Advance uhplc system

Atglistatin of tissue preparations (>10 mg) was extracted twice according to Folch et al.35 (link) using chloroform/methanol/water (2/1/0.6, v/v/v) containing 500 nmol l⁻¹ BHT and 15 pmol internal standard (NM-421, Atglistatin derivative) per sample. Extraction was performed under constant shaking for 60 min at RT. After centrifugation at 1,000 g for 15 min at RT the organic phase was collected. Combined organic phases of the double-extraction were dried under a stream of nitrogen and dissolved in chloroform/methanol/2-propanol (2/1/12, v/v/v) for liquid chromatography–mass spectrometry analysis. Chromatographic separation was performed using an Advance-UHPLC system (Bruker, Billerica, Massachusetts, USA), equipped with a Kinetex C18 column (2.1 × 50 mm, 1.7 μm; Phenomenex, Torrance, California, USA). Solvent A and B consisted of methanol/water (1/1, v/v) and 2-propanol, respectively, containing 0.1% formic acid and 10 mmol l⁻¹ ammonium acetate. An EVOQ Elite mass spectrometer (Bruker) equipped with an electro spray ionisation (ESI) source was used for detection. Analyte ions were monitored in multiple reaction monitoring mode (Atglistatin, Qualifier 284→239, Quantifier 284→224; NM-421, Qualifier 270→255, Quantifier 270→227). Atglistatin from 25 pmol l⁻¹ to 250 nmol l⁻¹ was used for calibration.

NAPE species were separated on a Waters Acquity UPLC BEH C₈ (1.7 μm, 1 mm x 100 mm; Milford, MA, USA) column, thermostatted to 50°C in an Advance UHPLC system (Bruker Daltonics, Bremen, Germany). Mobile phase A was deionized water containing 1 vol% of 1 M aqueous ammonium formate and 0.1 vol% formic acid; mobile phase B was acetonitrile/2‐propanol (5:2, v/v) with the same concentrations of additives. The binary gradient started with 85% B and linearly increased to 100% B over 15 min. 100% B were held for 3 min before the column was re‐equilibrated with 85% B for another 12 min. The flow rate was 100 μL/min, the autosampler tray was kept at 10°C and 2 μL of sample were injected.

The LC–MS analysis was performed using an Advance UHPLC system (Bruker, Bremen, Germany) with a Kinetex XB-C18 column (100 × 2.1 mm, 1.7 μm, Phenomenex, Palo Alto, U.S.A.) coupled to an EVOQ Elite TripleQuad mass spectrometer equipped with an electrospray ionisation source (ESI) (Bruker, Bremen, Germany). The method was performed as previously described [46 ,47 (link)] using formic acid (0.05%) in water and formic acid (0.05%) in acetonitrile as mobile phase A and B, respectively. Ionisation was obtained by electrospray ionisation in positive ionisation mode with spray voltage of 3500 V and probe and cone temperatures at 400 and 350°C, respectively. Parent ion to product ion transitions were monitored for one aromatic (2-phenylethyl), three indolic (indol-3-ylmethyl, N-methoxy-indol-3-ylmethyl, 4-methoxy-indol-3-ylmethyl) and nine aliphatic (3-methylthiopropyl, 3-methylsulfinylpropyl, 4-methylthiobutyl, 4-methylsulfinylbutyl, 5-methylsulfinylpentyl, 7-methylthioheptyl, 7-methylsulfinylheptyl, 8-methylthiooctyl, 8-methylsulfinyloctyl) glucosinolates. Quantification of individual glucosinolates was done using p-hydroxybenzyl glucosinolate as internal reference. Further details and transitions can be found in [46 ,47 (link)].