simple performance, low cost, short response time and high sensitivity, it has been extensively applied to the determination of ions (21,22), drugs (23,24), noxious gases (25,26), proteins (27,28), nucleic acids (29,30), etc. In this study, the interaction among methyl orange (MO), QNS and Pd(II) was investigated and trace amounts of QNS were determined. The experiments showed that QNS could react with MO to form a ion-pair complex (31), which further formed six-membered rings chelate complexes with Pd(II), leading to the enhancement of RRS and a new RRS spectrum appeared with the maximum peak at ~ 360 nm. The detection limit (3σ) ranged from 6.8 to 12.6 ng/ mL. Under optimum conditions, determination of QNS at trace amounts in pharmaceutical preparations and human urine samples was successfully performed. The results obtained were in good agreement with those obtained using the official and reference methods.
Experimental
spectra were all 400 V. A UV/Vis 8500 spectrophotometer (Tianmei, Shanghai, China) was used to record the absorption spectra. A pHS-3C meter (Shanghai Scientific Instruments Company, China) was used to measure the pH of the solution. The stock solution of quinolone antibiotics (Huamei Biology Technology Co. Ltd.) was 200.0 μg/mL. The working solution was further diluted to 8.0 μg/mL with doubly distilled water. The stock solution of MO (Sanland Chemical Cc. Ltd) was 1.0 × 10 3 mol/L. The stock solutions were further diluted to 2.0 × 10 4 mol/L with doubly distilled water as the working solution. The stock solution of PdCl2 (Shanghai Reagent Factory, China) was 1.0 × 10 3 mol/L. It was prepared by dissolving 0.0887 g PdCl2 in 0.50 mL of concentrated hydrochloric acid and diluting to 100 mL with water. The stock solution was diluted to 2.0 × 10 4 mol/L as working solution with doubly distilled water. Britton–Robinson (BR) buffer solutions at different pH values (2.7–6.5) were prepared by mixing the mixed acid (composed of 0.04 mol/L H3PO4, HAc and H3BO3) with 0.2 mol/L NaOH in proportion. The buffer was used to control the acidity of the interacting system. All other reagents were of analytical reagent grade and were used without further purification. Doubly distilled water was used throughout. General procedure Into a 10.0 mL calibrated flask were added 1.00 mL of pH 3.8–4.2 BR solution, 1.00 mL of MO solution, an appropriate amount QNS solution and 1.50 mL PdCl2 solution in turn. The mixture was then diluted to the mark and mixed thoroughly. After 15 min, the RRS spectra of the system were recorded with synchronous scanning at λex = λem. Then, the scattering intensities (IRRS) for the complexes, and the scattering intensities (I0RRS) for the reagent blank were measured at their own maximum wavelengths, ΔIRRS = IRRS – I0RRS.
Results and discussion RRS spectra Figure 1(a) showed the RRS spectra of MOO–QNS–Pd(II) systems. It could be seen that the RRS intensities of QNS, Pd(II) and MO themselves were very weak within the range 220–800 nm. The binary systems of MOO–QNS, MO–Pd(II) and QNS–Pd(II) could only result in a small change in the RRS spectra. However, when they reacted further with Pd(II) to form ternary systems, remarkably enhanced RRS spectra were observed. The five different reaction systems had similar spectral characteristics because their maximum RRS peaks were all at 360 nm and the other smaller peaks were located at 610 nm. However, the intensities of reaction systems were different and the order of their intensities was CIP > PIP > NOR > LOM > SAR. Furthermore, the enhanced RRS intensities were directly proportional to the concentrations of QNS. Taking CIP as an example, the quantitative relationship between IRRS and the concentration of CIP is shown in Fig. 1(b). Hence, the RRS method could be used for the determination of trace amounts of QNS.
Apparatus and reagents
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A Hitachi F-2500 spectrofluorophotometer (Tokyo, Japan) was used to record the RRS and fluorescence spectra. The slit width was 5 nm in the RRS spectra and 10 nm in the fluorescence spectra. The photomultiplier tube (PMT) voltages of the above three
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Optimum experimental conditions Effect of pH. The effects of different buffer solutions on RRS intensity of the reaction systems were tested with BR buffer, HAc–NaAc, Na2HPO4–C6H8O7 and HCl–NaAc. The results showed
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Determination of quinolones by resonance Rayleigh scattering reaction acidity for the first two systems and pH 4.2 for last three systems. The appropriate amount was 1.0 mL. Effect of MO concentration. The effect of MO concentration on the RRS intensity of the reaction systems was investigated. The results showed that the RRS intensity was gradually enhanced with increasing MO concentrations. When the MO concentration was in the range of 1.0 × 10 5–4.0 × 10 5 mol/L, the RRS intensity reached a maximum and remained stable. If the amount of MO was not adequate, the reaction was incomplete, and the RRS intensities were low. If the amount of MO was excessive, the RRS intensities decreased slightly because of the formation of MO dimer by self-aggregation. Therefore, 2.0 × 10 5 mol/L was chosen as the most suitable MO concentration. Effect of Pd(II) concentration. According to the experimental results, when a small amount of Pd(II) existed, the reaction was incomplete, and the RRS intensity was low. The ΔIRRS of the reaction systems reached a maximum and remained constant in the range 2.0 × 10 5–5.0 × 10 5 mol/L. So the concentration of Pd(II) used was 3.0 × 10 5 mol/L.
Figure 1. RRS spectra. (a) The RRS spectra of MO–QNS–Pd(II) systems. (1–18) MO, Pd(II), LOM, PIP, SAR, NOR, CIP, MO–Pd(II), MO–LOM, MO–PIP, MO–SAR, MO–NOR, MO–CIP, LOM–Pd(II), PIP–Pd(II), SAR–Pd(II), NOR–Pd(II), CIP–Pd(II); (19) MO–SAR–Pd (II); (20) MO–LOM–Pd(II); (21) MO–NOR–Pd(II); (22) MO–PIP–Pd(II); (23) MO–CIP–Pd(II). 5 5 cMO: 2.0 × 10 mol/L, cPd(II): 3.0 × 10 mol/L, cQNS: 0.8 μg/mL, pH 3.8 (CIP, NOR), 5 5 pH 4.2 (LOM, PIP, SAR). cMO: 2.0 × 10 mol/L, cPd(II): 3.0 × 10 mol/L, cQNS: 0.8 μg/mL, pH 3.8 (CIP, NOR), pH 4.2 (LOM, PIP, SAR). (b) The relationship between RRS intensity 5 5 and the concentrations of CIP. cMO: 2.0 × 10 mol/L, cPd(II): 3.0 × 10 mol/L, cQNS (1 → 5): 0.4, 0.8, 1.2, 1.6, 2.0 μg/mL, pH 3.8.
that the sensitivity and stability of the systems were best in BR buffer. Therefore, BR buffer solution was chosen to control the pH of the solutions. Figure 2 showed the dependence of ΔIRRS on the pH of the solution. It was found that the optimum pH ranges were 3.3–4.4 for MO–CIP–Pd(II) and MO–NOR–Pd(II) systems, and 3.8–5.1 for MO–PIP–Pd(II), MO–LOM–Pd(II) and MO–SAR–Pd(II) systems. Therefore, pH 3.8 was chosen as
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Reaction time and the stability. Under optimum conditions, the reaction time and stability were studied by determining the RRS intensities of the reaction systems every 5 min for 2.5 h immediately after mixing thoroughly. The results showed that the RRS intensity reached a maximum after ~ 15 min and then remained stable for 2 h at room temperature (20–25°C). Thus, the assay was completed after 15 min.
