Defluorinated sparfloxacin as a new photoproduct identified by liquid chromatography coupled with UV detection and tandem mass spectrometry

Article abstract of DOI:10.1128/aac.42.5.1151

Photodegradation of sparfloxacin was observed by means of high-pressure liquid chromatography with UV detection and liquid chromatography coupled with UV detection and tandem mass spectrometry (LC-MS/MS). Three products were detected. Comparison with an independently synthesized derivative of sparfloxacin revealed the structure of one product which is believed to be 8- desfluorosparfloxacin. The second product is likely to be formed by the splitting off of a fluorine and a cyclopropyl ring. Thus, photodefluorination of quinolone antibacterial agents is found and proved for the first time by LC-MS/MS.

Full text of DOI:10.1128/aac.42.5.1151

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1998, p. 1151–1159
0066-4804/98/$04.00ϩ0
Copyright © 1998, American Society for Microbiology
Vol. 42, No. 5
Defluorinated Sparfloxacin as a New Photoproduct Identified
by Liquid Chromatography Coupled with UV Detection
and Tandem Mass Spectrometry
1
2
2
MICHAEL ENGLER, GUIDO RUSING, FRITZ SORGEL, AND ULRIKE HOLZGRABE1*
¨
¨
Pharmazeutisches Institut der Universita¨t Bonn, D-53115 Bonn,1 and Institute for Biomedical and
Pharmaceutical Research, D-90562 Nu¨rnberg-Heroldsberg,2 Germany
Received 24 June 1997/Returned for modification 20 December 1997/Accepted 10 February 1998
Photodegradation of sparfloxacin was observed by means of high-pressure liquid chromatography with UV
detection and liquid chromatography coupled with UV detection and tandem mass spectrometry (LC-MS/MS).
Three products were detected. Comparison with an independently synthesized derivative of sparfloxacin
revealed the structure of one product which is believed to be 8-desfluorosparfloxacin. The second product is
likely to be formed by the splitting off of a fluorine and a cyclopropyl ring. Thus, photodefluorination of
quinolone antibacterial agents is found and proved for the first time by LC-MS/MS.
RP-18 (7 ␮m by 125 mm); loop, 20 ␮l; mobile phase, acetonitrile-formic acid
Phototoxicity is one of the major adverse effects of modern
(0.2% in water; 50:50), isocratic; flow rate, 1 ml/min. Running of the detector in
fluoroquinolone antibacterial agents (9, 12, 17). Two alterna-
the auto-spectrum mode gave UV spectra every second.
LC-MS/MS. LC-MS/MS experiments were performed on a Perkin-Elmer
tive models (5) have been discussed as being the reason for
phototoxicity: first, the formation of stable toxic photoproducts
leading to skin reactions (15), and second, the formation of
singlet oxygen (16), which nonspecifically injures the body. In
1975, Detzer and Huber (1) first isolated dimeric photoprod-
ucts of nalidixic acid, a prototype quinolone antibacterial
agent. Many papers concerning the photolability of modern
fluoroquinolones appeared, and these mostly described the
loss of antibiotic activity during the course of irradiation (5–8,
13). It was hypothesized that a high degree of fluorination may
result in a low photostability and, in line with this, in the
formation of various photoproducts, which might cause ad-
verse effects. However, little is known about the structures of
these photodegradation products.
According to the hypothesis that low stability is connected
with a high degree of fluorination, we have chosen sparfloxacin
with a fluorine substituent at the 6 and the 8 positions as a
representative of the highly fluorinated gyrase inhibitors of the
newest class of drugs. The aim of the present study is to gain
more insight into the process of photodegradation. Therefore,
sparfloxacin has been irradiated in aqueous solution, and the
structures of the photoproducts that were obtained were elu-
cidated by means of liquid chromatography coupled with UV
detection (photodiode array detector) and tandem mass spec-
trometry (LC-MS/MS). Additionally, an analog compound
which is missing a fluorine atom at position 8 has been synthe-
sized and spectroscopically characterized.
Sciex API III Plus biomolecular mass analyzer equipped with an IonSpray in-
terface. Adjustments were as follows: orifice 60 volts; split, 5/1; collision energy,
