Metal Complexes of Quinolone Antibiotics and Their Applications: An Update

Quinolones are synthetic broad-spectrum antibiotics with good oral absorption and excellent bioavailability. Due to the chemical functions found on their nucleus (a carboxylic acid function at the 3-position, and in most cases a basic piperazinyl ring (or another N-heterocycle) at the 7-position, and a carbonyl oxygen atom at the 4-position) quinolones bind metal ions forming complexes in which they can act as bidentate, as unidentate and as bridging ligand, respectively. In the polymeric complexes in solid state, multiple modes of coordination are simultaneously possible. In strongly acidic conditions, quinolone molecules possessing a basic side nucleus are protonated and appear as cations in the ionic complexes. Interaction with metal ions has some important consequences for the solubility, pharmacokinetics and bioavailability of quinolones, and is also involved in the mechanism of action of these bactericidal agents. Many metal complexes with equal or enhanced antimicrobial activity compared to the parent quinolones were obtained. New strategies in the design of metal complexes of quinolones have led to compounds with anticancer activity. Analytical applications of complexation with metal ions were oriented toward two main directions: determination of quinolones based on complexation with metal ions or, reversely, determination of metal ions based on complexation with quinolones.


Introduction
The generic term "quinolone antibiotics" refers to a group of synthetic antibiotics with bactericidal effects, good oral absorption and excellent bioavailability [1,2]. Nalidixic acid (1-ethyl-1,4-dihydro-7-OPEN ACCESS methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid, Figure 1), the first compound of the series, was introduced in therapy in the 1960s [3]. The clinical use of nalidixic acid was limited by its narrow spectrum of activity. Several modifications were made on the basis nucleus in order to enlarge the antibacterial spectrum and to improve the pharmacokinetics properties, two of these considered as being major: introduction of a piperazine moiety or another N-heterocycles in the position 7 and introduction of a fluoride atom at the position 6. Thus, the new 4-quinolones, fluoroquinolones, have been discovered starting in the 1980s. Taking into account the chemical structure of the basis nucleus (Figure 2), the quinolone are classified in four groups (Table 1) [4][5][6].  Based on their antibacterial spectrum and their pharmacokinetic properties, the quinolones are classified in four generations [7][8][9] (Table 2). Table 2. Generations of quinolones based on their antibacterial spectrum and pharmacokinetic properties.
Older quinolones have greater activity against DNA-gyrase than against topoisomerase IV in Gram negative bacteria and greater activity against topoisomerase IV than against DNA-gyrase in Gram positive bacteria. Newer quinolones equally inhibit both enzymes [16][17][18].

