Plasma polyunsaturated fatty acid profile
®
HEAT, SEPARATION, -20, REGRESSION, SIL
Plasma samples were thawed on ice, to 50 µL of the sample, 2.5 µL of an internal standard mixture (each standard concentration was 10 µg/mL in MeOH/H2O (80:20 v/v)) and 500 µL of ice-cold Hexane/2-Propanol (3:2) were added. Samples were vortexed at -20 °C for 10 mins and finally centrifuged at 14,680 rpm, for 10 mins at 4°C. 500 µL of supernatant were dried under nitrogen and were reconstituted in 1 mL of MeOH/H2O (80:20 v/v) and injected in UHPLC-MS/MS. ALA, α-linoleic acid-d14 (ALA-d14), LA, linoleic acid-d4 (LA-d4), EPA, Eicosapentaenoic acid-d5 (EPA-d5), DHA, Docosahexaenoic acid-d5 (DHA-d5), ARA, Arachidonic acid-d8 (ARA-d8) standards were purchased from Cayman Chemical Company (Michigan, USA). Other reagents were all purchased from Merck.UHPLC-MS/MS analysis was carried out with a Shimadzu Nexera (Shimadzu, Milan, Italy) UHPLC system consisting of two LC 30 AD pumps, a SIL 30AC autosampler, a CTO 20AC column oven, a CBM 20A controller, and the system was coupled online to a triple quadrupole LCMS 8050 (Shimadzu, Kyoto, Japan) equipped with an Electrospray Ionization (ESI) source.The separation was carried on a Kinetex® EVOTM C18 (100 × 2.1 mm; 2.6 µm, 100Å) protected with a SecurityGuardTM precolumn (5 × 2.1 mm) (Phenomenex, Bologna, Italy). The column temperature was set at 50°C, a flow rate of 0.5 mL/min was used, mobile phase consisted of (A): 5 mM CH3COONH4 in H2O (v/v) and (B): ACN. The following gradient was used: 0 min, 60%B, 6.5 min, 60%B, 6.6 min, 98%B, hold for 1 min, returning to 60% in 0.1 min. 2 were injected.The ESI was operated in negative mode. Interface temperature, desolvation line temperature, and heat block temperature were set to 300 °C, 250 °C and 400 °C, respectively. Nebulizing gas, drying (N2) and heating gas (air) was set to 3, 10 and 10 L/min, respectively. MS/MS analysis was conducted in scheduled multiple reaction monitoring (MRM), and the transitions were optimized by flow injection analysis of the corresponding standard compounds.Three replicates of each sample were performed. Samples were analysed in a random order to prevent any variation in mass spectrometer sensitivity and QC were randomly inserted in the batch to monitor system stability over time.Calibration standards were prepared in MeOH/H2O (80:20 v/v) and internal standards were added to all calibration standards at concentrations of 25 ng/mL. Calibration curves in the range of 5–300 ng/mL for EPA, DHA and ARA, 5–500 ng/mL for ALA and 0.5–1.5 µg/mL for LA, with five concentration levels, were generated from regression analysis (R2 ≥ 0.999) and apply to quantitation of free fatty acids in plasma.For sensitivity evaluation, limits of detection (LODs) and quantification (LOQs) were calculated by the ratio between the standard deviation (SD) and analytical curve slope multiplied by 3 and 10, respectively.Repeatability was established by triplicate injections of QC samples with the same chromatographic conditions and analysed on the same day and within two consecutive days. Results were expressed as relative standard deviation % (RSD %) for concentration and retention time. Recovery was assessed by spiking a known amount of each internal standard solution at low, medium, and high concentration range in samples, which were subsequently extracted and analysed as previously described. The efficiency was calculated by comparing the internal standard peak areas of standards and real samples.