Polyvinylidene fluoride syringe filter

Conditioned medium (FS03CM) was prepared from an anaerobic bacterium, FS03, which is closely related to Terrisporobacter spp., and had been isolated from soil in a previous study [12 (link)] following the method described by Pahalagedara, Jauregui, Maclean, Altermann, Flint, Palmer, Brightwell and Gupta [27 (link)]. Briefly, colonies of isolate FS03 grown on sheep blood agar (SBA) were used to inoculate the cooked meat glucose starch (CMGS) medium (45 mL) (Fort Richard Laboratories, New Zealand) supplemented with yeast extract (0.0005%), hemin (0.1%), and vitamin K (1%), and incubated inside a 35 °C anaerobic chamber for 48 h. After the incubation period, the culture was centrifuged at 10,000× g for 40 min at 4 °C, and the supernatant was filter-sterilized using a 0.22 µm polyvinylidene fluoride syringe filter (Millipore, Ireland). The sterile conditioned media was aliquoted and stored frozen at −20 °C until use.

All hair samples were washed using methanol, deionized water and methanol in sequence and then dried at laboratory temperature. Each sample was cut into small pieces (1–2 mm length) and weighted. Each analysis was performed using approximately 10 mg of hair, to which 5 μL of 1 μg/mL EtG-d₅ and 95 μL deionized water were added. Then, the samples were quickly spun down and incubated at 4 °C for 15 h. After incubation, the samples were briefly vortexed and centrifuged (10,000 rpm, 30 min, 4 °C). The supernatants were filtered with a 0.45-μm polyvinylidenefluoride syringe filter (4 mm, Millipore, Molsheim, France) and 30 μL was injected into the HPLC-MS/MS system.