Chemical Properties of Quinolones Related to Complexation Process
Most quinolone molecules are zwitterionic, based on the presence of a carboxylic acid function at the 3-position and a basic piperazinyl ring (or another N-heterocycle) at the 7-position. Both functions are weak and give a good solubility for the quinolones in acidic or basic media.
Protonation equilibria of quinolones have been studied in aqueous solution using potentiometry, 1 H-NMR spectrometry and UV spectrophotometry [19,20]. For a quinolone molecule with the general structure depicted in Figure 3, two proton-binding sites can be identified. In solution, such a molecule exists in four microscopic protonation forms, two of the microspecies being protonation isomers.  [20][21][22]). The microspeciation of drug molecules is used to depict the acid-base properties at the molecular level (macroconstants) and at the submolecular level (microconstants). The macroconstants quantify the overall basicity of the molecules. The values for pKa 1 , correlated with the acid function of carboxyl group, fall in the range 5.33-6.53, while the values for pKa 2 , correlated with the basic function of the piperazinic group, fall in the range 7.57-9. 33. Table 3 contains the protonation constant values for norfloxacin and ofloxacin, two representative quinolones.  [23] The microconstants describe the proton binding affinity of the individual functional groups and are used in calculating the concentrations of different protonation isomers depending on the pH. The quinolones exist mainly in the zwitterionic form between pH 3 and 11. The positively charged form QH 2 + is present in 99.9% at pH 1. At pH 7.4 all microspecies are present in commensurable concentrations. Quinolone microspeciation has been correlated with bioavailability of quinolone molecules, serum protein binding and antibacterial activity [20]. The microspeciation is also important in the synthesis of metal complexes, the quinolone molecules acting as ligand in the deprotonated form (Q − ) in basic conditions, and in the zwitterionic form (QH ± ) in neutral, slightly acidic or slightly basic medium. In strongly acidic medium, quinolones form ionic complexes in their cation form (QH 2 + ). Quinolones form metal complexes due to their capacity to bind metal ions. In their metal complexes, the quinolones can act as bidentate ligand, as unidentate ligand and as bridging ligand. Frequently, the quinolones are coordinated in a bidentate manner, through one of the oxygen atoms of deprotonated carboxylic group and the ring carbonyl oxygen atom [ Figure 4
The stability of chelates is greater in solvents with lower dielectric constant [26] and is pH dependent; the affinity of lomefloxacin for the Ca 2+ and Mg 2+ ions decreases in the order: anion>zwitterion>>cation [27].
Tables 4-6 present a selection of the chelates obtained in solid state with quinolone acting as bidentate ligand through the pyridone oxygen and one carboxylate oxygen, and the type of experiments carried out for investigating their biological activity. The tables include those chelates in which the quinolones are the only bidentate ligands; complexes with other bidentate co-ligands (e.g., 2, 2'-bipyridine, 1,10-phenantroline), and their biological activity are not discussed here.            antimicrobial activity [105] The first review regarding the interactions of metal ions with quinolone was published ten years ago and discussed selected crystal structures of quinolone-metal compounds, different physico-chemical methods of characterization, as well as some results of bioactivity test [21]. The structural characteristics of a part of fluoroquinolone complexes and their biological activity were reviwed four years ago [106]. A recent comprehensive review [107] presented the structures and the biological activity of complexes of some quinolones with Cu(II), Ni(II), Co(II) and Zn(II) and analysed the influence of the second ligand on biological activity.
In one report, norfloxacin acts as bidentate ligand through two carboxylate oxygen atoms ( Figure 6) in complexes with Co(II) and Fe(III) ions [108]. A quite rare coordination mode of quinolones occurs in a bidentate fashion via the piperazine nitrogen atoms. This coordination was reported in complexes of general formula [PtCl 2 (L)] (Figure 7) formed by ciprofloxacin, levofloxacin, ofloxacin, sparfloxacin, and gatifloxacin with Pt(II) [109], and could be explained through the basicity both of N4 nitrogen from piperazine ring and of N1 nitrogen, the last one evidenced in recent studies [110].

Chelates Introduced into the Polyoxometalates (POMs) Surface
Quinolone molecules are excellent multidentate ligands able to construct metal-organic polymers with medical applications, due to the higher electronic cloud density of oxygen and nitrogen atom [111]. Such hybrid organic-inorganic materials have been obtained by introducing a quinolone chelate into the surface of a polyoxometalate anion. The polyoxometalates (POMs) are known as anti-tumor, antiviral, and antibacterial inorganic medical agents, and the modifying of their surface with such compounds with biological activity is aimed to ameliorate their properties.
Generally, these complexes were obtained by hydrothermal reaction of a quinolone with a metal salt and a polyoxometalate (in the acidic form or as ammonium salt) with adjusting the pH.
One of the simplest compound of this series is ) were used as inorganic building blocks in compounds constructed from PW 12 or SiW 12 clusters and two M(Quin) 2 chelates. The PW 12 or SiW 12 clusters and quinolone molecule as chelating bidentate organic ligands coordinate the metal ions together (Figure 8). The binuclear metal clusters are connected to the POM clusters, bound as unidentate or as bridging bi-dentate inorganic ligands, forming a 1D chain architecture, as shown in Figure 9.

Metal Complexes with Quinolone Acting as Unidentate Ligand
The quinolones bearing a piperazinyl ring in the 7-position could form complexes where the terminal piperazinyl nitrogen (N4) is involved in the coordination to the metal ion. This coordination mode was reported for complexes with transition metals Ag(I), Au(III), and Ru(III). The structure proposed for the complex Ag 2 (Nf) 2 (NO 3 ) 2 [116] is presented in Figure 10.  Figure 11(b)] were obtained [117].     4 (DMSO) 2 ]·2H 2 O revealed that the cadmium ion is heptacoordinated; the coordination environment consists in two cinoxacinate ions acting as tridentate chelate and bridging ligands, one as bidentate chelate ligand, and one water molecule [33].
In polymeric complexes, different modes of coordination are simultaneously possible. In the case of two Fe(II) complexes, norfloxacin adopts different modes of coordination depending on the synthesis conditions. In the structure of Fe(H-Nf) 2 (SO 4 )⋅2H 2 O, Fe(II) is surrounded by two norfloxacinate anions bound as bidentate ligand coordinated through the pyridone oxygen and one carboxyl carboxylate oxygen and two norfloxacin molecules coordinated as unidentate ligand by two oxygen atoms from two different carboxylate [ Figure 14(a)]. In the other complex, Fe(Nf) 2 ⋅4H 2 O, two molecules are bound as bidentate ligand, and two as unidentate ligand coordinated through piperazine nitrogen [ Figure 14