25 V for product ion scan; collision gas thickness, 280 ϫ 1013 atoms/cm2; neb-
ulizer pressure, 50 lb/in2; curtain gas flow, 0.6 liters/min. Chromatographic con-
ditions were as follows: loop, 200 ␮l; other conditions, see above.
Sample preparation and irradiation procedure. Two milligrams of sparfloxa-
cin was dissolved in 100 ␮l of 0.1 M NaOH, and this solution was diluted with 900
␮l of water. This solution was irradiated for 8 h in a quartz cuvette placed at a
distance of 1 cm from a high-pressure mercury lamp (Philips HPK 125 W with
solidex glass filter; ␭ ϭ 248.2 to 578.0 nm; energy ϭ 1.63 to 60.89 W at different
wavelengths). For observation of the degradation process, a sample of 20 ␮l was
taken every hour and was diluted with water to a final concentration of 20 ␮g/ml
for HPLC with UV detection and 40 ␮g/ml for LC-MS/MS.
LC-MS/MS experiments. First, a Q1 scan was recorded, i.e., masses from m/z
50 to m/z 1,000 were registered over a certain period of time (the recording is
comparable to a chromatogram). The contour plot of the Q1 scan and the
extracted spectra revealed the m/z values and retention times of the chromato-
graphically separated compounds. For each compound, a new run (product ion
scan of the pseudomolecular ion) was performed, giving the fragmentation pat-
tern of each compound.
Synthesis of the reference substance, 8-amino-1-cyclopropyl-1,4-dihydro-7-
(2,6-dimethyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid, compound 10.
The reference substance, compound 10, has been synthesized by the methods
described in the literature (3, 11) (Fig. 1). 1H nuclear magnetic resonance
(NMR) spectra were recorded on a Varian EM 360A spectrometer (60 MHz;
tetramethylsilane was used as the internal standard) or a Varian XL 300 spec-
trometer (299,956 MHz). Melting points were determined (with a Gallenkamp
melting point apparatus) in capillary tubes and are uncorrected. Reagents were
purchased from common commercial suppliers and were used as received. All
solvents have been distilled and dried by appropriate methods. Organic solutions
were dried over anhydrous magnesium sulfate and were concentrated with an
IKA rotary evaporator at low pressure. Infrared (IR) spectra were measured in
KBr or Nujol on a Perkin-Elmer PE-298 instrument.
2,4-Dichloro-5-fluoro-3-nitrobenzoic acid, compound 1. A solution of 30 g
(144 mmol) of 2,4-dichloro-5-fluorobenzoic acid (3) in 360 ml of concentrated
sulfuric acid was heated at 70°C. Sixty milliliters of fuming nitric acid was added
dropwise over a period of 3 h. After the addition of the acid, the reaction mixture
was stirred for 2 h at 70°C and at room temperature overnight. The mixture was
poured into ice and the white precipitate was filtered, washed with water, and
dried over P2O5 to yield 31.5 g (86%) of compound 1; melting point, 183°C; 1H
NMR (60 MHz, methanol-d4) (ppm) ␦ 8.0 (d, 1 H, 9 Hz); IR (cmϪ1) 3000, 1720,
1560, 1240, 1120.
2,4-Dichloro-5-fluoro-3-nitrobenzoyl chloride, compound 2. A mixture of 21 g
(83 mmol) of compound 1 and 70 ml of thionylchloride was refluxed for 1 h. The
excess thionylchloride was removed in vacuo, and the yellow residue was crys-
tallized overnight at room temperature to give 21.8 g (91%) of compound 2,
which was used without further purification for the next step.
Part of this work was already presented as a poster at the
ACS Conference, New Orleans, La., 1996.)
MATERIALS AND METHODS
Materials. Sparfloxacin was generously provided by Rhoˆne-Poulenc Rorer
GmbH. Acetonitrile (Acros) was of high-pressure liquid chromatography
(HPLC) grade, and all other reagents used were of analytical grade.
HPLC with UV detection. HPLC was performed with a Kontron HPLC pump
420 equipped with a Perkin-Elmer PE LC 480 Autoscan diode array detector.
The chromatographic conditions were as follows: column, Merck LiChrosorb
* Corresponding author. Mailing address: Pharmazeutisches Institut
der Universita¨t Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany.
Phone: 49-228-732-845. Fax: 49-228-739-038. E-mail: holzgrabe@uni
-bonn.de.
Ethyl (2,4-dichloro-5-fluoro-3-nitrobenzoyl)acetate, compound 4. A total of
1.82 g (76 mmol) of magnesium was treated with 100 ml of ethanol and 1 ml of
tetrachloromethane. After the reaction started, 12.16 g (76 mmol) of malonic
1151

