Applications

Phenyl-Hexyl

Strata-X-Drug B 33 µm Polymeric Strong Cation, 30 mg / 3 mL, Tubes, 50/Pk

Load: Samples from hydrolysis Using b-Glucoronidase as below To 200 µL Urine (calibrator, control) in a 1.5 mL micro centrifuge tube, add 20 µL combined IS (Internal Standards) spiking solution. Vortex 10-15 seconds followed by adding 100 µL 0.1 M Ammonium acetate buffer (pH 4.0) to each tube, mix/vortex another 15 seconds. Add 40 µL ß-Glucoronidase solution (100,000 units/mL). Vortex 10-15 seconds. Incubate for 2 hours in a shaker at 55 °C to complete hydrolysis of the glucuronides. Add 400 µl of 0.1% Formic acid and vortex for another 60 seconds. Centrifuge the hydrolyzed samples at 21,000 g for 10 minutes (to separate any proteins). Collect supernatant for further cleanup via Solid Phase Extraction (SPE). Wash 1: 1 mL 0.1% FA Wash2: 1 mL 30% methanol Dry: 3 to 4 mins at high vacuum (~10” of Hg) Elute: 2x500 uL 2% ammonium hydroxide/methanol: acetonitrile (1:1) Blow Down: To dryness under nitrogen at 40-45o C Reconstitute: With 200 uL of 85/15 (A/B) of LC mobile phase (Note: 1. No conditioning or equilibration needed in extraction) 2.Total of 15 internal standard used for the quantitation of 41 analytes.

Methanol

0.1% Formic acid/water

Load: Samples from hydrolysis Using b-Glucoronidase as below To 200 µL Urine (calibrator, control) in a 1.5 mL micro centrifuge tube, add 20 µL combined IS (Internal Standards) spiking solution. Vortex 10-15 seconds followed by adding 100 µL 0.1 M Ammonium acetate buffer (pH 4.0) to each tube, mix/vortex another 15 seconds. Add 40 µL ß-Glucoronidase solution (100,000 units/mL). Vortex 10-15 seconds. Incubate for 2 hours in a shaker at 55 °C to complete hydrolysis of the glucuronides. Add 400 µl of 0.1% Formic acid and vortex for another 60 seconds. Centrifuge the hydrolyzed samples at 21,000 g for 10 minutes (to separate any proteins). Collect supernatant for further cleanup via Solid Phase Extraction (SPE). Wash 1: 1 mL 0.1% FA Wash2: 1 mL 30% methanol Dry: 3 to 4 mins at high vacuum (~10” of Hg) Elute: 2x500 uL 2% ammonium hydroxide/methanol: acetonitrile (1:1) Blow Down: To dryness under nitrogen at 40-45o C Reconstitute: With 200 uL of 85/15 (A/B) of LC mobile phase (Note: 1. No conditioning or equilibration needed in extraction) 2.Total of 15 internal standard used for the quantitation of 41 analytes.

0.1% Formic acid/water

30% methanol

Evaporate to dryness