Ionic Complexes
Based on the basic function of the N4 pyperazinyl atom, quinolones are protonated in acidic medium, forming ionic chlometalates, generally obtained by slow evaporation of an acidic solution of complex and metal salt. Most of these complexes were tested for their antimicrobial activity (see Subsection 4.3).
The chloroantimonates (III) obtained with nalidixium C 12 H 13 N 2 (nalidixium cation) and ciprofloxacinium ions have the general formulae (C 12

Pharmaceutical Aspects
Some chelates of quinolones with trivalent cations have shown an improved solubility compared to that of the free ligand, and this behaviour could be advantageous for pharmaceutical formulation. The hydrochlorides of the aluminium (III) complexes of ciprofloxacin and norfloxacin were reported [48,133]. Both complexes are more soluble than the antibiotics themselves. The complexes can be used for developing more dose-efficient formulations, such as compressed tablet dosage forms [48,134]. The pharmacodynamic properties of ciprofloxacin are not drastically affected upon complexation with aluminium. The complex [(HCl·Cf) 3 Al] showed a longer post-antibiotic effect (PAE) compared to that the free ciprofloxacin [135].
The solubility studies of a bismuth (III) complex of norfloxacin, [Bi(C 16 H 18 FN 3 O 3 ) 4 (H 2 O) 2 ] (BNC) in different pH buffers indicated that the solubility of the BNC was higher than that of norfloxacin until pH 6.5. Above this pH value, a significant decrease in the solubility of BNC was observed, while the solubility of norfloxacin did not change significantly. The increased solubility can be an advantage for the antibacterial activity of the bismuth complex [49].

Biopharmaceutical and Pharmacokinetic Implications
Reducing the oral bioavailability of quinolones in the presence of multivalent cations is the main consequence of the metal ions-quinolones interaction, and it was reported for the first time in 1985 [136]. A reduction in ciprofloxacin biavailability in healthy human subjects was observed at co-administration with ferrous salts and a combination of multi-vitamin and mineral preparation. In correlation with UV-Vis spectra features, the formation of a 1:3 ferric ion-ciprofloxain complex was proposed as the cause of the reduction in ciprofloxacin biovailability [137]. A strong correlation between the reduction in oral bioavailability of norfloxacin in the presence of divalent and trivalent cations and the magnitude of formation constants measured in vitro was established (Ca 2+ < Mg 2+ < Zn 2+ ~ Fe 2+ < Al 3+ ). A marked difference between the effect of Zn 2+ and Fe 2+ was observed in vivo, namely a greater reduction in norfloxacin absorption with co-administration of Fe 2+ . The oxidation of Fe 2+ to Fe 3+ in gastrointestinal tract was proposed as possible explanation [138].
Several mechanisms were proposed in order to explain the decreased biovailability of quinolone in the presence of metal ions. The first hypothesis was that the reduction of quinolone absorption is due to the formation of insoluble and unabsorbable chelates in the gastrointestinal tract [139][140][141]. On the contrary, in other studies it was observed that the solubility of lomefloxacin increases in the presence of Ca 2+ , Mg 2+ , Al 3+ şi Fe 3+ ions [142]. This means that the reduction of the gastric absorption of lomefloxacin at co-administration with these metal ions, are not caused by the precipitation, but by a decrease of the octanol-water partition cofficient. Only for Bi 3+ , solubility and thus absorption of lomefloxacin, decresed as a result of formation of species with low solubility [143]. The permeability through intestinal mucosa of fluoroquinolone alone and in the presence of metal ions was studied in vitro. The effect of Ca 2+ , Mg 2+ , Fe 2+ was tested with ciprofloxacin, while the effect of Al 3+ was tested with ciprofloxacin, norfloxacin and ofloxacin. The experimental data revealed that the fluoroquinolone-metal ion combinations resulted in a reduced intestinal permeability compared to that of the corresponding fluoroquinolone, leading to a reduction of fluoroquinolone bioavailability [144].