Sensitivity and selectivity of the method Sensitivity of the RRS method. Under optimum conditions, the ΔIRRS values of the ternary systems were measured at their maximum scattering wavelengths. Calibration graphs of ΔIRRS against QNS concentration were constructed. The regression equation, linear range, correlation coefficient and detection limit are listed in Table 1. It can be seen from Table 1 that the method had high sensitivity. The detection limits (3σ) ranged from 6.8 to 12.6 ng/mL, and were lower than or comparable with that of other common analytical methods, as shown in Table 2 (take the CIP and NOR as examples). Therefore, the method was very suitable for determining trace amounts of QNS. Selectivity of the RRS method. Taking the MO–CIP–Pd(II) system as an example, the effects of foreign substances on the determination of CIP were investigated. As shown in Table 3, most metal ions, acid radical anions, amino acids, carbohydrate and some other organics and inorganics barely affect the determination of CIP within a permissible error of ± 5%. Whereas, Fe (III) and I interfered greatly with the determination of CIP. Fortunately, the amount of Fe(III) and I in pharmaceutical preparations and urine samples is extraordinary low and it did not affect the selectivity of this method. In addition, 50 times the amount of
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Figure 2. Effect of pH on the RRS intensity of MO–QNS–Pd(II) systems. (1) MO–CIP–Pd(II), (2) MO–NOR–Pd(II), (3) MO–PIP–Pd(II), (4) MO–LOM–Pd(II), (5) 5 5 MO–SAR–Pd(II). cQNS: 0.8 μg/mL; cPd(II): 3.0 × 10 mol/L; cMO: 2.0 × 10 mol/L.
Effect of ionic strength. The effects of ionic strength on the ΔIRRS of the reaction systems were studied by adding different concentrations of NaCl. The results showed that the ΔIRRS remained constant when the ionic strength was < 6.0 × 10 4 mol/L. ΔIRRS decreased gradually when the concentration of NaCl was > 6.0 × 10 4 mol/L. The possible reason for this phenomenon might be the effect of electrostatic shielding of charges, which would reduce the affinity of MO for QNS.
M. Qiao et al. Table 1. Correlation coefficient, linear ranges and detection limits for calibration graphs System MO–CIP–Pd(II) MO–PIP–Pd(II) MO–NOR–Pd(II) MO–LOM–Pd(II) MO–SAR–Pd(II)
Regression equation
Correlation coefficient (r)
ΔI = 51.3 + 846.0c ΔI = 15.9 + 832.4c ΔI = 21.3 + 793.5c ΔI = 14.0 + 727.3c ΔI = 6.9 + 352.3c
Linear range (μg/mL)
0.9999 0.9997 0.9987 0.9979 0.9977
Detection limit (ng/mL)
0.023–2.0 0.023–2.0 0.024–2.4 0.026–2.0 0.042–2.4
6.8 6.9 7.4 7.9 12.6
Table 2. Results of comparison for the analysis of QNS with different methods Detected substance CIP
NOF
Method
Reagent
Linearity (μg/mL) Detection limit (ng/mL)
SPE-UV F FIA-CL HPLC Voltammetric RRS RRS RRS K-SP SF FIA-CL HPLC EC RRS RRS RRS
– 4-Chloro-7-nitrobenzofurazan Peroxynitrous acid – Cetyltrimethylammonium bromide Erythrosine Pd(II) and Eosin Y Pd(II) and Methyl Orange KMnO4 4-Chloro-7-nitrobenzofurazan Peroxynitrous acid – Cd(OH)2 microcrystals Erythrosine Pd(II) and Eosin Y Pd(II) and Methyl Orange
0.05–0.3 0.023–0.5 0.03–3.3 0.09–2.0 0.033–6.6 0.0574–5.6 0–2.4 0.023–2.0 1.0–12.0 0.03–8.0 0.03–3.2 0.01–2.0 0.2–265.0 0.0596–4.4 0–2.4 0.024–2.4
12.0 7.0 14.9 30 16.5 17.0 9.4 6.8 30.0 9.2 19.8 5.0 156.5 18.0 12.8 7.4
Ref (7) (9) (10) (13) (32) (33) (18) This work (34) (9) (10) (13) (35) (33) (18) This study
EC, electrochemical; F, spectrofluorimetric; FIA-CL, flow-injection chemiluminescence; HPLC, high-performance liquid chromatography; K-SP, kinetic spectrophotometric; SPE-UV, solid-phase extraction UV spectrophotometric method.
Table 3. Effects of coexistent substances (cCIP: 1.6 μg/mL) Coexistent species Concentration (μg/mL) Relative error (%) Al2(SO4)3 Mg(AC)2 CuSO4 MnSO4 CaCl2 ZnSO4 NH4Cl KBr KH2PO4 NiSO4 CdCl2 HgCl2 SnCl2 Na3PO4 FeCl2 NH4Fe(SO4)2 Co(NO3)2 NH4F
16.0 16.0 32.0 80.0 160.0 32.0 160 80.0 160.0 100 40 60 20 200 20 6.0, 80.0a 150 40
1.9 2.0 4.4 4.5 2.3 4.9 1.5 5.0 2.8 4.4 3.8 4.2 3.9 2.4 3.4 4.8 2.0 4.2
Coexistent species Cystine Valine Glycine Phenylanine Sucrose Glucose KI Maltose Soluble starch Ascorbic acid Urea Uric acid Creatine Creatinine Hippuric acid Citric acid Oxalic acid EDTA
Concentration (μg/mL) Relative error (%) 60 16.0 16.0 13.2 160.0 160.0 2.0 128.0 160.0 160.0 160.0 20 100 120 200 160 140 800
3.8 3.5 4.5 5.4 5.0 3.6 4.8 1.5 2.6 2.6 2.3 3.6 2.8 3.0 2.5 2.8 4.0 3.5
a
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1.0 mL of 0.01 mol/L EDTA was added.
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Determination of quinolones by resonance Rayleigh scattering Fe(III) did not interfere after addition of 1.0 mL of 0.01 mol L EDTA. Therefore, the method showed good selectivity. Formation of the ternary complex The structures of the investigated QNS are given in Table 4. Because they had the same parent structure and only some substituted groups were different, we took CIP as an example to study the interaction of MO and Pd(II). The composition ratio of the ternary complex was determined by Job’s method (36,37) and the molar ratio method (38). The results showed that the ratio of MO : CIP : Pd(II) was 1 : 1 : 1. This finding was consistent with previous results (19) in which CIP and Pd(II) formed a 1 : 1 chelate. Scheme 1 shows the protonation/deprotonation equilibria of QNS with a piperazinyl substituent. As indicated, in general, two charged species exist in the system in the form of H2Z+, Z , or together with a zwitterionic species, designated as HZ. According to the literature (39,40), the pKa1 of QNS ranged from 5.42 to 5.94. So, QNS chiefly existed as H·QNS+ under the experimental conditions. The calculation indicated that the distribution ratio of H·CIP+ reached 99.1%. At this point, the structure of MO exists as an azo form (Scheme 2), which is more stable than the quinoid form, that is, MO chiefly existed as MO and its distribution ratio reached 71.6%. So H·CIP+ could react with MO to form a neutral ion-pair complex (31) via electrostatic forces. When some Pd(II) solution
was added to the ion-pair complex, the ketonic and carboxylic oxygen atoms coordinated with Pd(II) and formed a stable six-membered ring chelate. Finally, the chelate formed square–planar complexes with the chloride ligands. The structure of the ternary complex was speculated as shown in Scheme 3.
Effects of the formation of ternary complex on spectra characteristics The effect on IR spectra. Figure 3 showed the IR spectra of free CIP, MO and the ternary complex. Contrasting curve c and
Scheme 3. The structure of the ternary complex.
