Quantitation of total vitamin D₂ and D₄ in UV-exposed mushrooms using HPLC with UV detection after novel two-step...

A robust method for quantitation of total vitamin D₂ and D₄ in mushrooms by high performance liquid chromatography with UV detection (HPLC-UV) was developed to analyze mushrooms exposed to UV light. A two-step solid phase extraction (SPE) (silica, carbon black) removed chromatographic interferences typically resolved only with mass spectrometric detection (LC-MS) and allowed quantitation of all vitamin D and pre-D analytes. The vitamin and pre-vitamin forms of D₂, D₄ and D₃ (internal standard), as well as other photoisomers and sterols were resolved. Results for six types of UV-exposed mushrooms were comparable to LC-MS. Screening of ten additional types of UV-exposed mushrooms without the IS confirmed lack of interference with the IS. The limit of quantification (µg/100 g fresh weight) was 0.4 for vitamin D and 0.9 for pre-vitamin D. Mushrooms do not have to be dried, and separatory funnels and large solvent volumes were also eliminated from sample preparation.