Determination of fortified and endogenous folates in food-based Standard Reference Materials by liquid chromatography-tandem mass spectrometry.

The National Institute of Standards and Technology (NIST) is developing a wide variety of Standard Reference Materials (SRMs) to support measurements of vitamins and other nutrients in foods. Previously, NIST has provided SRMs with values assigned for the folate vitamer, folic acid (pteroylglutamic acid), which is fortified in several foods due to its role in prevention of neural tube defects. In order to expand the number of food-based SRMs with values assigned for folic acid, as well as additional endogenous folates, NIST has developed methods that include trienzyme digestion and isotope-dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Sample preparation was optimized for each individual food type, but all samples were analyzed under the same LC-MS/MS conditions. The application of these methods resulted in folic acid values for SRM 1849a Infant/Adult Nutritional Formula and SRM 3233 Fortified Breakfast Cereal of (2.33 ± 0.06) µg/g and (16.0 ± 0.7) µg/g, respectively. In addition, the endogenous folate vitamer 5-methlytetrahydrofolate (5-MTHF) was detected and quantified in SRM 1849a Infant/Adult Nutritional Formula, candidate SRM 1549a Whole Milk Powder, and candidate SRM 1845a Whole Egg Powder, resulting in values of (0.0839 ± 0.0071) µg/g, (0.211 ± 0.014) µg/g, and (0.838 ± 0.044) µg/g, respectively. SRM 1849a Infant/Adult Nutritional Formula is the first food-based NIST SRM to possess a reference value for 5-MTHF and the first certified reference material to have an assigned 5-MTHF value based on LC-MS/MS. The values obtained for folic acid and 5-MTHF by LC-MS/MS will be incorporated into the final value assignments for all these food-based SRMs.

Analysis of 5-methyltetrahydrofolate in serum of healthy children.

5-Methyltetrahydrofolate (5MTHF) is the active one-carbon donor and the principal circulating form of plasma folates. It is involved in a number of metabolic and neurodevelopmental processes and analysis of cerebrospinal fluid (CSF) 5MTHF is of great importance in the diagnosis of cerebral folate deficiency (CFD). Serum 5MTHF levels and the 5MTHF serum/CSF ratio may be important additional parameters for the understanding of CFD. We developed a HPLC method for the measurement of 5MTHF in serum and established reference values for the pediatric population. Serum samples from 64 healthy children were extracted with Sep-Pak C18 cartridges and 5MTHF was separated by RP-HPLC and quantified by electrochemical detection. 5MTHF was separated from other folates and detected after 8.7 min with linearity of up to 1600 nmol/L. The detection limit was 4.5 nmol/L and recovery during solid-phase extraction for low and high concentrations of 5MTHF was 66 and 62%, respectively. Within-run imprecision (13.5%) was slightly higher than run-to-run imprecision (8.5%). 5MTHF levels in healthy children were found to be age-dependent, decreasing from 158.0 nmol/L in newborns to 60.1 nmol/L in children older than 16 years. The method we describe is sensitive, selective, and reliable for the analysis of 5MTHF from 400 microL of serum.

Determination of folates in human plasma using hydrophilic interaction chromatography-tandem mass spectrometry.

Folic acid is an essential nutrient, and folate deficiency is associated with a variety of disorders including neural tube defects (during pregnancy) and heart disease. A fast, sensitive, and robust HPLC-tandem mass spectrometry (LC-MS-MS) method was developed for the quantification of free folic acid, tetrahydrofolate, 5'-methyltetrahydrofolate, and 5'-formyltetrahydrofolate in human plasma. Sample preparation required only acetonitrile precipitation of proteins followed by filtration instead of solid-phase extraction or solvent-solvent extraction as in other methods. The rapid and streamlined sample handling procedure minimized degradation of the highly unstable folate species. Hydrophilic interaction chromatography was used for additional sample cleanup on-line, and baseline separation and detection of all four folate species was achieved in less than 30 min. The folate species were detected using negative ion electrospray-tandem mass spectrometry with multiple reaction monitoring of the diagnostic fragment ions of each deprotonated molecule. The predominately organic (hydrophobic) solvent system combined with the microbore flow rate (50 microL/min) used for the chromatography resulted in enhanced electrospray signal response compared to reversed-phase HPLC using a wider bore column. The recovery of all folate species (from spiked plasma) was >97% over a concentration range from 300 pg/L to 12 mg/L with intraday precision (RSD, n = 5) of 3.7-6.5%. Stability studies were carried out for spiked samples in order to define storage and handling conditions. The folic acid limit of quantification (LOQ) in human plasma was 80 pmol/L +/- 10%, and the limit of detection (LOD) was 37.5 pmol/L. The LOQ and LOD for tetrahydrofolate, 5'-methyltetrahydrofolate, and 5'-formyltetrahydrofolate were 1250, 400, and 360 pmol/L of plasma and 425, 165, and 140 pmol/L of plasma, respectively.

Evaluation of pteroylglutamic acid and n-5-methyltetrahydrofolic acid reference standards in a new "No-Boil" radioassay for serum folate.

Serum folate, pteroylglutamic acid standards, and N-5-methyltetrahydrofolate standards extract differently during sample preparation for radiometric assay. B/B0 values for each material were determined before and after an alkaline extraction procedure. Serum folate most closely resembled N-5-methyltetrahydrofolate standards in that it exhibited a slightly greater ability to displace iodinated folate derivative in competition for beta-lactoglobulin binder. Pteroylglutamic acid standards displaced folate derivative less well, so that serum folate values were lower than when N-5-methyltetrahydrofolate was used as the standard material.