Acquity ultra high performance liquid chromatographic system

FFPE tumour tissue (5–10 mg) from three available individuals (cases 1 and 24 with dual NF1/DLST variants, and case 3 with dual NF1/MDH2 variants) were immersed in 500 mL LC-MS/MS-grade methanol containing isotope-labelled internal standards and processed as previously described (19 (link)). An analysis of metabolites was carried out with an AB Sciex 5500 QTRAP mass spectrometer coupled to an Acquity ultra-high-performance liquid chromatographic system (Waters) as previously described (19 (link)). We compared the data with that from a series of tumours carrying either NF1 or DLST mutations previously analysed in our laboratory.

LC-UV/MS analysis was carried out on a Waters Acquity ultra-high performance liquid chromatographic system (Milford, MA, USA) equipped with a photodiode-array detector (PDA) and a single quadrupole mass spectrometer (Acquity SQ Detector) with an electrospray ionization source (ESI) operating in negative mode. An Agilent Poroshell EC-C18 column (150 mm × 4.6 mm, 2.7 µm) held at 50° C was used for the separation with 1 mM ammonium fluoride (NH4F) in water (solvent A) and acetonitrile (solvent B) as mobile phases. The flow rate was 0.6 mL/min with an elution gradient as follows: 0–2 min 75% A, 2–5 min 75–68% A, 5–14 min 68% A, 14–16 min 68–50% A, 16–18 min 50% A, 18–18.5 min 50–10% A, 18.5–22 min 10% A, 22–22.5 min 10–75% A, and 22.5–28 min 75% A. The injection volume was 5 µL. The mass acquisition window was from 50 to 2000 Da.