26


May/June 2011


Analysis of Aminoglycosides with a Zwitterionic HILIC Stationary Phase and Mass Spectrometry Detection


by Wen Jiang*, Patrik Appelblad, Tobias Jonsson, Petrus Hemström Merck SeQuant AB, Tvistevägen 48, S-90736, Umeå, Sweden * Corresponding author: Tel: +46 90 154985; Fax: +46 90 154883; E-mail: wen.jiang@merckgroup.com


A method was developed to analyse three model aminoglycosides (neomycin, apramycin, and kanamycin) using a zwitterionic HILIC (hydrophilic interaction liquid chromatography) stationary phase and mass spectrometry (MS) detection. High separation efficiency and good peak shape were obtained with an optimal combination of mobile phase composition and gradient profile. Gradient elution proved to be reproducible and a good way to combine both HILIC and electrostatic interactions to obtain unique selectivity. The possible lower limit of quantification (LLOQ) was under 0.25 µg/mL using HILIC coupled with a single quadrupole ESI-MS instrument.


Introduction Aminoglycosides are bactericidal antibiotics that have amino-modified sugar in their structures [1-3]


. This particular group of


antibiotics are widely used as clinical and veterinary medicines to treat infections caused by gram-negative or some gram-positive organisms, and are classified as bactericidal agents because of their interference with bacterial replication. However, theses antibiotics can also cause varying degree of ototoxicity and nephrotoxicity. McGlinchey et al. described in a recent review that the overuse of antibiotics and exposure to the food animals are the two major routes attributed to the antibiotic resistance [3]


.


Therefore, it is very important to develop sensitive and reliable analytical methods for determining and monitoring aminoglycosides residue in different sample matrices.


Aminoglycosides are normally very hydrophilic and carry several amino groups, which mean they are positively charged at neutral pH condition. It is very difficult to separate them by reversed-phase liquid chromatography (RPLC) unless an ion-pair reagent or pre- column derivatization is applied. Furthermore, aminoglycosides usually have no chromophores in the molecular backbone, which limits the use of light absorption detection. Despite these technical barriers,


scientists have developed several LC methods [2,3]


, e.g. RPLC with pre-column derivatization and UV or fluorescent detection [4-7]


, ion-pair


RPLC with evaporative light scatter detection (ELSD) or mass spectrometry (MS) [8,9]


chromatography with electrochemical or fluorescent detection [11,12]


.


emerged in recent years as an alternative technique for the separation and detection low levels of aminoglycosides [12-15]


HILIC is a straightforward chromatographic separation mode, which is suitable for separation of polar and hydrophilic compounds without any sample derivatization or ion-pair reagent in the mobile phase. It is ideal to combine the HILIC with MS because HILIC uses large proportion of organic solventthat enhances the ionization and desolvation in ESI-MS detection. Through literature searching, it was found that most published HILIC-MS methods for aminoglycoside separation are based on a zwitterionic HILIC stationary phases with ESI- MS in positive mode [12-14]


. As yet the


separation mechanism on such HILIC phases has not been clarified. In this work, we aim to understand what are the most critical factors affecting separation efficiency, selectivity and detection sensitivity under HILIC conditions.


Experimental Chemicals and Reagents:


Neomycin, apramycin, kanamycin and ammonium acetate were all purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile (HPLC grade) was obtained from Merck (Darmstadt, Germany). Formic acid was


, and ion . HILIC-MS has also


from J.T. Bakers (Deventer, The Netherlands). Water was purified by a bench-top Milli-Q water purification system from Merck Millpore (Billerica, MA, USA).


LC-MS Instrumentation and Conditions HPLC Pump: Shimadzu LC-20AD HPLC Pump


Autosampler: Shimadzu SIL-20AC Column: SeQuant ZIC®


-HILIC,


100 x 2.1 mm, 3.5 µm/100 Å (Merck, P/N 1504410001)


Mobile Phase: A: acetonitrile with 1 w% formic acid


B: 150 mM ammonium acetate with 1.5 w% formic acid


Flow Rate: 0.4 mL/min Injection: 20 µL Temperature: 50 ˚C UV Detection: Shimadzu


SPD-20A UV/Vis Detector


MS Detection: Shimadzu LCMS-2010EV; ESI in positive mode; neomycin: m/z 615; Apramycin: m/z 540; Kanamycin: m/z 484; capillary voltage: 4 kV; Block temperature 250 ˚C; spray gas: nitrogen at 1.5 L/min; detector voltage: 2 kV.


Data Acquisition: Shimadzu LCMSsolution Chromatography Software; Version 3.30


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