Lysosomes Are Sites of Fluoroquinolone Photosensitization in Human Skin Fibroblasts: A Microspectrofluorometric Approach*

The fluoroquinolone antibiotics are widely used despite their strong phototoxicity under solar UV irradiation. Although they are known as good photodynamic photosensitizers, other factors than production of activated oxygen species may play a role in the effectiveness of the phototoxic effect. Subcellular localization is one of the important parameters that may determine this strength. Using microspectrofluorometry, it is shown that norfloxacin, ofloxacin, lomefloxacin, ciproflaxin and BAYy3118 are readily incorporated into lysosomes of HS68 human skin fibroblasts although weak staining of the whole cytoplasm also occurs especially with norfloxacin. Consistent with their photoinstability in solutions, the fluoroquinolones under study are readily photobleached by UVA in the HS68 fibroblasts. The BAYy3118 derivative that has the fastest bleaching rate also shows the strongest phototoxicity toward HS68 fibroblasts. Photosensitization with these fluoroquinolones induces lysosomal membrane damage as shown by the increased rate of leakage of the lysosomal probe lucifer yellow as compared to that observed with untreated cells.     

Alteration of the endocytotic pathway by photosensitization with fluoroquinolones

The endocytotic pathway is profoundly altered by the UVA-induced photosensitization of HS 68 fibroblasts by the fluoroquinolone (FQ) antibiotics lomefloxacin, BAYy 3118, norfloxacin and ciprofloxacin, which preferentially localize in lysosomes. The endocytosis of low-density lipoproteins (LDL) loaded with two carbocyanine dyes compatible for effective Forster-type resonance energy transfer (FRET), namely 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) as the donor and 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as the acceptor, has been used as a model system. Binding of LDL to their cell surface receptors is impaired by irradiation with 10 J cm(-2) of UVA and/or treatment with 250 microM BAYy 3118 during 2 h. Perturbation of the plasma membrane by the FQ is revealed by the change in the rate of exchange of DiO from the LDL to the cell membrane as compared to untreated cells. The lysosomal degradation of LDL, demonstrated by the disappearance of FRET between DiO and DiI, is partly inhibited by the FQ. The actin filament network, involved in the fusion of mature endosomes with lysosomes, is readily destroyed upon photosensitization with the four FQ. However, actin depolymerization can be avoided by incubation of the cells with trans-epoxysuccinyl-1-leucylamido-(4-guanidino)butane, an inhibitor of lysosomal cathepsins prior to FQ photosensitization. All these data suggest that several components of the endocytotic pathway are impaired by photosensitization with these FQ.     

Photogenotoxicity of Skin Phototumorigenic Fluoroquinolone Antibiotics Detected Using the Comet Assay

Abstract— The fluoroquinolone(FQ) antibiotics photosensitize human skin to solar UV radiation and are reported to photosensitize tumor formation in mouse skin. As tumor initiation will not occur without genotoxic insult, we examined the potential of ciprofloxacin, lomefloxacin, fle-roxacin, BAYy3118 (a recently developed monofluori-nated quinolone) and nalidixic acid to photosensitize DNA damage in V79 hamster fibroblasts in vitro. Cells were exposed to 37.5 kj/m₂ UVA (320-400 nm; glass filtered Sylvania psoralen + UVA (PUVA) tubes; calibrated Waldmann radiometer) at 4A_oC in the presence of FQ and immediately afterwards embedded in agarose, lysed and placed in an electrophoretic field at pH 12. Under these denaturing conditions, the presence of DNA single-strand breaks (SSB), alkali-labile sites (ALS) and double-strand breaks (DSB) can be visualized as DNA migrating away from the nucleus (characteristic "comet" appearance) after staining with a specific fluorochrome. At FQ concentrations that induced minimal loss of cell viability (neutral red uptake assay) the compounds tested induced comets with a rank order of BAYy3118 norfloxacin ciprofloxacin lomefloxacin fleroxacin nalidixic acid. If cells were incubated after treatment for 1 h at 37_oC, the comet score decreased, suggesting efficient removal of SSB/ALS/DSB. Addition of the DNA polymerase, inhibitor, aphidicolin, to cells treated with either ciprofloxacin alone or ciprofloxacin + UVA resulted in an accumulation of SSB due to the endo/exonuclease steps of excision repair. We have demonstrated that the FQ are photogenotoxic in mammalian cells but that FQ-pho-tosensitized SSB are efficiently repaired. Preliminary evidence that ciprofloxacin photosensitizes the formation of DNA lesions warranting excision repair may indicate production of more mutagenic lesions.     

