Prominence lc system

It was kindly performed by specialist personel (Helinho) in the AB-Sciex Laboratory located in Sao Paulo, SP, Brazil. Plasma samples from 59 breast cancer patients were compared to control group composed of 93 healthy menopaused volunteers (Supplementary Note). Samples were injected onto a Shimadzu Prominence LC system coupled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument with an acquisition scan rate of 100 spectra/sec and stable mass accuracy of ~2 ppm.
Flow Injection Analysis (FIA) was performed using isocratic elution with Methanol/Water (90/10) with 5.0 mM of ammonium formate. Flow rate and injection volumes were 0.025 mL/min and 50 μL respectively.
No ion source or declustering potential (50 V and −40 V) optimization was performed. The following ionization parameters were applied: CUR = 20 psi, GS1 = 20 psi, GS2 = 15 psi, Temp = 250oC, IS = 5000 V (–4000V). MS scan ranging from m/z 100 to 1200 with accumulation time of 0.25 s and product ion scan from m/z 100 to 1200 and accumulation time of 0.03 s were the adopted parameters during survey and dependent scans respectively.

The analysis was conducted on a SHIMADZU Prominence LC system (Kyoto, Japan) coupled with a 5500 QTRAP mass spectrometer (AB SCIEX, Foster City, CA, USA), which includes a LC-20AD_XR solvent delivery system, a DGU-20A_3R automatic degasser, a SIL-20A_XR autosampler and a CTO-20AC column oven. Chromatographic separation was performed at 40 °C on a Waters UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) The mobile phase consisted of a binary solvent system with ACN (A) and 0.1% formic acid in water (B), and run under the following parameters: 35% A at 0.01–0.5 min, 35–90% A at 0.5–9 min, 90% A at 9–11 min, 90–35% A at 11–12 min and 35% of A for 4 min to re-equilibrate the column. The injection volume was 2 μL and the flow rate was set to 0.3 mL/min.
The mass spectrometric detection was operated in the multiple reaction monitoring (MRM) mode with the negative electrospray ionization (ESI). The optimized ESI parameters included ion spray voltage of 4500 V, temperature of 550 °C, curtain gas of 0.24 Mpa, nebulizer gas of 0.38 Mpa and heater gas of 0.38 Mpa. The parameters of MRM transitions, declustering potential (DP) and collision energy (CE) of nine analytes were optimized using a syringe infusion pump and are listed in Table 2. Data acquisition and analysis were processed by the Analyst software (V.1.6.2) from AB SCIEX (Concord, ON, Canada).

Sphingosine-1-phosphate was extracted from 10 μL of plasma after dilution with 50μL with 30mM citric Acid/40mM sodium phosphate buffer pH 4.0 and spiking with 5uL internal standard (S1P-C17 base, Avanti LM-6002) to a concentration of 0.23μM.[24 (link)] After thorough mixing the sample was extracted with 275 μL of 1-butanol (Fisher A399), spun down and 220 μL were collected and dried down. The dried down samples were reconstituted for LCMS analysis in 125 μL of 50% methanol, 1% formic acid in 5mM ammonium formate. Samples were spun down and 10μL injected onto LCMS consisting of Shimadzu Prominence LC system and 4000 QTrap (ABSciex) in Turbo Ion Spray Mode. Separation was accomplished on a Luna 3μm C18 100Å 50x2.0mm column (Phenomenex) with 1.5 minute gradient from LC buffer A (50% methanol, 1% formic acid in 5mM ammonium formate) to LC buffer B (89% methanol, 10% 2−propanol, 1% formic acid in 5mM ammonium formate) and S1P was quantified together with the internal standard (S1P-C17) using multiple-reaction-monitoring. Calibration was accomplished using a single point calibrator repeatedly assayed throughout the analysis.