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Folate Deficiency Biomarkers in Human Plasma on Raptor HILIC-Si by LC-MS/MS

PeakstR (min)Conc.
Precursor IonProduct Ion
1.5-Formyl tetrahydrofolate2.2350474.2327.0
2.Folic acid2.2325442.2295.0
3.5-Methyltetrahydrofolic acid2.2525460.3313.0
ColumnRaptor HILIC-Si (cat.# 9310A5E)
Dimensions:50 mm x 3.0 mm ID
Particle Size:2.7 µm
Temp.:30 °C
Diluent:20 mM Ammonium acetate in acetonitrile:water (80:20) containing 10 mg/mL 2-mercaptoethanol
Inj. Vol.:5 µL
Mobile Phase
A:50:50 Water:acetonitrile, 20 mM ammonium acetate
B:20:80 Water:acetonitrile, 20 mM ammonium acetate
Time (min)Flow (mL/min)%B
Max Pressure:344 bar
Ion Mode:ESI+
Sample Preparation1. Aliquot 380 μL of human plasma (K2EDTA, 2x charcoal stripped) containing 100 μg/mL 2-mercaptoethanol and add 20 μL fortification solution.
2. Vortex for 2 min and centrifuge for 2 min at 4000 rpm.
3. Condition EVOLUTE EXPRESS WAX 30 mg SPE plate (Biotage 604-0030-PX01) with 1 mL methanol and then equilibrate with 1 mL 2% formic acid in water. Apply vacuum to dry the plate completely.
4. Load 400 μL of sample onto the plate and apply vacuum to initiate the flow.
5. Wash the plate with 1 mL water. Apply vacuum to dry the plate completely.
6. Elute samples with 300 μL 5% ammonium hydroxide in methanol containing 10 mg/mL 2-mercaptoethanol. Apply vacuum for elution.
7. Evaporate extracts to dryness under nitrogen at 30 °C.
8. Reconstitute in 200 μL mobile phase B containing 10 mg/mL 2-mercaptoethanol.

Note: The whole sample preparation process was performed under dim light.