1152
ENGLER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Synthesis of the reference compound, compound 10. Et, ethyl.
acid diethylate, dissolved in a mixture of 110 ml of ethanol and 300 ml of toluene,
was added dropwise. After the addition was completed the mixture was stirred at
70°C for 2 h and cooled to Ϫ10°C, and 21.8 g (76 mmol) of acid chloride,
compound 2, dissolved in 110 ml of toluene was added slowly. Stirring for 2 h at
0°C and overnight at room temperature completed the reaction. The reaction
mixture was cooled to 0°C, and under vigorous stirring, an ice-cold mixture of 200
ml water and 240 ml of concentrated sulfuric acid was added. After extraction
with toluene, washing of the combined organic layers with saturated sodium
FIG. 2. Photoproducts of sparfloxacin.

VOL. 42, 1998
IDENTIFICATION OF DEFLUORINATED SPARFLOXACIN
1153
isomer), 0.97-0.81 (m, 4 H, cyclopropyl CH2); IR (cmϪ1) 3450, 3180, 3030, 1670,
1620, 1560, 1540, 1360.
TABLE 1. UV data obtained from the HPLC run in acetonitrile-
formic acid (0.2%; 50:50) showing the hypsochromic shift
of the absorption maximum
Ethyl 7-chloro-1-cyclopropyl-1,4-dihydro-6-fluoro-8-nitro-4-oxo-3-quinolinecar-
boxylate, compound 7. Ten grams (25.6 mmol) of compound 6 and 2.9 g (25.6
mmol) of potassium tert-butoxide dissolved in 250 ml of dioxane were refluxed
for 4 h. The mixture was evaporated to half of its volume and was poured into a
mixture of 200 ml of ice water, 40 ml of concentrated hydrochloric acid, and 75
ml of dichloromethane. The organic layer was separated, washed with water,
dried, and evaporated. Recrystallization of the residue from ethanol gave 7.3 g
(80%) of brown crystals, compound 7; melting point, 172°C; 1H NMR (60 MHz,
dimethyl sulfoxide [DMSO]-d6 ϩ trifluoroacetic acid) (ppm) 8.75 (s, 1 H, ACH),
8.37 (d, 1 H, 10 Hz, aromatic H), 4.30 (q, 2 H, OOCH2), 2.70 (m, 1 H,
cyclopropyl CH), 1.35 (t, 1 H, CH3), 1.30-1.0 (m, 4 H, cyclopropyl CH2); IR
(cmϪ1) 3090, 2990, 1730, 1600, 1540, 1340.
Compound
␭
(nm)
␭
max2
(nm)
␭
max3
(nm)
max1
Sparfloxacin
364
292
218
Photoproduct [MϩH]ϩ with
a mass of 375 Da
353
286
227
Compound 10
350
341
288
284
242
227
Photoproduct [MϩH]ϩ with
a mass of 335 Da
Ethyl 1-cyclopropyl-1,4-dihydro-7-(2,6-dimethyl-1-piperazinyl)-6-fluoro-8-ni-
tro-4-oxo-3-quinolinecarboxylate, compound 8. A mixture of 2.8 g (7.9 mmol) of
compound 7 and 3.6 g (32 mmol) of 2,6-dimethylpiperazine was refluxed in 100
ml of acetonitrile for 2 h. After stirring overnight at room temperature, the
yellow precipitate was filtered and the mother liquid was concentrated. Recrys-
tallization of both residues from ethanol yielded 3 g (88%) of compound 8;
melting point, 195°C; 1H NMR (300 MHz, CDCl3) (ppm) 8.56 (s, 1 H, ACH),
8.20 (d, 1 H, 11.3 Hz, aromatic H), 4.34 (q, 2 H, 7 Hz, OOCH2), 3.85 (m, 1 H,
