Vitamin D4 in mushrooms

Abstract

An unknown vitamin D compound was observed in the HPLC-UV chromatogram of edible mushrooms in the course of analyzing vitamin D(2) as part of a food composition study and confirmed by liquid chromatography-mass spectrometry to be vitamin D(4) (22-dihydroergocalciferol). Vitamin D(4) was quantified by HPLC with UV detection, with vitamin [(3)H] itamin D(3) as an internal standard. White button, crimini, portabella, enoki, shiitake, maitake, oyster, morel, chanterelle, and UV-treated portabella mushrooms were analyzed, as four composites each of a total of 71 samples from U.S. retail suppliers and producers. Vitamin D(4) was present (>0.1 µg/100 g) in a total of 18 composites and in at least one composite of each mushroom type except white button. The level was highest in samples with known UV exposure: vitamin D enhanced portabella, and maitake mushrooms from one supplier (0.2-7.0 and 22.5-35.4 µg/100 g, respectively). Other mushrooms had detectable vitamin D(4) in some but not all samples. In one composite of oyster mushrooms the vitamin D(4) content was more than twice that of D(2) (6.29 vs. 2.59 µg/100 g). Vitamin D(4) exceeded 2 µg/100 g in the morel and chanterelle mushroom samples that contained D(4), but was undetectable in two morel samples. The vitamin D(4) precursor 22,23-dihydroergosterol was found in all composites (4.49-16.5 mg/100 g). Vitamin D(4) should be expected to occur in mushrooms exposed to UV light, such as commercially produced vitamin D enhanced products, wild grown mushrooms or other mushrooms receiving incidental exposure. Because vitamin D(4) coeluted with D(3) in the routine HPLC analysis of vitamin D(2) and an alternate mobile phase was necessary for resolution, researchers analyzing vitamin D(2) in mushrooms and using D(3) as an internal standard should verify that the system will resolve vitamins D(3) and D(4).

Figure 1. HPLC chromatograms and UV spectra of vitamin D components in a mixed mushroom extract.

Chromatography on a Vydac® ODS column developed using (A) acetonitrile:methylene chloride (70∶30) (the solvent system used previously for quantitation of vitamin D₂ [14]), showing co-migration of the putative vitamin D₄ with vitamin D₃ in this system; (B) developed with acetonitrile:methanol (1∶1) mobile phase, showing separation of the peak containing putative vitamin D₄ and vitamin D₃ into two components.

Figure 2. High resolution mass spectral comparison of putative vitamin D₄ isolated from mushroom.

(A) Spectrum of HPLC-purified mushroom isolate corresponding to vitamin D₄ with structure and breakdown products highlighted. (B) Spectrum of vitamin D₄ standard.

Figure 3. Spectral analysis of putative dihydroergosterol in a mushroom isolate.

(A) High resolution mass spectrum of purified mushroom isolate corresponding to dihydroergosterol. (B) Gas chromatogram of products obtained following derivatization of the purified mushroom isolate with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). (C) Low resolution GC-MS of derivatized mushroom product at t = 17.20 min corresponding to dihydroergosterol with structure and breakdown products highlighted. (D) Low resolution GC-MS of commercially available ergosterol standard following derivatization with BSTFA.

Figure 4. Relationship between the vitamin D₄ and vitamin D₂ concentrations in ten types of mushrooms ( Table 1 ).

Data for vitamin D₂ were previously reported .

Figure 5. Structure of six forms of vitamin D their sterol precursors.