Modified EMR-lipid method combined with HPLC-MS/MS to determine folates in egg yolks from laying hens supplemented with different amounts of folic acid.

Egg yolks are a good source of folates. However, the method for analyzing the naturally occurring folates in egg yolks is complicated and time-consuming. In this study, a simplified pre-treatment method followed by validated HPLC-MS/MS was developed to determine native folates in eggs from laying hens treated with different amounts of folic acid. The modified enhanced matrix removal -lipid method to purify samples showed good performance in lipid elimination, reduction of steps and time savings. According to experimental analysis, yolks contained total folate amounts ranging from 147 to 760 µg/100 g when laying hens' diet was supplemented with folic acid from 0 to 10 mg/kg. Four folate vitamers were detected in egg yolks: 5-methyltetrahydrofolate accounted for 91-98% of total folates, whereas folic acid, 5-formyltetrahydrofolate and 10-formylfolic acid together accounted for 2-9%. Therefore, laying hens efficiently converted folic acid in feed into 5-methyltetrahydrofolate in eggs with little folic acid deposition.

Improved folate monoglutamate extraction and application to folate quantification from wild lentil seeds by ultra-performance liquid chromatography-selective reaction monitoring mass spectrometry.

Folates are important micronutrients in lentils (Lens culinaris Medik.). In this work, the folate extraction workflow in ascorbate-containing buffer was optimized and validated, and the concentrations of eight folate monoglutamates in cultivated and six wild lentil species, grown under field or greenhouse conditions, were quantified by ultra-performance liquid chromatography and mass spectrometry (UPLC-MS). In general wild lentil species had higher folate concentrations than cultivated genotypes. Lens tomentosus had the highest folate concentration with median values of 439.7 and 360.9 µg/100 g in the field and greenhouse, followed by Lens orientalis with 416.6 and 327.6 µg/100 g, respectively. A significant effect (P < 0.05) of growing conditions was observed in four out of six wild lentil species, with seeds from the field having higher folate concentration (6% to 45%) compared with the greenhouse. MeFox, an oxidation product of 5-methyltetrahydrofolate, was present in all lentil species at concentrations 2.2 to 5.6 times higher than the total folates.

Effect of extrusion on folic acid concentration and mineral element dialyzability in Great Northern beans (Phaseolus vulgaris L.).

Great Northern beans (GNB) contain appreciable magnesium (Mg), potassium (K), phosphorus (P), and iron (Fe), together with the heat-labile vitamin, folate, and the anti-nutritional compound phytate. Thus, the objective was to increase dialyzability of essential mineral elements while degrading phytate and minimizing destruction of folate through extrusion of GNB. Extrusion resulted in significant (p < 0.05) increases in dialyzability of Mg, P, K, and Fe by as much as 50%, 30%, 5%, and 79%, respectively, while decreasing cadmium (Cd) dialyzability. Screw speed (SS) had a significant quadratic effect on dialyzability of all elements. Low MC resulted in a significant reduction (46%) in phytate, although this was accompanied by as much as 24% destruction of folate. In conclusion, low barrel temperature, medium MC and high SS were identified as the optimum conditions to maximize essential mineral element dialyzability and folate retention while minimizing phytate and dialyzable Cd.

Simultaneous measurement of folate cycle intermediates in different biological matrices using liquid chromatography-tandem mass spectrometry.

