Journal of Antimicrobial Chemotherapy (1999) 43, 434-435
© 1999 The British Society for Antimicrobial Chemotherapy
Correspondence |
A reverse-phase, isocratic high-performance liquid chromatography assay for levofloxacin
J Antimicrob Chemother1999; 43: 434–435
Bristol Centre for Antimicrobial Research & Evaluation, Southmead Health Services NHS Trustand the University of Bristol, Department of Medical Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, UK
Sir,
Levofloxacin, the L-isomer of ofloxacin, was licensed for use in the USA in 1996 and has recently become availablein the UK. It has a broad spectrum of in-vitro activity and has been shown to be more active thanofloxacin. 1 The mean peak plasma concentrations of levofloxacin (t max 1–2 h) after 250 mg and 500 mg oral doses have been reported to be 2.8 and 5.2mg/L respectively. 2 As with ofloxacin, the oral and iv preparations have been formulated to allow for interchangeand, with a half-life of approximately 8 h, once-daily dosing may be feasible when treatingpatients with mild to moderate infections. 3 We have previously developed a simple high-performance liquid chromatography (HPLC)assay for ofloxacin 4 and describe here a modification of that technique for the assay oflevofloxacin.
The stationary phase was Spherisorb 5 ODS in a stainless steel column, 25 cm x 4.6 mm (HPLC Technology, Macclesfield, UK), heated to 50°C (column block heater, HPLCTechnology, Macclesfield, UK). The mobile phase was 0.16% ortho-phosphoric acid, adjusted to pH 3 with tetrabutylammonium hydroxide; 50 mL of acetonitrilewere added to the 1 L solution after the pH adjustment. Serum samples were mixed with equalvolumes of methanol, allowed to stand for 5 min and centrifuged at 25,000g for 5 min; 20 µL of the supernatants were injected on to the column. The flow rate was 1.5 mL/min. Detectionwas by fluorescence (excitation wavelength, 310 nm; emission wavelength, 467 nm; modelLC240, Perkin Elmer, Beaconsfield, UK).
A chromatogram of levofloxacin is shown in the Figure. The reproducibility of the assay,expressed as the percentage coefficient of variation (% CV), was < 4% when the assay was repeated six times with aqueous and serum samples spiked with 0.9or 4.6 mg/L of levofloxacin. The detection limit (in serum), defined as a levofloxacinconcentration equivalent to a peak three-fold greater than that of the base-line noise, was 0.05mg/L. Linearity and serum recovery were investigated by assaying aqueous and serum specimenscontaining levofloxacin at concentrations of 0, 0.4, 0.9, 1.9, 4.6 and 8.0 mg/L. When the peakheight of levofloxacin was plotted against drug concentration and a regression analysisperformed, the correlation between these two variables for both the aqueous and serum levofloxacin samples (r = 0.996 and 0.982 respectively) was good. The percentage serum recovery (serum peakheight/ aqueous peak height x 100) approached 100% at each drug concentration tested. The accuracy of the assay wasinvestigated by assaying serum samples containing 1.5, 3.0 or 5.0 mg/L of levofloxacin witha single standard of 1.9 mg/L. Accuracy, expressed as the percentage error ((measuredconcentration - target concentration)/target concentration x 100) was 0.7%, 6.7% and 0.2% for the 1.5, 3.0 and 5.0 mg/L samples respectively. The assay isspecific. Following the assay of 22 commonly used antibiotics (including other fluoroquinolones) and antifungal agents, as well as samples containing unknown drugs which were obtained from24 patients, no chromatographic peaks that could potentially interfere with that of levofloxacin,with the exception of the peak for ofloxacin which, in any event, would not be co-administered with levofloxacin, were observed. We conclude that the technique describedhere is a rapid assay for levofloxacin than can be routinely performed in clinical diagnosticlaboratories.
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Notes
* Tel: +44-(0)117-959-5654; Fax: +44-(0)117-959-7213; E-mail: staff{at}bcare.org.uk 
References
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George, J. & Morrissey, I. (1997). The
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ciprofloxacin, sparfloxacin and cefotaxime against Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 39, 719–23.
2 . Fish, D. N. & Chow, A. T. (1997). The clinical pharmacokinetics of levofloxacin. Clinical Pharmacokinetics 32, 101–19.[Web of Science][Medline]
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Ernst, M. E., Ernst, E. J. & Klepser, M. E. (1997). Levofloxacin and trovafloxacin:
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4 . White, L. O., MacGowan, A. P., Lovering, A. M., Reeves, D. S. & MacKay, I. G. (1987). Apreliminary report on the pharmacokinetics of ofloxacin, desmethyl ofloxacin and ofloxacinN-oxide in patients with chronic renal failure. Drugs 34, Suppl. 1, 56–61.
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