A validated ultra-high-performance liquid chromatography–tandem mass spectrometry method for the selective analysis of free and total folate in plasma and red blood cells

• Determination of short- and long-term folate status starting from 300 μl of whole blood.
• Use of an integrated plasma and RBC sample preparation.
• Reliable RBC lysis coupled to controlled deoxygenation of hemoglobin.
• Full method validation based on EMA-guidelines.
• Verification of method performance using CRM-materials for both plasma and RBCs.

A stable isotope dilution LC–MS/MS method is the method of choice for the selective quantitative determination of several folate species in clinical samples. By implementing an integrated approach to determine both the plasma and red blood cell (RBC) folate status, the use of consumables and time remains limited.Starting from a single 300 μl whole blood sample, the folate status in plasma and RBCs can be determined after separating plasma and RBCs and sequential washing of the latter with isotonic buffer, followed by reproducible lysis using an ammonium-based buffer. Acidification combines both liberation of protein bound folates and protein precipitation. Sample cleanup is performed using a 96-well reversed-phase solid-phase extraction procedure, similar for both plasma and RBC samples. Analyses are performed by UHPLC–MS/MS.Method validation was successfully performed based on EMA-guidelines and encompassed selectivity, carry-over, linearity, accuracy, precision, recovery, matrix effect and stability. Plasma and RBC folates could be quantified in the range of 1–150 nmol/l and 5–1500 nmol/l, respectively.This method allows for the determination of 6 folate monoglutamates in both plasma and RBCs. It can be used to determine short and long term folate status in both normal and severely deficient subjects in a single analytical sequence.