Detection and Prevention of Ocular Phototoxicity of Ciprofloxacin and Other Fluoroquinolone Antibiotics†

Fluoroquinolone (FLQ) drugs are a potent family of antibiotics used to treat infections including ocular infections. To determine if these antibiotics may be phototoxic to the eye, we exposed human lens epithelial cells to 0.125–1 mm FLQs (ciprofloxacin [Cipro], lomefloxacin [Lome], norfloxacin [Nor] and ofloxacin [Ofl]), the precursor quinolone nalidixic acid (Nalid) and UVA radiation (2.5 J cm⁻²). Based on fluorescence confocal microscopy, FLQs are diffused throughout the cytoplasm and preferentially located in the lysosomes of lens epithelial cells. Neither FLQ exposure alone nor UVA exposure alone reduced cell viability. However, with exposure to UVA radiation the FLQs studied (Cipro, Nor, Lome and Ofl) induced a phototoxic reaction that included necrosis, apoptosis, loss of cell viability as measured by MTS, and membrane damage as determined by the lactate dehydrogenase assay. Both Nalid and all FLQs studied (Cipro, Nor, Lome and Ofl) photopolymerized the lens protein α-crystallin. Phototoxic damage to lens epithelial cells and/or α-crystallin will lead to a loss of transparency of the human lens. However, if precautions are taken to filter all UV radiation from the eye while taking these antibiotics, eye damage may be prevented.     

Flow-Injection Chemiluminescence Determination of Fluoroquinolones

ABSTRACT

A new flow-injection CL method was developed for the determination of fluoroquinolones including ofloxacin, norfloxacin, ciprofloxacin and lomefloxacin in pharmaceutical preparations, based on the chemiluminescence reaction of sulphite with cerium(IV) sensitized by these compounds. The linear ranges are 0.04 to 4.0 μg ml^?1 for ofloxacin and 0.4 to 40.0 μg ml^?1 for norfloxacin, ciprofloxacin and lomefloxacin, respectively. The detection limits are 0.016 μg ml^?1 for ofloxacin and 0.16 μg ml ^?1 for norfloxacin, ciprofloxacin and lomefloxacin, respectively. The relative standard deviations (RSD) are 2.1 to 2.6% (n=10) for these fluoroquinolones. The analytical procedure has been applied to the determination of the fluoroquinolones in pharmaceutical commercial formulations. The results are in agreement with those obtained by the official methods.     

Photohaptenic Properties of Fluoroquinolones

Abstract— Although quinolone antibacterial agents have both phototoxicity and photoallergenicity, the latter's potency has been poorly investigated compared with the former's. Some of the photoallergic chemicals serve as photohap-tens, which lead to T-cell-mediated immune reactions after photobinding to protein by UVA radiation. We examined the photohaptenic potential of fluoroquinolones, including lomefloxacin (LFLX), ciplofloxacin, norfloxacin, ofloxacin, levofloxacin, fleroxacin, enoxacin and spar-floxacin (SPFX). The absorption spectra of the quinolo-nes were altered by UVA irradiation, with an exception of SPFX that seems to be photostable toward UVA. Bovine serum albumin and murine epidermal cells were coupled with these fluoroquinolones other than SPFX by exposure to UVA. Subcutaneous inoculation of fluoro-quinolone-photomodified epidermal cells induced and elicited a delayed-type hypersensitivity reaction in mice. However, epidermal cells incubated with LFLX without UVA exposure also induced and elicited a significant hypersensitivity reaction to a lesser degree than LFLX-pho-tomodified epidermal cells. Furthermore, there was cross-reactivity between LFLX-photomodified epidermal cells and simply LFLX-incubated cells. This suggests that cells can be weakly modified with LFLX even in the dark and that UVA irradiation promotes this modification. Our study demonstrated that fluoroquinolones have photohaptenic properties to which their photoallergenicity is probably ascribed.     

IS RHODAMINE 123 A PHOTOSENSITIZER?

