Evoq elite mass spectrometer

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.