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THIN-LAYER CHROMATOGRAPHYWITH DENSITOMETRY FOR THEDETERMINATION OF DIFLOXACINAND ITS PHOTODEGRADATIONPRODUCTS. KINETIC EVALUATION OFTHE DEGRADATION PROCESS ANDIDENTIFICATION OF PHOTOPRODUCTS BYMASS SPECTROMETRYUrszula Hubicka a , Barbara Żuromska-Witek a , Paweł Żmudzki b ,

Barbara Matwiej a & Jan Krzek aa Department of Inorganic and Analytical Chemistry , JagiellonianUniversity Medical College, Faculty of Pharmacy , Kraków , Polandb Department of Medicinal Chemistry , Jagiellonian UniversityMedical College, Faculty of Pharmacy , Kraków , PolandAccepted author version posted online: 09 Apr 2013.Publishedonline: 16 Jul 2013.

To cite this article: Urszula Hubicka , Barbara Żuromska-Witek , Paweł Żmudzki , Barbara Matwiej& Jan Krzek (2013) THIN-LAYER CHROMATOGRAPHY WITH DENSITOMETRY FOR THE DETERMINATIONOF DIFLOXACIN AND ITS PHOTODEGRADATION PRODUCTS. KINETIC EVALUATION OF THE DEGRADATIONPROCESS AND IDENTIFICATION OF PHOTOPRODUCTS BY MASS SPECTROMETRY, Journal of LiquidChromatography & Related Technologies, 36:17, 2431-2445

To link to this article: http://dx.doi.org/10.1080/10826076.2013.790768

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THIN-LAYER CHROMATOGRAPHY WITH DENSITOMETRY FORTHE DETERMINATION OF DIFLOXACIN AND ITSPHOTODEGRADATION PRODUCTS. KINETIC EVALUATION OF THEDEGRADATION PROCESS AND IDENTIFICATION OFPHOTOPRODUCTS BY MASS SPECTROMETRY

Urszula Hubicka,1 Barbara Zuromska-Witek,1 Paweł Zmudzki,2

Barbara Matwiej,1 and Jan Krzek1

1Department of Inorganic and Analytical Chemistry, Jagiellonian University MedicalCollege, Faculty of Pharmacy, Krakow, Poland2Department of Medicinal Chemistry, Jagiellonian University Medical College,Faculty of Pharmacy, Krakow, Poland

& TLC-densitometric method was developed for determination of difloxacin (DIF) in the presence itsphotodegradation products. Silica gel TLC F254 plates were used as the stationary phase and methylenechloride:methanol:2-propanol:ammonia 25% (4:4:5:2, v=v=v=v) as the mobile phase. The elaboratedmethod meets the acceptance criteria for specificity, linearity, sensitivity, accuracy, and precision. Thephotodegradation process of DIF followed kinetics of the first order reaction for the substrate. Potentialphotodegradation products of DIF identified by UPLC-MS=MS are: 7-(2-aminoethylamino)-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, 7-amino-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, 6-fluoro-1-(4-fuorophenyl)-7-(3-hydroxypiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, 6-fluoro-1-(4-fuorophenyl)-4-oxo-7-(piperazin-1-yl)-1,4--dihydroquinoline-3-carboxylic acid.

Keywords difloxacin, kinetic evaluation, mass spectrometry, method validation,photodegradation, TLC

INTRODUCTION

Difloxacin (DIF) is 6-fluoro-1-(4-fluorophenyl)-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic acid. It belongs to a group of fluoroquino-lones of antibacterial activity, including gram-positive and gram-negativebacteria, as well as mycoplasma and chlamydia, used in veterinary medicine.