Because of conflicting reports on the photoxicity of rhodamine 123 (Rh 123), we have undertaken a study of Rh 123 photosensitization in several in vitro systems. First, Rh 123 is not a photodynamic agent and does not react with singlet oxygen. Second, when bound to cytochrome c (Cyt c), Rh 123 photosensitizes ferro Cyt c but not ferri Cyt c degradation by an oxygen-independent process. When delivered to skin fibroblasts where it specifically stains mitochondria, Rh 123 photosensitizes membrane damage. These results are consistent with the hypothesis that Rh 123 is a phototoxic stain. The lack of photosensitivity of Rh 123-stained mitochondria in some cell lines might therefore be due to specific structural features.     

Photogenotoxicity of fluoroquinolones in Chinese hamster V79 cells: dependency on active topoisomerase II

The Chinese hamster V79 lung cell in vitro micronucleus assay was adapted to detect and quantify phototoxicity and photogenotoxicity of fluoroquinolones. Using this assay, the quinolones were ranked in terms of decreasing phototoxicity: clinafloxacin > lomefloxacin, sparfloxacin > trovafloxacin, nalidixic acid, ofloxacin, ciprofloxacin > enoxacin, norfloxacin. This rank order agrees well with published studies utilizing various other phototoxicity models and establishes this approach as a fast and sensitive way to characterize the phototoxic potential of quinolones. Nearly complete inhibition of phototoxicity was observed if the cells were pretreated for as little as 1 min with 10-20 mM sodium azide prior to the addition of quinolone. An identical azide effect was seen in unirradiated quinolone- and etoposide-treated cells. These findings are consistent with a model in which sodium azide renders DNA topoisomerase II catalytically inactive. In this state, topoisomerase II cannot initiate DNA strand cleavage and the DNA/topoisomerase complex becomes insensitive to quinolones and other topoisomerase II inhibitors. The fact that azide reduces both UV-dependent and UV-independent toxicity and clastogenicity strongly suggests a common mechanism of toxicity dependent on the formation of topoisomerase-induced DNA double-strand breaks.     

UV light induced photodegradation of liposome encapsulated fluoroquinolones: An MS study

Fluoroquinolone antibacterial agents are among the drugs most commonly causing phototoxic side effects. The phototoxicity may be originated in formation of reactive oxygen species upon ultraviolet exposure. Researches aiming the liposomal encapsulation of fluoroquinolones, expecting an increase in their therapeutic index, enhance the importance of studies on physicochemical properties and photostability of liposomal preparations. We studied the photodegradation of ciprofloxacin, ofloxacin and lomefloxacin by mass spectrometry upon various doses of UV irradiation. Lomefloxacin, the most phototoxic fluoroquinolone among them, was encapsulated into small unilamellar and multilamellar liposomes. Impact of vesicle structure and lipid composition – the presence of unsaturated fatty acid containing dioleoyl-phosphatidylcholine in dipalmitoyl-phosphatidylcholine liposomes – on the lomefloxacin photolysis was investigated; the structure of the main photoproducts was identified by mass spectrometry. It was found that the presence and type of lipids influence the ways of photodegradation process.     

A flow injection chemiluminescence method for the determination of fluoroquinolone derivative using the reaction of luminol and hydrogen peroxide catalyzed by gold nanoparticles

Based on the enhancement of chemiluminescence (CL) of luminol-hydrogen peroxide-gold nanoparticles system by fluoroquinolones (FQs), a novel and rapid CL method is reported for the determination of FQs derivatives. Under the optimum conditions, the CL intensity is proportional to the concentration of FQs derivative in solution. The corresponding linear regression equations are established over the range of 0.08-1.28 μg/mL for norfloxacin, 0.013-1.32 μg/mL for ciprofloxacin, 0.014-1.4 μg/mL for lomefloxacin, 0.029-1.46 μg/mL for fleroxacin, 0.02-1.0 μg/mL for ofloxacin and 0.01-1.44 μg/mL for levofloxacin, respectively. The limits of detection (S/N = 3) are 3.2, 9.5, 7.0, 9.0, 8.0, and 8.0 ng/mL with the relative standard deviation (n = 11) 4.3, 1.5, 1.9, 1.3, 1.6 and 2.1% for norfloxacin, ciprofloxacin, lomefloxacin, fleroxacin, ofloxacin and levofloxacin, respectively. This proposed method has been applied to detect FQs derivatives in human urine successfully.     

