Investigation on Ruthenium ( II ) Bipyridine / AgIII Complexes Chemiluminescence System and Its Application for Sensitive Norfloxacin and Ofloxacin Detection

The reaction mechanism of Ag complex ([Ag(HIO6)2]) with ruthenium(II) bipyridine (Ru(bpy)3) in acid medium was investigated. A novel flow injection chemiluminescence (CL) analysis method was developed for the detection of norfloxacin (NFLX) and ofloxacin (OFLX) in commercial drug, milk and human urine samples based on Ag-ruthenium(II) bipyridine in acidic solution. Under optimal conditions, CL intensities were proportional to drug concentrations in the range of 1.6 × 10 to 8 × 10 g mL for NFLX and 1.0 × 10 to 4 × 10 g mL for OFLX. The detection limit was 1.0 × 10 g mL for NFLX and 0.8 × 10 g mL for OFLX. On the basis of the CL, UV and fluorescence spectra, possible reaction mechanisms were proposed.


Introduction
Norfloxacin (NFLX) and ofloxacin (OFLX) belong to the third generation of quinolone antibiotics with a broad spectrum of activity widely used in human and veterinary medicine.NFLX and OFLX inhibit the activity of DNA helicase and topoisomerase IV in digestive tract pathogenic bacteria, achieving rapid bactericidal effects.The abuse of quinolones in animal husbandry has led to serious food safety problems. 1It is, therefore, necessary to rapidly and sensitively detect fluoroquinolones in biological samples and foods.
Flow-injection chemiluminescence (CL) methods have received much attention for analytical purposes due to their advantages of low cost, excellent sensitivity and rapid analysis.Several CL methods within various reaction systems have been used for the analysis of NFLX and OFLX, including Ru(bpy) 3 2+ -Ce IV -H 2 SO 4 , 2 cerium(IV)sulfite-terbium(III), 3 hydrogen peroxide-sulfuric acid, 4 luminol-hydrogen peroxide-nano gold, 5,6 Ce IV -Na 2 SO 3 , 7 Ce IV -Na 2 S 2 O 4 8,9 and Ag III -H 2 SO 4 . 10,11The detection limit of these methods ranges from 1.1 × 10 −10 to 2.61 × 10 −8 g mL −1 .The dynamic linear range can be as low as 10 times, and as high as 400 times.
In our previous studies, 12,13 we have been focusing on the interactions between the bis(hydrogenperiodato) argentate(III) complex anion, [Ag(HIO 6 ) 2 ] 5-, and bioactive and medically important molecules in alkaline medium.Based on the Ag-luminol-NaOH CL system, a variety of sensitive detection methods for small biological molecules [14][15][16] and drug molecules 17,18 have been developed.However, the oxidation properties and applications of Ag III in acidic medium require further analysis.We found that the oxidizing ability of Ag III in acidic medium was stronger than that in alkaline medium.Ag III in acidic medium can not only oxidize fluoroquinolones, but can also oxidize water to oxygen, 11 and oxidize ruthenium(II) to ruthenium(III).As a result, the chemical reaction and luminescence mechanism of Ag III -Ru II -sulfuric acid system was studied in detail using UV, fluorescence and CL spectra.Due to fluoroquinolones sensitizing this system, a new method for the determination of NFLX and OFLX in pharmaceutical and biological samples based on the Ag III -Ru II -sulfuric acid system was proposed.ultrapure water was used throughout.The Ag III complex was synthesized according to previously described procedures. 14The concentration of stock solution was determined according to the molar absorptivity (ε = 1.26 × 10 4 mol −1 L cm −1 ) at 362 nm.Solutions were stored at room temperature and diluted to working concentrations in pure water.
Stock solutions of 1 mg L −1 OFLX and NLFX (Institute for the Control of Pharmaceutical and Biological Products) were prepared by dissolving 10 mg in 0.5 mL 0.1 mol L −1 hydrochloric acid and diluting to 10 mL with pure water.

