J w hp 5ms column

The DnFPPS activity was determined by transforming IPP and DMAPP to produce FPP. We put 2 mM MgCl₂, 5 mM of DL-Dithiothreitol, 50 µM of DMAPP, 50 µM of IPP, 50 mM of MOPS (pH 7.5) and an appropriate amount of the DnFPPS (0.483 mg/mL) into the reaction mixture with a total volume of 200 µL. The product of the transformed colonies harboring pET-28a was used as a negative control. After incubating overnight at 37 °C, the mixture was extracted with l-butanol saturated with water, and the diphosphate group was hydrolyzed with potato acid phosphatase at 37 °C.The hydrolyzed products were extracted with n-hexane, filtered with a 0.22 μm membrane filter, loaded on an Agilent J&W HP-5MS column, eluted with N₂ at 1.2 mL/min, detected by Gas Chromatography (GC) (Models 7890B; Agilent Technologies, Palo Alto, CA, USA). The temperature of the oven was first kept at 80 °C for 1 min, then increased to 220 °C at a rate of 10 °C/min, and finally held at this temperature for 10 min. The injector and transmission line temperatures were set at 200 and 250 °C, respectively. The detection of the analytes was performed using a mass spectrometer (MS) (Models 5977 A; Agilent Technologies) and the Nist11 database was used to analyze the compounds.

Gas Liquid Chromatography (GLC) analyses were performed using a Dani GC 1000 instrument (Dani Instruments S.p.A., Cologno Monzese, Italy) equipped with a programmed temperature vaporizer injector and recorded with a Dani DDS 1000 data station. The following capillary columns were used: (i) Agilent J&W HP-5ms column (30 m × 0.25 mm i.d. × 0.25 μm); (ii) Agilent J&W DB-5 column (30 m × 0.25 mm i.d. × 1 μm); (iii) Alltech AT-35 FSOT column (30 m × 0.25 mm i.d. × 0.25 μm). Chiral-GLC analyses were carried out through a CyclodexB (30 m × 0.25 mm × 0.25 μm) or a Chiraldex G-TA (20 m × 0.25 mm × 0.25 μm) column. EI-MS spectra were recorded at 70 eV by GLC-MS and performed on an Agilent 6890 N gas-chromatograph interfaced with an Agilent 5973 N mass detector. Elemental analyses were acquired with an Elementar Vario Micro Cube in CHNS mode.
¹H NMR spectra were recorded on a Varian Gemini 200 or a Bruker 400 MHz spectrometer using tetramethylsilane as an internal standard. ¹H NMR spectral data of the synthetized compounds are reported in Supplementary Materials section.

The metabolomic sample was prepared from 0.2 g of feces as described previously (50 (link), 51 (link)). Metabolites were assayed by gas chromatography-mass spectrometry (GC-MS) using an Agilent 7890A GC system coupled to an Agilent 5975C inert mass selective detector system (Agilent Technologies, Santa Clara, CA). One microliter of prepared sample was injected into an Agilent J&W Hp-5 MS column in the pulsed splitless mode. The injection and interface temperatures were set to 250°C. The initial GC oven temperature was first maintained at 60°C for 5 min, then increased to 70°C for 2 min, further increased to 200°C at 10°C/min, maintained for 10 min, increased at a rate of 4°C/min to 300°C, and maintained at 300°C for 2 min. The carrier gas was helium at a flow rate of 1.0 ml/min. The solvent delay was set to 5 min 25 s. The measurements were made with electron impact (EI) ionization (70 eV) in the full-scan mode (50 m/z to 600 m/z).
Metabolites were identified using the NIST databases (https://www.nist.gov/) (score > 80). Orthogonal partial least-squares-discriminant analysis (OPLS-DA) and variable importance in the projection (VIP) calculations were performed using SIMCA software (version 14.1; Sartorius Stedim Biotech, Umeå, Sweden).