Fast, Accurate LC-MS/MS Method for Folate Deficiency Biomarkers in Plasma

• Strong retention on a Raptor HILIC-Si column prevents matrix interference.
• Complete separation from phospholipids ensures accurate results in a quick, 5-minute analysis.
• Divert matrix to waste to keep your MS source clean and reduce downtime for maintenance.

Folate deficiency is considered a risk factor for a wide range of human health problems, including neural tube defects in newborns, cardiovascular diseases, Alzheimer’s disease, and certain forms of cancer. Plasma levels of folic acid and its metabolites (5-formyl tetrahydrofolate and 5-methyltetrahydrofolic acid) are used as biomarkers to diagnose folate deficiency. However, LC-MS/MS methods for folate deficiency biomarkers in plasma can be very challenging because these small, polar compounds are not retained well on traditional reversed-phase LC columns. Retention can be improved using a HILIC method, but in this case, the column must provide strong enough retention to prevent coelution with the phospholipid components in the plasma sample matrix. Although a good sample preparation protocol will help, 100% removal of phospholipids is very difficult, and even low levels of phospholipids can interfere with target analytes, compromise quantitation, and contaminate the MS source.

Using a HILIC approach with a Raptor HILIC-Si column is a much better alternative for LC-MS/MS methods for folate deficiency biomarkers in plasma because you can quickly and completely separate the matrix interferences from the target analytes. The increased retention obtained on a Raptor HILIC-Si column ensures good separation of folate deficiency biomarkers from phospholipids and allows labs to accurately quantitate these important compounds, even at just 25–50 ng/mL, with no ion suppression from matrix interferences. In addition, more resolution between analytes and matrix components lets you divert matrix to waste, which keeps your MS cleaner longer. The LC-MS/MS method for folate deficiency biomarkers in plasma shown here allows folic acid, 5-formyl tetrahydrofolate, and 5-methyltetrahydrofolic acid to be accurately analyzed with no matrix interference in a fast, 5-minute analysis.

	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion
1.	5-Formyl tetrahydrofolate	2.23	50	474.2	327.0
2.	Folic acid	2.23	25	442.2	295.0
3.	5-Methyltetrahydrofolic acid	2.25	25	460.3	313.0

Column	Raptor HILIC-Si (cat.# 9310A5E)
Dimensions:	50 mm x 3.0 mm ID
Particle Size:	2.7 µm
Temp.:	30 °C
Standard/Sample
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 0.00 0.5 100 3.00 0.5 0 3.20 0.5 0 3.21 0.5 100 5.21 0.5 100
Max Pressure:	344 bar

Detector	MS/MS
Ion Mode:	ESI+
Mode:	MRM
Instrument	UHPLC
Sample Preparation	1. 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.
Notes	Endogenous peaks for phospholipids and lysophospholipids are displayed because they are common matrix interferences and are known to suppress ionization efficiency.

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