Determination of eight fluoroquinolones in groundwater samples with ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction prior to high-performance liquid chromatography and fluorescence detection

An ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction (US-IL-DLLME) procedure was developed for the extraction of eight fluoroquinolones (marbofloxacin, norfloxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, oxolinic acid and nalidixic acid) in groundwater, using high-performance liquid chromatography with fluorescence detection (HPLC-FD). The ultrasound-assisted process was applied to accelerate the formation of the fine cloudy solution using a small volume of disperser solvent (0.4 mL of methanol), which increased the extraction efficiency and reduced the equilibrium time.     

Interaction between fluoroquinolones and bovine serum albumin studied by affinity capillary electrophoresis.

The interactions between eight fluoroquinolone antibiotics (ciprofloxacin, enoxacin, fleroxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, pefloxacin) and bovine serum albumin (BSA) were studied by affinity capillary electrophoresis (ACE). The binding constants were estimated by the change of migration times of the analytes through the change of concentration of BSA in the buffer solution. The yield binding constants were between 3.19 x 10(4) and 1.21 x 10(5) M(-1). These were related with the structures of fluoroquinolones, and agreed with the results obtained by other techniques. The obtained binding constants may help us in gaining some insights on possible drug/protein interactions and in early evaluation of the drugs' pharmacokinetic profiles during drug discovery.     

Induced and photoinduced DNA damage by quinolones: ciprofloxacin, ofloxacin and nalidixic acid determined by comet assay

Quinolones are degraded by light with loss of their antimicrobial activity, generating active species or radicals. Evidence exists that some fluoroquinolones (lomefloxacin, fleroxacin and enoxacin) induce damage to the cellular membrane and DNA cleavage by photosensitization. In this study, the genotoxic potential of the quinolones ofloxacin, nalidixic acid and ciprofloxacin (three antimicrobials frequently used in therapy) was evaluated upon irradiation with UV light by using the comet assay on cells of the Jurkat line. The results demonstrate that there are significant differences between the control groups (positive control with 50 microM H2O2, negative controls without drug and with and without irradiation) and the groups of irradiated quinolones (ofloxacin 2.76 x 10(-5) M, nalidixic acid 2.15 x 10(-4) M and ciprofloxacin 2.01 x 10(-5) M), indicating that, at the dose of irradiation employed (necessary to produce 50% photodegradation), the photodecomposition of the quinolones enhanced DNA damage. The unirradiated drugs also exhibited genotoxicity significantly different from that of the negative control.     

Extension of multi-residue methodology to include the determination of quinolones in food.

The multi-residue procedure for basic drugs described elsewhere by this laboratory has been evaluated for quinolone and fluoroquinolone antibiotics. The fluoroquinolones are a relatively new class of drug and an increasing number of licensed products containing these compounds are becoming available for use in animal husbandry. This, along with the possibility of the development of antibiotic resistant human pathogens, make it an important class of drug for which methodology is required for the monitoring of residues in food. Validation data are presented for a range of compounds including the quinolones; oxolinic acid and nalidixic acid, and the fluoroquinolones; flumequine, ciprofloxacin, danofloxacin, enoxacin, enrofloxacin, lomefloxacin, marbofloxacin, norfloxacin, ofloxacin and sarafloxacin. Foods for which the method was validated included poultry muscle (chicken and turkey), egg, chicken liver, honey, cattle muscle and pig muscle. This application of the multi-residue procedure further demonstrates the importance and wide-ranging usefulness of the technique.     

