2.4 Instrumental analysis

The derivatized samples were processed by on-line pre-concentration and desalting coupled to ultra-high-performance liquid chromatography high resolution Orbitrap mass spectrometry (UHPLC-HRMS), using the EQuan interface and the Chromeleon 7.2 Software (Thermo Fisher Scientific, Waltham, MA, U.S.A., and Dionex Softron GMbH, part of Thermo Fisher Scientific, Germany). A PAL RTC autosampler (Zwingen, Switzerland) was used for sample injection into a 2-mL stainless steel (SST) loop. A Thermo Dionex UltiMate^™ 3000 pump was used for on-line pre-concentration and desalting, and a Thermo Dionex UltiMate^™ 3000 RS pump for subsequent UHPLC separation.

The pre-concentration step consisted in loading 2 mL of the sample onto a Thermo HyperSep Retain PEP column (hydrophilic lipophilic balance, 20 mm x 2.1 mm, 40–60 μm particle size) at a flow rate of 2,000 μL min⁻¹. After the loading step, the on-line aqueous mobile phase (HPLC water) was left to flow for 1 min at 2,000 μL min⁻¹ to allow salt removal. Analytes were then back-flush eluted at 450 μL min⁻¹ with the analytical mobile phase (A: 2.5 mM CH₃COONH₄ in HPLC water; B: ACN). Chromatographic separation was achieved by a Thermo Hypersil Gold C18 column (100 mm x 2.1 mm, 1.9 μm particle size, 175 Å pore size) thermostated at 35 °C and fitted with a 0.2 μm column pre-filter. Details on the chromatographic gradient elution programs are provided in SI (S3 Table).

The FMOC-derivatized analytes were detected in negative electrospray ionization mode using a Thermo Q-Exactive Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, U.S.A.) operated in full scan MS mode (scan range: 200–600 m/z). Orbitrap parameters were as follows: resolution was set at 70,000 full width at half maximum (FWHM; value at 200 m/z), automated gain control (AGC) at 3E6, and injection time (IT) at 100 ms. Further details including source parameters are provided in SI (S3 Table). LC-MS data quantification was carried out using Xcalibur 3.0 software (Thermo Scientific).