Milli q uf plus apparatus

All chemicals, including methanol, formic acid, and acetonitrile, were purchased from Sigma-Aldrich (Milan, Italy). Ultra-pure water was obtained using a Milli-Q ® UF-Plus apparatus (Millipore, Bedford, MA, USA). For UHPLC-QTOF-HRMS quantitative analysis, ketamine-d4 was purchased from LGC Promochem (Milan, Italy) (purity >99%, concentration 1 mg/mL). Methoxpropamine was kindly provided by the Italian National Institute of Health (methanolic solution at a 0.02 mg/ mL concentration, purity provided by the supplier >99%). All working solutions were prepared in methanol at 1 μg/mL and stored at À20 C until used. MXPr was dissolved in Tween 80 (2%) and ethanol (5%), brought to the final volume with saline (0.9% NaCl). Mice were treated by intraperitoneal (i.p.) injection (volume of 4 μl/g) in subsequent days and divided into two groups (4 males and 4 females for each group).

(trimethylsilyl) trifluoroacetamide (MSTFA) were provided by Sigma-Aldrich (Milan, Italy). βglucuronidase from Escherichia coli was purchased from Roche Life Science (Indianapolis, IN, USA) and ammonium iodide, was from TCI Co., Ltd. (Tokyo, Japan). C-18 endcapped Solid-Phase Extraction (SPE) cartridges were provided by UCT Technologies (Bristol, PA, USA). Ultra-pure water was obtained from a Milli-Q® UF-Plus apparatus (Millipore, Bedford, MA, USA). Stock standard solutions were stored at -20 °C until used.. Two working solution mixtures were prepared in methanol at final concentrations of respectively 1 µg mL -1 (working solution A, composed by all reference substances with the exception of androsterone and etiocholanolone) and 10 µg mL -1 (working solution B, composed by androsterone and etiocholanolone). These concentrations were chosen according to the upper limit of their endogenous concentration interval [9, 22] . Two separate internal standard working solutions were prepared in methanol for testosterone-d3 and 17αmethyltestosterone at the final concentration of 10 µg mL -1 .

All steroid standards were purchased as pure powders from Steraloids (Newport, RI, USA); DHT was provided by LGC Promochem (Milan, Italy). Methanol, methyl tert-butyl ether (TBME), ethyl acetate, 17α-methyltestosterone, dithioerythritol and N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) were provided by Sigma-Aldrich (Milan, Italy). β-glucuronidase from Escherichia Coli was purchased from Roche Life Science (Indianapolis, IN, USA). Ultra-pure water was obtained using a Milli-Q® UF-Plus apparatus (Millipore, Bedford, MA, USA).
Standards solutions were prepared in Methanol at the concentration of 1 mg/mL. Then, three working solution mixtures were prepared by dilution. Two internal standards were used: testosterone-d 3 served for the quantification of all the target analytes, with the exception of A and Etio which were quantified by 17α-methyltestosterone. The list of the studied steroids, together with details about each working solution are reported in Table 1. All standard solutions were stored at 10°C until used.