Mechanism of Action of Quinolones
The DNA-binding capacity of quinolone complexes was studied in relation with the mechanism of action of quinolones. Experimental data suggested an interaction of quinolone-Mg 2+ complex with DNA and gyrase and not a direct interaction of free quinolones with DNA, and a model for the ternary complex was proposed. In this model, Mg 2+ acts as a bridge between the phosphate groups of the nucleic acid and the carbonyl and carboxyl moieties of norfloxacin, with additional stabilization arising from stacking interactions between the condensed rings of the drug and DNA bases [145].
Interaction of an oligonucleotide duplex and ciprofloxacin in the absence and in the presence of Mg 2+ was studied and a model of the ternary Cf-Mg 2+ -duplex adduct orientation was proposed. Docking carried out on this model sustained the orientation of the CFX-Mg 2+ in the minor groove of DNA [146].
From the experimental results, it was concluded that the metal ion plays an intermediary role in the interaction between quinolone and DNA, and the metal complex of quinolone can interact with DNA by an intercalative binding model [155,156]. In vitro experiments demonstrated the hypothesis that, on the one hand, DNA gyrase cannot bind quinolones in the absence of DNA, and on the other hand, the quinolone-gyrase-DNA complex is formed in the presence of Mg 2+ .
Magnesium and related metal ions affect the stability and function of topoisomerases: they reduce the stability of protein thus increasing the structural flexibility required for the structural changes involved in catalytic cycle [157,158]. On the other side, the divalent metal ions (especially Mg 2+ ) might play a role in enzyme poisoning due to their ability to bind the topoisomerase II-directed drugs, including quinolones [158]. The coordination environment proposed for Mg 2+ bound to topoisomerase IV consists in two C3/C4 oxygen atoms from a quinolone molecule chelated and four water molecules. Two of these water molecules are involved in hydrogen bonds with serine side chain hydroxyl group and with serine glutamic acid side chain carboxyl group. It was suggested that the interaction between quinolone and topoisomerases is mediated by this water-metal ion "bridge" [159]. Mutations of one of both amino acid residues that disrupt the bridge function partially or total, and thus the proteinquinolone interaction, are the most common causes of quinolone resistance [160].