Simultaneous determination of fluoroquinolone and tetracycline antibacterials in sewage sludge using ultrasonic-assisted extraction and HPLC-MS/MS

A reliable method was proposed for the simultaneous determination of five fluoroquinolones (FQs) and two tetracyclines (TCs) in sewage sludge using ultrasonic-assisted extraction (USE) followed by SPE cleanup and high-performance liquid chromatography-mass spectrometry (HPLC-MS)/MS analysis with electrospray ionisation (ESI) in a positive mode. The USE conditions (e.g. extraction solvent, pH, and extraction cycles) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) parameters were optimised. Quantification was performed by internal standard calibration in multiple reaction monitoring mode. Recoveries of the antibacterials ranged from 41 to 123%, with relative standard deviations within 17%. The sample-based limits of quantification were 10–63?ng?g^?1 dry weight (dw) for FQs (ciprofloxacin, enrofloxacin, lomefloxacin, norfloxacin, and ofloxacin) and 250–500?ng?g^?1 dw for TCs (tetracycline and oxytetracycline). The method was applied to determine the antibacterials in sewage sludge and sediment samples were collected from the Pearl River Delta, China. Ciprofloxacin, norfloxacin, and ofloxacin were frequently detected, ranging from 1052 to 17740?ng?g^?1 dw in dewatered sludge samples, 585–3545?ng?g^?1 dw in untreated solids, and 98–258?ng?g^?1 dw in an urban stream sediment sample, respectively. Lomefloxacin and enrofloxacin were also occasionally detected.     

Evaluation of phototoxic and photoallergic potentials of 13 compounds by different in vitro and in vivo methods

Phototoxic side effects of pharmaceutical and cosmetic products are of increasing concern for patients, dermatologists and the chemical industry. Moreover, the need of new chemicals and drugs puts pressure on pre-clinical test methods for side effects, especially interactive adverse-effects with UV-light. So, the predictive potential of different established test methods, which are used regularly in our departments in order to detect the phototoxic potential of chemicals, were analyzed. Namely the fibroblast 3T3 test, the photo hen's egg test, a guinea pig test for measuring acute photoreactions, and a modified Local Lymph Node Assay, the Integrated Model for the Differentiation of Skin Reactions. Various agents with different photoreactive potential were tested: quinolones like Bay y 3118, ciprofloxacin, enoxacin, lomefloxacin, moxifloxacin, ofloxacin, sparfloxacin, as well as promethazine, chlorpromazine, 8-methoxypsoralen and olaquindox serving as control. Special emphasis was taken to evaluate the capability of the employed test procedures to predict phototoxic side effects in patients. Following our results, both in vitro assays were useful tools to detect photoirritancy while the photoallergic potentials of tested compounds were exclusively detected by an in vivo assay. As long as no in vitro model for photoallergy is available, the UV-IMDS should be considered to evaluate photoallergic properties of a supposed photoreactive agent.     

Fluoroquinolone Antimicrobials: Singlet Oxygen, Superoxide and Phototoxicity

The fluoroquinolone antibacterial agents possess photo-sensitizing properties that lead to phototoxic responses in both human and animal subjects. The phototoxicity order reported in humans is: fleroxacin > lomefloxacin, pefloxacin > ciprofloxacin ? enoxacin, norfloxacin and ofloxacin. Studies both in vivo and in vitro have related this phototoxicity to the generation of reactive oxygen species including hydrogen peroxide and the hydroxyl radical. We determined the quantum yields of singlet oxygen generation (φ_Δ,) by detection of the singlet oxygen (¹O₂) luminescence at 1270 tun for several fluoroquinolones, naphthyridines and other structurally related compounds. All the fluoroquinolones examined have low φ_Δ values ranging from 0.06 to 0.09 in phosphate buffer at pD 7.5. We also determined the ¹O₂ quenching constants for these compounds and their values were on the order of 10⁶M^?1 s¹, except for lomefloxacin whose rate constant was 1.8 × 10⁷M^?1 s^?1. The φ_Δ values were significantly decreased in a solvent of lower polarity such as methanol (0.007 ≤φ_Δ≤ 0.02). The production of ¹O₂ by these antibiotics did not correlate with the order reported for their phototoxicity. We also measured the photogeneration (λ > 300 nm) of superoxide by these antibacterials in dimethylsulfoxide using electron paramagnetic resonance and the spin trap 5,5-dimethyl-l-pyrroiine N-oxide. Although there is not a one-to-one correspondence between the relative rates of superoxide generation and the phototoxicity ranking of the fluoroquinolones, the more phototoxic compounds tended to produce superoxide at a faster rate. Nevertheless, the magnitudes of the observed differences do not appear sufficient to explain the range of fluoroquinolone phototoxicity potencies in human and animal subjects in general and the high activity of fleroxacin and lomefloxacin in particular. For these latter drugs the photoinduced loss of the F₈ atom as fluoride and the concomitant generation of a highly reactive carbene at C-8 provide a more plausible mechanism for their potent phototoxic and photocarcinogenic properties.     