Address correspondence to Jan Krzek, Department of Inorganic and Analytical Chemistry, MedicalCollege of Jagiellonian University, 9 Medyczna Str, 30-688 Krakow, Poland. E-mail: jankrzek@cm-uj.krakow.pl

Journal of Liquid Chromatography & Related Technologies, 36:2431–2445, 2013Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2013.790768

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Compared to other fluoroquinolones its molecule has two fluorine atoms;therefore, its biological activity is substantially greater.[1,2]

The main direction of the analytical studies of veterinary fluoroquino-lones, including difloxacin is the determination of residues in foodproducts, which seems to be reasonable, taking into account the abuse ofthese drugs in the treatment of both animals and humans.

The most commonly employed methods to determine fluoroquino-lones, their impurities, and metabolites are based on liquid chromato-graphy—with different detection techniques—mainly spectrophotometricin UV,[3,4] fluorescence,[4–9] electrochemical (ECD),[10] and chemilumi-nescence (CL).[11]

Liquid chromatography coupled with mass spectrometry (LC-MS)method is often used for the determination of residues of fluoroquinolonesin the fish meat, chicken meat and Royal Jelly.[9,12,13] Capillary electro-phoresis coupled to tandem mass spectrometry (CE-MS=MS) has been usedfor the identification and quantification of eight quinolones in milk.[14]

Two-dimensional TLC technique for the analysis of six fluoroquino-lones in biological fluids and edible animal tissues has been applied.[15]

TLC methods were also used for the determination of fluoroquinolonesin pharmaceutical products and in stability studies for the assessment oftheir photodegradation.[16–18]

There are publications describing transformations of DIF and other fluor-oquinolones during natural and technical processes of photolysis in aqueoussolution in the presence of humic substances and other additives, such as acet-one, hydrogen peroxide, phosphates, fluorides, and sulfates.[19–22]

The purpose of this work was to develop a new TLC-densitometricmethod for the determination of difloxacin and its photodegradation pro-ducts. In addition, the kinetic evaluation of photodegradation of DIF dur-ing exposure to UVA and identification of photoproducts by massspectrometry was also the aim of this study.

EXPERIMENTAL

Materials and Methods

ReagentsMethanol, methylene chloride, 25% ammonia, and 2-propanol of

analytical grade were purchased from Chempur (Piekary Slaskie, Poland)or POCH (Gliwice, Poland). HPLC grade methanol, acetonitrile, andformic acid (98%) were purchased from J.T. Baker. HPLC grade waterwas obtained from HLP 5 (HYDROLAB Poland) apparatus and was filteredthrough a 0.2-mm filter before use. Analytical grade copper (II) sulfate

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pentahydrate was manufactured by POCH (Gliwice, Poland, cat. numberACRS 42361).

Standard Solutions and SubstanceDifloxacin hydrochloride (DIF) – assay �98% (HPLC), cat no. D2819

Sigma-Aldrich (Germany) was used. Solutions of difloxacin in methanolwere prepared at concentrations from 0.72 mg mL�1 to 117.5mg mL�1.

Copper Salt SolutionsTo prepare the solution of copper ion at 0.1 M concentration the fol-

lowing salt was used: (1.2488 g) CuSO4 � 5H2O. The aforementioned givenweighed portion of salt was transferred to a 50-mL flask and filled withwater to required volume. Used directly for testing, the obtained solutionwas diluted with the same solvent to a final concentration of 0.025 M.

Preparation of Samples for Tests in SolutionsTwo milliliters of 98.0 mg mL�1 aqueous solution of DIF were measured

off on quartz dishes of 4 cm diameter, and 0.2 mL of water or of salt sol-ution with 0.025 M metal ion concentration was added. The dishes weresealed with a quartz lid. The solutions were exposed to UVA radiation fora maximum of 168 hr and 20mL were taken every 24 hr for analysis andspotted onto TLC plate. For each sample a dark control sample wasprepared, which was protected with aluminum foil before irradiation.

Irradiation ConditionsIrradiation was conducted in a climatic chamber KBF-ICH 240 APT.

lineTM; (Binder GmbH, Tuttlingen, Germany) at 20�C and 60% humidityusing UVA radiation (320–400 nm) with maximum emission at 365 nm. Theintensity of radiation was determined by means of radiometer typeVLX-3 W, Vilber Lourmat, with a sensor CX-365, to be each time of3.89 W=m2. The distance of the samples to radiation source was 13 cm.

