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Journal of Chromatography A, 1163 (2007) 333–336 Short communication Separation of seven fluoroquinolones by microemulsion electrokinetic chromatography and application to ciprofloxacin, lomefloxacin determination in urine Shoulian Wei a,b, Jiesheng Lin a, Haifang Li b, Jin-Ming Lin b,∗ a Department of Light Industry & Chemistry, Zhaoqing University, Zhaoqing 526061, China b Department of Chemistry, Tsinghua University, Beijing 100084, China Received 5 June 2007; received in revised form 26 June 2007; accepted 28 June 2007 Available online 30 June 2007 A simple, reliable microemulsion electrokinetic chromatography (MEEKC) method is developed for the simultaneous separation of seven fluoroquinolones (FQs). The best separation is achieved in a carrier electrolyte containing 1% (v/v) heptane, 100 mmol/L sodium dodecyl sulfate(SDS), 10% (v/v) 1-butanol, and 8 mmol/L phosphate–sodium tetraborate buffer at pH 7.30. The proposed method was directly applied to thedetermination of ciprofloxacin (CPF) and lomefloxacin (LMF) in urine samples of subjects administered either with CPF or LMF.
2007 Elsevier B.V. All rights reserved.
Keywords: Microemulsion electrokinetic chromatography; Fluoroquinolones; Ciprofloxacin; Lomefloxacin MEEKC [12] is a relatively new CE technique that often provides higher resolving power. In this study, we develop a In generally, following oral administration, fluoroquinolones selective MEEKC method for the separation of CPF, LMF, (FQs) are rapidly and completely absorbed from the gastroin- norfloxacin (NF), ofloxain (OF), fleroxacin (FL), gatifloxacin testinal tract and reach the maximal plasma concentration in (GTF) and sparfloxacin (SPF) and determination of CPF or LMF 1–2 h [1–3]. But how long it takes to reach the maximal urine in urine samples.
concentration has not been investigated, and this may be usefulto know in clinical pharmacology. Therefore, there is a neces- sity to develop analytical methods that would allow the directlydetermination of FQs in urine and make it suitable for routine analysis in clinical laboratories.
Several techniques such as spectrophotometry [4], spec- A separation was performed with a Beckman (Fullerton CA, trofluorometry [5], electrochemical detection [6], flow-injection USA) PACE/MDQ capillary electrophoresis system. 32 Karat chemiluminescence [7], capillary electrophoresis (CE) [8] and Software V.7.0 was used for instrument control and data analysis.
high-performance liquid chromatography (HPLC) [9–11] have An untreated fused-silica capillary of 56.9 cm (46.6 cm from been developed for the determination of FQs in urine sam- inlet to detector) × 75 ␮m I.D. (Yongnian Optical Fiber Factory, ples. However, the literature available on determination of FQs Hebei, China) was used. The detection wavelength was 280 nm.
in urine is very short and commonly suffers from long pre- Samples were injected by applying a pressure of 3.4 kPa for 5 s.
treatment of the samples and low resolving power.
2.2. Chemicals and materials All chemicals were of analytical grade. SDS was obtained ∗ Corresponding author. Tel.: +86 10 62792343; fax: +86 10 62792343.
from Sigma (St. Louis, MO, USA). CPF, LMF, OF and NF were E-mail address: (J.-M. Lin).
obtained from the Institute to Pharmaceutical and Biomaterial 0021-9673/$ – see front matter 2007 Elsevier B.V. All rights reserved.

Author's personal copy
S. Wei et al. / J. Chromatogr. A 1163 (2007) 333–336 Authentication of China (Beijing, China). GTF, FL and SPFwere kindly donated by the Institute to Pharmaceutical and Bio-material Authentication of Zhaoqing (Guangdong, China). CPFand LMF tablets were purchased from local drugstore.
2.3. Standard solutions Stock solutions of NF, CPF, OF, LMF, SPF, FL and GTF (1.0 × 10−3 mol/L) were prepared using 0.01 mol/L NaOH andstored under light-protecting condition at 4 ◦C. Standard work-ing solutions were daily prepared by mixing and diluting thestock solution in water.
Fig. 1. MEEKC electropherogram of FQs standard mixture each at 2.4. Preparation of the microemulsion 5.0 × 10−5 mol/L (except for OF: 1.0 × 10−5 mol/L). Separation condi-tions: 1% (v/v) heptane + 100 mmol/L SDS + 10% (v/v) 1-butanol + 8 mmol/L The microemulsion was prepared by adding 1% (v/v) hep- phosphate–sodium tetraborate buffer at pH 7.30. The applied voltage was 22 kV,temperature was 20 ◦C. Peaks: 1 = FL; 2 = LMF; 3 = OF; 4 = GTF; 5 = CPF; tane, 10% (v/v) 1-butanol, 2.88 g (100 mmol/L) SDS, 0.125 g 6 = NF; 7 = SPF.
