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.
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 300μl 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 1-150nmol/l and 5-1500nmol/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.
Laboratory of Toxicology, Department of Bioanalysis, Ghent University, Ottergemsesteenweg 460, B-9000 Gent, Belgium. Electronic address: filip.kiekens@UGent.be.
Filip
F
Kiekens
Laboratory of Toxicology, Department of Bioanalysis, Ghent University, Ottergemsesteenweg 460, B-9000 Gent, Belgium. Electronic address: jeroen.vandaele@UGent.be.
Jeroen
J
Van Daele
Laboratory of Functional Plant Biology, Department of Physiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium. Electronic address: dieter.blancquaert@UGent.be.
Dieter
D
Blancquaert
Laboratory of Functional Plant Biology, Department of Physiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium. Electronic address: dominique.vanderstraeten@UGent.be.
Dominique
D
Van Der Straeten
Laboratory of Toxicology, Department of Bioanalysis, Ghent University, Ottergemsesteenweg 460, B-9000 Gent, Belgium. Electronic address: willy.lambert@UGent.be.
Willy E
WE
Lambert
Laboratory of Toxicology, Department of Bioanalysis, Ghent University, Ottergemsesteenweg 460, B-9000 Gent, Belgium. Electronic address: christophe.stove@UGent.be.
Christophe P
CP
Stove
D001774:Blood Chemical Analysis / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004912:Erythrocytes / Q000737:chemistry*; D005492:Folic Acid / Q000031:analogs & derivatives / Q000032:analysis* / Q000097:blood*; D005971:Glutamates / Q000032:analysis; D006801:Humans; D010949:Plasma / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D005971:Glutamates; C083563:folate monoglutamate; D005492:Folic Acid
Chromatography; Folate; Mass spectrometry; Plasma; Polyglutamylation; Red blood cell