In-Well Hydrolysis Using UCT’s Refine Ultra-Filtration Technology

Published on: 
The Application Notebook, The Application Notebook-06-01-2018, Volume 36, Issue 6
Pages: 422

UCT's Refine™ Ultra-Filtration technology allows for sample precipitation and filtration to occur simultaneously in the individual column/well. A novel, hydrophobically treated, submicron frit combination facilitates the removal of sample proteins without a complicated extraction.


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 °C.

d) Further dilute sample by adding 500 µL D.I. H2O.

e) Mix 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 min or until filtrate is collected.

3. Positive Pressure: Apply 2–5 psi using positive pressure for up to 5 min or until filtrate is collected.


LC–MS/MS: Agilent™ 1200 HPLC and AB Sciex™ 4000 Q Trap (MS/MS)

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

Column flow rate: 0.30 mL/min


Table I: Percent reduction in analyte suppression following use of Refine™ plate compared to dilute and shoot preparation for hydrolyzed urine samples.


Using the above procedure, the Refine™ Ultra-Filtration technology allowed for a reduction in instrumental back pressure 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.

Figure 1: Back pressure per sample injection of Refine™ Ultra Filtration technology versus a dilute and shoot preparation for hydrolyzed urine samples.

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