12 February / March 2020 Table 1: MRM transitions for target analytes and phospholipids. Target analytes FNN
FNN-D5 NFNN
NFNN-D6 Phospholipid Lysophosphatidylcholine (18:2) Lysophosphatidylcholine (18:1) Lysophosphatidylcholine (18:0) Lysophosphatidylcholine (20:4)
Phosphatidylcholine (30:1) Phosphatidylcholine (34:2) Phosphatidylcholine (34:1) Phosphatidylcholine (36:3) Phosphatidylcholine (36:2)
Phosphatidylcholine (38:6) Phosphatidylcholine (38:5) Phosphatidylcholine (38:4) Q1/Q3 232.0/159.0
237.0/159.0 204.0/109.0
210.1/161.0 Q1/Q3 520.3/184.1 522.4/184.1 524.4/184.1 544.3/184.1
704.5/184.1 758.6/184.1 760.6/184.1 784.6/184.1 786.6/184.1
806.6/184.1 808.6/184.1 810.6/184.1
the following gradient: MP-B was ramped from 70 to 90% over 1.75 min and held isocratic for 0.5 min after which the column was re-equilibrated at 70% MP-B for 0.75 min. Column flow rate was 0.70 mL/min at a column temperature of 60°C; the LOQ was achieved using an injection volume of 8 µL. A triple quadrupole mass spectrometer was operated under positive electrospray ionisation (ESI) conditions with detection in multiple-reaction monitoring (MRM) mode for the transitions outlined in Table 1.
Sample Preparation
Mouse whole brain (CD-1 strain) was purchased from BIOIVT. Cerebellum was harvested from whole brain by dissection (Figure 3A). Either dissected cerebellum or whole brain were treated with water (10 µL per mg of tissue), after which ceramic beads (Matrix D, MP Biomedicals™) were added (Figure 3B) and the sample was homogenised (Figure 3C). An aliquot of homogenate (10 µL) was fortified with internal standard spiking solution (25 µL) and 5% NH4
OH (165 µL), then vortexed (1
Reference standard and internal standard (IS) stock solutions were provided by Altasciences (100 µg/mL FNN and NFNN in MeOH, 100 µg/mL FNN-D5
, and NFNN-D6 in MeOH). LC-MS/MS Instrumentation
Chromatographic separations were conducted on a C18 column (2.1 x 100 mm, 2.0 µm) with 10 mM NH4
HCO3 , pH 10.0 (MP-A), and MeOH (MP-B), delivered using an UPLC system under
min) and centrifuged (2 min, 738 g). The entire sample homogenate was loaded onto SLE plates using synthetic SLE sorbent (Chem Elut S 96-well plate, 200 mg, Agilent Technologies) for SLE extraction followed with the procedure shown in Figure 4. In order to prevent analyte loss during evaporation, a keeper consisting of HCl in MeOH was added in the collection plate prior to the elution step, which will create the analytes salt form. Consequently, this eliminated well to well variations previously noticed from the evaporation step.
Figure 3: Mouse brain pre-treatment, including A) dissection and weighing, B) Lysing buffer (water) and ceramic bead addition; C) homogenisation with MP Biomedicals FastPrep-96 homogeniser at 1,600 rpm for 40 seconds.
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