TLC ConditionsIn the first stage of the study conditions for the separation of DIF and

its photodegradation products were found. TLC was performed on pre-coated TLC sheets of silica gel 60 with fluorescent indicator on aluminum(10� 10 cm, cut from 20� 20 cm, Art. 1.0554, Merck, Germany). Twentymicroliters of obtained solutions were applied using a Linomat V (Camag,Switzerland) sample applicator as 8 mm bands with distance of 10.0 mmfrom the plate bottom, 10 mm from the edge of the plate, and 8 mmbetween the edges of the bands. 20mL of nonirradiated DIF solution was

Determination of Difloxacin 2433

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applied on the plates at the same time. Chromatograms were developed toa distance of 95 mm with methylene chloride:methanol:2-propanol:ammo-nia 25% (4:4:5:2, v=v=v=v) as mobile phase immediately after its prep-aration in a glass chromatographic chamber (18� 9� 18 cm in size,Sigma–Aldrich, USA). The plate was dried at room temperature for30 min. Registration of spots on chromatograms was achieved by meansof a Camag TLC Scanner 3 with winCats 1.3.4 software at 294 nm in absor-bance mode. The slit dimensions were 6� 0.4 mm and scanning speed was20 mm=s. For identification, absorption spectra within the range200–400 nm were recorded. Identification of constituents was done by com-parison of position of peaks on chromatograms on the basis of retardationfactors (RF) and absorption spectra. Percentage ratio of constituent (%i)was calculated from quotient of peak area (Ai) to the sum of all peak areas(RA) on chromatograms according to the formula %i¼ (Ai=RA)100.

Method Validation

The method was validated for specificity, linearity, limit of detection,limit of determination, accuracy, precision, and robustness according toICH guidelines.[23,24]

SpecificitySpecificity of the method was assessed by comparing chromatograms of

the pure standard substance, chromatograms of DIF solution after UVAexposure for 96 hr and blank chromatogram. In obtained chromatograms,the retardation factor (RF) and resolution factor (Rs) values of the analyzedsubstances, peak areas, shape, and purity of the peaks were taken intoaccount. Peak purity was estimated by comparing the spectra at three pointsacross each peak, that is, peak start, peak apex, and peak end.

LinearityThe calibration plot for the method was constructed by analysis of seven

solutions containing different concentrations of DIF in the range59.0–117.5 mg mL�1. Solutions were applied to a plate in the amounts of20 mL. Further analytical procedure was as described in the TLC Conditionssection. Linearity was assessed in triplicate on the basis of the relationshipbetween peak areas and concentration, in micrograms per band. Linearand quadratic model were fitted to the calibration data. To evaluate thegoodness of the fit, residuals were found and determination coefficient(R2) was computed. Next, to determine whether the residuals have normaldistribution, the Shapiro-Wilk statistical test was used. Lack-of-fit test, basedon ANOVA comparison of within-group and between-group variance of

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regression residuals, was also applied to calibration data. ANOVA (Mandel’sfitting test) compared the linear and quadratic models, whereas AkaikeInformation Criterion (AIC) determined the complexity of models. Statisti-cal data were calculated and plotted using Statistica v. 10 and R-projectopen-source software.[25]

Limit of Detection (LOD) and Limit of Quantification (LOQ)For the determination of LOD and LOQ, calibration curve of low con-

centrations of DIF in the range 0.72–4.9 mg mL�1 was constructed. LODand LOQ were calculated on the basis of the slope (a) of the calibrationline and the standard error of the estimate (Se), using formulas LOD¼ 3.33.3 Se=a and LOQ¼ 10 Se=a.

AccuracyAccuracy for DIF was carried out for three concentration levels (mg=

band), 80% (1.25 mg), 100% (1.57mg), and 120% (1.88 mg) on the DIFsolutions after UV exposure to which DIF standard was added. Percent ofrecovery was calculated using the following formula:

R ¼ ðCd=CoÞ100

where Cd is the amount of DIF determined and Co is the amount of DIFadded. For each level, three determinations were completed.

