Short communicationOn-line sample cleanup and chiral separation of gemifloxacin in a urinary solution using chiral crown ether as a chiral selector in microchip electrophoresis
Introduction
As a result of an increasing concern about the physiological, sensory, and toxicological properties of chiral compounds, many analytical methods including high-performance liquid chromatography and capillary electrophoresis (CE) have been applied to separate, identify, and quantify chiral compounds [1], [2]. Microchip electrophoresis is based on the same separation principle as CE [3], [4] and can be superior to CE in the incorporation of complex channels and parallel processing systems for high throughput analyses owing to modern micromachining technology. For example, precolumn [5], postcolumn [6], or two-dimensional [7] microchip electrophoresis devices could be easily fabricated by coupling microchannels. Very fast separation within a few milliseconds could also be achieved by constructing a very short separation channel of 200 μm [8]. Recently, high speed chiral separation of fluorescein isothiocyanate-labeled amino acids has been demonstrated using cyclodextrin as a chiral selector in microchip electrophoresis with a short separation channel of 7 cm [9].
Chiral crown ether, (+)-18-crown-6-tetracarboxylic acid (18C6H4; Fig. 1a), is quite efficient in resolving chiral primary amines [10], [11], [12]. 18C6H4 can form diastereomeric complexes with protonated primary amine enantiomers and its chiral recognition stems from the difference in the complex formation energies. Alkaline metals such as sodium and potassium can also form strong complexes with 18C6H4, which is detrimental to capillary electrophoretic separation of amine enantiomers [13]. Thus, alkaline metal ions should be avoided as a buffer constituent when 18C6H4 is used as a chiral selector. Biological samples usually contain sodium and potassium ions at about 1 wt.% concentration [14]. Such metal ions not only in the run buffer but also in the sample solution could spoil the separation efficiency significantly [15]. Furthermore, direct injection of biological samples containing salts into a mass spectrometer for further analysis can cause electrospray instability and sensitivity reduction [16]. Various sample pretreatment methods such as solid phase extraction and dialysis often have to be applied to clean up such salts [16], [17], [18], [19], [20], [21], [22].
In this paper, we investigated channel-coupled microchip electrophoresis for the chiral separation of gemifloxacin [15], [23], [24], which is a primary amine and chiral antibacterial agent as shown in Fig. 1b, in a urinary solution using 18C6H4 as a chiral selector. In the first channel, highly mobile metal ions in a gemifloxacin sample, monitored by indirect detection, were removed to the waste and then gemifloxacin enantiomers were separated using a run buffer containing 18C6H4 in the second channel. Channel-coupled microchip electrophoresis will be suitable for desalting from the sample solution, considering its simplicity, analysis time, and solvent compatibility.
Section snippets
Materials
18C6H4 was obtained from RStech (Daejeon, Korea) and Fluka (Buchs, Switzerland). Gemifloxacin was acquired from LG Chemical (Daejeon, Korea). Bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (Bis–Tris), potassium chloride, and quinine were from Sigma (St. Louis, MO, USA). Sodium chloride was from Junsei Chemical (Tokyo, Japan). Citric acid (CA) was from Yakuri (Osaka, Japan). All reagents were of analytical grade and were used without further purification. Deionized water was obtained from a
Results and discussion
The cleanup of metal ions was performed using the first separation channel including reservoirs A, B, C, and D. Monitoring of metal ions was conducted with an indirect detection method. A mixture of 50 mM KCl, 50 mM NaCl, and 100 μM gemifloxacin was separated with the run buffer of (50 mM Bis–Tris + 10 μM quinine)/CA of pH 4.0. Bis–Tris was used as a run buffer component to prevent the competitive binding with the chiral selector 18C6H4 [13]. 10 μM quinine was added to the run buffer as a chromophore
Conclusions
The chiral separation of gemifloxacin in saline solution using 18C6H4 as a chiral selector was investigated using a channel-coupled microchip electrophoresis device. Metal ions hampering the complex formation of primary amine analytes with 18C6H4 were removed in the first separation channel using the difference in electrophoretic mobilities of metal and amine ions. In the second separation channel the enantiomers of gemifloxacin were successfully separated with a run buffer of (50 mM Bis–Tris +
Acknowledgements
This work was supported by the International Mobile Telecommunications 2000 R&D Project (Ministry of Information and Communication) and the BK21 Program of Korea.
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Present address: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.