The folate cycle is an essential metabolic pathway in the cell, involved in nucleotide synthesis, maintenance of the redox balance in the cell, methionine metabolism and re-methylation reactions. Standardised methods for the measurement of folate cycle intermediates in different biological matrices are in great demand. Here we describe a rapid, sensitive, precise and accurate liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method with a wide calibration curve range and a short run time for the simultaneous determination of folate cycle metabolites, including tetrahydrofolic acid (THF), 5­methyl THF, 5­formyl THF, 5,10­methenyl THF, 5,10­methylene THF, dihydrofolic acid (DHF) and folic acid in different biological matrices. Extraction of folate derivatives from soft and hard tissue samples as well as from adherent cells was achieved using homogenisation in buffer, while extraction from the whole blood and plasma relied on the anion exchange solid-phase extraction (SPE) method. Chromatographic separation was completed using a Waters Atlantis dC18 2.0 × 100 mm, 3-µ column with a gradient elution using formic acid in water (0.1% v/v) and acetonitrile as the mobile phases. LC gradient started with 95% of the aqueous phase which was gradually changed to 95% of the organic phase during 2.70 min in order to separate the selected metabolites. The analytes were separated with a run time of 5 min at a flow rate of 0.300 mL/min and detected using a Waters Xevo-TQS triple quadrupole mass spectrometer in the multiple reaction monitoring mode (MRM) at positive polarity. The instrument response was linear over a calibration range of 0.5 to 2500 ng/mL (r2 > 0.980). The developed bioanalytical method was thoroughly validated in terms of accuracy, precision, linearity, recovery, sensitivity and stability for tissue and blood samples. The matrix effect was compensated by using structurally similar isotope labelled internal standard (IS), 13C5­methyl THF, for all folate metabolites. However, not all folate metabolites can be accurately quantified using this method due to their high interconversion rates especially at low pH. This applies to 5,10­methylene THF which interconverts into THF, and 5,10­methenyl­THF interconverting into 5­formyl­THF. Using this method, we measured folate cycle intermediates in mouse bone marrow cells and plasma, in human whole blood; in mouse muscle, liver, heart and brain samples.

Effect of Freezing, Thermal Pasteurization, and Hydrostatic Pressure on Fractionation and Folate Recovery in Egg Yolk.

In this study, the impact of pasteurization and freezing of raw material, as performed at a commercial scale, on egg yolk fractionation and folate recovery was assessed. Freezing induced denaturation of the lipoproteins in egg yolk, which prevented further fractionation of the yolk. Thermal pasteurization of egg yolk at 61.1 °C for 3.5 min as well as high hydrostatic pressure (HHP) treatment (400 MPa for 5 min) did not change (p < 0.05) the composition of egg yolk or yolk fractions after their recovery by centrifugation. Expressed as dry matter, folate in pasteurized yolk was measured to be 599 µg/100 g, while its concentration reached 1969.7 µg/100 g for pasteurized granule and 1902.5 µg/100 g for HHP-treated granule. Folate was not detected in plasma, emphasizing the complete separation of yolk folate into granule. Further, we studied the effect of HHP on different dilutions of egg yolk, which were then fractionated. Egg yolk was diluted with water at different concentrations (0.1, 1.0, 10, 25, and 50%), HHP-treated at 400 MPa for 5 min, and centrifuged. Characterization of the compositions of the separated granule and plasma followed. Folate was stable under the HHP conditions used. However, HHP caused separation of folate from the yolk structure into water-soluble plasma. After HHP processing, the amount of folate detected in the plasma fraction was significantly (p < 0.05) higher (1434.9 µg/100 g) in the 25% diluted samples but was significantly (p < 0.05) lower in HHP-treated granule samples. Native sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that phosvitin, α-livetin, and apovitellenin VIa were the proteins most resistant to HHP. This study confirms that dilution of egg yolk before HHP treatment can significantly (p < 0.05) change the composition of granule and plasma fractions after centrifugal fractionation of egg yolk.

Combined Labelled and Label-free SERS Probes for Triplex Three-dimensional Cellular Imaging.

Cells are complex chemical systems, where the molecular composition at different cellular locations and specific intracellular chemical interactions determine the biological function. An in-situ nondestructive characterization of the complicated chemical processes (like e.g. apoptosis) is the goal of our study. Here, we present the results of simultaneous and three-dimensional imaging of double organelles (nucleus and membrane) in single HeLa cells by means of either labelled or label-free surface-enhanced Raman spectroscopy (SERS). This combination of imaging with and without labels is not possible when using fluorescence microscopy. The SERS technique is used for a stereoscopic description of the intrinsic chemical nature of nuclei and the precise localization of folate (FA) and luteinizing hormone-releasing hormone (LHRH) on the membrane under highly confocal conditions. We also report on the time-dependent changes of cell nuclei as well as membrane receptor proteins during apoptosis analyzed by statistical multivariate methods. The multiplex three-dimensional SERS imaging technique allows for both temporal (real time) and spatial (multiple organelles and molecules in three-dimensional space) live-cell imaging and therefore provides a new and attractive 2D/3D tracing method in biomedicine on subcellular level.

SERS encoded nanoparticle heterodimers for the ultrasensitive detection of folic acid.

