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Analysis of Ofloxacin Corneal Deposits by Microbore HPLC – Tandem MS
with Electrospray Ionisation
B.Sinnaeve, T. Decaestecker, J. Van Bocxlaer
Laboratory of Medical Biochemistry and Clinical Analysis, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium
4. Results and discussion
Nowadays, ophthalmic formulations of new antibiotics of the fluoroquinolon family,
Summation of the protonated molecular ion [M+H+] and the two most intense
including ofloxacin, are frequently used for the topical treatment of external ocular
product ions (m/z
261.1+318.1+362.1 for ofloxacin and m/z
infections and corneal ulcer. Despite of the safe character of this class of drugs, a
for pefloxacin) permitted the construction of linear response curves (between 0.5
white crystalline deposit has been reported for ciprofloxacin and norfloxacin. At the
µg/L or 5 pg on column and 5 mg/L, respectively, 50 ng on column) with
moment, this precipitation has not yet been reported on ofloxacin eyedrops. In this
correlation coefficients between 0.9991 and 0.9994. The limit of detection
case we investigated the corneal precipitate of a 6-year-old-boy with vernal
(retaining full scan spectral identity confirmation potential) was 0.4 µg/L and the
keratoconjunctivitis (VKC), treated with topical ofloxacin 0.3% eyedrops. Because
limit of quantitation 0.5 µg/L. The reconstructed ion fragmentogram obtained for
of the extremely small sample amount, provided by corneal scraping, a very
the clinical sample showed two peaks which could be identified as the internal
sensitive and specific method was needed with the possibility of an unambiguous
standard pefloxacin (t = 21.06 min.) and ofloxacin (t = 18.44 min.) (Fig.1) . A
identification of ofloxacin, supposed to be present in the precipitate. In this respect,
diagnostic CID product ion spectrum (prominent peaks: m/z 362.2 [M+H]+; 318.1;
tandem Q-TOF mass spectrometry combined with micro LC was chosen.
261.1) was obtained (Fig.2) which, in combination with the relative retention time, allowed unequivocal confirmation of the presence of ofloxacin in the corneal scraping. Relating the sample peak area ratio to the standards, revealed an
µg/L. As such, we can substantiate the presence
of at least 125 pg of ofloxacin in the corneal scraping.
Development of an, in essence, qualitative procedure to positively indentify ofloxacin in the corneal deposits. Nevertheless, the method was extended with a quantitative dimension, to substantiate the amount of ofloxacin in the precipitate.
3. Materials and methods
Column: Inertsil ODS-3 C18 (5µm, 1000 µm I.D. x 15 cm; LC Packings)
Mobile Phase: 90/10 water/acetonitrile brought to pH 3.0 with formic acid;
HPLC: Ultimate Micro Pump HPLC System (LC Packings)
Autosampler: FAMOS (LC Packings), 10 µl injection
Mass Spectrometer: Micromass Q-TOF hybrid mass spectrometer
Ion Source: orthogonal electrospray source (Z-spray®) in positive ion mode
Fig.1: Reconstructed ion fragmentograms obtained for the clinical sample
Collision energy: 28 eV for ofloxacin, 26 eV for pefloxacin
Pefloxacin was chosen as internal standard, because of its structural similarity to and small mass difference with the analyte, ofloxacin. Working standard solutions
of ofloxacin were prepared in the concentration range 0.5 µg/L–5 mg/L by dilution with 90/10 water/acetonitrile, acidified with formic acid to pH 3.0. The internal
standard was present in a final concentration of 0.5 mg/L. The injection parameters were optimised in the microliter pick up mode, meaning that exactly 10 µl of the
samples could be injected, without any sample loss. This method is particularly
suited for limited sample volumes, as in our case of the extremely restricted
50 75 100 125 150 175 200 225 250 275 300 325 350 375 400
quantity of low dosed corneal deposits. We opted specifically for Micro LC (1 mm ID) because of the improved sensitivity obtained by this approach. The Q-TOF
Fig.2: Diagnostic product ion spectrum of ofloxacin in the clinical sample
instrument was operated in the MS/MS mode, selecting both protonated molecular ions m/z
362.1 for ofloxacin and m/z
334.1 for pefloxacin. Qualitative identity
confirmation of ofloxacin was based on a combination of relative retention time and the full scan product ion spectrum, which a Q-TOF is able to produce for very low
We clearly showed that the precipitate, removed by corneal scraping from the 6-
year old boy with VKC, contained at least 125 pg of ofloxacin. Since treatment had been discontinued for seven days and in view of the short half-life of
To the corneal deposits, surgically removed from the 6-year old boy by scraping,
ofloxacin, our findings indicate that, at least in the treatment of VKC associated
100 µL acetonitrile was added. After ultrasonication, it was placed overnight in a
ulcers, deposits can occur after the topical use of ofloxacin.
lab shaker. Subsequently, 150 µL of water, acidified with formic acid to pH 3.0, together with the internal standard pefloxacin, was added.
Finally, 10 µL was injected in the LC-MS/MS system.
The authors would like to thank Ing. Sofie Vande Casteele for her practical assistance in performing the mass spectrometric analyses. This work was supported by grant GOA-120501.99 (Eigen Onderzoeksfonds).
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