PrecisionThe repeatability of the method was determined by analysis of six repli-

cates of standard solutions from individual weightings. The study was donefor three concentration levels of DIF: 75% (1.18mg=band); 100% (1.57 mg=band), and 150% (2.35mg=band). The intermediate precision was obtainedfor the same concentration of freshly prepared solutions by different ana-lysts who performed the analysis over a period of one week. The resultswere expressed as the relative standard deviation (RSD).

RobustnessUnder conditions of the developed method, comparison of results

obtained after changing of stationary phase from TLC to HPTLC (Merck,Germany) plates was performed. The impact of small changes in the con-tent of ammonia in the mobile phase (�5% from the initial composition)on the separation of DIF and its degradation product was also checked.

Determination of Difloxacin 2435

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Preparation of Samples for UPLC=MS=MS AnalysisExtraction of the spots of DIF and its photodegradation products was

made with TLC-MS Interface (Camag, Switzerland) into vials with meth-anol. Flow rate was 0.3 mL=min and extraction time 1 min. Prior toUPLC=MS analysis, extracts were concentrated to a volume of 0.5 mL at60�C.

UPLC=MS=MS Analysis10mL of each extract was injected onto an ACQUITY UPLC system. The

UPLC-MS=MS system consisted of a Waters ACQUITY UPLC (WatersCorporation, Milford, MA, USA) coupled to a Waters TQD mass spec-trometer (electrospray ionization mode ESI-tandem quadrupole). Chroma-tographic separations were carried out using the Acquity UPLC BEH(bridged ethyl hybrid) C18 column; 2.1� 100 mm, and 1.7mm particle size.The column was maintained at 40�C, and eluted under gradient from 95%of eluent A and 5% of eluent B to 100% of eluent B over 10.0 min, at a flowrate of 0.3 mL min�1. Eluent A: water=formic acid (0.1%, v=v); eluent B:acetonitrile=formic acid (0.1%, v=v). Chromatograms were made usingWaters ek PDA detector. Spectra were analyzed at 294 nm with 1.2 nm res-olution and sampling rate 20 points=s. MS detection settings of WatersTQD mass spectrometer were as follows: source temperature 150�C, deso-lvation temperature 350�C, desolvation gas flow rate 600 L h�1, cone gasflow 100 L h�1, capillary potential 3.00 kV, and cone potential 20 V.Nitrogen was used for both nebulizing and drying gas. The data wereobtained in a scan mode ranging from 50 to 1000 m=z in 0.5 s intervals. Dataacquisition software was MassLynx V 4.1 (Waters).

RESULT AND DISCUSSION

An important part of the stability studies of pharmaceutical substancesare photostability studies, which aim to test whether exposure to lightchanges the quality and quantity of the pharmaceutical product.[26,27] Tomeet these requirements, development of new analytical methods isrequired, which could be useful for the studies of appearing changes.

In the first stage of studies, conditions for the separation of DIF and itsphotodegradation products were established. The mobile phase foundexperimentally as methylene chloride:methanol:2-propanol:ammonia25% (4:4:5:2, v=v=v=v) enables good separation of analyzed substances.The obtained values of retardation factors were: for DIF RF�0.43, and forpeaks of degradation products 0.25, 0.32, and 0.39. Example chromato-gram is shown in Figure 1.

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Registration of peak areas for DIF and degradation products wascarried out in UV at 294 nm. The analytical wavelength chosen for the den-sitometric registration in UV corresponded to the absorption maximum foranalyzed substances (Figure 2).

FIGURE 2 Absorption spectra recorded under UV: DIF-difloxacin, 1,2,3-photodegradation products.

FIGURE 1 Densitogram of separation of DIF and its photodegradations product (1, 2, 3).