Table 4. Structures of quinolones studies and their pKa
QNS
Substituents at position R1
PIP CIP NOR LOM SAR
–C2H5 –C2H5 –C2H5
pKa
X
R6
Y
R8
R3`
N CH CH CH CH
– F F F F
N CH CH CH CH
– – – F –
– – – -CH3 –
pKa1
pKa2
5.42 5.86 5.94 – 5.62
8.18 8.24 8.22 – 8.18
Figure 3. IR spectra of CIP, MO and MO–CIP–Pd(II). (a) CIP, (b) MO–CIP–Pd(II), (c) MO.
Scheme 1. Protolytic equilibria of QNS.
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Scheme 2. The structure of MO.
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M. Qiao et al. curve b, the absorption band in the 1039 cm 1 region is attributed to -SO3Na, which shifted to 1035 cm 1, and the vibration intensity was decreased. It was shown that -NH+3 bound with SO3Na, that is, [H·CIP]+ could react with MO to form a neutral ion-pair complex. Comparing curve a with curve b, we observed that the absorption band in the region around 1700 cm 1 belonging to the free carboxylic acid disappeared (13), which indicated that the Pd(II) ion had reacted with the carboxylate oxygen. The absorption band in the 1625 cm 1 region attributed to the ketone group in the free ligand spectra was shifted to 1631 cm 1 in the palladium complexes, which was a good indication that this group was coordinated to the Pd(II) ion also (13). In curve b, two characteristic absorption bands around 1631 and 1400 cm 1 were observed, which could be attributed to asymmetric and symmetric ν(O–C–O), respectively (13). The frequency difference [Δν = νas(COO ) – νs(COO )] was 231 cm 1. It was reported that if Δν was > 200 cm 1, this group probably bound to metal ions in a monodentate way (41). The conclusion showed a good agreement with experimental results.
of QNS. However, the sensitivity of the method was not high enough. Taking CIP as an example, the effects of MO and Pd(II) on fluorescence spectra were investigated. As shown in Fig. 5, the fluorescence intensity of CIP was quenched in the presence of MO due to formation of the ion-pair complex, and the fluorescence intensity of CIP was also quenched in the presence of Pd (II) due to the formation of a six-membered ring chelate. However, the fluorescence intensity was quenched markedly in the presence of MO and Pd(II). It was interesting that a new spectrum appeared at 571 nm. The reason for this might be ascribed to the formation of a ternary complex. The effects of MO on fluorescence spectra of CIP were investigated when the concentration of CIP and Pd(II) were fixed. A gradual reduction in the emission intensity was observed (Fig. 6a) with increasing MO concentration, whereas the RRS intensity increased (Fig. 6b). The effects of Pd(II) on fluorescence spectra of CIP were similar to MO. This phenomenon revealed that the energy transferred between fluorescent and scattering.
The effect on absorption spectra. As shown in Fig. 4, Pd(II) had no absorption in the range 220–600 nm (curve 1). MO had a maximum absorption wavelength at 473 nm and a smaller absorption peak at 274 nm (curve 2). CIP had a maximum absorption wavelength at 276 nm (curve 3). Comparing curve 2 with curve 4, it was observed that the absorption of MO–CIP system (curve 4) was actually a superposition of curves 2 and 3. Thus, it could be seen that formation of ion-pair complex had no effect on the absorption spectra. Comparing curve 3 and curve 5, we could see that the maximum absorption wavelength of CIP shifted to 280 nm. The absorbance was reduced from 0.79 to 0.62. This indicated that CIP could interact with Pd(II) and form a complex. Contrasting curve 4 and curve 6, the absorption spectra of the ternary system were changed markedly: the maximum absorption wavelength at 276 nm shifted to 281 nm, whereas the maximum absorption wavelength at 476 nm shifts to 467 nm. The absorbance reduced greatly.
The effect on RRS spectra. It can be seen from Fig. 1(a) that the formation of the ternary complexes results in great enhancement of RRS and new RRS spectra appear. The possible reasons for RRS enhancement are described below.
The effect on fluorescence spectra. The five types of quinolone drugs had similar fluorescence spectra. Their maximum excitation wavelength (λex) and maximum emission wavelength (λem) were located in the range 277–283 nm and 442–459 nm, respectively. Their fluorescence intensities (F) were different and reduced in the order CIP > PIP > NOR > LOM > SAR. So fluorescence spectrometry (FL) could be used for the determination
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Figure 4. Absorption spectra. (1) Pd(II), (2) MO, (3) CIP, (4) MO–CIP, (5) CIP–Pd(II), (6) 5 5 MO–CIP–Pd(II) cMO: 2.0 × 10 mol/L, cPd(II): 3.0 × 10 mol/L, cCIP: 8.0 μg/mL, pH 3.8.
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Figure 5. Fluorescence spectra. cMO: 2.0 × 10 4.0 μg/mL, pH 3.8.
5
mol/L, cPd(II): 1.0 × 10
5
mol/L, cCIP:
Figure 6. Fluorescence quenching spectra and RRS spectra. cCIP: 4.0 μg/mL, cPd(II): 5 6 6 5 5 1.0 × 10 mol/L, cMO(1 → 6): 4.0 × 10 , 8.0 × 10 , 1.2 × 10 , 1.6 × 10 , 5 5 2.0 × 10 , 2.4 × 10 mol/L.
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Determination of quinolones by resonance Rayleigh scattering
Figure 7. Comparison between fluorescence spectrum and RRS spectrum of MO–CIP–Pd(II) system. (1) Fluorescence spectrum, (2) RRS spectrum.
Fluorescence-scattering resonance energy transfer. From Fig. 7 (a), we can see that the RRS spectrum partially overlaps with the fluorescence spectrum. This means that the RRS spectrum and fluorescence spectrum are at the same frequency, and can resonate and exchange energy with each other. So the IRRS increased on reduction of F (Fig. 7b). The ternary complexes result in great enhancement of RRS and the appearance of new RRS spectra because of the marked quenching of fluorescence intensity. Enhancement of hydrophobicity. MO , H·CIP+ and Pd(II) are all hydrophilic and can easily dissolve in aqueous solution so that they cannot form a hydrophobic interface with water. When H·CIP+ reacts with MO to form a neutral ion-pair complex, the hydrophobicity of the complex increases to some extent. But the increase is still small because of the existence of a carboxyl group. However, when the ion-pair complex further reacts with Pd(II) and forms a ternary complex, the carboxyl is coordinated by the Pd(II) ion and their hydrophobicity is increased significantly so that a hydrophobic liquid–solid interface appeared. The formation of the hydrophobic interface is favorable to the enhancement of RRS signal. Enlargement of molecular volume. According to the Rayleigh scattering formula, an increase in the volume of the scattering molecule is advantageous to the enhancement of scattering intensity. If the molecular volume is difficult to estimate, the formula can be simplified as I = kI0Mc (42), that is, when the incident light intensity (I0), the concentration of the solution (c) and (k) are constant, the scattering intensity (I) is proportional to the molecular weight of the particle. The molecular weight of QNS is of 303.3–385.36. Taking CIP as an example, when it reacts with MO and Pd(II) to form a ternary complex, the molecular weight is changed from 331.35 to 813.0. Hence, enlargement of the molecular volume is another reason for the enhancement of scattering intensity. Affected by all the above factors, the intensity of RRS is greatly enhanced.
