Cypex CYP2C8 QC Assays

Activity: Paclitaxel 6a-hydroxylase

Incubation conditions
Assay buffer: 0.1 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 200 µl
Stop reagent: acetonitrile (100 µl)

HPLC conditions
Column: Hypersil BDS-C18 (3 µm) 100 x 4 mm
Temperature: 40°C
Mobile phase: 20 mM potassium phosphate pH 7/acetonitrile 57.5/42.5 (v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 8.5 min
Injection volume: 75 µl
Detection: UV, l = 230 nm
Retention times:
      6a-hydroxypaclitaxel          4.5 min
      paclitaxel                        18.5 min

The substrate, paclitaxel, and metabolite, 6alpha-hydroxypaclitaxel are both available from Cypex Ltd.

Effect of phosphate concentration on paclitaxel turnover by CYP2C8R Bactosomes
Paclitaxel 6a-hydroxylation by CYP2C8 is sensitive to the concentration of phosphate in the incubation.

This assay was carried out at 25 µM paclitaxel at a P450 concentration of 2 pmol per 200 µl.

Made under licence from BTG International Ltd (AU730155, EP0914446, US6566108 and other patents pending).

United States Patent Nos. 5,420,027 or 5,240,831, Canada Patent No. 2100245 and other patents pending.