Fisons ea 1108 chns o element analyzer

Elemental compositions of the HSs were determined using a Fisons EA-1108 CHNS-O Element Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) at University of Naples Federico II. The amounts of total carbon, nitrogen, and sulfur were determined. Roughly 2–5 mg of the sample was measured on a precision balance and mixed with an oxidizer (vanadium pentoxide [V₂O₅]) in a tin capsule. The container and the sample were combusted in a reactor at 1000 °C, and, after melting, the tin container promotes a violent reaction in the oxygen-enriched atmosphere. The products of this reaction (CO₂, SO₂, NO₂) are carried by the carrier gas (helium) to the packed columns. The measured signal was quantified with a thermal conductivity detector (calibrated against pre-analyzed standards), and the elemental content of the sample was calculated in weight percentage. We corrected the measured data for moisture and ash content, and we calculated the oxygen content [30 (link)].

Three dose-levels of the test article were prepared by diluting the thawed stock solution in 0%, 25 % or 50 % distilled water. These dose-levels were tested against 100 % distilled water as the control vehicle. The respective dilutions resulted in a daily dosage of the test article of 0 (distilled water), 250, 500 and 1000 mg TOS/kg bw/day, based on the analyzed TOS content of the test article of 21.75 %. These dose levels were equivalent, respectively, to 112,500, 225,000 and 450,000 FTU/kg bw/day, based on the known activity and percentage TOS of the product described in section 2.1. The test dose-levels were selected based on the OECD limit dose of 1000 mg/kg/day (and dilutions thereof) on the basis that enzymes are not considered toxic by the oral route [25 (link),26 (link)]. Dosing formulations were prepared and used within the analytically established range of stability of the test article and refrigerated at -4 °C before use. Dose formulations were collected on three occasions during the experiment (beginning, middle, end), frozen, and analyzed together for concentration of protein (mg/mL) by CHNS Elemental analysis using a nitrogen/protein analyzer (Fisons EA 1108 CHNS-O Element Analyzer, Thermo Fisher Scientific, USA) to verify target dose levels.