Antimicrobial Activity
The consequence of interaction with metal ions on the biological activity of quinolones was approached in the first instance as a negative phenomenon, and some evidences of reduction in the antimicrobial activity of quinolones in the presence of metal ions [161,162] support this assumption. Two possible mechanisms were proposed for explaining the reduction of ciprofloxacin activity by metal cations. First of these, especially valid for chelates with 1:1 stoichiometry, could be a decreased permeation of the antibiotic into bacterial cells, while the second one is the formation of an inactive chelate [25].
However, for many chelates of quinolones obtained in solid state, an equal or superior activity was observed compared to that of parent drugs. Selected results expressed as minimal inhibitory concentration (MIC, μg mL −1 ) or as the inhibition diameter zone (mm) are presented in Tables 7 and 8. Increased biological activity of metal chelates was explained by the overtone concept of cell permeability and chelation theory. Upon chelation, the polarity of a metal ion is reduced due to the partial sharing of positive charge with the donor groups of ligand and as a consequence of overlap with the ligand orbitals. Chelation increases the delocalization of π electrons over the whole chelate ring and thus increases the lipophilic nature of the central ion. This increased in lipophilicity enhances the passage of complex through the lipid membranes and the penetration in cells [163][164][165]. Table 7. Minimal inhibitory concentration (MIC, μg mL −1 ) of the drugs for some assayed bacteria.    11 10 In fact, many more factors should be considered for metal complexes with antimicrobial activity: (i) the nature of the metal ion; (ii) the nature of the ligands; (iii) the chelate effect; (iv) the total charge of the complex; (iv) the nature of the ion neutralizing the ionic complex; and (vi) the nuclearity of the metal center in the complex [28,29,[89][90][91]107]. A detailed comment of the effect of these factors on the biological activity of metal-quinolone complexes was made in a recent review [107].
The results obtained in some particular bacterial strains (Mycobacterium tuberculosis and Helycobacter pylori), which have not been included in Tables 7 and 8, are worth emphasizing distinctively. Fluoroquinolones have been used successfully in helping cure multidrug-resistant tuberculosis, and studies in mice suggest that they can be considered as first line drugs to shorten the duration of therapy [166]. The main drawback with these agents is the high level of resistance, mainly associated with mutation at gyrA or gyrB genes [167,168]. Metal coordination to quinolones can be used not only as strategy to enhance their activity, but also to overcome the drug resistance. The complex of Cu(II) with ciprofloxacin having general formula [Cu(Cf) 2 (BF 4 ) 2 ]·6H 2 O exhibited a significant enhancement in the antitubercular activity comparing to ciprofloxacin alone [169]. A series of Pd(II) and Pt(II) complexes with general formula [MCl 2 (L)] (where L = ciprofloxacin, levofloxacin, ofloxacin, sparfloxacin, and gatifloxacin) were evaluated against Mycobacterium tuberculosis virulent strain H37Rv. The Pd(II) and Pt(II) complexes with sparfloxacin and the Pt(II) complex with gatifloxacin were the most active within each series in inhibiting bacterial growth, while the least active complexes of the series were the Pd(II) complex with ciprofloxacin and the Pt(II) complex with ofloxacin. Complexes have not shown better antitubercular activity than free gatifloxacin, but their activity was good and, except the complex of Pd(II) with ciprofloxacin, all of them were more active than rifampicin [79]. The results are in agreement with the in vitro activities of the parent drugs against M. tuberculosis isolated: ciprofloxacin < or = ofloxacin < sparfloxacin < gatifloxacin [170].
Fluoroquinolones from new generations, like levofloxacin, moxifloxacin, gatifloxacin or sitafloxacin have demonstrated efficacy in Helicobacter pylori eradication, in third-line or second-line triple therapy, in combination with a proton pump inhibitor (PPI) and amoxicilin [171,172]. Bismuth-containing quadruple therapy (omeprazole, bismuth, metronidazole and tetracycline) is an alternative first choice treatment for H. pylori [173]. Good results were also obtained with quadruple therapy of bismuth subcytrate-moxifloxacine-tetracycline-lansoprazole (BMTL) with high eradication rate and relatively mild side effects [174]. Starting from these premises, a series of bismuth-fluoroquinolone complexes [Bi(Flq) 3 (H 2 O) 2 ] (Flq: norfloxacin, ofloxacin, ciprofloxacin, sparfloxacin, lomefloxacin, pefloxacin, gatifloxacin) were evaluated for their anti-H. pylori activity, and were found to be more potent against all strains of H. pylori used, comparing to the parent FLQs. Moreover, the synthesized complexes also showed high potency against some fluoroquinolone-resistant strains of H. pylori. [50].

Antifungal and Antiparasitic Activity
Altough quinolones themselves does not exhibit antifungal activity some of complexes generated by newer fluoroquinolones act not only as antimicrobial agents, but have also shown antifungal activity. Complexes with 1:1 stoichiometry of levofloxacin with Cr(III), Fe(III), Co(II), Ni(II), Cu(II), Th(IV), Mn(II), Zn(II) and UO2(II) have proved an antifungal effect higher than the free ligand against Candida albicans [82]. Complexes of gatifloxacin with Ni(II), Cu(II), Zn(II), Cd(II), Fe(III), Ca(II), Mg(II),Cr(III), Mn(II) and Co(II) having a stoichiometry 1:2 (metal: ligand) have excellent activity as compared to standard drug toward the fungi Trichophyton rubrum, Candida albicans and Fusarium solani [100].
The  2 ] displayed a considerable antiparasitic activity against Trypanosoma cruzi. The corresponding complexes of norfloxacin have a differentiated activity: the Mn(II) complex did not improve the anti-parasitic effect of the free norfloxacin, while the Co(II) complex displayed a 4-fold higher activity than norfloxacin ligand [53].
A new field of research was opened starting to the synthesis of organometallic ruthenium complexes of some quinolone antibacterial agents. The organometallic ruthenium complex of ofloxacin [(η 6 -p-cymene)RuCl(O,O-oflo)]·2.8H 2 O has a "piano-stool" structure with quinolone acting as bidentate ligand coordinated to the metal through the ring carbonyl and one of the carboxylic oxygen atoms [175]. The complex interacts with DNA and provokes DNA shrinkage. It is moderately active against Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Plasmodium falciparum.

