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 Sciex^TM 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.

UCT, LLC

2731 Bartram Rd, Bristol, PA 19007

tel: (800) 385-3153

Website: www.unitedchem.com