Instrumentation
The flow-injection analysis (FIA) CL system (Xi'an Remax Electronic Science-Tech Co. Ltd., shown in Figure 1) consisted of two peristaltic pumps, a six-way injection valve and photomultiplier tube detector.
[Ag(HIO 6 ) 2 ] 5-and Ru (bpy) 3 2+ solution were delivered by pump P1.Solutions were mixed and produced a baseline signal.Sample solutions were injected in a six-way injection valve with a sample loop by P2.Sample solution, which was carried by the [Ag(HIO 6 ) 2 ] 5-stream,were mixed with acidic ruthenium(II) bipyridine solution to produce enhanced CL signals.CL was detected through a photomultiplier tube detector (PMT, operated at −600 V).
UV absorbance was assessed using TU-1901 UV-Vis spectrophotometry (Beijing Purkinje General Instrument Co. Ltd.).Fluorescence spectra were obtained using an F-7000 fluorescence spectrophotometer (Hitachi).CL spectral information was achieved using 20 narrow band interference filters (360-670 nm), which were inserted between the cuvette and photomultiplier tube.

Drug capsules
Ten capsules of the same batch number (drug labeled quantity 0.1 g per granules) were randomly selected and taken as the total mass.It was dissolved 1/10 of the total mass (equivalent to the mass of a single capsule) in a small volume of 0.1 mol L −1 hydrochloric acid and diluted the sample in water to the required concentration.

Milk
Milk samples (1 mL) purchased from a local market were diluted to 10 mL in water.From diluted samples, 4 mL was added to 1 mL of 20% trichloroacetic acid, and centrifuged at 14000 rpm for 10 min.Supernatants were collected and analyzed.

Urine samples
Urine was added to 0.2 g PbO 2 to remove uric acid, thiourea, and ascorbic acid.Samples were vortexed for 5 min and centrifuged at 4000 rpm for 10 min.Supernatants were collected, 1 mL of which was diluted to a final volume of 10 mL in distilled water for analysis.

Kinetics of the CL reaction
Investigation of the CL reaction kinetic curves was performed using the static CL analysis method.Briefly, in a 10-mL beaker, 0.2 mL of fluoroquinolones and 0.2 mL H 2 SO 4 (0.2 mol L −1 ) were mixed, and 0.

UV spectra
UV spectra for different systems (Figures 3 and 4) were examined to obtain further information regarding the CL mechanism of the [Ag(HIO 6 ) 2 ] 5− -H 2 SO 4 -fluoroquinolones-Ru(bpy) 3 2+ system.Figure 3 shows that Ru II displayed absorption bands at approximately 288 nm (curve a).Ag III possesses a characteristic absorption band at 357 nm (curve b).When Ag III was mixed with acid, the absorption peak of [Ag(HIO 6 ) 2 ] 5− almost disappeared (curve d).The curve c was the sum of curves a and b, which showed that Ru II was minimally oxidized by Ag III .When sulfuric acid was added to the system, peaks characteristic of Ru II and Ag III disappeared (curve e), indicating that Ru II is easily oxidized by Ag III in acid medium.
Figure 4 indicates that NFLX alone has a maximum absorption peak at a wavelength of approximately 277 nm (curve a).Following mixing of NFLX with Ag III , the [Ag(HIO 6 ) 2 ] 5− color slowly faded and the absorption peak of Ag III decreases gradually (curve c).However, when acid and NFLX were mixed with Ag III , the absorption peak and color of Ag III disappeared rapidly (curve d).Figures 3 and 4 display the oxidative ability of the Ag III complex is improved through the addition of acidic medium as a catalyst.

Fluorescence spectra
Fluorescence emission spectra of NFLX and OFLX were observed in Figures 5A and 5B.As shown in    the fluorescence peaks at 475 and 590 nm decreased rapidly (curve c). Figure 5B shows that the acidic OFLX produced fluorescence emission at 505 nm (curve a).When [Ag(HIO 6 ) 2 ] 5− was added into acidic OFLX, the fluorescence peak at 505 nm decreased gradually, but the fluorescence peak at approximately 430 nm gradually increased (curves b and c).This demonstrates that OFLX is oxidized to form a fluorescent product near 430 nm.

CL spectra
The mechanism of the CL reaction was investigated using the CL spectra (Figure 6).From Figure 6 (curves a and b) it can be seen that the maximal CL spectrum was located at about 610 nm, the characteristic wavelength of Ru(bpy) 3 2+ *. 19 In addition, relatively weak CL emission at 490 and 430 nm was also observed in the two CL systems.According to previous study, 11 the [Ag(HIO 6 ) 2 ] 5− -H 2 SO 4 system produces CL emission at 490 nm, which may have been caused by the excited state (O 2 ) 2 .CL emission at 440 nm (Figure 6, curve b) may have occurred due to OFLX intermediate product energy transfer, the fluorescence peak of which is 430 nm (Figure 5, curve c).

