Off-line Coupling of Capillary Electrophoresis and MALDI-MS for Analysis of Fluoroquinolone Antibiotics in Milk Samples

V. SPRINGER; P. EK; J. JACKSEN; A.G. LISTA; A. EMMER

Charlottesvile, Virginia

Simposio; ?29th International Symposium on MicroScale Bioseparation; 2013

Fluoroquinolones (FQs) are antibiotics used in human and veterinary medicine for the treatment of infections. Their massive use has become a serious problem since residues can be found in the environment and in food of animal origin. This could be directly toxic or induce pathogens resistant to antibiotics and allergic hypersensitivity reactions in humans. Taking this into account, several organizations (e.g. FDA, EU and WHO) have established tolerance levels for these compounds in different samples in order to protect human health. Therefore, there is a substantial need for sensitive and selective analytical methodologies for control of FQ levels in e.g. food. In the present work, an off-line capillary electrophoresis (CE) matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) method combined with a sample pretreatment using multiwalled carbon nanotubes (MWCNTs) for the determination of ciprofloxacin, norfloxacin and ofloxacin was developed (fig. 1). The aim was to optimize the analysis scheme for determination of the antibiotics in milk samples. Thus, a micro solid phase extraction (μSPE) pretreatment procedure utilizing MWCNTs as sorbent material, the CE method and the MS analysis were all investigated in view of CE resolution and sensitivity, and the MS signal quality. Several CE buffers and additives such as surfactants were evaluated along with μSPE, CE and MS parameters. In addition, the use of poly(diallyldimethylammonium chloride) (PDDA) as a capillary coating was introduced in order to minimize the MS signal suppression caused by CE buffer components. Finally, a custom-made setup was adapted to facilitate CE fraction deposition onto a MALDI plate for further MS analysis. It could be concluded that 20 mM ammonium phosphate containing 2 mM CTAB at pH 3 gave satisfying CE resolution, and the best CE sensitivity (UV detection) and MS signal-to-noise values comparing different buffers (fig. 2 and 3). Nevertheless, when CE fractions were collected and analyzed with MALDI-MS a severe reduction in signal-to-noise values was observed. This was probably caused by signal suppression due to accumulation of CTAB. The use of a PDDA coating gave maintained CE resolution with significantly improved MS sensitivity even after fractionation (fig. 2 and 3), though. Since the accepted maximum residue levels of FQs are within the ppb domain a pretreatment step was crucial. Optimizing the amount of MWCNTs, sample and elution volumes, and pH and type of eluent rendered a preconcentration factor of 40 possible, which was sufficient for detection of sample concentrations of 100ppb for all three FQs with possibilities to obtain even lower limits. For successful fractionation the suitable collection starting and ending times were estimated from the migration time as detected with UV detection. Different times for each fraction collection were also investigated to accomplish best separation possible between the three FQs for analysis of spiked milk samples (fig. 4). In summary, an analysis procedure including μSPE preconcentration, CE separation and fraction collection followed by MALDI-MS analysis for the analysis of fluoroquinolones has been developed. Using the optimized methodology the three antibiotics could be detected in milk samples at a concentration of 100 ppb.