Determination of Difloxacin 2437

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Method Validation

For estimation of reliability of the developed method according to ICHrecommendations the following parameters were determined: specificity, lin-earity, limits of detection and quantitation, recovery, and robustness.[23,24]

The developed method is specific against studied components. Thereare no peaks with RF values on the recorded blank chromatogram wherethe studied components occur. Under established chromatographic con-ditions, spots on chromatogram for DIF and its degradation products wereobtained which, after densitometric registration, gives quite well resolvedsymmetrical peaks easy to identify and determine. Resolution between peakof DIF and peak 3 was Rs�0.8. Good correlation between UV spectra atthree points across each peak indicated that separated compounds werefree of any interference and pure.

Regression results are presented in Table 1. ANOVA comparisonbetween linear and quadratic models gives a statistic of 1.119 with p-valueof 0.3041, which is higher than 0.05 hence, the null hypothesis that linearand quadratic models are equal should not be rejected at 0.95 significancelevel. Determination coefficients for linear and quadratic models are high(>0.98). The distribution of the residuals can well be approximated with anormal distribution as it is shown by p-values of the normality tests(Shapiro-Wilk). The lack-of-fit test for both models indicates their goodfit. Computed AIC values for linear and quadratic model show no differ-ences. The a coefficient of the linear equation is not statistically significant(p-value> 0.05). Based on regression analysis, it was assumed that the cali-bration data fitted well to the linear model (Figure 3). Linearity range was1.18–2.35 mg=band.

TABLE 1 Calibration Data for Determination of DIF

Equation Coefficients

Calibration Curve:

Linear y¼ aþbx Quadratic y¼ aþbxþ cx2

a 1479.0 (p¼ 0.0525) �2180.0 (p¼ 0.5448)b 12916.0 (p< 0.001) 17277.0 (p< 0.001)c – �1236.0 (p¼ 0.3041)TestsR2 0.9825 0.9835AIC 339.1 339.8Shapiro-Wilk 0.9558 (p¼ 0.4363) 0.9293 (p¼ 0.1335)Lack of fit 1.2324 (p¼ 0.3454) 1.2455 (p¼ 0.3368)ANOVA between models (Mandel’s test) Linear vs quadratic 1.119 (p¼ 0.3041)

a-y-intercept.b-Slope of regression line.c-Quadratic term.

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Based on parameters of the calibration curve constructed at low con-centrations of DIF, the LOD and LOQ (mg per band) values were 0.01and 0.03, respectively. These low values indicated satisfactory sensitivity ofthe method.

Accuracy of the method expressed as % recovery at three concentrationlevels was from 101.06% to 110.94%. Good precision and intermediateprecision with % RSD less than 2.01% was observed. Detailed results were

FIGURE 3 Linear calibration curve and residual plot of DIF.

Determination of Difloxacin 2439

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presented in Table 2. In all the deliberately varied chromatographic con-ditions (content of ammonia in the mobile phase, change of stationaryphase), the retention parameters of DIF and its photodegradation productsremained unchanged.

Photodegradation of Difloxacin in Solutions

Chromatograms recorded for dark control samples showed only onepeak with RF� 0.43 corresponding to DIF. Whereas, in chromatogramsrecorded for test solutions with and without metal ions exposed to UVAradiation for 24 hr, 48 hr, 96 hr, 120 hr, and 168 hr three additional peaksof photodegradation products of DIF with retardation factors: 0.25, 0.32,and 0.39 were observed beside the peak of DIF (Figure 1). First changes giv-ing evidence of DIF photodegradation were recorded after 24 hr reaching27.92% for samples without copper ions and 5.36% for samples with copperions. Degradation increased during prolonged time of irradiation, reachingafter 168 hr 75.76% for samples without copper ions and 34.84% forsamples with copper ions.

Comparing photodegradation results for the solutions containingcopper ions, it can be stated that lower degradation occurs in them in com-parison with analogous solutions, which did not contain metal ions.

