Determination of folate vitamers in food and in Italian reference diet by high-performance liquid chromatography (original) (raw)
Related papers
Analytical and bioanalytical chemistry, 2003
Stable isotope dilution assays were developed for the quantification of the folate vitamers 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, tetrahydrofolate, 10-formylfolate and pteroylglutamic acid in food samples by using deuterated isotopomers as internal standards. Vitamers and their labeled analogues were analyzed simultaneously by HPLC/MS/MS using selected reaction monitoring, which allowed a higher specificity than other methods published previously. Sample preparation involved treatment by protease in sequence with alpha-amylase and rat serum deconjugase, followed by anion exchange chromatography. The detection limits for 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, tetrahydrofolate, 10-formylfolate and pteroylglutamic acid were found to be 0.5, 1.2, 1.5, 0.6 and 2.6 microg/100 g fresh weight, respectively. Using the new method, folate contents were determined in meat, cereals, and vegetables. Data were in good agreement with literature data, except results for br...
Food Chemistry, 1997
The folate content of 30 commodities of vegetables, fruits and berries, including a few processed products, was determined by high-performance liquid chromatography (HPLC). HPLC was used with a combination with fluorescence and ultraviolet detectors to analyze folate monoglutamate derivatives and their distribution after extraction at pH 6.0 and deconjugation with hog kidney deconjugase at pH 4.9. 5Methyltetrahydrofolate was the main derivative in all foods studied, but tetrahydrofolate, 5-formyltetrahydrofolate and IO-formylfolic acid were also detected. The sum of the monitored folate derivatives (as ug folic acid) in vegetables ranged from 9 to 114 ug per 100 g and that of berries and fruits from 3 to 36 ug per 100 g. The variation in folate content, which was studied by analyzing raw potato, carrot and cabbage bought from retail shops three times a year, was small. Some of the studied processed vegetable foods were also reasonably good sources of folate. The results obtained with HPLC are rather similar to the previously reported values for vegetables determined by a microbiological method. Several measures for method improvement and quality control of the analysis allowed reliable determination of the main folate forms, particularly S-methyltetrahydrofolate, in a wide range of plant-derived foods. 0 1997 Elsevier Science Ltd
Third EU MAT intercomparison study on food folate analysis using HPLC procedures
Food Chemistry, 1996
Three samples (milk powder, lyophilized pig's liver and wholemeal flour), a 5methyltetrahydrofolic acid (5-MTHF) calibrant and two deconjugase enzymes (purified hog kidney and human plasma) were circulated to three laboratories taking part in the study. The objectives were to optimize the deconjugation step in these foods and to improve the between-laboratory agreement in HPLC results for folates. The predominant natural folate form in milk powder was 5-MTHF, together with appreciable amounts of folic acid. In pig's liver 5-MTHF was found to represent about one-third of the total folate content found. For these two foods, results from one laboratory of the sum of the folate vitamers agreed favourably with the microbiological data. 5-MTHF was most successfully determined by all three laboratories. There was little or no agreement found for the other folate vitamers detected.
Analytical Biochemistry, 2003
A comparison study on folate quantitation was carried out between the recently developed stable isotope dilution assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the frequently used HPLC with fluorimetric detection (LC-FD). By applying LC-MS-MS, spinach, wheat bread, beef, and blood plasma were found to contain 159.2, 19.8, 1.2, and 5.6 lg/100 g total folates, respectively, whereas the respective quantitative data obtained by LC-FD were 95.5, 16.2, 0.7, and 6.8 lg/100 g. In all samples, LC-MS-MS revealed superior selectivity and precision and circumvented the shortcomings of conventional LC techniques, i.e., ambiguous peak assignment as well as high detection limits for 5-formyltetrahydrofolate, 10-formylfolic acid, and folic acid. The affinity chromatography columns used in this study showed excellent cleanup performance and permitted detection limits as low as 0.1, 0.5, 0.1, 0.08, and 0.1 lg/100 g for tetrahydrofolate (H 4 folate), 5-methyl-H 4 folate, 5-formyl-H 4 folate, 10-formylfolate, and pteroylglutamic acid, respectively. Thus, a 10-fold higher sensitivity compared to solid-phase anion-exchange cartridges was achieved. However, affinity chromatography columns revealed a significantly higher affinity toward the natural vitamers than to the racemic isotopomeric standards, which has to be considered when applying the latter in stable isotope dilution assays.