Analytical application Determination of QNS in pharmaceutical preparation Ten CIP hydrochloride tablets (0.25 g/tablet) were weighed and powdered in a pestle and mortar. A mass of powder equivalent to one tablet was weighed and dissolved into 100.0 mL water and impurities removed by filtering. Then, 1.00 mL of filtrate was pipetted into a 250 mL volumetric flask and diluted to volume with doubly distilled water. Suitable amounts of the solution were measured according to the general procedure described above. The results obtained by our method and the official method (43) are listed in Table 5. Five LOM hydrochloride capsules (0.10 g/capsule) were dissolved in 100.0 mL water and impurities removed by filtering. Then, 1.00 mL of filtrate was pipetted into a 250 mL volumetric flask and made up to volume with doubly distilled water. Suitable amounts of the solution were measured using the RRS method. The results are listed in Table 5 and compared with those obtained using the official method (44). One milliliter of NOR eye drops were pipetted into a 500 mL volumetric flask and made up to volume using doubly distilled water. Suitable amounts of the solution were measured using the RRS method. The accuracy was tested by comparison with the standard addition method. The results are listed in Table 6. It can be seen from Table 5 that results agreed with those obtained using the official method. Table 6 shows that the RRS method had good accuracy and repeatability. The satisfactory result indicated that the proposed method could be used to determine QNS in pharmaceutical formulations. This would provide a simple and new method for quantitative tests of QNS.
Determination of CIP in human urine In this experiment, five urine samples were collected before drug administration as blank samples. Five healthy volunteers received a single oral dose of 250 mg CIP after an overnight fast.
Table 5. Results for the determination of CIP in tablet and LOM in capsules by the RRS method and the comparison of it with official method Sample
Label claim (g/piece)
CIP tables
0.25
LOM capsules
0.10
RRS Official RRS Official
Found (g/piece) 0.242 0.248 0.099 0.102
0.255 0.250 0.105 0.104
0.242 0.255 0.100 0.103
0.248 0.250 0.256 0.238 0.104 0.105 0.098 0.102
Copyright © 2014 John Wiley & Sons, Ltd.
Average (g/piece) 0.247 0.249 0.103 0.102
RSD (%) 2.2 2.9 2.8 0.2
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Method
M. Qiao et al. Table 6. Results for the determination of NOR in eye drops Sample
Label claim (mg/mL)
NOR in eye drops
Found (mg/mL)
3.00
2.97
Added (mg/mL) 1.00 2.00 4.00 6.00
Found total amount (mg/mL) 3.85 4.72 6.77 9.10
4.08 4.86 6.94 8.84
3.92 4.75 6.87 8.93
4.05 5.07 6.72 8.79
4.01 5.01 6.97 9.04
Average (mg/mL)
RSD (%)
3.98 4.88 6.85 8.94
Recovery (%)
2.3 3.2 1.6 1.5
101.0 95.5 97.0 99.5
Table 7. Results for the determination of CIP in human urine samples (n = 5) Samplea
No.1 No.2 No.3
Times (h) 0–6 6–12 12–24
Found (μg/mL) RRS 138.2 27.6 9.2
SP
b
139.8 28.6 10.6
Added (μg/mL) 40.0 40.0 40.0
Total amount (μg/mL) RRS
SP
178.7 67.3 48.7
180.6 67.8 49.9
RSD (%) RRS 2.2 2.0 1.8
Recovery (%)
SP 2.4 2.1 2.2
RRS
SP
101.3 99.3 98.8
102.0 98.0 98.2
a
No CIP were detected in any of the blank urine samples; spectrophotometry.
b
The volunteers were asked to drink sufficient and comparable amounts of water through all collection periods to ensure sufficient urine production. The urine samples of individuals were gathered within 0–24 h and the urinary volumes were recorded. In order to ensure the sample concentrations of the drug were within the linear range of determination, the urine samples were diluted and analyzed by the RRS method. The spectrophotometric method (45) (taking Eosin Y as a probe) was used as a reference method. The results are given in Table 7. The average recovery obtained with the RRS method was 98.8–101.3%, and the RSD was 1.8–2.2%. In addition, the test showed that there were 106.5 mg of the original drug in urine; the cumulative excretion rate reached 42.6%. The results were in agreement with those given in the literature (46). The results showed that both methods (RRS and spectrophotometry) yield values within the same range when tested using adequate statistical procedures. The method allowed the directly determination of QNS in urine and was suitable for routine analysis in clinical laboratories. This may provide guidance for studying the pharmacokinetics of CIP.
Conclusions We have successfully provided a new, rapid and facile RRS method for the determination of trace amounts of QNS with high sensitivity and selectivity. Under the optimized conditions, a linear correlation has been established between the RRS intensity and the concentration of QNS in certain ranges, with the detection limits (3σ) ranging from 6.8 to 12.6 ng/mL. The mechanism of the reactions were discussed in terms of fluorescence-scattering resonance energy transfer, hydrophobicity and molecular size. This method had been satisfactorily used for the determination of QNS in real samples. Acknowledgement
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This paper was supported by the financial supports from the National Natural Science Foundation of China (No. 21175015) and the Special Fund of Chongqing key Laboratory (CSTC) and
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the Fundamental Research Funds for the Central Universities (XDJK2013A022).
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Copyright © 2014 John Wiley & Sons, Ltd.
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Revised: 05 May 2014,
Accepted: 07 May 2014
Published online in Wiley Online Library: 27 June 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2714
A rapid and sensitive resonance Rayleigh scattering spectra method for the determination of quinolones in human urine and pharmaceutical preparation Man Qiao,a Yaqiong Wang,a Shaopu Liu,a Zhongfang Liu,a Jidong Yang,b Jinghui Zhua and Xiaoli Hua* ABSTRACT: A new method based on resonance Rayleigh scattering (RRS) was proposed for the determination of quinolones (QNS) at the nanogram level. In pH 3.3–4.4 Britton–Robinson buffer medium, quinolones such as ciprofloxacin, pipemidic acid (PIP), lomefloxacin (LOM), norfloxacin (NOR) and sarafloxacin (SAR) were protonated and reacted with methyl orange (MO) to form an ion-pair complex, which then further formed a six-membered ring chelate with Pd(II). As a result, new RRS spectra appeared and the RRS intensities were enhanced greatly. RRS spectral characteristics of the MO–QNS–Pd(II) systems, the optimum conditions for the reaction, and the influencing factors were investigated. Under optimum conditions, the scattering intensity (ΔI) increments were directly proportional to the concentration of QNS with in certain ranges. The method had high sensitivity, and the detection limits (3σ) ranged from 6.8 to 12.6 ng/mL. The proposed method had been successfully applied for the determination of QNS in pharmaceutical formulations and human urine samples. In addition, the mechanism of the reaction system was discussed based on IR, absorption and fluorescence spectral studies. The reasons for the enhancement of scattering spectra were discussed in terms of fluorescence-scattering resonance energy transfer, hydrophobicity and molecular size. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: quinolones; Pd(II); methyl orange; resonance Rayleigh scattering
Introduction
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* Correspondence to: X. Hu, Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China. E-mail: [email protected] a
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
b
College of Chemical and Environmental Engineering, Chongqing Three Gorges University, Wanzhou, Chongqing 404100, China
Copyright © 2014 John Wiley & Sons, Ltd.