Absorption spectra registered directly from the chromatograms for thepeaks 2 and 3 are similar to DIF spectrum (kmax1� 294 nm and kmax2�330 nm) which suggests that the analyzed products may be similar in struc-ture to DIF (Figure 2). Absorption spectrum recorded for peak 1 has aslightly different shape with only one maximum at 294 nm (Figure 2).

TABLE 2 Validation of the Method

Parameter Difloxacin

RF 0.43� 0.03Limit of detection, (mg=band) 0.01Limit of quantitation, (mg=band) 0.03Linearity range, (mg=band) 1.18–2.35Precision level 75%: RSD¼ 1.96%

level 100%: RSD¼ 1.34%level 150%: RSD¼ 1.09%

Indirect precision level 75%: RSD¼ 2.01%level 100%: RSD¼ 1.48%level 150%: RSD¼ 1.54%

Recovery, (%) n¼ 3 level 80%: 101.06%level 100%: 110.94%level 120%: 101.11%

RSD – relative standard deviation.

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Kinetic Evaluation

Based on the chart of DIF concentration changes (ln c) intime-dependant manner (t), it was confirmed that photodegradation insolutions occurs according to the kinetics of first-order reaction(Figure 4). The calculated kinetic parameters k, t0,5, t0,1 confirm that DIFphotodegradation proceeds about 3.2-times slower in the presence of cop-per ions (Table 3).

Identification of Photodegradation Products

The identification of the products formed during the photodegrada-tion process of DIF was performed on a basis of molecular ions [MþH]þ

from UPLC=MS=MS analysis. The mass spectrum of spot with RF value 0.39indicated a molecular ion at m=z 376 amu which can be attributed to thefollowing compound 6-fluoro-1-(4-fuorophenyl)-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (DPP-3). The mass spectrum of spot

FIGURE 4 The ln c¼ f(t) graph of photodegradation of DIF.

TABLE 3 Kinetic Parameters of DIF Photodegradation in Solutions, With and Without the Presenceof Copper Ion

Sample Composition Rate Constant k � 10�3 (h�1) t0.5 (h) t0.1 (h) Correlation Coefficient

Without metal ion 8.4 82.5 12.5 0.9894Cu(II) 2.6 266.5 40.5 0.9684

Determination of Difloxacin 2441

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with RF value 0.32 indicated a molecular ion at m=z 386 amu which can beattributed to the following compound 6-fluoro-1-(4-fuorophenyl)-7-(3-hydroxypiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (DPP-2). How-ever, in the mass spectrum obtained for spot with RF value 0.25 two molecularions at m=z 317.25 amu and m=z 360.33 amu were observed, which can beassigned to the following chemical compounds: 7-(2-aminoethylamino)-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (DPP-1a)

TABLE 4 Photodegradation Products of DIF

Product Id RF (MþH)þ Proposed Structure

DPP-1a 0.25 360.33

DPP-1b 317.25

DPP-2 0.32 402.12

DPP-3 0.39 386.12

DIF 0.43 400.14

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and 7-amino-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-car-boxylic acid (DPP-1b). Difloxacin degradation products are shown inTable 4. The plausible photodegradation pathway of difloxacin under studiedconditions is shown in Figure 5.

CONCLUSIONS

The elaborated method complies with the acceptance criteria forquantitative methods and may be used for the determination of DIF inthe presence of its photodegradation products. The photodegradation

FIGURE 5 The plausible photodegradation pathway of difloxacin under studied conditions.

Determination of Difloxacin 2443

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process of DIF followed kinetics of the first order reaction for the substrate.Rate constants k and the times t0.1 and t0.5 confirm lower stability of DIF insolutions without copper ions than in their presence. Potential productsof photodegradation of DIF identified by UPLC-MS=MS are: 7-(2-aminoethylamino)-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4- dihydroquinoline-3-carboxylic acid, 7-amino-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, 6-fluoro-1-(4-fuorophenyl)-7-(3-hydroxypiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, and 6-fluoro-1-(4-fuorophenyl)-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylicacid.

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