In this paper, gold­silver nanoparticle (AuNP­AgNP) heterodimers were assembled with highly yield as an active SERS substrate, based on antigen­antibody immunoreaction. The developed SERS sensor has successful achieved the ultrasensitive detection of folic acid (FA) with the limit of detection (LOD) as 0.86 pg/mL. And the linear range was from 0.005 ng/mL to 1 ng/mL. The results also demonstrated that this developed method showed high specificity and excellent recovery for the human serum samples, indicating its promising potential in bio-diagnosis and the environmental monitoring.

Hybrid molecularly imprinted poly(methacrylic acid-TRIM)-silica chemically modified with (3-glycidyloxypropyl)trimethoxysilane for the extraction of folic acid in aqueous medium.

In the present study a hybrid molecularly imprinted poly(methacrylic acid-trimethylolpropane trimethacrylate)-silica (MIP) was synthesized and modified with (3-glycidyloxypropyl)trimethoxysilane (GPTMS) with posterior opening of epoxy ring to provide hydrophilic properties of material in the extraction of folic acid from aqueous medium. The chemical and structural aggregates of hybrid material were characterized by means of Fourier Transform Infrared (FT-IR), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Thermogravimetric analysis (TGA) and textural data. Selectivity data of MIP were compared to non-imprinted polymer (NIP) through competitive sorption studies in the presence of caffeine, paracetamol or 4-aminobenzamide yielding relative selectivity coefficients (k') higher than one unit, thus confirming the selective character of MIP even in the presence of structurally smaller compounds than the folic acid. The lower hydrophobic sorption by bovine serum albumin (BSA) in the MIP as compared to unmodified MIP proves the hydrophilicity of polymer surface by using GPTMS with opening ring. Under acid medium(pH 1.5) the sorption of folic acid onto MIP from batch experiments was higher than the one achieved for NIP. Equilibrium sorption of folic acid was reached at 120 min for MIP, NIP and MIP without GPTMS and kinetic sorption data were well described by pseudo-second-order, Elovich and intraparticle diffusion models. Thus, these results indicate the existence of different binding energy sites in the polymers and a complex mechanism consisting of both surface sorption and intraparticle transport of folic acid within the pores of polymers.

Ion pair-based dispersive liquid-liquid microextraction followed by high performance liquid chromatography as a new method for determining five folate derivatives in foodstuffs.

A novel technique for simultaneous determination of five folate derivatives in various food matrices was developed by ion pair-based dispersive liquid-liquid microextraction (IP-DLLME) combined with high-performance liquid chromatography (HPLC). In the proposed method, N-methyl-N,N-dioctyloctan-1-ammonium chloride (aliquat-336) was used as an ion-pair reagent. Effective variables of microextraction process were optimized. Under optimum conditions, the method yielded a linear calibration curve ranging from 1-200 ng g(-1) with correlation coefficients (r(2)) higher than 0.98. The relative standard deviation for the seven analyses was 5.2-7.4%. Enrichment factors for the five folates ranged between 108-135. Limits of detection were 2-4.1 ng g(-1). A comparison of this method with other methods described that the new proposed method is rapid and accurate, and gives very good enrichment factors and detection limits for determining five folate derivatives. The newly developed method was successfully applied for the determination of five folate derivatives in wheat flour, egg yolk and orange juice samples.

High performance liquid chromatography coupled to mass spectrometry for profiling and quantitative analysis of folate monoglutamates in tomato.

Folates are essential micronutrients for animals as they play a major role in one carbon metabolism. Animals are unable to synthesize folates and obtain them from plant derived food. In the present study, a high performance liquid chromatography coupled to mass spectrometric (HPLC-MS/MS) method was developed for the high throughput screening and quantitative analysis of folate monoglutamates in tomato fruits. For folate extraction, several parameters were optimized including extraction conditions, pH range, amount of tri-enzyme and boiling time. After processing the extract was purified using ultra-filtration with 10 kDa membrane filter. The ultra-filtered extract was chromatographed on a RP Luna C18 column using gradient elution program. The method was validated by determining linearity, sensitivity and recovery. This method was successfully applied to folate estimation in spinach, capsicum, and garden pea and demonstrated that this method offers a versatile approach for accurate and fast determination of different folate monoglutamates in vegetables.

Use of short chain alkyl imidazolium ionic liquids for on-line stacking and sweeping of methotrexate, flinic acid and folic acid: their application to biological fluids.

