β glucuronidase from helix pomatia type h1

The reference standards curcumin (≥98% HPLC), demethoxycurcumin (≥98% HPLC), bisdemethoxycurcumin (≥98% HPLC), piperine analytical standard and β-glucuronidase from Helix pomatia-type H-1 (partially purified powder) were purchased from Sigma-Aldrich (St. Louis, MOI, USA), The Netherlands). Tetrahydrocurcumin, and the Internal Standards (IS) curcumin-D6, (2E)-demethoxycurcumin-d7, (E,E)Bisdemethoxycurcumin-D8, Tetrahydrocurcumin-D6 and Piperine-D10 were purchased from Toronto Research Chemicals (Toronto, Canada). Dimethylsulfoxide (DMSO) was purchased from Merck (Darmstadt Germany), formic acid was purchased from Sigma Aldrich and, Methanol absolute (MeOH) and tert-butylmethyl ether (TBME)) was purchased from Biosolve BV (Valkenswaard, The Netherlands). Milli-Q water was obtained with an Elga Medico Pro watersystem. Blank human plasma (Omniplasma® (Octapharma GmbH, Langenfeld, Germany)) was used to prepare calibration and quality control (QC) samples. Omniplasma is a product produced from pooled Dutch obtained human plasma which underwent a solvent detergent ion and prion reduction step. Single blank human urine and feces samples were obtained from a healthy volunteer that followed a restriction diet for 5 days in which any products containing curcumin and piperine were avoided.

Analytical grade chemicals and reagents were used in this analysis including Quercetin (purity ≥ 95%), trans-resveratrol (purity ≥ 98%), fenofibric acid (FFA, purity ≥ 98%), and β-glucuronidase from Helix pomatia (Type H-1) (partially purified) purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS grade methanol (MeOH) and acetonitrile (ACN) purchased from JT Baker (Radnor, PA, USA) were used. Ethyl acetate from Merck Life Sciences. Pvt. Ltd. (Mumbai, India), and formic acid and ammonium acetate from Sisco Research Laboratories (SRL) Pvt. Ltd. (Mumbai, India).

β-Glucuronidase from Helix pomatia type H1 was obtained from Sigma Aldrich (St. Louis, MO, USA). The enzymatic solution was prepared weekly by disolving the β-glucuronidase purified powder in ammonium acetate 1M (acetic acid was added to pH = 5) to obtain a solution of 3500 U/mL.