Agilent 7693 als

Adenosine and cordycepin were quantified using the method described previously [26 (link)]. Briefly, whole GSC extract fermented with P. pentosaceus ON188 (ONE) were derivatized by N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA with 1% TMCS, Thermo) for trimethylsilylation [27 (link)]. A 0.5μL of derivatized mixture was injected using an Agilent 7693 ALS (Agilent Technologies, Wilmington, DE, USA) in splitless mode into an Agilent 7890B gas chromatograph (Agilent Technologies, Wilmington, DE, USA) for chromatographic separation using Rtx-5Sil MS column. Mass spectrometric analysis was conducted on a LECO Pegasus HT time-of-flight (TOF) mass spectrometer controlled by LECO ChromaTOF software version 4.50 (LECO, St. Joseph, MI, USA). Mass spectra were collected from 85 to 500 m/z at acquisition rate of 17 spectra/second of and detector voltage of 1800 V. Data pre-processing was conducted using ChromaTOF software upon data acquisition in which apex mass values, the entire spectrum, retention time, peak purity, and signal-to-noise ratio were acquired [28 (link)].

The derivatives (0.5 μL) were injected using an Agilent 7693 ALS (Agilent Technologies, Wilmington, DE, USA) in splitless mode. Chromatographic separation was carried out using an Agilent 7890B gas chromatograph (Agilent Technologies) equipped with an RTX-5Sil MS column (Restek, Gellefonte, PA, USA). The oven temperatures were programmed at 50°C for 1 min, ramped at 20°C/min to 330°C, and held constant for 5 min. Mass spectrometry analysis was performed on a Leco Pegasus HT time of flight mass spectrometer controlled by ChromaTOF software 4.50 version (LECO, St. Joseph, MI, USA). Mass spectra were acquired in the mass range of 85–500 m/z at an acquisition rate of 17 spectra/s.
Raw result files were collected and pre-processed using ChromaTOF software, and further process using Binbase, an in-house database. ChromaTOF-specific peg files were converted to generic *.txt result files and additionally as generic netCDF files for further data evaluation. More details can be found in previous reports [13 (link), 14 (link)]. A total of 85 out of 962 metabolic signals were finally reported with occurrence in 50% of the samples per study design group. The quality control was carried out with mixture of 31 pure reference compounds between every 10 samples [13 (link), 15 (link), 16 (link)].