Anticancer Activity
The anticancer activity of fluoroquinolones has been explored in the last years [176][177][178][179] based on their ability to block topoisomerase II, thus inhibiting the DNA repair activity. It is not surprising that numerous studies concerning the biological activity of quinolone metal complexes include their ability to interact with DNA, as a premise for anticancer activity (see Table 4).
Some  , along with the normal breast epithelial MCF-10A cell line, comparing to the parent drug, moxifloxacin. Both the parent ligand as well as its copper complex did not significantly inhibit the proliferation of non-tumorogenic MCF-10A breast epithelial cells. Moxifloxacin did not exhibit anti-proliferative effect against any of the breast cancer cell lines examined, instead, the Co(II) complexes showed differential anti-proliferative activity against the tested breast cancer cell lines [103].
The gold (III) complexes with general formula [AuCl 2 L]Cl (L = norfloxacin, levofloxacin, sparfloxacin) were tested against A20 (murine lymphoma), B16-F10 (murine melanoma) and K562 (human myeloid leukemia) tumor cell lines comparing to the normal cell lines L919 (murine lung fibroblasts) and MCR-5 (human lung fibroblasts). The free ligands did not showed significant activity in the tumor or normal cell lines, whereas the complexes are more active than the parent drugs, and they have with a similar cytotoxic activity [62].
Recent research has focused on increasing the antitumor activity of polyoxometalates (POMs) by introduction of medicine molecules into the POM surface [180], and such molecules could be quinolone chelates. The first compound obtained by modifying the surface of a POM with a quinolone chelate was the complex {[Co(PPA) 2 ]H 2 [SiW 12 O 40 ]}·HPPA·3H 2 O. The inhibitory effect against MCF-7 cells lines showed that the complex and pipemidic acid have shown high antitumor activity to MCF-7, whereas the parent compound SiW 12 exhibits no antitumor activity to MCF-7. Furthermore, the antitumor activity of complex was higher that that of its parent compounds, and this superiority could be explained from the synergism of POMs and Co-PPA [113]. = enrofloxacin; L 2 = pipemidic Acid; L 3 = norfloxacin; L 4 = enoxacin). The complexes 1, 3, and 4 exhibited a higher effect against SGC7901 lines comparing to the parent compound, while compound 2 showed no anti-SGC7901 activity [111].
The organometallic ruthenium complexes chlorido(η 6 -p-cymene)(nalidixicato-κ 2 O,O)ruthenium(II) and chlorido(η 6 -p-cymene)(cinoxacinato-κ 2 O,O)ruthenium(II) were investigated as anticancer agents in human A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon carcinoma) cells by means of the colorimetric MTT assay and compared to the tumor-inhibiting properties of the respective ligands. Even though the compounds were shown to be mostly non-cytotoxic to the various cell lines, the complexes and all the ligands are inactive in the three cell lines [181].