Possible CL reaction mechanism
To study whether Ru(bpy) 3 3+ can be directly reduced to Ru(bpy) 3 2+ * by fluoroquinolones, it was prepared Ru(bpy) 3 3+ using PbO 2 and observed the CL spectra of the Ru(bpy) 3 3+ -fluoroquinolones-H 2 SO 4 system. 20The CL intensity was too small to observe, indicating that the fluoroquinolones reactive intermediates should be reacted with Ru(bpy) 3 3+ rather than the actual original form of fluoroquinolones.
Both UV absorption and fluorescence spectra suggested that chemical reactions had occurred between [Ag(HIO 6 ) 2 ] 5− and Ru(bpy) 3 2+ in addition to [Ag(HIO 6 ) 2 ] 5− and fluoroquinolones in H 2 SO 4 medium.The mechanism of luminescence can be summarized as follows: ruthenium(II) is first oxidized to ruthenium(III) by Ag III ; then, ruthenium(III) is reduced to excited ruthenium(II) by the fluoroquinolones active intermediates, which are produced through the reaction of Ag III and fluoroquinolones; and finally, excited ruthenium(II) emits characteristic spectra at about 610 nm and meanwhile become ground state ruthenium(II).
Based on our experiments and previous studies, 21,22 taking OFLX as an example, a specific CL mechanism is described in Scheme 1.

Optimization of experimental conditions
Experimental conditions were optimized through orthogonal experiments using three factors and three levels.Ag III as the oxidant, H 2 SO 4 as the acid medium and Ru(bpy 1.0 × 10 −4 mol L −1 Ag III , 0.3 mol L −1 H 2 SO 4 for OFLX and 0.2 mol L −1 for NFLX.
The calibration graph of OFLX was linear in the range of 1 × 10 −9 -4 × 10 −6 g mL −1 .The equation of linear regression was I = 965.92× 10 6 C + 23.875 (r = 0.9991), and the detection limit was 0.8 × 10 −9 g mL −1 .Relative standard derivation for OFLX (n = 7) was 1.10%.Within 180 min, the 7 measurements of CL intensity had a variation of 1.65%.Both the detection limit and linear range of our assays were performed to a higher level than previous methods (Table 1).Particularly for the dynamic linear range, the present study displayed nearly 5000 times.Such a wide dynamic linear range is rare in CL analysis.

Sample analysis
The proposed method was applied to determine the content of NFLX and OFLX in capsules and tablets.The results and recoveries are shown in Table 2 along with the results obtained using the UV method, which were in excellent agreement.
To evaluate the validity of the proposed method for the determination of milk and urine sample, recovery studies were performed using matrix labeling working curves.The recovery values of milk for NFLX were in the range of 98.51-105.17%with RSDs of 3.17-3.58%.
Urine samples were taken from two healthy volunteers.NFLX and OFLX (200 mg) were taken orally by the subjects and assessed after 2 h.Results are presented in Table 3.The recovery values for NFLX and OFLX were in the range of 97.2-105.6%with RSDs of 0.35-3.58%.

Conclusions
This work demonstrates a new CL system composed of Ag III complexes as oxidant and acidic ruthenium(II) as a CL reagent.Fluoroquinolones drugs could dramatically enhance CL intensity in this system.Thus, accurate and sensitive detection of OFLX and NFLX in drug, milk and  human urine samples was achieved.An advantage of this method was that the dynamic linear range was up to 3-4 orders of magnitude and the detection limit was as low as 0.8 × 10 −9 g mL −1 .The possible CL enhance mechanism of the system was deeply researched.
The UV spectra, fluorescence spectra and CL spectra show that the Ag III complexes exhibit strong oxidation ability in acidic media, which not only increases the rate of drug oxidation, but also oxidizes water to oxygen and ruthenium(II) to ruthenium(III).It can be concluded that the Ag III complexes are similar to strong oxidants potassium permanganate, that is, they possess oxidation ability in both acidic and alkaline media, but display stronger oxidative ability in acidic media.
Our work broadens the range of applications of Ag III reagents and species of chemiluminescence systems.

Table 1 .
Comparison of various CL systems for the determination of fluoroquinolones

Table 3 .
Determination results of ofloxacin (OFLX) and norfloxacin (NFLX) in milk and urine sample