Xbridge beh amide column

During batch culture using glucose as the sole carbon source, C. acetobutylicum cells were collected at 8, 16 and 24 h by centrifugation at 10,000×g for 3 min at − 10 °C. The resulting cell pellets were quenched immediately with 500 μL solution mixture of methanol, acetonitrile and water (40:40:20, v/v, − 40 °C), and then frozen in liquid nitrogen for preparing crude extracts. According to our previous study [22 (link)], LC–MS/MS analysis was conducted for ATP quantification with an ACCELA HPLC system (Thermo Scientific, CA) equipped with an XBridge BEH Amide column (100 mm × 2.1 mm I.D., 2.5 μm, Waters, Ireland). Mass monitoring was achieved using a TSQ Quantum Ultra triple quadrupole mass analyzer (Thermo Scientific, CA) equipped with a heated electrospray ionization source (HESI). NADH assay was performed using a commercial kit (Sigma, MO). Cell pellets were first lysed using a Qiagen Tissue Lyser LT (Qiagen, Germany) at 50 oscillations/s for 3 min in the NADH extraction buffers (Sigma, MO), the resulting lysate was then used for NADH quantification at 450 nm with an iMark™ microplate reader (Bio-Rad, CA).

LC/MS was performed to detect 15N-labeled isotopes of metabolites in the de novo pyrimidine synthesis pathway. LC separation was achieved using a Vanquish UHPLC system (Thermo Fisher Scientific) and an Xbridge BEH Amide column (2.1 mm × 150 mm × 2.5 mm particle size, 130 A °pore size; Waters, Milford, MA), column temperature 25°C. Solvent A is 95:5 water:acetonitrile with 20 mM ammonium acetate and 20 mM ammonium hydroxide at pH 9.4, and solvent B is acetonitrile. Flow rate was 150 mL/min. The LC gradient was 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B. 25 min, stop run. Injection volume was 5 µL. The Q-Exactive Plus mass spectrometer was operated in negative ion mode scanning from m/z 70-1000 with a resolution at 140,000. Data were analyzed by using El-Maven.

A quadrupole-orbitrap mass spectrometer (Q Exactive, Thermo Fisher Scientific, San Jose, CA) operating in a positive ion mode was coupled to vanquish UHPLC system (ThermoFisher Scientific, San Jose, CA) with electrospray ionization and used to scan from m/z 70 to 1000 at 1 Hz and 75,000 resolution. The LC separation was achieved on a XBridge BEH Amide column (2.1 mm × 150 mm, 2.5 μm particle size, 130 Å pore size; Waters, Milford, MA) using a gradient of solvent A (95:5 water: acetonitrile with 20 mM ammonium acetate and 20 mM ammonium hydroxide, pH 9.45) and solvent B (acetonitrile). Flow rate was 150 μL/min. The LC gradient was: 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B; 30 min, 85% B. Autosampler temperature was 5°C, and injection volume was 3 μL. Data were analyzed using the MAVEN software.

To obtain an accurate determination of the amount of sucrose, fructose, and glucose in a complex matrix, a sugar analysis was done using liquid chromatography coupled with mass spectrometry.
Sugars were extracted as previously described [28 (link)]. After thawing, the samples were diluted in 50/50 (v/v) LC/MS water/acetonitrile and were quantified using high-performance liquid chromatography coupled with mass spectrometry (Waters, Beaver Dam, WI, USA).
Metabolites were separated on an XBridge BEH Amide column (length: 150 mm; internal diameter: 4.6 mm; particle size: 3.5 µm; WATERS). The column temperature was set at 75 °C. The flow was 0.4 mL/min and the solvent were acetonitrile with 0.1% formic acid (D) and ultra-pure water (B) + 0.1% formic acid. The elution gradient was as follows: 0 min at 80% D + 20% B, then 50% D + 50% B for 23 min, level at 80% D and 20% D for 2 min. The injection volume was 5 µL, and the injector temperature was 7 °C. Each analysis took 25 min.
Mass spectrometric detection was performed with an ISQ™ EC-LC Quadrupole with a heated electrospray source (HESI–II) operated in the negative ionization mode (Thermofisher Scientific). Metabolites were identified and quantified (ng/g wet weight) using Chromeleon 7.2.10 software (Thermofisher scientific, Waltham, MA, USA).

Liquid chromatography and mass spectrometry were carried out on an Agilent 1260 Infinity pump coupled to a QTRAP5500 (AbSCIEX). The chromatographic separation of NAD⁺ metabolites was achieved using an XBridge Beh Amide column (130Å, 3.5 µm, 201 mm × 100 mm; Waters, Australia) at a flow rate of 0.2 mL/min with a total run time of 30 min. The mobile phase consisted of ACN with 20 mM ammonium acetate and 20 mM NH₄OH (A) and 100% ACN (B). The initial composition of the mobile phase was 15% A and 85% B, which was ramped up to 30% A after 10 min and held for 3 min before being increased again to 70% A for 5 min and 30 s before returning to initial levels and being maintained for the duration of the run. An injection volume of 2.5 µL was used for all samples. The ion source voltage was set at 4500 V, the capillary temperature was set at 350 °C and the pressure was maintained at 45 bar. Data were acquired and analysed with Analyst 1.6.2 and MultiQuant software, respectively.