cyclopropyl CH), 2.94-2.72 (m, 6 H, piperazinyl CH and piperazinyl CH2), 1.36
(t, 3 H, 7.1 Hz, OCH2CH3), 1.13-1.02 (m, 10 H, piperazinyl CH3 and cyclopropyl
CH2); IR (cmϪ1) 3080, 2960, 1730, 1700, 1540, 1320.
1-Cyclopropyl-1,4-dihydro-7-(2,6-dimethyl-1-piperazinyl)-6-fluoro-8-nitro-4-
oxo-3-quinolinecarboxylic acid, compound 9. A total of 1.57 g (3.6 mmol) of
compound 8 was treated with a solution of 40 ml of acetic acid, 30 ml of water,
and 5 ml of concentrated sulfuric acid. The mixture was heated at 100°C for 2 h
and poured onto ice. The pH was adjusted to 4 to 5 with 2 M NaOH, and the
solution was extracted with chloroform. The combined organic layers were dried
and concentrated, and the yellow oil was triturated with benzine for crystalliza-
tion. Subsequently, the yellow solid was filtered and washed with benzine to give
1.46 g (94%) of compound 9; melting point, 255 to 260°C; 1H NMR (300 MHz,
DMSO-d6 ϩ trifluoroacetic acid) (ppm) 8.78 (s, 1 H, ACH), 8.28 (d, 1 H, 11 Hz,
aromatic H), 3.71 (m, 1 H, cyclopropyl CH), 3.28-3.15 (m, 6 H, piperazinyl CH
chloride solution, and drying of the layers over magnesium sulfate, the evapo-
ration of the solvent yielded 35.6 g of a mobile oil. This oil was treated with 300
ml of water and 800 mg of p-toluenesulfonic acid, and the mixture was refluxed
for 3 h. The mixture was stirred overnight at room temperature and was extracted
with dichloromethane. The combined organic layers were dried and evaporated
to yield 23.4 g (95%) of compound 4 as a yellow oil, which was used without
further purification.
Ethyl 2-(2,4-dichloro-5-fluoro-3-nitrobenzoyl)-3-(cyclopropylamino)-acrylate,
compound 6. A solution of 10 g (31 mmol) of compound 4, 8.8 g (60 mmol) of
triethyl ortho-formate, and 10.2 g (100 mmol) of acetic anhydride was refluxed
for 4 h. The solvent was removed under reduced pressure, with the residue been
dissolved in 50 ml of ethanol. After cooling to 0°C, a solution of 1.8 g (31 mmol)
of cyclopropylamine dissolved in 20 ml of ethanol was added dropwise. The
mixture was stirred for 36 h and the precipitate was filtered and recrystallized
from ethanol to give 3.36 g (28%) of a yellow powder, compound 6; melting
point, 142°C; 1H NMR (300 MHz, CDCl3) (ppm) mixture of E and Z isomers
11.01 (d, 1 H, 13.7 Hz, NH; E isomer), 9.78 (d, 1 H, 13 Hz, NH; Z isomer), 8.36
(d, 1 H, 14.5 Hz, ACH; Z isomer), 8.27 (d, 1 H, 14.7 Hz, ACH; E isomer), 7.17
(d, 1 H, 8.1 Hz, aromatic H; Z isomer), 7.11 (d, 1 H, 7.9 Hz, aromatic H; E
isomer), 3.99 (q, 2 H, 7 Hz, OOCH2; E isomer), 3.92 (q, 2 H, 7 Hz, OOCH2; Z
isomer), 3.01 (m, 1 H, cyclopropyl H; E isomer), 1.03 (t, 3 H, 7.1 Hz, CH3; E
FIG. 3. Product ion spectrum of sparfloxacin.

1154
ENGLER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 4. Fragmentation pathway of sparfloxacin under MS/MS conditions. Numbers in brackets are m/z’s.

