Development of simple method for the determination of 5-Methyl tetrahydrofolate in Dried Blood Spot by High Performance Liquid Chromatography (HPLC)
Abstract
Folate (Reduced) or folic acid (oxidized), is a water-soluble vitamin necessary for thymidylate synthase, methionine synthase, serine hydroxymethyltransferase, and folate deficiency associated with megaloblastic anemia, neural tube defect, and coronary heart disease. The available folate data is minimal due to a dearth of suitable, accurate, field-friendly blood collection and estimation methods. Conventional assays like microbiological and competitive protein-binding radioassay methods are used to estimate the total folate in serum and whole blood. In erythrocytes, folate exists mainly as 5-methyltetrahydrofolate polyglutamate and indicates the long-term storage of folate status. Therefore, the study aimed to develop a simple method for estimating 5-methyl tetrahydrofolate (5-MTHF) by high-performance liquid chromatography (HPLC) in dried blood spots (DBS) as an indicator of folate status. The present study results indicate standard 5-methyltetrahydrofolate with different concentrations with linear regression was showed R2-0.98, and the minimum detection limit of a standard was 20 pg. The inter-and intra-assay variations were found to be less than 10%. A good correlation (R2-0.964) between the DBS and blood and the recovery of added standard in the DBS sample was more than 80%. A minimum of three DBS punches equivalent to 20mL of blood was required for the analysis, and the competitive protein-binding radioassay showed slightly high folate compared to the HPLC method. The stability of 5-MTHF in DBS was also tested periodically. DBS technique and HPLC method are simple, sensitive, not expansive, and safe compared to the microbiological and competitive binding assay for assessing sub-clinical folate status in a field survey on a large population.
Keywords:
DBS, Finger prick blood spot, HPLC, 5-Methyltetrahydrofolate, Whole bloodDOI
https://doi.org/10.25004/IJPSDR.2021.130312References
Kubo Y, Fukuoka H, Kawabata T, Shoji K, Mori C, Sakurai K, Nishikawa M, Ohkubo T, Oshida K, Yanagisawa N, Yamashiro Y. Distribution of 5-Methyltetrahydrofolate and Folic Acid Levels in Maternal and Cord Blood Serum: Longitudinal Evaluation of Japanese Pregnant Women. Nutrients. 2020;12(6):1633.
Martins-Júnior HA, Wang AY, Alabourda J, Pires MA, Vega OB, Lebre DT. A validated method to quantify folic acid in wheat flour samples using liquid chromatography: tandem mass spectrometry. Journal of the Brazilian Chemical Society. 2008;19(5):971-7.
Czeizel AE, Dudás I, Vereczkey A, Bánhidy F. Folate deficiency and folic acid supplementation: the prevention of neural-tube defects and congenital heart defects. Nutrients. 2013;5(11):4760- 4775.
Caudill MA, Cruz AC, Gregory III JF, Hutson AD, Bailey LB. Folate status response to controlled folate intake in pregnant women. The Journal of nutrition. 1997;127(12):2363-2370.
Bailey SW, Ayling JE. The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake. Proceedings of the National Academy of Sciences. 2009;106(36):15424-9.
Lamers Y, Prinz-Langenohl R, Brämswig S, Pietrzik K. Red blood cell folate concentrations increase more after supplementation with [6 S]-5-methyltetrahydrofolate than with folic acid in women of childbearing age. The American journal of clinical nutrition. 2006;84(1):156-61.
Farrell CJ, Kirsch SH, Herrmann M. Red cell or serum folate: what to do in clinical practice?. Clinical Chemistry and Laboratory Medicine (CCLM). 2013;51(3):555-69.
Guthrie R, Susi A. A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics. 1963;32(3):338-43.
Kopp M, Rychlik M. Quantitation of 5-methyltetrahydrofolic acid in dried blood spots and dried plasma spots by stable isotope dilution assays. PLoS One. 2015 Nov 25;10(11):e0143639.
Suva MA. A brief review on dried blood spots applications in drug development. J. Pharm. Biosci. 2014;1:17-23.
Lehmann S, Delaby C, Vialaret J, Ducos J, Hirtz C. Current and future use of “dried blood spot” analyses in clinical chemistry. Clinical Chemistry and Laboratory Medicine (CCLM). 2013 Oct 1;51(10):1897-909.
Maase R, Skrinska V, Younes N, Hassan L, Mitri R, Matern D, Rinaldo P, Turgeon C. A rapid screening method for the measurement of neonatal total homocysteine in dried blood spots by liquid chromatography-tandem mass spectrometry. International Journal of Neonatal Screening. 2017;3(4):32.
McDade TW, Williams S, Snodgrass JJ. What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography. 2007;44(4):899-925.
O'Broin S, Kelleher B. Optimization of erythrocyte folate extraction. Clinical chemistry. 2001;47(12):2181-3.
O'Broin SD, Gunter EW. Screening of folate status with use of dried blood spots on filter paper. The American journal of clinical nutrition. 1999;70(3):359-67.