Standardisation of HPLC techniques for the determination of naturally-occurring folates in food
Food Chemistry, 1999
The aim of this work was to evaluate current in-house HPLC procedures for the determination of naturally-occurring folates in food, and to identify problem areas for further improvement. Five intercomparison studies were completed over the period 1990± 1997 in which nine participants from six countries took part. Through careful validations and detailed discussions held at evaluation meetings, possible biases and sources of systematic error have been identi®ed and reduced. The use of ascorbic acid and nitrogen¯ushing during extraction, sample clean-up using strong anion exchange columns, spectrophometrically calibrated standards and¯uorescence detection are all recommended. Both in-house hog kidney and human plasma deconjugase enzymes gave similar results to the circulated common hog kidney enzyme which was prepared from fresh pig's kidneys. The most consistently reported values were for 5-CH 3 H 4 -PteGlu, and to a lesser extent, for H 4 PteGlu. Four candidate reference materials (CRM 121, wholemeal¯our; CRM 421, milk powder; CRM 485, lyophilised mixed vegetables, and CRM 487, lyophilised pig's liver) have been proposed with both indicative values (mean uncertainty) for 5-CH 3 H 4 -PteGlu in CRM 421 (0.25; 0.02 mg/kg) and CRM 485 (2.14; 0.42 mg/kg), and information values (mean; range) for 5-CH 3 H 4 -PteGlu in CRM 121 (0.04; 0.03±0.08 mg/kg) and CRM 487 (2.6; 1.9±3.8 mg/kg). Certi®ed values are also given for total folate by microbiological assay: CRM 121 (0.50; 0.07 mg/kg), CRM 421 (1.42; 0.14 mg/kg), CRM 485 (3.15; 0.28 mg/kg), and CRM 487 (13.4; 1.3 mg/kg). Average recovery of 5-CH 3 H 4 -PteGlu, added prior to extraction and deconjugation, was 91% (84±95%) for the four CRMs. The average within-and betweenlaboratory variations were 6 and 15% for the determination of 5-CH 3 H 4 -PteGlu by HPLC, and 9 and 18% for the determination of total folate by microbiological assay. These CRMs will be used for quality control of folate measurements for nutritional labelling, and validation of new techniques. Further methodology work is required for the HPLC analyses of folate forms other than 5-CH 3 H 4 -PteGlu. #
Comparison of UPLC and HPLC for Analysis of Dietary Folates
2011
Ultra performance liquid chromatography (UPLC) using small sub-2 μm particles and high performance liquid chromatography (HPLC) were compared for separation and determination of the most common dietary folates; 5-methyltetrahydrofolate, tetrahydrofolate, 5-formyltetrahydrofolate, 10-formylfolic acid and folic acid. Two UPLC columns—Acquity BEH C18 and Acquity HSS T3, and two HPLC columns with similar surface chemistry—Xbridge C18 and Atlantis d18 were tested. When using UPLC, the signal-to-noise ratio could be improved by a factor of 2–50 for different folate derivatives and the run time could be reduced fourfold without sacrificing separation efficiency. The applicability of UPLC to real food samples was demonstrated.
