Improved Stable Isotope Dilution Assay for Dietary Folates Using LC-MS/MS and Its Application to Strawberries

Abstract

Folates play an important role in the human body and a deficiency of this vitamin can cause several diseases. Therefore, a reliable analytical method is crucial for the determination of folate vitamers in strawberries and other dietary folate sources. A stable isotope dilution LC-MS/MS method for analyzing folates in food was developed and validated. The folate vitamers Pteroylmonoglutamic acid, tetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate were quantified using 13C-labeled internal standards. Validation of the assay was accomplished by determining linearity, precision, recovery, limit of detection, and limit of quantification and revealed to be a precise, sensitive, and accurate method to determine folate vitamers. Strawberries are worldwide consumed and known to be a good dietary source of nutritive compounds. Using this method, folate concentrations in selected commercial strawberry cultivars and experimental breeding lines grown in Germany and Australia were investigated. Total folates varied from 59 to 153 μg/100 g on fresh weight basis. Furthermore, folate content after lyophilizing or freezing did not show any significant differences compared to fresh strawberries. However, significant losses of total folates in pureed strawberries could be observed after 5 days of storage with only 16% of the original concentration retained. In summary, some of the investigated strawberry cultivars/breeding lines can be considered as rich dietary sources of natural folates.

Comparison of ESI‐MS/MS and APCI‐MS methods for the quantification of folic acid analogs in C. elegans

“…Both methods resulted in high intraday and interday precision, 6.26% and 8.32% for ESI‐MS/MS and 11.5% and 11.29% for APCI‐MS. These results are comparable with works from Striegel et al and Garratt et al…”

“…Both methods resulted in high intraday and interday precision, 6.26% and 8.32% for ESI-MS/MS and 11.5% and 11.29% for APCI-MS. These results are comparable with works from Striegel et al and Garratt et al 33,34 We additionally investigated the recovery and the matrix effects related to folates. For the analysis of recovery, we spiked pteroic acid (100 ng) into the C. elegans extracts (n = 4).…”

“…The selection of unique and intense fragment ions in the ESI‐MS/MS method resulted in a better LOQ ranging from 6 pmol to 12 nmol, which is ≈20 fold lower compared with previous published data . The ESI‐based method was comparable with that of Striegel et al; however, we were able to detect four more analogs of FA . The new APCI‐MS approach was less sensitive when compared with the ESI‐MS/MS, but the LOQs for most of the intermediates were comparable to the work of Chen et al Nevertheless using the APCI‐based method, we retrieved the concentration of DHF, which was not possible using the ESI ion source.…”

“…Additionally, all spectra had ion clusters that indicated the pteridine ring is the main target for protonation and decomposition. The CID fragmentation often results in the cleavage between the para‐amino benzoic acid and the glutamate moiety leading to the formation of product ions such as, m/z 295.10 (C 14 H 11 N 6 O 2+ ) in FA, m/z 297.10 (C 14 H 13 N 6 O 2+ ) in DHF, m/z 299.15 (C 14 H 15 N 6 O 2+ ) in THF, and m/z 313.04 in 5MTHF, which were already described by Striegel et al The daughter ion at m/z 335.20 (C 17 H 15 N 6 O 2+ ) in ML, as well as the product ion at m/z 310.14 (C 15 H 16 N 7 O + ) in MN and the product ion at m/z 327.15 (C 15 H 15 N 6 O 3+ ) in FO, belongs to the same ion series (Figures and ).…”

“…The product ion spectra of 5MTHF is composed of ions at m/z 378.21 (C 18 H 24 N 3 O 6 + ) and at m/z 177.07. Both belong to a specific ion series of the tetrahydrofolate containing molecules . Although we were not able to assign a structure to the ion at m/z 337.23, we propose the elemental composition for this ion as C 16 H 21 N 2 O 5 + (Figure D).…”