207
Quinolones (QNS) are a large family of structurally related antibacterial agents that are widely used in the treatment of a variety of infections. The first quinolone, nalidixic acid, was accidentally discovered in 1962 (1). Since then, several modifications have been made to its chemical structure in an attempt to improve its antibacterial activity and pharmacological properties, leading to the development of four generations of quinolone compounds (2). Pipemidic acid (PIP) belongs to the first generation. PIP has moderate activity against aerobic Gram-negative bacteria and very little or no activity against aerobic Gram-positive bacteria, anaerobes or atypical pathogens. It is less used nowadays because of its poor oral bioavailability and limited distribution into systemic tissues (2,3). Ciprofloxacin (CIP), lomefloxacin (LOM) and norfloxacin (NOR) are second-generation quinolone derivatives which are obtained by the addition of a fluorine group at the 6-position of the basic quinolone structure. Because their activity against Gram-negative bacteria is remarkably expanded compared with first-generation QNS, they have been widely used in the treatment of urinary and respiratory tract infections, with good localized action on infected sites, and also in gastrointestinal diseases (4,5). Sarafloxacin (SAR) is a third-generation QNS and its activity against Gram-positive bacteria, anaerobes, Gram-negative bacteria and atypical pathogens have been largely improved (2). Because of their gradual enhanced activity against bacteria and pathogens, in recent decades, QNS have been frequently used as antimicrobials in veterinary medicine,
clinical medicine and food-producing animal husbandry. The wide applications have raised public health concerns because their residues may persist in edible animal tissues and result in the development of drug-resistant bacterial strains or allergies (6). Therefore, there is a need to establish a rapid and sensitive analytical method for the determination of trace amounts of QNS in pharmaceutical formulations and biological fluids to ensure human health. Various methods such as spectrophotometry (7), spectrofluorimetry (8,9), chemiluminescence (10,11), highperformance liquid chromatography (12,13) and electrochemical methods (14) have been used to determine QNS. Among these, Pascual-Reguera et al. (7) reported a solid-phase extraction UV spectrophotometric method for the determination of CIP with a detection limit of 12.0 ng/mL. The method improves the sensitivity of spectrophotometry, but is too complex to operate. Ulu et al. (9)
M. Qiao et al. present a highly sensitive spectrofluorimetric method for the determination of fluoroquinolone drugs based on the derivatization of fluoroquinolone with 4-chloro-7-nitrobenzofurazan, but the method is time consuming and requires a large amount of solvent. Chemiluminescence is a rapid and specific method, but its reproducibility and stability are relatively poor (10). Although the electrochemical method exhibits good sensitivity, it is not suitable for routine analysis. Nowadays, analysis of QNS is dominated by high-performance liquid chromatography, which offers high accuracy and efficiency, but requires too much time and costly instruments and skilled operators. Thus, it is important that a more sensitive, simple, economical and selective method is found for the determination of QNS. Because metal ions play a vital role in a vast number of biological processes, the interaction of metal ions with biologically active ligands, for example drugs, has attracted increasing attention. Many biologically active compounds act via chelation (15), and previous studies have shown that quinolone–metal complexes have stronger antibacterial activities than QNS themselves. Also, they can overcome cross-resistance between QNS and other antibiotic agents. For example, norfloxacin–Zn(II) and norfloxacin–Fe(III) complexes show increased activity over norfloxacin alone against Gram-negative Escherichia coli and Bacillus dysenteriae (16). Co(II), Ni(II), Mn(II) Cr(III), La(III) and UO2 (VI) complexes with sparfloxacin show remarkably higher antimicrobial activity than free quinolone ligands (16). Ciprofloxacin–Cu exhibits significant enhancement in the antitubercular activity (17). However, to the best of our knowledge, there are few reports of quinolone–Pd(II) complexes (18,19). Resonance Rayleigh scattering (RRS) spectroscopy belongs to the phenomenon of light scattering and can be obtained simply on a common low-cost fluorescence meter. When the wavelength of Rayleigh scattering is located at or close to the molecular absorption band, RRS is produced. For species that aggregate, enhancements in RRS of several orders of magnitude can be observed at wavelengths characteristic of these species (20). Owing to the various advantages of the RRS technique, such as simple performance, low cost, short response time and high sensitivity, it has been extensively applied to the determination of ions (21,22), drugs (23,24), noxious gases (25,26), proteins (27,28), nucleic acids (29,30), etc. In this study, the interaction among methyl orange (MO), QNS and Pd(II) was investigated and trace amounts of QNS were determined. The experiments showed that QNS could react with MO to form a ion-pair complex (31), which further formed six-membered rings chelate complexes with Pd(II), leading to the enhancement of RRS and a new RRS spectrum appeared with the maximum peak at ~ 360 nm. The detection limit (3σ) ranged from 6.8 to 12.6 ng/ mL. Under optimum conditions, determination of QNS at trace amounts in pharmaceutical preparations and human urine samples was successfully performed. The results obtained were in good agreement with those obtained using the official and reference methods.
Experimental
spectra were all 400 V. A UV/Vis 8500 spectrophotometer (Tianmei, Shanghai, China) was used to record the absorption spectra. A pHS-3C meter (Shanghai Scientific Instruments Company, China) was used to measure the pH of the solution. The stock solution of quinolone antibiotics (Huamei Biology Technology Co. Ltd.) was 200.0 μg/mL. The working solution was further diluted to 8.0 μg/mL with doubly distilled water. The stock solution of MO (Sanland Chemical Cc. Ltd) was 1.0 × 10 3 mol/L. The stock solutions were further diluted to 2.0 × 10 4 mol/L with doubly distilled water as the working solution. The stock solution of PdCl2 (Shanghai Reagent Factory, China) was 1.0 × 10 3 mol/L. It was prepared by dissolving 0.0887 g PdCl2 in 0.50 mL of concentrated hydrochloric acid and diluting to 100 mL with water. The stock solution was diluted to 2.0 × 10 4 mol/L as working solution with doubly distilled water. Britton–Robinson (BR) buffer solutions at different pH values (2.7–6.5) were prepared by mixing the mixed acid (composed of 0.04 mol/L H3PO4, HAc and H3BO3) with 0.2 mol/L NaOH in proportion. The buffer was used to control the acidity of the interacting system. All other reagents were of analytical reagent grade and were used without further purification. Doubly distilled water was used throughout. General procedure Into a 10.0 mL calibrated flask were added 1.00 mL of pH 3.8–4.2 BR solution, 1.00 mL of MO solution, an appropriate amount QNS solution and 1.50 mL PdCl2 solution in turn. The mixture was then diluted to the mark and mixed thoroughly. After 15 min, the RRS spectra of the system were recorded with synchronous scanning at λex = λem. Then, the scattering intensities (IRRS) for the complexes, and the scattering intensities (I0RRS) for the reagent blank were measured at their own maximum wavelengths, ΔIRRS = IRRS – I0RRS.
Results and discussion RRS spectra Figure 1(a) showed the RRS spectra of MOO–QNS–Pd(II) systems. It could be seen that the RRS intensities of QNS, Pd(II) and MO themselves were very weak within the range 220–800 nm. The binary systems of MOO–QNS, MO–Pd(II) and QNS–Pd(II) could only result in a small change in the RRS spectra. However, when they reacted further with Pd(II) to form ternary systems, remarkably enhanced RRS spectra were observed. The five different reaction systems had similar spectral characteristics because their maximum RRS peaks were all at 360 nm and the other smaller peaks were located at 610 nm. However, the intensities of reaction systems were different and the order of their intensities was CIP > PIP > NOR > LOM > SAR. Furthermore, the enhanced RRS intensities were directly proportional to the concentrations of QNS. Taking CIP as an example, the quantitative relationship between IRRS and the concentration of CIP is shown in Fig. 1(b). Hence, the RRS method could be used for the determination of trace amounts of QNS.
Apparatus and reagents
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A Hitachi F-2500 spectrofluorophotometer (Tokyo, Japan) was used to record the RRS and fluorescence spectra. The slit width was 5 nm in the RRS spectra and 10 nm in the fluorescence spectra. The photomultiplier tube (PMT) voltages of the above three
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Optimum experimental conditions Effect of pH. The effects of different buffer solutions on RRS intensity of the reaction systems were tested with BR buffer, HAc–NaAc, Na2HPO4–C6H8O7 and HCl–NaAc. The results showed
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Determination of quinolones by resonance Rayleigh scattering reaction acidity for the first two systems and pH 4.2 for last three systems. The appropriate amount was 1.0 mL. Effect of MO concentration. The effect of MO concentration on the RRS intensity of the reaction systems was investigated. The results showed that the RRS intensity was gradually enhanced with increasing MO concentrations. When the MO concentration was in the range of 1.0 × 10 5–4.0 × 10 5 mol/L, the RRS intensity reached a maximum and remained stable. If the amount of MO was not adequate, the reaction was incomplete, and the RRS intensities were low. If the amount of MO was excessive, the RRS intensities decreased slightly because of the formation of MO dimer by self-aggregation. Therefore, 2.0 × 10 5 mol/L was chosen as the most suitable MO concentration. Effect of Pd(II) concentration. According to the experimental results, when a small amount of Pd(II) existed, the reaction was incomplete, and the RRS intensity was low. The ΔIRRS of the reaction systems reached a maximum and remained constant in the range 2.0 × 10 5–5.0 × 10 5 mol/L. So the concentration of Pd(II) used was 3.0 × 10 5 mol/L.
