J. Sep. Sci. 2009, 32, 2993 – 3000 B. Stanley et al. 2993 Bob Stanley 1 Kevin A. Mehr 1 Trent Kellock 2 Jonathan D. Van Hamme 2 Kingsley K. Donkor 1 1 Department of Chemistry, Thompson Rivers University, Kamloops, BC., Canada 2 Department of Biological Sciences, Thompson Rivers University, Kamloops, BC., Canada Original Paper Separation and determination of closely related lantibiotics by micellar electrokinetic chromatography A sensitive micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination of four closely related lantibiotics: gallider- min, cinnamycin, duramycin and nisin. Factors affecting the separation of the lanti- biotics such as pH, phosphate buffer concentration, SDS concentration and wave- length for UV detection were investigated. By optimizing these experimental condi- tions, successful separation was achieved between class 1A lantibiotics (nisin and gallidermin) and class 1B lantibiotics (duramycin and cinnamycin). The four lantibi- otics were separated within 12 min in 50 mM phosphate buffer at pH 3.95 l 0.1 con- taining 80 mM SDS with UV detection of 214 nm. The LOD (S/N = 3) were 61 ng/mL for gallidermin, 57 ng/mL for cinnamycin, 55 ng/mL for duramycin and 58 ng/mL for nisin. The method was successfully applied to real samples such as fermentation broth, bovine colostrum and predrop beer. This method yielded satisfactory results, with quantitative recoveries of spiked lantibiotics in the three samples ranging from 86.1 to 99.6%. Keywords: Capillary electrophoresis / Cinnamycin / Duramycin / Gallidermin / MEKC / Nisin / Received: March 20, 2009; revised: May 9, 2009; accepted: May 11, 2009 DOI 10.1002/jssc.200900185 1 Introduction Certain microbial groups produce low molecular weight antimicrobial peptides as defence mechanisms against other microbes and to increase competitiveness [1, 2]. Lantibiotics produced by the Gram positive lactic acid bacteria are small antimicrobial peptides (a4 kDa) that contain a high content of thioether-bridged amino acid residues called lanthionine and 3-methyllanthionine. They also contain unsaturated amino acid residues dehy- droalanine and dehydrobutyrine [2 – 6]. Interestingly, lantibiotics produced by one strain are often active against closely related strains found in similar habitats [5 – 11], and have also been shown to have antimicrobial activity against other gram-positive bacteria such as staphylococci, streptococci, bacilli, clostridia and myco- bacteria [8, 12, 13]. The primary mode of action of lantibi- otics is the formation of pores in the bacterial cytoplas- mic membrane [3, 4, 10]. The formation of pores deener- gizes the membrane, causes an efflux of cellular metabo- lites such as adenosine triphosphate (ATP), and in some cases cell lysis [8, 14]. Some lantibiotics target lipid II in the bacterial cell wall thus inhibiting peptidoglycan syn- thesis [10, 15 – 17]. The broad bacteriocidal spectrum and limited resistance mechanisms in target organisms have made lantibiotics a very attractive alternative to tradi- tional antibiotics. Antibiotics are crucial to combat pathogenic bacteria [18]. However, there has been an increase in the preva- lence of pathogenic bacteria resistant to antibiotics such as penicillin, cephalosporins and quinolones [19, 20]. Even vancomycin, often hailed as the last line of defence against multi-drug resistant strains, is becoming less effective [21, 22]. Therefore, there is a need to develop new antimicrobial compounds with different modes of action such that pathogenic bacteria cannot employ existing resistance mechanisms [23 – 25]. Presently, there is growing concern with using syn- thetic chemicals as preservatives in food. As a result, more consumers are selecting less processed foods which may harbour harmful microorganisms that survive refrigeration. Many lantibiotics are safe for consumption and are excellent natural preservatives in food and bever- Correspondence: Dr. Kingsley K. Donkor, Department of Chem- istry, Thompson Rivers University, Box 3010, 900 McGill Road, Kamloops, BC., Canada V2C 5N3 E-mail: kdonkor@tru.ca Fax: +1-250-8285450 Abbreviations: MEKC, micellar electrokinetic chromatography; UV, ultraviolet i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com