Pfeiffer CM, Gregory 3rd JF. Enzymatic deconjugation of erythrocyte polyglutamyl folates during preparation for folate assay: investigation with reversed-phase liquid chromatography. Clinical chemistry. 1996;42(11):1847-54.
Arning E, Bottiglieri T. Quantitation of 5-methyltetrahydrofolate in cerebrospinal fluid using liquid chromatography-electrospray tandem mass spectrometry. In clinical applications of mass spectrometry in biomolecular analysis. Humana Press, New York.. 2016;175-182.
Berti C, Fekete K, Dullemeijer C, Trovato M, Souverein OW, Cavelaars A, Dhonukshe-Rutten R, Massari M, Decsi T, Van't Veer P, Cetin I. Folate intake and markers of folate status in women of reproductive age, pregnant and lactating women: a meta-analysis. Journal of nutrition and metabolism. 2012;2012:1-13.
Harrell Shreckengost C, Kim AW, Whitaker SH, Weng L, Pearce BD, Kaiser BN. Dried blood spot microbiological assay indicates high prevalence of folate deficiency in rural adult men and women in the Haitian Central Plateau. International health. 2019;11(6):487-95.
Horne DW. High-performance liquid chromatographic measurement of 5, 10-methylenetetrahydrofolate in liver. Analytical biochemistry. 2001;297(2):154-9.
Jastrebova J, Witthöft C, Grahn A, Svensson U, Jägerstad M. HPLC determination of folates in raw and processed beetroots. Food Chemistry. 2003;80(4):579-88.
Ohrvik VE, Witthoft CM. Human folate bioavailability. Nutrients. 2011;3(4):475-90.
Opladen T, Ramaekers VT, Heimann G, Blau N. Analysis of 5-methyltetrahydrofolate in serum of healthy children. Molecular genetics and metabolism. 2006 ;87(1):61-5.
Vardhanapu Mary Bharathi, Devanaboyina Narendra, Gadi Vijaya Lakshmi, Yendamuri Narayudu, Penumantra Venkata Kishore. Method development and validation for simultaneous estimation of l-methyl folate & escitalopram by RP-HPLC method. World journal of pharmaceutical and medical research.2019; 5(10):101-6.
Villalpando S, Montalvo-Velarde I, Zambrano N, García-Guerra A, Ramírez-Silva CI, Shamah-Levy T, Rivera JA. Vitamins A, and C and folate status in Mexican children under 12 years and women 12-49 years: a probabilistic national survey. Salud publica de mexico. 2003;45:508-19.
Obeid R, Schön C, Wilhelm M, Pietrzik K, Pilz S. The effectiveness of daily supplementation with 400 or 800 μg/day folate in reaching protective red blood folate concentrations in non-pregnant women: a randomized trial. European journal of nutrition. 2018;57(5):1771- 80.
Krishnaswamy K, Nair KM. Importance of folate in human nutrition. British Journal of Nutrition. 2001;85(S2):S115-24.
Hyland K, Surtees R. Measurement of 5-methyltetrahydrofolate in cerebrospinal fluid using HPLC with coulometric electrochemical detection. Pteridines. 1991;3(1-2):149-50.
Gregory III JF, Sartain DB, Day BP. Fluorometric determination of folacin in biological materials using high performance liquid chromatography. The Journal of nutrition. 1984;114(2):341-53.
Kalmbach R, Paul L, Selhub J. Determination of unmetabolized folic acid in human plasma using affinity HPLC. The American journal of clinical nutrition. 2011;94(1):343S-7S.
King MJ, Brima EI, Dainty JR, Barrett DA, Wright AJ, Willis G, Finglas PM. Estimation of the 5-methyltetrahydrofolate apparent volume of distribution in humans. The Journal of nutrition. 2012;142(2):389- 95.
O’Broin SD, Kelleher BP, Davoren A, Gunter EW. Field-study screening of blood folate concentrations: specimen stability and finger-stick sampling. The American journal of clinical nutrition. 1997;66(6):1398-405.
Giulidori P, Galli-Kienle M, Stramentinoli G. Liquid-chromatographic monitoring of 5-methyltetrahydrofolate in plasma. Clinical chemistry. 1981 Dec 1;27(12):2041-3.
Leeming RJ, Pollock A, Melville LJ, Hamon CG. Measurement of 5-methyltetrahydrofolic acid in man by high-performance liquid chromatography. Metabolism. 1990;39(9):902-4.
Truswell AS, Kounnavong S. Quantitative responses of serum folate to increasing intakes of folic acid in healthy women. European journal of clinical nutrition. 1997;51(12):839-45.
Fazili Z, Pfeiffer CM, Zhang M, Jain R. Erythrocyte folate extraction and quantitative determination by liquid chromatography–tandem mass spectrometry: comparison of results with microbiologic assay. Clinical chemistry. 2005 ;51(12):2318-25.
Wilson SD, Horne DW. High-performance liquid chromatographic determination of the distribution of naturally occurring folic acid derivatives in rat liver. Analytical biochemistry. 1984 ;142(2): 529-535.
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