UCT - In-Well Hydrolysis Using UCT’s Refine Ultra-Filtration Technology
UCT’s Refine Ultra-Filtration Technology allows for sample precipitation and filtration to occur simultaneously in the individual column or well. A novel, hydrophobically treated, submeter frit combination facilitates the removal of sample proteins without a complicated extraction.
Procedure
Sample Extraction
a) Place a collection plate under the Refine Ultra-Filtration Plate
b) Add 250 μL of urine and 250 μL Abalonase Ultra Enzyme working solution.
c) Hydrolyze for 30 min at 55°.
d) Further dilute sample by adding 500 μL D.I. H2O.
e) Mix the sample. This can be executed via vortexing at maximum speed or multiple pipette aspirations and dispenses.
f) Filter the sample using one of the following techniques:
1. Centrifuge: For 5 min at 500 g or until filtrate is collected.
2. Vacuum: Apply vacuum at ~20” of Hg for up to 5 minor until filtrate is collected.
3. Positive Pressure: Apply 2–5 psi using positive pressure for up to 5 min or until filtrate is collected.
Instrumental
LC-MS/MS: AgilentTM 1200 HPLC and AB SciexTM 4000 Q Trap (MS/MS)
Figure 1: Backpressure per sample injection of Refine Ultra Filtration technology versus a dilute and shoot preparation for hydrolyzed urine samples.
Column: UCT Selectra® DA HPLC Column 100 × 2.1 mm, 3 μm
Guard Column: UCT Selectra DA Guard Column 10 × 2.1 mm, 3 μm
Injection Volume: 10 μL
Mobile Phase A: D.I. H2O + 0.1% Formic Acid
Mobile Phase B: MeOH + 0.1% Formic Acid 0.30 mL/min
Column Flow rate: 0.30 mL/min
Conclusion
Using the above procedure, the Refine Ultra-Filtration technology allowed for a reduction in instrumental backpressure and removal of unwanted matrix components in urine samples. Overall, this allows for an end user to have enhanced analyte selectivity and an increase in HPLC/UPLC column life.
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