Accela hplc autosampler system

Plasma concentrations of corticosterone (n = 90) were analyzed using LC-MS technique as already described (27 (link)). In brief, after protein precipitation, the supernatant was dried and stored at −20°C. For LC–MS/MS analysis, samples were dissolved in MeOH/H₂O (50/50) adding DXM (100 ng/ml), vortexed for 30 s, sonicated for 2 min, and centrifuged at 4°C for 2 min at 14,000 rpm. Subsequently, samples were transferred to mass spectrometry analysis using an Accela HPLC/autosampler system (Thermo Fisher Scientific) coupled to the LTQ Orbitrap high-resolution hybrid mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany). At various concentrations between 5 and 500 ng/ml, the intra-assay CVs were 13.05–4.64%. The inter-assay CV for 100 ng/ml (n = 20) was 5.57%.

Deuterated leucine was measured with ESI LC-MS on a high resolution LTQ-Orbitrap instrument (Thermo, San Jose, USA) and an Accela HPLC-autosampler System (Thermo).
The mobile phases A and B consisted of 0.1% formic acid in water/acetonitrile (A = 0% acetonitrile, B = 80% acetontrile v/v). A simple gradient at a flow rate of 0.4 ml/min was used: 4 min at 100% A, then linear to 100% B in 5 min, followed by 5 min equilibration at 100% A. Leucine was separated with a Synergi Hydro-RP 80A 4 µm, 75×3 mm i.d. column (Phenomenex) at slightly elevated temperature (30°C). This HPLC method gives baseline separation of isoleucine and leucine. The injection volume was 10 µl.
The LTQ-Orbitrap instrument was operated in positive mode at a mass resolution setting of 30000. The instrument was scanned from m/z 131 to 137 to measure leucine (m/z 132.1019), 15N-leucine (m/z 133.0989), and d3-leucine (m/z 135.1207). The 15N labeled leucine was used as an internal standard. At 30k resolution setting the 15N and d3 isotopes are well resolved from other natural isotopes (e.g. 13C, 17O and 18O). The natural abundance of the 15N isotope of leucine is only 0.37%, and the amount of 15N leucine was selected to have a minimal contribution of the 15N isotope from leucine from ApoB leucine.