Surveyor ms pump plus

The LC system consisted of a quaternary, low-pressure mixing pump with vacuum degassing, type Surveyor MSpump Plus and an autosampler with temperature controlled tray and column oven, type Autosampler Plus, both from ThermoFisher Scientific (Breda, The Netherlands). Chromatographic separation was achieved on a Zorbax Eclipse Plus column (100 mm × 3.0 mm i.d., dp: 3.5 μm) in combination with a guard column of the same type (13 mm × 3.0 mm i.d., dp: 3.5 μm), both from Agilent (Diegem, Belgium). The temperatures of the column oven and autosampler tray were set a 45°C and 5°C, respectively. Mobile phase A consisted of 0.1% glacial acetic acid in water whereas mobile phase B was ACN. Following gradient elution program was run: 0–3.5 min (92% A, 8% B), 3.5-4.0 min (linear gradient to 80% A), 4.0-8.0 min (80% A, 20% B), 8.0-8.5 min (linear gradient to 92% A), 8.5-13.0 min (92% A, 8% B). Flow rate was set at 500 μL/min.

Qualitative and quantitative analyses were carried out by LC–MS/MS using a LC system Surveyor MS PUMP PLUS (Thermo Fisher Scientific, Monza, Italy) coupled with an LTQ ion-trap mass spectrometer (Thermo Fisher Scientific, Monza, Italy) equipped with an ESI source operating in the negative mode. The column used was a XSelect HSS T3 XP (100 Å, 2.5 μm, 2.1 × 100 mm; Waters, Sesto San Giovanni, Italy) with a flow rate of 0.15 mL/min. A gradient elution was performed, and the mobile phase was a mixture of water containing 0.1% formic acid (A) and acetonitrile (B). The elution gradient was set as follows: 0–1 min (5% B), 1–10 min (5–100% B), 10–15 min (100% B), 15–25 min (100–5% B). The operating conditions for MS analysis were as follows: spray voltage, −5 kV; capillary temperature, 250 °C; sheath gas and auxiliary gas flow, 60 and 5 arbitrary units, respectively; tube lens, −110 V; total ion current (TIC); base peak-dependent mass range, m/z 230–1500; collision energy, 20 eV.
Compounds in the extract (injection: 25 μg) were identified by comparison of the retention times and mass spectra with those of authentic compounds (injection: 5 or 10 ng) commercially available at a high grade of purity.

An LTQ XL Linear Ion Trap coupled with Surveyor MS Pump Plus (Thermo Scientific, Waltham, MS, USA) was used with sheath and auxiliary gas flow at 40 and 10 arbitrary units, respectively, capillary temperature 275 °C, isolation width 1.0, with source voltage at 3.5 kV (negative ionization) and 5 kV (positive ionization). Separations were achieved using a Kinetex F5 LC column (2.6 μm, 150 × 2.1 mm, Phenomenex) and mobile phases (A) water and (B) 95% CH₃CN, both containing 2 mM formic acid and 3.6 mM ammonium formate. The gradient (0.2 mL min⁻¹) was A 75–30% over 6 min, 30–75% A over 3 min, and held at 75% A for 2 min, with 20 μL full-loop injections.
Oxidized standards (10 μg mL⁻¹) of MC-LR (9), [ADMAdda⁵]MC-LR (3) and [DMAdda⁵]MC-LR (1) were scanned in negative ionization mode. A list of target molecular ions was generated based on full-scan mass spectra. MS/MS spectra (negative ionization) were obtained using 20% CE for m/z 207 (MMPB), m/z 193 (2R-methyl-3S-hydroxy-4-phenylbutanoic acid (MHPB)) and m/z 291 (2-methyl-3-oxo-4R-methyl-5S-acetyloxy-6-phenylhexanoic acid (MOMAPH)). MS/MS/MS spectra were obtained for additional characterization of m/z 291 (MOMAPH) using 35% CE of the dominant product ion in the MS/MS spectrum (m/z 231). Positive ionization was also used for MS/MS characterization of m/z 293 (MOMAPH).

All extracted samples were analyzed in triplicate for TTX using LC-MS/MS, according to the method of Lee et al. [34 (link)]. TTX was quantified using a triple-quadrupole mass spectrometer (TSQ Quantum Discovery Max; Thermo Electron, San Jose, CA, USA) coupled to an HPLC system. The HPLC unit consisted of a Surveyor MS Pump Plus and Surveyor AS Plus (Thermo Electron, San Jose, CA, USA). Chromatographic separations were performed on a TSK Gel-amide-80 column (250 × 0.2 mm, 5 μm; Tosoh Bioscience, Tokyo, Japan) preceded by a guard column cartridge (4.0 × 0.2 mm; Tosoh Bioscience). Eluents A (0.1% formic acid) and B (methanol) were used for the linear gradient elution, with 10 µL of injection volume. The mass spectrometer was operated with SRM, detecting in positive ionization mode, with the product ions at m/z 302, 256, and 162 from the parent ion at m/z 320 for quantification of the TTX (Supplementary Figure S1). The calibration curve was generated with TTX standard solutions (6.25, 12.5, 25, 50, and 100 ng/mL), which were >0.99 of the correlation coefficient (r²) with good linearity. The limit of detection (LOD) and the limit of quantitation (LOQ) were determined using a signal-to-noise ratio (S/N = 3) and a concentration of LOD × 3, respectively. LOD and LOQ were 0.01 μg/g and 0.03 μg/g, respectively. The TTX concentrations in the samples are expressed as μg/g wet weight.