Methotrexate (MTX) is widely used for the treatment of many types of cancer. Folinic acid (FNA) and folic acid (FA) were usually simultaneously supplemented with MTX to reduce the side effects of a folate deficiency. This study, for the first time, included on-line sample preconcentration by stacking and sweeping techniques under reduced or enhanced electric conductivity in the sample region using short chain alkyl imidazolium ionic liquids (ILs) as micelle forming agents for analyte focusing. Both analyte focusing by micelle collapse (AFMC) and sweeping-MEKC had been investigated for the comparison of their effectiveness to examine simultaneously MTX, FNA and FA in plasma and urine under physiological conditions. In sweeping-MEKC, the sample solution without micelles was hydrodynamically injected as a long plug into a fused-silica capillary pre-filled with phosphate buffer containing 3.0 mol/L of 1-butyl-3-methylimidazolium bromide (BMIMBr). Using AFMC, the analytes were prepared in BMIMBr micellar matrix and hydrodynamically injected into the phosphate buffer without IL micelles. The conductivity ratio between BGE and sample (γ, BGE/sample) was optimized to be 3.0 in sweeping-MEKC and 0.33 in AFMC resulting the adequate separation of analytes within 4.0 min. To reduce the possibility of BMIMBr adsorption, an appropriate rinsing protocol was used. The limits of detection were calculated as 0.1 ng/mL MTX, 0.05 ng/mL FNA and 0.05 ng/mL FA by sweeping-MEKC and 0.5 ng/mL MTX, 0.3 ng/mL FNA and 0.3 ng/mL FA by AFMC. The accuracy was tested by recovery in plasma and urine matrices giving values ranging between 90 and 110%. Both stacking and sweeping by BMIMBr could be successfully used for the rapid, selective and sensitive determination of pharmaceuticals in complex matrices due to its fascinating properties, including high conductivity, good thermal stability and ability to form different types of interactions by electrostatic, hydrophobic, hydrogen bonding and π-π interactions. In sweeping-MEKC, the using of BMIMBr enhanced the γ factor, k retention factor and the injected amount of sample. Consequently, this technique offers particular potential for higher sensitivity by giving 22- and 5-fold sensitivity enhancement factors (SEFs) of MTX compared to CZE and AFMC, respectively.

A "three-in-one" sample preparation method for simultaneous determination of B-group water-soluble vitamins in infant formula using VitaFast(®) kits.

VitaFast(®) test kits designed for the microbiological assay in microtiter plate format can be applied to quantitative determination of B-group water-soluble vitamins such as vitamin B12, folic acid and biotin, et al. Compared to traditional microbiological methods, VitaFast(®) kits significantly reduce sample processing time and provide greater reliability, higher productivity and better accuracy. Recently, simultaneous determination of vitamin B12, folic acid and biotin in one sample is urgently required when evaluating the quality of infant formulae in our practical work. However, the present sample preparation protocols which are developed for individual test systems, are incompatible with simultaneous determination of several analytes. To solve this problem, a novel "three-in-one" sample preparation method is herein developed for simultaneous determination of B-group water-soluble vitamins using VitaFast(®) kits. The performance of this novel "three-in-one" sample preparation method was systematically evaluated through comparing with individual sample preparation protocols. The experimental results of the assays which employed "three-in-one" sample preparation method were in good agreement with those obtained from conventional VitaFast(®) extraction methods, indicating that the proposed "three-in-one" sample preparation method is applicable to the present three VitaFast(®) vitamin test systems, thus offering a promising alternative for the three independent sample preparation methods. The proposed new sample preparation method will significantly improve the efficiency of infant formulae inspection.

An approach for quantitative analysis of vitamins D and B9 and their metabolites in human biofluids by on-line orthogonal sample preparation and sequential mass spectrometry detection.

An approach for quantitative analysis of two vitamins with different polarities (vitamins D and B9) and their metabolites is presented here. The approach is based on an experimental setup based on hyphenation of an automated workstation for preparation of liquid samples and an LC-MS/MS system with a triple quadrupole mass spectrometer. This configuration enabled development of an orthogonal protocol for sequential SPE retention of analytes with different polarities for subsequent elution and chromatographic separation prior to detection. The resulting method was validated by application to three human biofluids. Estimation of recovery factors in the SPE step led to values from 85.2 to 100% for vitamin D and metabolites and from 93.1 to 100% for vitamin B9 and metabolites (folic acid and folates). The influence of sample matrix variability by analysis of human serum, urine and breast milk was minimized with a complete optimization of the SPE step. The utility of the proposed configuration is shown by the sensitivity and precision of the method, expressed as limits of detection (between 0.2 and 0.30 ng mL(-1) or 4 and 60 pg on-column) and within-laboratory reproducibility (lower than 6.7%, as relative standard deviation). The present application represents an example of determination methods involving targeted analysis of compounds with different polarities using a single aliquot of the sample.

