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The combination of monolithic columns and modern HPLC/MS/MS instrumentation for high chromatographic throughput and sensitive detection of beta-agonists

Beta-agonists are group A substances of the European guideline 93/257/EWG (revision 98/536/EG) having an anabolic effect and not licensed. Thus, they have to be determined in veterinarian samples like muscle, liver, retina, and urine. The coupling of High Pressure Liquid Chromatography (HPLC) and Electrospray Ionization Tandem Mass Spectrometry (ESI-MS-MS) seems to be the analytical method of choice for such thermally labile and polar substances. Classical HPLC/ESI-MS-MS instrumentation allows the determination with satisfying detection limits, accuracy, and reproducibility. Due to the high number of substances that have to be analyzed in a single run, longer chromatographic separation times have to be accepted. Monolithic columns are made of a single piece of porous silica with a defined pore structure. Thus, lower back pressure in the column allows the use of higher flow rates and flow gradients, without loss in chromatographic resolution. In the following work the possibility of increasing sample throughput and sensitivity using monolithic columns at higher flow rates and a modern mass spectrometer, which is optimized to work with such high flow rates is evaluated.

High Pressure Liquid Chromatography

: Agilent 1100 series binary pump, column oven, and well-plate autosampler

columns: Inertsil ODS-3 150x2.1 mm at a flow rate of 200 μL/min Merck Chromolith SpeedROD RP-18e 50x4.6 mm at flow rates of 1000 μL/min and 2000 μL/min

eluent A: H2O+5mM NH4formiate pH=4.0 eluent B: acetonitrile+5% H2O

gradients (A/B):

column oven temperature of 25C

injection of 20 μl Mass Spectrometry:

Applied Biosystems API 4000 LC/MS/MS System with Turbo V source in positive ion mode 500C at 200 μl/min 700C at 1000 and 2000 μl/min (without split)

detection
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