Figure 1. RRS spectra. (a) The RRS spectra of MO–QNS–Pd(II) systems. (1–18) MO, Pd(II), LOM, PIP, SAR, NOR, CIP, MO–Pd(II), MO–LOM, MO–PIP, MO–SAR, MO–NOR, MO–CIP, LOM–Pd(II), PIP–Pd(II), SAR–Pd(II), NOR–Pd(II), CIP–Pd(II); (19) MO–SAR–Pd (II); (20) MO–LOM–Pd(II); (21) MO–NOR–Pd(II); (22) MO–PIP–Pd(II); (23) MO–CIP–Pd(II). 5 5 cMO: 2.0 × 10 mol/L, cPd(II): 3.0 × 10 mol/L, cQNS: 0.8 μg/mL, pH 3.8 (CIP, NOR), 5 5 pH 4.2 (LOM, PIP, SAR). cMO: 2.0 × 10 mol/L, cPd(II): 3.0 × 10 mol/L, cQNS: 0.8 μg/mL, pH 3.8 (CIP, NOR), pH 4.2 (LOM, PIP, SAR). (b) The relationship between RRS intensity 5 5 and the concentrations of CIP. cMO: 2.0 × 10 mol/L, cPd(II): 3.0 × 10 mol/L, cQNS (1 → 5): 0.4, 0.8, 1.2, 1.6, 2.0 μg/mL, pH 3.8.
that the sensitivity and stability of the systems were best in BR buffer. Therefore, BR buffer solution was chosen to control the pH of the solutions. Figure 2 showed the dependence of ΔIRRS on the pH of the solution. It was found that the optimum pH ranges were 3.3–4.4 for MO–CIP–Pd(II) and MO–NOR–Pd(II) systems, and 3.8–5.1 for MO–PIP–Pd(II), MO–LOM–Pd(II) and MO–SAR–Pd(II) systems. Therefore, pH 3.8 was chosen as
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Reaction time and the stability. Under optimum conditions, the reaction time and stability were studied by determining the RRS intensities of the reaction systems every 5 min for 2.5 h immediately after mixing thoroughly. The results showed that the RRS intensity reached a maximum after ~ 15 min and then remained stable for 2 h at room temperature (20–25°C). Thus, the assay was completed after 15 min.
Sensitivity and selectivity of the method Sensitivity of the RRS method. Under optimum conditions, the ΔIRRS values of the ternary systems were measured at their maximum scattering wavelengths. Calibration graphs of ΔIRRS against QNS concentration were constructed. The regression equation, linear range, correlation coefficient and detection limit are listed in Table 1. It can be seen from Table 1 that the method had high sensitivity. The detection limits (3σ) ranged from 6.8 to 12.6 ng/mL, and were lower than or comparable with that of other common analytical methods, as shown in Table 2 (take the CIP and NOR as examples). Therefore, the method was very suitable for determining trace amounts of QNS. Selectivity of the RRS method. Taking the MO–CIP–Pd(II) system as an example, the effects of foreign substances on the determination of CIP were investigated. As shown in Table 3, most metal ions, acid radical anions, amino acids, carbohydrate and some other organics and inorganics barely affect the determination of CIP within a permissible error of ± 5%. Whereas, Fe (III) and I interfered greatly with the determination of CIP. Fortunately, the amount of Fe(III) and I in pharmaceutical preparations and urine samples is extraordinary low and it did not affect the selectivity of this method. In addition, 50 times the amount of
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Figure 2. Effect of pH on the RRS intensity of MO–QNS–Pd(II) systems. (1) MO–CIP–Pd(II), (2) MO–NOR–Pd(II), (3) MO–PIP–Pd(II), (4) MO–LOM–Pd(II), (5) 5 5 MO–SAR–Pd(II). cQNS: 0.8 μg/mL; cPd(II): 3.0 × 10 mol/L; cMO: 2.0 × 10 mol/L.
Effect of ionic strength. The effects of ionic strength on the ΔIRRS of the reaction systems were studied by adding different concentrations of NaCl. The results showed that the ΔIRRS remained constant when the ionic strength was < 6.0 × 10 4 mol/L. ΔIRRS decreased gradually when the concentration of NaCl was > 6.0 × 10 4 mol/L. The possible reason for this phenomenon might be the effect of electrostatic shielding of charges, which would reduce the affinity of MO for QNS.
M. Qiao et al. Table 1. Correlation coefficient, linear ranges and detection limits for calibration graphs System MO–CIP–Pd(II) MO–PIP–Pd(II) MO–NOR–Pd(II) MO–LOM–Pd(II) MO–SAR–Pd(II)
Regression equation
Correlation coefficient (r)
ΔI = 51.3 + 846.0c ΔI = 15.9 + 832.4c ΔI = 21.3 + 793.5c ΔI = 14.0 + 727.3c ΔI = 6.9 + 352.3c
Linear range (μg/mL)
0.9999 0.9997 0.9987 0.9979 0.9977
Detection limit (ng/mL)
0.023–2.0 0.023–2.0 0.024–2.4 0.026–2.0 0.042–2.4
6.8 6.9 7.4 7.9 12.6
Table 2. Results of comparison for the analysis of QNS with different methods Detected substance CIP
NOF
Method
Reagent
Linearity (μg/mL) Detection limit (ng/mL)
SPE-UV F FIA-CL HPLC Voltammetric RRS RRS RRS K-SP SF FIA-CL HPLC EC RRS RRS RRS
– 4-Chloro-7-nitrobenzofurazan Peroxynitrous acid – Cetyltrimethylammonium bromide Erythrosine Pd(II) and Eosin Y Pd(II) and Methyl Orange KMnO4 4-Chloro-7-nitrobenzofurazan Peroxynitrous acid – Cd(OH)2 microcrystals Erythrosine Pd(II) and Eosin Y Pd(II) and Methyl Orange
0.05–0.3 0.023–0.5 0.03–3.3 0.09–2.0 0.033–6.6 0.0574–5.6 0–2.4 0.023–2.0 1.0–12.0 0.03–8.0 0.03–3.2 0.01–2.0 0.2–265.0 0.0596–4.4 0–2.4 0.024–2.4
12.0 7.0 14.9 30 16.5 17.0 9.4 6.8 30.0 9.2 19.8 5.0 156.5 18.0 12.8 7.4
Ref (7) (9) (10) (13) (32) (33) (18) This work (34) (9) (10) (13) (35) (33) (18) This study
EC, electrochemical; F, spectrofluorimetric; FIA-CL, flow-injection chemiluminescence; HPLC, high-performance liquid chromatography; K-SP, kinetic spectrophotometric; SPE-UV, solid-phase extraction UV spectrophotometric method.