Determination of methotrexate and folic acid by ion chromatography with electrochemical detection on a functionalized multi-wall carbon nanotube modified electrode.

A simple and sensitive method was developed for simultaneous determination of methotrexate (MTX) and folic acid (FA) by ion chromatography with electrochemical detection (IC-ECD). Quaternary amine functionalized multi-wall carbon nanotubes (q-MWNTs) modified glassy carbon electrode (GCE) was used as an amperometric sensor to determine MTX and FA. The electrochemical behaviors of MTX and FA at the q-MWNTs/GCE were studied by cyclic voltammetry (CV). Results indicated that this modified electrode exhibited electrocatalytic oxidation toward MTX and FA with high sensitivity, stability and long life. Good separation of MTX and FA was demonstrated by IC on an anion-exchange column with phosphate buffer solution (PBS) as eluent. Under the optimal conditions, the linear ranges were from 0.01 to 20mg/L for both MTX and FA with correlation coefficients r ≥ 0.9994. The obtained detection limits (LODs) for MTX and FA were 0.2 and 0.4 µg/L (S/N=3), respectively. The method has been successfully applied to the determination of MTX and FA in plasma and urine of patients of rheumatoid arthritis.

Online pre-column derivatization with chromatographic separation to determine folic acid.

A simple, sensitive, and selective online pre-column derivatization high-performance liquid chromatographic method was developed and validated for the first time to determine trace levels of folic acid (FA). An oxidant cerium (IV) trihydroxyhydroperoxide packed reactor was used for pre-column oxidation and was combined by column switching with a C18 analytical column for sample enrichment and separation. The method was based on oxidative cleavage of FA into highly fluorescence products, 2-amino-4-hydroxypteridine-6-carboxaldehyde and the corresponding 2-amino-4-hydroxypteridine-6-carboxylic acid, during the flow of 0.04 M phosphate buffer (pH 3.5) containing the analyte through packed reactor at a flow rate of 0.2 mL/min and 40°C. The fluorescent products were enriched on the head of the analytical column for the final separation. The separation was performed at room temperature using a mobile phase consisting of phosphate buffer (0.04 M, pH 3.5) and acetonitrile (90:10, v/v). The eluents were monitored at emission and excitation wavelengths of 463 and 367 nm, respectively. The method showed excellent recovery, precision and accuracy with detection limits of 0.067 ng/mL from 500 µL of sample FA. The developed method was successfully applied to the determination of FA in pharmaceutical formulations and showed a recovery of 99.31% and a relative standard deviation of 1.72%.

Purity determination and uncertainty evaluation of folic acid by mass balance method.

Folic acid is one of the most important nutrient substances for human beings, especially for the pregnant women and infants. Therefore the purity determination of folic acid is particularly important. The mass balance method was employed to determine the purity of folic acid, by using the measures of high performance liquid chromatography (HPLC), Karl Fischer titration and other conventional approach. The moisture quantification of folic acid was a major problem since it is a thermally unstable substance and it is apt to contain crystal water. Therefore, a novel improved Karl Fischer method was established for accurate determination of the water content in folic acid, whose repeatability (RSD=2.9%) was significantly better than that of the original direct injection method (RSD=12%). The purity of folic acid certified reference material (CRM) determined by mass balance method was 90.9% with an expanded uncertainty of 0.35%, and the content of water (the major impurity) was 8.5%, with an expanded uncertainty of 0.32%.

Development, optimization and validation of a sub-minute analytical enantioselective high performance liquid chromatographic separation for a folic acid precursor in normal phase mode.

