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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 of 46 MRMs in 3 periods using dwell times of 50-120 ms dependent on flow rate
2 to 3 internal standards were used in each period
Determination of group A substances
For doubtless determination of betaagonists, 4 identification points have to be achieved (guideline 93/256/EWG). That means in case of HPLC/MS/MS 2 MRMs (1 precursor ion and two product ions) have to be measured. The ratio of quantifier and qualifier MRM must be stable with a tolerance of 20 to 50% independent of relative intensity. Retention times must be higher than 2 times of the dead volume of the column and relative retention times are allowed to shift by 2.5%.
Results
By using monolithic columns at higher flow rates, the time for analysis can be reduced drastically (Figures 1-3). At flow rates below 1000 μL/min peak shape on this column is not satisfying (not shown). In the case of analysed -agonists the time of analysis dropped from 43 minutes to 15 minutes (1000 μL/min) or 8 minutes (2000 μL/min), respectively, including equilibration time (Figure 4). Due to optimised gas streams in the ion source and the possibilities of using higher temperatures for evaporation, similar or even lower limits of detection (LOD) were achieved for analyzed compounds at 1000 μL/min (Table). Little loss of sensitivity must be tolerated when flow rates of 2000 μL/min or higher are used. LODs of 1000 and 2000 μL/min are comparable when compounds elute with higher organic content due to easier evaporation of such solvents.
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