Table 3. Effects of coexistent substances (cCIP: 1.6 μg/mL) Coexistent species Concentration (μg/mL) Relative error (%) Al2(SO4)3 Mg(AC)2 CuSO4 MnSO4 CaCl2 ZnSO4 NH4Cl KBr KH2PO4 NiSO4 CdCl2 HgCl2 SnCl2 Na3PO4 FeCl2 NH4Fe(SO4)2 Co(NO3)2 NH4F
16.0 16.0 32.0 80.0 160.0 32.0 160 80.0 160.0 100 40 60 20 200 20 6.0, 80.0a 150 40
1.9 2.0 4.4 4.5 2.3 4.9 1.5 5.0 2.8 4.4 3.8 4.2 3.9 2.4 3.4 4.8 2.0 4.2
Coexistent species Cystine Valine Glycine Phenylanine Sucrose Glucose KI Maltose Soluble starch Ascorbic acid Urea Uric acid Creatine Creatinine Hippuric acid Citric acid Oxalic acid EDTA
Concentration (μg/mL) Relative error (%) 60 16.0 16.0 13.2 160.0 160.0 2.0 128.0 160.0 160.0 160.0 20 100 120 200 160 140 800
3.8 3.5 4.5 5.4 5.0 3.6 4.8 1.5 2.6 2.6 2.3 3.6 2.8 3.0 2.5 2.8 4.0 3.5
a
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1.0 mL of 0.01 mol/L EDTA was added.
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Determination of quinolones by resonance Rayleigh scattering Fe(III) did not interfere after addition of 1.0 mL of 0.01 mol L EDTA. Therefore, the method showed good selectivity. Formation of the ternary complex The structures of the investigated QNS are given in Table 4. Because they had the same parent structure and only some substituted groups were different, we took CIP as an example to study the interaction of MO and Pd(II). The composition ratio of the ternary complex was determined by Job’s method (36,37) and the molar ratio method (38). The results showed that the ratio of MO : CIP : Pd(II) was 1 : 1 : 1. This finding was consistent with previous results (19) in which CIP and Pd(II) formed a 1 : 1 chelate. Scheme 1 shows the protonation/deprotonation equilibria of QNS with a piperazinyl substituent. As indicated, in general, two charged species exist in the system in the form of H2Z+, Z , or together with a zwitterionic species, designated as HZ. According to the literature (39,40), the pKa1 of QNS ranged from 5.42 to 5.94. So, QNS chiefly existed as H·QNS+ under the experimental conditions. The calculation indicated that the distribution ratio of H·CIP+ reached 99.1%. At this point, the structure of MO exists as an azo form (Scheme 2), which is more stable than the quinoid form, that is, MO chiefly existed as MO and its distribution ratio reached 71.6%. So H·CIP+ could react with MO to form a neutral ion-pair complex (31) via electrostatic forces. When some Pd(II) solution
was added to the ion-pair complex, the ketonic and carboxylic oxygen atoms coordinated with Pd(II) and formed a stable six-membered ring chelate. Finally, the chelate formed square–planar complexes with the chloride ligands. The structure of the ternary complex was speculated as shown in Scheme 3.
Effects of the formation of ternary complex on spectra characteristics The effect on IR spectra. Figure 3 showed the IR spectra of free CIP, MO and the ternary complex. Contrasting curve c and
Scheme 3. The structure of the ternary complex.
Table 4. Structures of quinolones studies and their pKa
QNS
Substituents at position R1
PIP CIP NOR LOM SAR
–C2H5 –C2H5 –C2H5
pKa
X
R6
Y
R8
R3`
N CH CH CH CH
– F F F F
N CH CH CH CH
– – – F –
– – – -CH3 –
pKa1
pKa2
5.42 5.86 5.94 – 5.62
8.18 8.24 8.22 – 8.18
Figure 3. IR spectra of CIP, MO and MO–CIP–Pd(II). (a) CIP, (b) MO–CIP–Pd(II), (c) MO.
Scheme 1. Protolytic equilibria of QNS.
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Scheme 2. The structure of MO.
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M. Qiao et al. curve b, the absorption band in the 1039 cm 1 region is attributed to -SO3Na, which shifted to 1035 cm 1, and the vibration intensity was decreased. It was shown that -NH+3 bound with SO3Na, that is, [H·CIP]+ could react with MO to form a neutral ion-pair complex. Comparing curve a with curve b, we observed that the absorption band in the region around 1700 cm 1 belonging to the free carboxylic acid disappeared (13), which indicated that the Pd(II) ion had reacted with the carboxylate oxygen. The absorption band in the 1625 cm 1 region attributed to the ketone group in the free ligand spectra was shifted to 1631 cm 1 in the palladium complexes, which was a good indication that this group was coordinated to the Pd(II) ion also (13). In curve b, two characteristic absorption bands around 1631 and 1400 cm 1 were observed, which could be attributed to asymmetric and symmetric ν(O–C–O), respectively (13). The frequency difference [Δν = νas(COO ) – νs(COO )] was 231 cm 1. It was reported that if Δν was > 200 cm 1, this group probably bound to metal ions in a monodentate way (41). The conclusion showed a good agreement with experimental results.
of QNS. However, the sensitivity of the method was not high enough. Taking CIP as an example, the effects of MO and Pd(II) on fluorescence spectra were investigated. As shown in Fig. 5, the fluorescence intensity of CIP was quenched in the presence of MO due to formation of the ion-pair complex, and the fluorescence intensity of CIP was also quenched in the presence of Pd (II) due to the formation of a six-membered ring chelate. However, the fluorescence intensity was quenched markedly in the presence of MO and Pd(II). It was interesting that a new spectrum appeared at 571 nm. The reason for this might be ascribed to the formation of a ternary complex. The effects of MO on fluorescence spectra of CIP were investigated when the concentration of CIP and Pd(II) were fixed. A gradual reduction in the emission intensity was observed (Fig. 6a) with increasing MO concentration, whereas the RRS intensity increased (Fig. 6b). The effects of Pd(II) on fluorescence spectra of CIP were similar to MO. This phenomenon revealed that the energy transferred between fluorescent and scattering.
The effect on absorption spectra. As shown in Fig. 4, Pd(II) had no absorption in the range 220–600 nm (curve 1). MO had a maximum absorption wavelength at 473 nm and a smaller absorption peak at 274 nm (curve 2). CIP had a maximum absorption wavelength at 276 nm (curve 3). Comparing curve 2 with curve 4, it was observed that the absorption of MO–CIP system (curve 4) was actually a superposition of curves 2 and 3. Thus, it could be seen that formation of ion-pair complex had no effect on the absorption spectra. Comparing curve 3 and curve 5, we could see that the maximum absorption wavelength of CIP shifted to 280 nm. The absorbance was reduced from 0.79 to 0.62. This indicated that CIP could interact with Pd(II) and form a complex. Contrasting curve 4 and curve 6, the absorption spectra of the ternary system were changed markedly: the maximum absorption wavelength at 276 nm shifted to 281 nm, whereas the maximum absorption wavelength at 476 nm shifts to 467 nm. The absorbance reduced greatly.
The effect on RRS spectra. It can be seen from Fig. 1(a) that the formation of the ternary complexes results in great enhancement of RRS and new RRS spectra appear. The possible reasons for RRS enhancement are described below.
The effect on fluorescence spectra. The five types of quinolone drugs had similar fluorescence spectra. Their maximum excitation wavelength (λex) and maximum emission wavelength (λem) were located in the range 277–283 nm and 442–459 nm, respectively. Their fluorescence intensities (F) were different and reduced in the order CIP > PIP > NOR > LOM > SAR. So fluorescence spectrometry (FL) could be used for the determination
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Figure 4. Absorption spectra. (1) Pd(II), (2) MO, (3) CIP, (4) MO–CIP, (5) CIP–Pd(II), (6) 5 5 MO–CIP–Pd(II) cMO: 2.0 × 10 mol/L, cPd(II): 3.0 × 10 mol/L, cCIP: 8.0 μg/mL, pH 3.8.
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Figure 5. Fluorescence spectra. cMO: 2.0 × 10 4.0 μg/mL, pH 3.8.