Determination of Quinolones Based on Complexation with Metal Ions
The capacity to form complexes with different metal ions has been applied in the analysis of quinolones in pharmaceutical formulations or in biological samples through spectrophotometric, spectroflurimetric and atomic absorption spectrometric methods. Most of the spectrophotometric methods developed for analysis of quinolones are based on the formation of yellow or orange-yellow chelates with Fe 3+ in acid medium. The structure of such a complex is depicted in Figure 16. Generally, these methods are simple, rapid, efficient and inexpensive. Ciprofloxacin, ofloxacin and norfloxacin have been determined colorimetrically in tablets based on their amber coloured complex with Fe(III) that exhibited a maximum at 370 nm [182]. The complex with Fe 3+ showing a maximum absorption of 435 nm allowed spectrophotometric determination of ciprofloxacin in tablets and in solution for infusion [183]. Based on the complexation with Fe 3+ , some flow injection (FI) spectrophotometric methods for determination of norfloxacin in drug formulations were developed. The coloured Fe(III) complexes absorb at 430 nm [184] or 440 nm [185]. Ofloxacin has been also determined by a flow-injection spectrophotometric method by measuring the absorbance of its complex with Fe 3+ at 420 nm. The method was applied for analysis of ofloxacin in pharmaceuticals and human urine [186]. Ciprofloxacin formed with Fe(III) a brown-red complex whose absorbance was monitored at 447 nm, and the developed method was used for determination of ciprofloxacin in drug formulations [187]. A sequential injection spectrophotometric method was developed for analysis of ciprofloxacin and norfloxacin by measuring the absorbance of the corresponding complexes at 447 nm and 430 nm, respectively [188].
Chelates with Fe(II) and Cu(II) with maximum absorptions placed below 400 nm were also applied in the spectrophotometric analysis of quinolones. Based on the yellow-coloured chelate with Fe(II) with absorbance at 358 nm, norfloxacin has been determined both in pure form and in tablet form [189]. Norfloxacin, ciprofloxacin and sparfloxacin have been determined in formulations and spiked biological fluids (plasma and urine) via their Cu(II) complexes [190].
Coloured ion-association complexes were applied in developing the new visible spectrophotometric methods for determination of quinolones. Ciprofloxacin and norfloxacin have been determined in pharmaceutical tablets via formation of a ternary complex with eosin and palladium (II) which showed an absorption maximum at 545 nm [191]. Ofloxacin generates with Al(III) and erythrosin an ion-association complex between {Al III (OFX)} cation and (ERY) anion. The ternary complex has an effective molar absorptivity at 555 nm, allowed spectrophotometrically determination of ofloxacin and other quinolone antibiotics (norfloxacin, enoxacin and levofloxacin) in pharmaceutical preparation [192]. Ion-association complexes formed with [Cr(NCS) 4 (NH 3 ) 2 ] -(Reineckate anion) displaying a maximum absorption at 524 nm were used for determination of ofloxacin [193] and norfloxacin [194].
A spectrophotometric method related to the interaction of quinolones with metal ions was developed based on the oxidation of quinolones with ammonium vanadate in sulphuric acid medium, followed by the development of a greenish blue colour measured at 766 nm, which has been attributed to vanadium(IV). The method was applied for determination of amifloxacin, ciprofloxacin, difloxacin, enoxacin, enrofloxacin, lomefloxacin, levofloxacin, norfloxacin, ofloxacin and pefloxacin in pharmaceutical dosage forms [195].
Modification of the fluorescent properties of quinolones in the presence of different metal ions has attracted the interest for studying the interaction of quinolones with antacids [196] and for development of spectrofluorimetric methods, applied in determination of quinolones in bulk, in biological fluids and in pharmaceutical formulations.
Determination of quinolones by spectrofluorimetric methods is based on: (i) the enhancement of quinolone fluorescence in the presence of metal ions (i.e., Al 3+ , Cu 2+ , Au 3+ etc.); (ii) fluorescence sensitization of Tb 3+ or Eu 3+ in the presence of quinolone or (iii) quenching the fluorescence of a Tb 3+ chelate after the addition of quinolone.
Interaction of a series of quinolones (sparfloxacin, oxolinic acid, flumequine and enrofloxacin) with Al 3+ was used to analyse them in pharmaceutical dosage forms or in biological fluids [197]. Norfloxacin has been also determined as its fluorescent complex with Al 3+ in serum [198] and in pharmaceutical preparations [199].
Formation of Y(III) fluorescent complexes underlying spectrofluorimetric methods for determination of norfloxacin in eye drops [200] and enrofloxacin in pharmaceutical formulations and its residue in milk [201].
The enhancement of luminescent properties of Tb(III) sorbates with ciprofloxacin and norfloxacin in zeolite was used for determination of these quinolones in biological fluids [202]. The enhancement effect of some quinolones on the fluorescence intensity of Tb(III)-sodium dodecylbenzenesulfonate system allowed the determination of enoxacin in pharmaceutical samples [203] and danofloxacin in milk [204]. Based on the sensitized fluorescence of Tb(III) enhanced by silver nanoparticles ciprofloxacin was dosed in pharmaceutical formulations [205], whereas pipemidic acid and lomefloxacin have been determined in pharmaceutical forms, urine and serum samples [206]. Europium (III)-sensitized fluorescence in the presence of quinolones was also applied for determination of quinolones ciprofloxacin, norfloxacin and gatifloxacin in pharmaceutical and serum samples [207] as for determination of ulifloxacin, the active metabolite of prulifloxacin in human serum and urine [208]. An optical sensor using Tb(III) and Eu(III) was constructed for analysis of norfloxacin and gatifloxacin in pharmaceutical and serum samples [209].
Quencing the fluorescence of an Eu(III)-β-diketone complex in micellar solution after the addition of pefloxacin underlying a time-resolved fluorimetric method for determination of pefloxacin in serum [210].
Apart from the main analytical applications in determination of quinolones in pharmaceutical forms and in biological samples, the fluorescent complexes were used also for other purposes. In this regard, fluorescence studies of Au(III)-norfloxacin system were carried out in order to study the association of Au 3+ ions with cationic, zwitterionic and anionic forms of the drug [211]. Cu(II)-ofloxacin interaction, studied by means of ofloxacin fluorescence quenching experiments in the presence of Cu(II), was evaluated for its environmental impact [212].
Forming the metal complexes was the basis of some indirect methods for analysis of quinolones using atomic absorption spectrometry (AAS). Flow injection-fast atomic absorption spectroscopy (FI-AAS) was applied for determination of norfloxacin based on the complexation reaction with Fe(III), via measuring the absorbance of Fe 3+ [213]. The formation of ion associated in the presence of cobalt sulphate was used for AAS determination of some fluoroquinolones in pharmaceutical dosage forms and biological fluids [195]. Ion-pair complexes formed with Reineckate anion allowed AAS determination of gatifloxacin, moxifloxacin and sparfloxacin in pharmaceutical formulations [214].

