Agilent 1100 series liquid chromatograph

For sugar sand, sugars were extracted before analysis. Briefly, 2 g of sample was vortexed for 5 min with 10 mL of ethanol diluted to 80% vol. with nanopure water. The mixture was heated for 10 min at 70 °C in a water bath with constant agitation, vortexed again for 20 s and then centrifuged at 1000× g for 5 min. The supernatant was dried under reduced pressure in a rotavapor apparatus. The total sugar content was determined using the phenol–sulfuric acid method [28 (link)].
Sugars extracted from sugar sand and sugars in maple syrup samples were identified using a Waters Sugar-Pak I column (300 × 6.5 mm, 10 μm) on an Agilent 1100 series liquid chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a refractive index detector (Agilent, 1260 Infinity). The isocratic solvent system contained calcium disodium EDTA (50 mg/L). Injected sample volume was 50 μL, the flow rate was 0.5 mL/min for a sample run time of 30 min and the column temperature was set at 90 °C.

Semi-preparative high performance liquid chromatography was performed on Agilent 1100 Series liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with diode array detector (DAD; λ=520 nm, λ=450 nm), autosampler, and fraction collector; conditions: injection volume, 600 μL (2 mg/mL, methanol); column Zorbax Eclipse XDB C18 (250 mm x 9.4 mm; 5 μm); mobile phase (6 mL/min), water (40 %) and methanol (60 %). Sephadex-LH-20 purchased from GE Helthcare (Uppsala, Sweden) was used for column chromatography. Preparative TLC was performed by using silica gel P/UV254 with CaSO₄ (Machery-Nagel, Germany, 2 mm layer of adsorbent). Analytical TLC was performed on silica gel (Silica gel 60, layer 0.20 mm, Alugram Sil G, Mashery-Nagel, Germany). All spectroscopic measurements were performed with a double beam UV-Vis spectrophotometer model Cary 300 (Agilent Technologies, Santa Clara, USA) with 1.0 cm quartz cells. Fluorescence measurements were carried out using a RF-1501 PC spectrofluorometer (Shimadzu, Japan) equipped with a 150 W Xenon lamp source with a 1.0 cm path length quartz cell. Color reaction was measured spectrophotometrically on ELISA (2100C) 96-well microplate reader (Rayto, China).

The MCs used for dosing solutions were obtained from Enzo Life Sciences (MCLR, MCLY, MCYR) or Beagle Bioproducts (MCLA, MCRR). MCs were reconstituted into stock solutions using Picopure^® water at a target concentration of 1 g/L. To ensure 100% reconstitution of the dry MCs, stock solutions were sonicated for one hour, followed by overnight refrigeration and an additional hour of sonication. Individual dosing solutions were prepared for each dosing group by diluting stock solutions with Picopure^® water. Dosing concentrations were calculated assuming a 0.2 mL dose volume, or 0.4 mL in the case of high MCRR dose levels, and a mouse weight equivalent to the average weight of mice in each group being dosed.
The concentration of each stock solution was verified using an Agilent 6210 series time of flight mass spectrometer (MS-TOF) coupled to an Agilent 1100 series liquid chromatograph (LC). MC stock solutions were quantified against a reference material purchased from an independent vendor for each MC with two separate methods (i.e., external calibration curves and standard addition curves). All stock solutions were verified to be within 25% of the target concentrations except for MCRR (Table 1). For MCRR, the concentration in the stock solution was verified to be lower than the target, so it was assumed to be 0.85 g/L in dosing solution preparation.