5
mol/L, cPd(II): 1.0 × 10
5
mol/L, cCIP:
Figure 6. Fluorescence quenching spectra and RRS spectra. cCIP: 4.0 μg/mL, cPd(II): 5 6 6 5 5 1.0 × 10 mol/L, cMO(1 → 6): 4.0 × 10 , 8.0 × 10 , 1.2 × 10 , 1.6 × 10 , 5 5 2.0 × 10 , 2.4 × 10 mol/L.
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Determination of quinolones by resonance Rayleigh scattering
Figure 7. Comparison between fluorescence spectrum and RRS spectrum of MO–CIP–Pd(II) system. (1) Fluorescence spectrum, (2) RRS spectrum.
Fluorescence-scattering resonance energy transfer. From Fig. 7 (a), we can see that the RRS spectrum partially overlaps with the fluorescence spectrum. This means that the RRS spectrum and fluorescence spectrum are at the same frequency, and can resonate and exchange energy with each other. So the IRRS increased on reduction of F (Fig. 7b). The ternary complexes result in great enhancement of RRS and the appearance of new RRS spectra because of the marked quenching of fluorescence intensity. Enhancement of hydrophobicity. MO , H·CIP+ and Pd(II) are all hydrophilic and can easily dissolve in aqueous solution so that they cannot form a hydrophobic interface with water. When H·CIP+ reacts with MO to form a neutral ion-pair complex, the hydrophobicity of the complex increases to some extent. But the increase is still small because of the existence of a carboxyl group. However, when the ion-pair complex further reacts with Pd(II) and forms a ternary complex, the carboxyl is coordinated by the Pd(II) ion and their hydrophobicity is increased significantly so that a hydrophobic liquid–solid interface appeared. The formation of the hydrophobic interface is favorable to the enhancement of RRS signal. Enlargement of molecular volume. According to the Rayleigh scattering formula, an increase in the volume of the scattering molecule is advantageous to the enhancement of scattering intensity. If the molecular volume is difficult to estimate, the formula can be simplified as I = kI0Mc (42), that is, when the incident light intensity (I0), the concentration of the solution (c) and (k) are constant, the scattering intensity (I) is proportional to the molecular weight of the particle. The molecular weight of QNS is of 303.3–385.36. Taking CIP as an example, when it reacts with MO and Pd(II) to form a ternary complex, the molecular weight is changed from 331.35 to 813.0. Hence, enlargement of the molecular volume is another reason for the enhancement of scattering intensity. Affected by all the above factors, the intensity of RRS is greatly enhanced.
Analytical application Determination of QNS in pharmaceutical preparation Ten CIP hydrochloride tablets (0.25 g/tablet) were weighed and powdered in a pestle and mortar. A mass of powder equivalent to one tablet was weighed and dissolved into 100.0 mL water and impurities removed by filtering. Then, 1.00 mL of filtrate was pipetted into a 250 mL volumetric flask and diluted to volume with doubly distilled water. Suitable amounts of the solution were measured according to the general procedure described above. The results obtained by our method and the official method (43) are listed in Table 5. Five LOM hydrochloride capsules (0.10 g/capsule) were dissolved in 100.0 mL water and impurities removed by filtering. Then, 1.00 mL of filtrate was pipetted into a 250 mL volumetric flask and made up to volume with doubly distilled water. Suitable amounts of the solution were measured using the RRS method. The results are listed in Table 5 and compared with those obtained using the official method (44). One milliliter of NOR eye drops were pipetted into a 500 mL volumetric flask and made up to volume using doubly distilled water. Suitable amounts of the solution were measured using the RRS method. The accuracy was tested by comparison with the standard addition method. The results are listed in Table 6. It can be seen from Table 5 that results agreed with those obtained using the official method. Table 6 shows that the RRS method had good accuracy and repeatability. The satisfactory result indicated that the proposed method could be used to determine QNS in pharmaceutical formulations. This would provide a simple and new method for quantitative tests of QNS.
Determination of CIP in human urine In this experiment, five urine samples were collected before drug administration as blank samples. Five healthy volunteers received a single oral dose of 250 mg CIP after an overnight fast.
Table 5. Results for the determination of CIP in tablet and LOM in capsules by the RRS method and the comparison of it with official method Sample
Label claim (g/piece)
CIP tables
0.25
LOM capsules
0.10
RRS Official RRS Official
Found (g/piece) 0.242 0.248 0.099 0.102
0.255 0.250 0.105 0.104
0.242 0.255 0.100 0.103
0.248 0.250 0.256 0.238 0.104 0.105 0.098 0.102
Copyright © 2014 John Wiley & Sons, Ltd.
Average (g/piece) 0.247 0.249 0.103 0.102
RSD (%) 2.2 2.9 2.8 0.2
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Method
M. Qiao et al. Table 6. Results for the determination of NOR in eye drops Sample
Label claim (mg/mL)
NOR in eye drops
Found (mg/mL)
3.00
2.97
Added (mg/mL) 1.00 2.00 4.00 6.00
Found total amount (mg/mL) 3.85 4.72 6.77 9.10
4.08 4.86 6.94 8.84
3.92 4.75 6.87 8.93
4.05 5.07 6.72 8.79
4.01 5.01 6.97 9.04
Average (mg/mL)
RSD (%)
3.98 4.88 6.85 8.94
Recovery (%)
2.3 3.2 1.6 1.5
101.0 95.5 97.0 99.5
Table 7. Results for the determination of CIP in human urine samples (n = 5) Samplea
No.1 No.2 No.3
Times (h) 0–6 6–12 12–24
Found (μg/mL) RRS 138.2 27.6 9.2
SP
b
139.8 28.6 10.6
Added (μg/mL) 40.0 40.0 40.0
Total amount (μg/mL) RRS
SP
178.7 67.3 48.7
180.6 67.8 49.9
RSD (%) RRS 2.2 2.0 1.8
Recovery (%)
SP 2.4 2.1 2.2
RRS
SP
101.3 99.3 98.8
102.0 98.0 98.2
a
No CIP were detected in any of the blank urine samples; spectrophotometry.
b
The volunteers were asked to drink sufficient and comparable amounts of water through all collection periods to ensure sufficient urine production. The urine samples of individuals were gathered within 0–24 h and the urinary volumes were recorded. In order to ensure the sample concentrations of the drug were within the linear range of determination, the urine samples were diluted and analyzed by the RRS method. The spectrophotometric method (45) (taking Eosin Y as a probe) was used as a reference method. The results are given in Table 7. The average recovery obtained with the RRS method was 98.8–101.3%, and the RSD was 1.8–2.2%. In addition, the test showed that there were 106.5 mg of the original drug in urine; the cumulative excretion rate reached 42.6%. The results were in agreement with those given in the literature (46). The results showed that both methods (RRS and spectrophotometry) yield values within the same range when tested using adequate statistical procedures. The method allowed the directly determination of QNS in urine and was suitable for routine analysis in clinical laboratories. This may provide guidance for studying the pharmacokinetics of CIP.
Conclusions We have successfully provided a new, rapid and facile RRS method for the determination of trace amounts of QNS with high sensitivity and selectivity. Under the optimized conditions, a linear correlation has been established between the RRS intensity and the concentration of QNS in certain ranges, with the detection limits (3σ) ranging from 6.8 to 12.6 ng/mL. The mechanism of the reactions were discussed in terms of fluorescence-scattering resonance energy transfer, hydrophobicity and molecular size. This method had been satisfactorily used for the determination of QNS in real samples. Acknowledgement
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This paper was supported by the financial supports from the National Natural Science Foundation of China (No. 21175015) and the Special Fund of Chongqing key Laboratory (CSTC) and
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the Fundamental Research Funds for the Central Universities (XDJK2013A022).
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