Review The analysis of folate and its metabolic precursors in biological samples Eoin P. Quinlivan a , Andrew D. Hanson b , Jesse F. Gregory a, * a Department of Food Science and Human Nutrition, University of Florida, Gainesville, FL 32611, USA b Department of Horticultural Sciences, University of Florida, Gainesville, FL 32611, USA Available online 5 October 2005 Introduction History Lucy Wills first discovered in 1931 [1] a component of yeast capable of curing ‘‘pernicious anaemia of pregnan- cy.’’ However, it was another 15 years [2] before the struc- ture of folate was elucidated (Fig. 1). This delay in identifying the structure of the ‘‘Wills factor’’ was due in part to the diversity of folate species resulting from differ- ential substitution of one-carbon units on nitrogens 5 and 10, the length of the glutamyl side chain, and/or the oxida- tion state of the pterin ring. The diverse biological and chemical characteristics of the various folate forms still complicate the task of assaying endogenous folate. The tra- ditional approach for analyzing folates has been to simplify the analysis by determining either the one-carbon species (after hydrolysis of the poly-c-glutamyl chain) or the c-glu- tamate chain length (normally after removal of the pterin moiety). Methods are now appearing whereby both the one-carbon substituents and the poly-c-glutamate chain length can be determined simultaneously. We review most of the methods commonly used to assay folate and its pre- cursors, and we discuss the strengths and weaknesses of the various analytical approaches. It should be noted that the folate glutamate peptide is somewhat unusual in that it is a gamma peptide and not a-linked as with proteins. This means that a distinct set of enzymes is used for folate polyglutamate synthesis and hydrolysis. Nomenclature The International Union of Pure and Applied Chemis- try–International Union of Biochemistry (IUPAC–IUB) 1 Commission on Biochemical Nomenclature name [3] for the ‘‘factor’’ discovered by Wills is pteroylglutamic acid. However, the trivial names ‘‘folic acid’’ and ‘‘folate’’ are more commonly used. The name folic acid was coined in 1941 by Mitchell and coworkers [4] after the 4 tons of spin- ach leaves from which it was purified (Latin folium–leaf). The naming convention [1] for the vitamin (Fig. 1) is atom number s ðÞ ð Þ substituent group ½   atom numbers ð Þ dihydro=tetrahydro ½   folate=pteroylglutamate. 0003-2697/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.09.017 * Corresponding author. Fax: +1 352 392 4515. E-mail address: jfgy@ufl.edu (J.F. Gregory). 1 Abbreviations used: IUPAC–IUB, International Union of Pure and Applied Chemistry–International Union of Biochemistry; 10-CHO-DHF, 10-formyldihydrofolate; THF, tetrahydrofolate; DHN, dihydroneopterin; HMDHP, 6-hydroxymethyldihydropterin; DHM, dihydromonapterin; pABA, p-aminobenzoate; ADC, 4-amino-4-deoxychorismate; pABA-Glc, p-aminobenzoyl b-D-glucopyranoside; DHF, dihydrofolate; 5-CH 3 -THF, 5-methyltet- rahydrofolate; 10-CHO-THF, 10-formyltetrahydrofolate; 5-CHO-THF, 5-formyltetrahydrofolate; 5-HN@CH-THF, 5-formiminotetrahydrofolate; 5,10- CH@THF, 5,10-methenyltetrahydrofolate; 5,10-CH 2 -THF, 5,10-methylenetetrahydrofolate; FBC, folate binding column; FBP, folate binding protein; LC–MS/MS, liquid chromatography–tandem mass spectrometry; SPE, solid-phase extraction; ELLSA, enzyme-linked ligand sorbent assay; G6PD, glucose-6-phosphate dehydrogenase; GC–MS, gas chromatography–mass spectrometry; pABGlu, p-aminobenzoylglutamic acid; DDATHF, 5,10- dideazatetrahydrofolic acid; PDDF, 10-propargyl-5,8-dideazafolate; RP–HPLC, reverse-phase HPLC; IP–HPLC, reverse-phase ion pair chromatography; LC–MS, liquid chromatography–mass spectrometry; C/V curve, current/voltage curve; LC–FD, liquid chromatography–fluorescence detection; m/z, mass-to-charge ratio; SRM, single reaction monitoring; ESI, electrospray ionization; APCI, atmospheric pressure chemical ionization; MALDI, matrix- assisted laser desorption/ionization; HILIC, hydrophilic interaction chromatography; HILIC–MS/MS, HILIC–tandem mass spectrometry; FdUMP, 5-fluorodeoxyuridylate; TS, thymidylate synthase; MCW, methanol–chloroform–water; TLC, thin-layer chromatography. www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 348 (2006) 163–184