FIG. 5. Product ion spectrum of the photoproduct [MϩH]ϩ with a mass of 375 Da.
FIG. 6. Product ion spectrum of the photoproduct [MϩH]ϩ with a mass of 335 Da.
1155

1156
ENGLER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 7. Product ion spectrum of the photoproduct [MϩH]ϩ with a mass of 405 Da.
and piperazinyl CH2), 1.24-1.04 (m, 10 H, piperazinyl CH3 and cyclopropyl
CH2); IR (cmϪ1) 3020, 1720, 1600, 1550, 1320, 1260.
formed with the nonirradiated sparfloxacin. The fragmentation
of the ion spray ionization technique applied is different from
that of electron impact-mass spectrometry. The product ion
spectrum of sparfloxacin is displayed in Fig. 3, and the derived
fragmentation pathway is shown in Fig. 4. The pseudomolecu-
lar ion [MϩH]ϩ (m/z 393) can split off of a water molecule
(loss of 18 Da) or decarboxylate (loss of 44 Da). The resulting
oxoquinoline ion (m/z 349) loses different fragments of the
piperazine ring. Interestingly, no fragmentations concerning
the aromatic ring system and the substituents at N-1 are ob-
served. The peaks m/z 98, 84, and 58 can be explained by the
presence of a pseudomolecular ion with a positive charge on
the piperazine nitrogen atom.
From the Q1 scan of the irradiated samples, a chromato-
gram was obtained. The chromatogram showed three chro-
matographically well separated compounds with pseudomo-
lecular ions of 375, 335, and 405 Da. The subsequent product
ion scans of these pseudomolecular ions gave the fragmenta-
tion patterns of all three photoproducts (Fig. 5 to 7).
As can be seen in Fig. 5, the spectra of the photoproduct
[MϩH]ϩ with a mass of 375 Da and sparfloxacin (Fig. 3)
exhibit corresponding mass differences. The masses of all frag-
ment ions of the photoproduct are decreased by 18 Da. A loss
of water may have occurred upon irradiation. Since the mass
difference of 18 Da is still found in the product ion spectrum of
the photoproduct [MϩH]ϩ with a mass of 375 Da, a conver-
sion of the carboxylic function is impossible. Thus, the loss of
water is unlikely. Alternatively, a photochemical replacement
of one fluorine atom of sparfloxacin with a hydrogen must be
8-Amino-1-cyclopropyl-1,4-dihydro-7-(2,6-dimethyl-1-piperazinyl)-6-fluoro-4-
oxo-3-quinolinecarboxylic acid, compound 10. One gram (2.5 mmol) of com-
pound 9 was dissolved in a solution of 30 ml of ethanol and 30 ml of acetic acid.
After the addition of 40 mg of Pd/C (10%) catalyst, the mixture was hydroge-
nated at room temperature and 1 bar for 24 h. After filtration and removal of the
solvent, a black oil was obtained. Treatment of the oil with benzine gave a black
solid, which was recrystallized from isopropanol with activated charcoal. A sec-
ond recrystallization from isopropanol yielded 312 mg (33%) of compound 10;
melting point, 259°C (decomposed); 1H NMR (300 MHz, DMSO-d6) (ppm) 8.70
(s, 1 H, ACH), 7.23 (d, 1 H, 12.1 Hz, aromatic H), 6.03 [s (br), 2 H, NH2], 3.77
(m, 1 H, cyclopropyl CH), 3.05-2.66 (m, 6 H, piperazinyl CH and piperazinyl
CH2), 1.25-0.97 (m, 10 H, cyclopropyl CH2 and piperazinyl CH3); IR (cmϪ1
3340, 3010, 1630, 1540; UV (nm) 242 maximum, 288, 350.
)
RESULTS AND DISCUSSION
Irradiation of sparfloxacin with UV light results in at least
three photoproducts (Fig. 2). As can be detected by HPLC, the
best irradiation time was found to be 4 to 8 h. After 4 h, the
sparfloxacin peak decreases significantly, whereas the other
peaks increase. Irradiation times longer than 8 h did not affect
the intensity of the sparfloxacin peak anymore. A total degra-
dation of the quinolone could not be achieved. A reason for
this might be the quenching effect in concentrated solutions
described by Morimura et al. (7). The UV spectra of all pho-
toproducts recorded during the HPLC run show a hypsochro-
mic shift of 6 to 8 nm of the absorption maximum (Table 1)
indicating the loss of a weak chromophoric group, such as a
fluorine atom.
In order to find similarities and changes in the MS spectra of
the photoproducts, an LC-MS/MS experiment was first per-