A sub-minute enantioselective normal phase high performance liquid chromatographic (HPLC) method for the analysis of a chiral precursor molecule employed frequently in folic acid syntheses was developed, optimized and successfully validated according to ICH-guidelines. It could be shown that ultra-high performance chromatography (UHPLC) can give significant advantages compared to traditional HPLC not only in reversed phase HPLC, but also for enantioselective separations in normal phase mode. Novel 3 µm-particle sizes allow developing analytical chromatographic methods completely resolving two enantiomers in the shortest time possible while preserving high efficiency and low detection limits. By offering increased resolution, sensitivity and speed, enantioselective UHPLC (eUHPLC) improves sample throughput, productivity and provides considerably faster access to information on enantiomeric purity also under non-aqueous conditions.

Pressurized liquid extraction and HPLC quantification of folic acid in fortified wheat flours.

A pressurized liquid extraction (PLE) method using phosphate buffer as solvent was applied for folic acid (FA) extraction from fortified wheat flours and was compared to a standard solid-liquid extraction (SLE) method. Extracted FA was quantified by reverse phase high-performance liquid chromatography (RP-HPLC) hyphenated with a phenyl column and an absorption photometric detector (λ = 280 nm). Detection and quantification limits were 0.12 and 0.4 ng, respectively, corresponding to 0.06 and 0.2 µg g(-1) of analyzed wheat flour. Equivalent FA contents were found by both extraction methods, but a single PLE allowed a total recovery of FA content, whereas at least three successive SLEs were needed to achieve a total recovery of FA. The obtained results indicated that PLE is a rapid and efficient technique for FA extraction from fortified wheat flour.

Fabrication of electrolytic cell for online post-column electrochemical derivatization in ion chromatography.

An electrolytic cell (EC), composed of two ruthenium-plated titanium electrodes separated by cation-exchange membranes, was fabricated and evaluated for online postcolumn derivatization in ion chromatography (IC). Folic acid (FA) and methotrexate (MTX) were preliminarily used as prototype analytes to test the performance of EC. After separation by an anion exchange column, FA and MTX, which emit very weak fluorescence when excited, were electrochemically oxidized online in the anode chamber of the EC. The compounds with strong fluorescence, which are oxidation products, were detected by the fluorescence detector. The phosphate buffer solution (100 mM KH(2)PO(4)) served as an optimal eluent for anion exchange chromatographic separation and a suitable supporting electrolyte for electro-oxidation, leading to ideal compatibility between IC separation and the postcolumn electrochemical derivatization. For the presently proposed method, the linear ranges were from 0.01 mg L(-1) to 5 mg L(-1) for both FA and MTX. The detection limits of FA and MTX were 1.8 and 2.1 µg L(-1), and the relative standard deviations (RSD, n=7) were 2.9% and 3.6%, respectively. The method was applied for the simultaneous determination of FA and MTX in the plasma of patients being treated for rheumatoid arthritis. The determination of MTX in the urine of the patients of diffuse large B cell lymphoma was also demonstrated.

Improved folate extraction and tracing deconjugation efficiency by dual label isotope dilution assays in foods.

A dual label stable isotope dilution assay was developed to trace the deconjugation efficiency of polyglutamic folate vitamers converted to their monoglutamic analogues. For this purpose, [(13)C(5)]-pteroylheptaglutamate was synthesized and added during extraction of foods as a tracer isotopologue along with [(2)H(4)]-5-methyltetrahydrofolate, [(2)H(4)]-5-formyltetrahydrofolate, [(2)H(4)]-tetrahydrofolate, [(2)H(4)]-10-formylfolate, and [(2)H(4)]-folic acid. The [(2)H(4)]-labeled folates were used as internal standards for the monoglutamates. Deconjugation converted the addition tracer [(13)C(5)]-pteroylheptaglutamate to the detection tracer [(13)C(5)]-folic acid, which was quantified along with unlabeled folic acid using [(2)H(4)]-folic acid as the internal standard. LC-MS/MS enabled the unequivocal differentiation of the three isotopologues. This tracing was used to optimize deconjugation efficiency, which was achieved by using 4-morpholineethanesulfonic acid buffer for extraction at pH 5.0 . The optimized assay revealed limits of detection for the folate vitamers ranging between 2.0 and 5.6 pmol per assay (equivalent to 2.2-6.6 µg/100 g dry mass), recoveries ranging between 98 and 105% and relative standard deviations in inter-assay precision ranging between 2 and 6%. The assay was applied to quantitate folates in spinach, beans, cheeses, bread, wheat germs, and yeast .