Naturally occurring folates in selected traditionally prepared foods in Southern India
Journal of Food Science and Technology, 2017
A wide range of Indian foods (cereals, pulses, vegetables and milk based preparations) were analysed for five folate vitamers naturally present in the foods (n = 44). A liquid chromatography-mass spectrometry (LC-MS/ MS) method using reversed phase chromatography and tandem mass spectrometry, coupled via positive mode electrospray ionization was used for the detection and quantification of the vitamers. The optimized LC-MS/MS method was capable of analysing the five most commonlyoccurring folates (folic acid, 5-methyl tetrahydrofolic acid, tetrahydrofolic acid, 10-formyl folic acid and 5-formyl tetrahydrofolic acid) in 20 min. Quantification of folates was performed using 13 C labelled internal standards. 5-methyl tetrahydrofolate was predominant in cereals, pulses and vegetable preparations. Fermented cereal preparations, beverages (coffee and tea) and green leafy vegetables were the main sources contributing to 5-formyl THF. Folic acid was identified in home-made yoghurt. All the values obtained in the present study using LC-MS/MS were compared to the total folate analysed using the microbiological assay in 2010 to generate data on the same foods. Findings suggest that the data obtained using both techniques showed agreement in the values (total folate calculated by adding the individual vitamers in the case of the LC-MS/MS values) particularly when foods were predominant in 5 methyl tetrahydrofolate. Keywords Folates Á Liquid chromatography-mass spectrometry Á Stable isotopes Á Internal standards Á Microbiological assay Abbreviations LC-MS/ MS Liquid chromatography-tandem mass spectrometry SRM Selected reaction monitoring HPLC High pressure liquid chromatography GCMS Gas chromatography mass spectrometry ESI Electrospray ionisation APCI Atmospheric pressure chemical ionisation m/z Mass to charge ratio R f Response factor SPE-SAX Solid phase extraction-strong anion exchange CID Collision induced dissociation CRM Certified reference materials & Jayashree Arcot
Tetrahydrofolate is the parent molecule of the folate coenzymes required for one carbon metabolism. Together with other unsubstituted folates such as dihydrofolate and folic acid, tetrahydrofolate represents the third pool of dietary folates following 5-methyltetrahydrofolate and formyl folates. Low intake of dietary folates and poor folate status are common problems in many countries. There is a critical need for reliable methods to determine folate in foods to accurately estimate folate intakes in populations. However, current values for folates in foods in databanks are often underestimated due to the high instability of several folate forms, especially tetrahydrofolate. The present review highlights the occurrence of unsubstituted folates in foods and their oxidation mechanisms and chemical behavior as well as interconversion reaction between tetrahydrofolate and 5,10-methylenetetrahydrofolate. The review shows also the important role of antioxidants in protecting folates during analysis and describes strategies to stabilize unsubstituted folates throughout all steps of the analytical procedure.
Journal of Chromatography A, 2007
A sensitive and reliable liquid chromatography-mass spectrometry (LC-MS) method to determine dietary folates was developed and validated. Folates were detected and quantified using positive electrospray ionisation (ESI) with selective ion monitoring of protonated ions [M+H] + . The effects of buffer nature and mobile phase composition on separation, peak shape and intensity of MS signal were investigated. The acidic-basic properties of folates were successfully used to predict possible ionisation patterns, but they were not sufficient to predict the intensity of MS signal and the proportion of different ionisation products, which indicated that other parameters, such as gas phase acidity/basicity of analytes and ion evaporation mechanisms might be important. The use of aqueous acetic acid as volatile buffer was found to be preferable compared to formic acid due to considerable gain in intensity of MS signal for all folate forms studied. Limits of quantifications were 0.3 ng/mL for 5-methyltetrahydrofolate and 0.6 ng/mL for tetrahydrofolate, 10-formylfolic acid, 5-formyltetrahydrofolate and folic acid when using 20 L injection. For 10-formylfolic acid, 5-formyltetrahydrofolate and folic acid the MS detection was found to be superior over commonly used fluorescence and UV detection in terms of selectivity and sensitivity. The method was successfully applied to analysis of folates in baker's yeast.