Acrodisc syringe filter

Stock, blank material, and standard solutions of hesperidin were prepared in DMSO-methanol 1 : 1 and filtered through an Acrodisc Syringe filter (13 mm, 0.2 μm, GHP minispike; Waters, USA). Standard curves were prepared by spiking blank milli Q water to yield final concentrations of 0.0019, 0.0076, 0.03, 0.488, 1.95, and 500 μg/mL of diosmin and hesperidin.

HA‐CBT conjugates were analyzed by a Viscotek 270max GPC system (Malvern Instruments Ltd., UK) equipped with an autosampler, a TDA 305 triple detection system, a UV detector 2600, a pump, a degasser, and OmniSec software. A combination of two TSKgel® GPC columns (G4000PW_XL and G3000PW_XL, 30 cm L × 7.8 mm ID) was eluted with triple‐filtered 0.2 M NaNO₃ and 0.01 M NaH₂PO₄ (pH 6.8) aqueous solution at a flow rate of 0.6 ml/min at 35°C. Samples were dissolved in the mobile phase solution and filtered through a 0.22 μm Acrodisc® syringe filter (Waters Corp., Milford, MA). A 100 μl of the polymer solutions was injected for each run. UV absorbance at 330 nm was used to confirm the conjugation of Gly‐CBT to HA.

Different extraction solvents were tested to extract as many pharmaceuticals as possible from the feces samples. Among others, extraction solvent tested included ACN, MeOH, water, mixture of ACN, MeOH and water, mixture of water and ACN or MeOH and Quecher extraction with the use of a mixture of water and an organic solvent (ACN or MeOH) combined with NaCl and MgSO₄. The different extraction solvents were tested on three replicated samples spiked with pharmaceuticals at 120 ng/g and a blank sample (analyte-free). In all cases the procedure was as follows: (i) weigh the sample, (ii) spike the sample with pharmaceuticals and homogenate the sample, (iii) leave to stand for 1 h, (iv) add the adequate volume of extraction solvent, (v) shake the sample in a vortex shaker, (vi) 20 min in a rotary shaker at room temperature (between 18 and 23 °C), (vii) 15 min of centrifugation at 4500 rpm at 4 °C, and (viii) filtrate the supernatant through an Acrodisc Syringe Filter (Waters, MA, USA) and transfer to an HPLC amber vial.
A calibration curve of the standard solution of a mixture of pharmaceutical at 0, 10, 25, 50, 100 and 250 ng/mL was prepared and injected also with the resultant extract to evaluate extraction efficiency.