(8 mmol/L) sodium phosphate (NaH2PO4·2H2O) to a 100 mLflask. This mixture was sonicated for 30 min to aid dissolution independent sources of drug-free urine. No interfering peaks in and an optically transparent microemulsion had formed. Before the retention time of CPF, LMF were observed in blank drug-free use, the microemulsion solutions were filtered through a 0.45- ␮m filter and degassed in an ultrasonic bath for 1 min. The bufferwas adjusted to pH 6.0–9.0 with 50 mM sodium tetraborate, if 3.2. Linearity of the method and limits of detection and 2.5. Sample preparation The calibration curves were constructed using response of peak areas (y) versus the urine sample concentra- 2.5.1. Drug administration tion (x, mol/L). The calibration curves for LMF and Eight healthy males and females adult volunteers were partic- CPF in the range of 1.2 × 10−6 − 5.0 × 10−4 mol/L were: ipated in the study. All subjects were healthy on the basis of their y = 1.93 × 109x + 8.55 × 103, and y = 9.82 × 108x + 3.58 × 103, medical history, clinical and laboratory examination. After the with r = 0.9987 and r = 0.9972, respectively. The detection lim- overnight fast, one 500 mg CPF tablet or LMF tablet of the test its (LODs, S/N = 3) were 0.95 and 0.97 ␮mol/L for LMF and was administered orally. The urine samples were respectively CPF, respectively. The quantification limits (LOQs, S/N = 9) collected at 0, 0.75 (0.5), 1.75 (1.5), 3.75 (2.75), 4.25(3.75), 5.25 were 2.85 and 2.91 ␮mol/L for LMF and CPF, respectively.
(4.5), 5.75 (5.5), 6.25 (6.0), 6.75 (7.0), 8.5 (8.0), 12.0 (12.0) hand stored at −20 ◦C until analysis.
3.3. Precision and accuracy 2.5.2. Urine Precision and accuracy were assessed for LMF and CPF at The frozen urine samples were thawed at room temperature three concentrations covering the linearity range (six replicate and centrifuged at 3500 × g for 15 min. The supernatants were analyses at each level during 5 consecutive days) by spiked urine.
transferred to clean glass tube and filtrated through a 0.45 ␮m The intra-day and inter-day precision were <5.0% and <7.0%, filter, and directly injected into the electrophoresis system.
3. Results and discussion
3.1. MEEKC profiles The standard microemulsion conditions [0.81% (w/w) hep- tane, 6.61% (w/w) 1-butanol, 3.31% (w/w) SDS, and 89.27%(w/w) 10 mmol/L sodium tetraborate buffer] were taken as thestarting point and performed a series of optimizations. The pHwas a critical factor in a series of optimizations. Thus, a pH inter-val of 7.0–9.5 was investigated. The best separation was achievedin a carrier electrolyte containing 1% (v/v) heptane, 100 mmol/LSDS, 10% (v/v) 1-butanol, and 8 mmol/L phosphate–sodiumtetraborate buffer at pH 7.30. Figs. 1 and 2 show the optimized Fig. 2. Electropherograms of a blank drug-free pooled urine; Analytical condi- MEEKC separation of a standard solution of all seven FQs and tions were as for Fig. 1.

Author's personal copy
S. Wei et al. / J. Chromatogr. A 1163 (2007) 333–336 Fig. 3. Electropherograms of: (A) urine 0.75 h and (B) 3.75 h after oral administration of one tablet with 500 mg CPF; (C) urine 4.0 h after oral administration of onetablet with 500 mg LMF. Analytical conditions were as for Fig. 1. Peaks 1, 2 and 3 were probably CPF metabolites.
respectively, for LMF and CPF. The intra-day and inter-dayaccuracy were in the range of 99.0–104.0% and 100.0–105.5%,respectively, for LMF and CPF. The above results indicated thatthe method was reliable, reproducible and accurate.
3.4. Urine samples The proposed method was applied to determination of CPF and LMF in urine samples. Fig. 3A and B shows an electro-pherogram of one healthy volunteer after oral administration of500 mg CPF tablet, collected at 0.75 h and 3.75 h, respectively.
Comparing the electropherogram obtained in Fig. 2, three newpeaks respectively at tR = 17.60 min, 19.72 min and 20.53 min Fig. 4. Urine concentration–time profiles after oral administration of one tablet can be observed in Fig. 3A and B. Their areas changed with the with (A) 500 mg CPF, (B) 500 mg LMF.
collection time and their spectra concordance with CPF. There-fore, Peaks 1, 2 and 3 were probably CPF metabolites. Fig. 3C results may be useful in clinical pharmacology for detecting the shows an electropherogram of another healthy volunteer after efficacy and side effects of LMF and CPF.
oral administration of 500 mg LMF tablet, collected at 3.75 h.
Comparing the electropherogram obtained in Fig. 1 and Fig. 2, few differences in migration times can be observed. These aredue to the time taken to remove the sample matrix from the This work was supported by the Program for Changjiang capillary, since the sample matrix is different.
Scholars and Innovative Research Team in Tsinghua University The urine concentration–-time profile of two healthy human (No. IRT0404) and the National Key Technology R & D Program volunteer after respectively receiving one table with 500 mg CPF and LMF were shown in Fig. 4. The concentration ofCPF and LMF in the urine respectively reached a maximum value of 39 mg/l and 49 mg/L after about 5 h oral administra-tion. Twelve hours after oral administration, the concentration [1] T. Nekvindov´a, J. Suchop´ar, Remedia 4 (1993) 206.
of CPF and LMF in the urine was, respectively, 2.5 mg/L and [2] K.G. Nabe, Chemotherapy 42 (1996) 1.
3.9 mg/L, which corresponded to a rapid elimination. The above [3] A.E. Struck, D.K. Kim, F.J. Frey, Clin. Pharmacokinet. 22 (1992) 116.
Author's personal copy
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[5] L.M. Du, Y.Q. Yang, Q.M. Wang, Anal. Chim. Acta 516 (2004) 237.
[10] G.J. Krol, G.W. Beck, T. Benham, J. Pharm. Biomed. Anal. 14 (1995) [6] M. Rizk, F. Belal, F.A. Aly, N.M. El-Enany, Talanta 46 (1998) 83.
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