Analysis of fluoroquinolone-mediated photosensitization of 2'-deoxyguanosine, calf thymus and cellular DNA: determination of type-I, type-II and triplet-triplet energy transfer mechanism contribution

Fluoroquinolone (FQ) antibacterials are known to exhibit photosensitization properties leading to the formation of oxidative damage to DNA. In addition, photoexcited lomefloxacin (Lome) was recently shown to induce the formation of cyclobutane pyrimidine dimers via triplet-triplet energy transfer. The present study is aimed at gaining further insights into the photosensitization mechanisms of several FQ including enoxacin (Enox), Lome, norfloxacin (Norflo) and ofloxacin (Oflo). This was achieved by monitoring the formation of DNA base degradation products upon UVA-mediated photosensitization of 2'-deoxyguanosine, isolated and cellular DNA. Oflo and Norflo act mainly via a Type-II mechanism whereas Lome and, to a lesser extent, Enox behave more like Type-I photosensitizers. However, the extent of oxidative damage was found to be relatively low. In contrast, it was found that cyclobutane thymine dimers represent the major class of damage induced by Enox, Lome and Norflo within isolated and cellular DNA upon UVA irradiation. This striking observation confirms that FQ are able to promote efficient triplet energy transfer to DNA. The levels of photosensitized formation of strand breaks, alkali-labile sites and oxidative damage to cellular DNA, as measured by the comet assay, were confirmed to be rather low. Therefore, we propose that the phototoxic effects of FQ are mostly accounted for energy transfer mechanism rather than by Type-I or -II photosensitization processes.     

Core-shell magnetic porous organic polymer for magnetic solid-phase extraction of fluoroquinolone antibiotics in honey samples followed by high-performance liquid chromatography with fluorescence detection

By monomer-mediated in-situ growth synthesis strategy, with hydroquinone and 1,3,5-tris(4-aminophenyl)benzene as monomers, a core-shell magnetic porous organic polymer was synthesized through a simple azo reaction. Based on this, a magnetic solid-phase extraction–high-performance liquid chromatography–fluorescence detection method was proposed for the analysis of fluoroquinolones in a honey sample. With ofloxacin, ciprofloxacin, enrofloxacin, lomefloxacin, and difloxacin as target analytes, factors affecting the extraction efficiency had been optimized. The LODs were 1.5–5.4 ng/L (corresponding to 0.23–0.81 μg/kg in honey). The linear range was 0.005–20 μg/L for difloxacin, 0.01–20 μg/L for ofloxacin, ciprofloxacin and lomefloxacin, and 0.02–20 μg/L for enrofloxacin. The enrichment factor was 84.4–91.7-fold with a high extraction efficiency of 84.4–91.7%. The method was assessed by the analysis of target fluoroquinolones in honey samples, and the recoveries for the spiked samples were 79.3–95.8%. The results indicated that the established magnetic solid-phase extraction–high-performance liquid chromatography–fluorescence detection method is efficient for the analysis of trace fluoroquinolones in honey.     

Chemiluminescence determination of some fluoroquinolone derivatives in pharmaceutical formulations and biological fluids using [Ru(bipy)(3)(2+)]-Ce(IV) system

A new chemiluminescence (CL) method using flow injection has been described for the rapid and sensitive determination of three fluoroquinolone derivatives, namely ofloxacin, norfloxacin and ciprofloxacin hydrochloride. The method is based on the CL reaction of the studied fluoroquinolones with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bipy)(3)(2+)] and Ce(IV) in sulfuric acid medium. Under the optimum conditions, the CL intensity is proportional to the concentration of the drugs in solution over the range 0.05-7.0 mug ml(-1) for norfloxacin, 0.05-6.0 mug ml(-1) for ciprofloxacin hydrochloride and 0.003-0.7 mug ml(-1) for ofloxacin. The limits of detection (s/n=3) were 3.1x10(-8) M norfloxacin, 2.6x10(-8) M ciprofloxacin hydrochloride and 5.5x10(-9) M ofloxacin. The method was applied successfully to the determination of these compounds in dosage forms and biological fluids.