Determination of Metal Ions Based on Complexation with Quinolones
Spectrophotometric and spectroflurimetric methods were developed for determination of metal ions based on their complexation with quinolones. Formation of a coloured chelate with norfloxacin, which exhibits an absorption maximum at 377 nm, was used for development of a spectophotometric method for determination of trace amounts of Fe(III) [215].
Norfloxacin was used as reagent for determination of neodymium, holmium and erbium in mixed rare earth through a derivative spectrophotometric method, based on the enhancement of absorption at 575 nm for neodymium, 450 nm for holmium, and 523 for erbium, respectively [216].
The complex between europium(III) and gatifloxacin in a co-luminiscence system Eu 3+ -La 3+gatifloxacin-sodium dodecylbenzene sulfonate was used for the determination of trace amounts of Eu 3+ in rare earth samples [217]. Quencing fluorescence of a terbium chelates in the presence of Hg 2+ was used for development of a highly sensitive and specific detection method of trace Hg 2+ in trace Hg 2+ in biological samples (urine) and environmental water [218].

Quinolone Metal Complexes as Labels or Probes for Various Purposes
The luminescent properties of Tb(III) and Eu(III) chelates of some quinolones (nalidixic acid, oxolinic acid, pipemidic acid, pefloxacin, norfloxacin, ofloxacin, ciprofloxacin and lomefloxacin) were characterized and the obtained reagents were proposed as labels for immunofluorimetric assay [219]. Based on the enhancement of the fluorescence intensity of the enoxacin-Tb 3+ complex, an environmentally friendly probe for determination of DNA (both single-stranded and double-stranded) was developed [220].

Conclusions
The metal ion -quinolone complexation represents a research field of increasing progress, having in view the consequences and applications of this process. Pharmaceutical profiles of quinolones can be improved by obtaining complexes with enhanced solubility. On the other side, pharmacokinetic interactions can occur at oral co-administration of quinolones and metal ions from mineral supplements and antacids. At the target site of their action, a quinolone-gyrase-DNA complex is formed in the presence of Mg 2+ ions.
Many metal ion-quinolone complexes obtained in the solid state have shown various biological effects: antimicrobial activity (sometimes equal or better than that of the parent quinolone), anticancer activity, and, in some cases, antifungal and antiparasitic activity.
Complexation with metal ions was harnessed in the development of spectrophotometric, spectroflurimetric and atomic absorbtion spectrometric methods for the determination of quinolones in pharmaceutical preparations or in biological samples. Conversely, trace metal ions can be determined using quinolones as complexing agents. It must be noted that the progresses in the field of quinolone complexes and their applications parallel the development of the newer fluoroquinolones with enlarged biological activity.