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, submicrometer 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 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.

Instrumental

LC–MS/MS: Agilent™ 1200 HPLC and AB Sciex™ 4000 Q Trap (MS/MS)
Column: 100 × 2.1 mm, 3-μm UCT Selectra® DA HPLC column
Guard column: 10 × 2.1, 3-μm UCT Selectra® DA guard column 
Injection volume: 10 μL
Mobile phase A: D.I. H₂O + 0.1% formic acid
Mobile phase B: Methanol + 0.1% formic acid
Column flow rate: 0.30 mL/min

Results

Conclusion

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 and UHPLC column life.

UCT, LLC
2731 Bartram Rd., Bristol, Pennsylvania 19007, USA
Tel.: (800) 385 3153
Website: www.unitedchem.com