VOL. 42, 1998
IDENTIFICATION OF DEFLUORINATED SPARFLOXACIN
1157
FIG. 8. Product ion spectrum of the reference compound, compound 10.
taken into account, because the UV spectrum of the photo-
product showed a corresponding shift (Table 1). The fact that
the masses of all the fragment ions showed a difference of 18
Da further supports the hypothesis of photodefluorination (2).
In order to ensure this hypothesis, the 8-desfluoro compound
(compound 10) has been synthesized, and mass and UV spec-
tra were recorded by using the conditions described above. As
can be seen in Fig. 5 and 8, the spectra of the photoproduct
[MϩH]ϩ with a mass of 375 Da and the synthesized compound
show nearly the same patterns of fragmentation, indicating
similar structures. The differences between the photoproduct
and compound 10 (fragment ions of m/z 274, 232, 219, 217, and
204) are due to the different positions of the amino group,
which results from a kind of “ortho effect” of the amino group
in the fragmentation pattern of compound 10 (Fig. 9). Accord-
ing to this observation, it seems likely that the fluorine atom in
the photoproduct takes the 6 position or, in turn, the fluorine
at position 8 was split off upon irradiation. In addition, the UV
spectrum of compound 10 shows nearly the same hypsochro-
and not yet finished. The mass spectrum of the product
[MϩH]ϩ with a mass of 335 Da (Fig. 6) exhibits the same
fragmentation pattern concerning the piperazine ring (com-
pare with Fig. 3 for sparfloxacin) and a loss of water ([MϩH]ϩ
with a loss of 18 Da). It is remarkable that there is no loss of
CO2 connected with the loss of water in this case, which indi-
cates the presence of a COOH group. Nevertheless, it seems
likely that the piperazine ring and the carboxylic group are still
present. The mass difference of 58 Da between sparfloxacin
([MϩH]ϩ with a mass of 393 Da) and the photoproduct might
be explained by the loss of two groups: first, a replacement of
fluorine with a hydrogen (18 Da), which is in agreement with
the hypsochromic shift in the UV spectrum, and second, sub-
stitution of the cyclopropyl group with a hydrogen (40 Da)
(Fig. 6). Since such a photodegradation has not yet been de-
scribed, further investigations must be performed. The infor-
mation which can be extracted from the product ion spectrum
of the photoproduct [MϩH]ϩ with a mass of 405 Da is rather
poor, because the signals are rather weak because of the high
degree of fragmentation (Fig. 7). The base peak of m/z 291 can
be explained by the loss of a neutral piperazine ring ([MϩH]ϩ
with a mass of 405 Da Ϫ 114 Da (neutral molecule) ϭ 291 Da).
In turn, a peak for a protonated piperazine is found at m/z 115.
Thus, it is likely that the piperazine ring is still present in this
photoproduct.
mic shift (␭
ϭ 288 [compound 10], and 292 [sparfloxacin])
max
as the photoproduct. The finding of defluorination of the dif-
luorinated quinolone supports the observation reported previ-
ously (10) that 6-monofluorinated quinolones are found to be
more photostable than 6,8-difluorinated quinolones. In addi-
tion, the defluorination is in accordance with recently reported
results obtained with irradiated lomefloxacin and fleroxacin
(4). The defluorinated products were identified by means of
19F NMR spectroscopy.
Taken together, the replacement of a fluorine atom with a
hydrogen atom in the quinolone skeleton of gyrase inhibitors
induced by UV light could be confirmed by LC-MS/MS as well
as with the UV spectra of two photoproducts. The photodeg-
radation of the piperazine ring (levofloxacin [18], ciprofloxacin
The elucidation of the structure of the photoproducts
[MϩH]ϩ with masses of 335 and 405 Da is more complicated

1158
ENGLER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 9. Fragmentation pathway of the reference compound, compound 10, under MS/MS conditions. Numbers in brackets are m/z’s.
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We thank Armin Haag for valuable help concerning the interpreta-
tion of the mass spectra.

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IDENTIFICATION OF DEFLUORINATED SPARFLOXACIN
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