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Development and Validation of an HPLC Method for the Determination of Thiamine and Riboflavin in Green Leafy Vegetables Using Clara‐Diastase
Author(s) -
del Carmen MondragónPortocarrero Alicia,
VázquezOdériz Lourdes,
RomeroRodríguez MaÁngeles
Publication year - 2011
Publication title -
journal of food science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.772
H-Index - 150
eISSN - 1750-3841
pISSN - 0022-1147
DOI - 10.1111/j.1750-3841.2011.02151.x
Subject(s) - diastase , riboflavin , leafy vegetables , thiamine , chemistry , chromatography , food science , high performance liquid chromatography , biochemistry
There is no literature on the use of the currently available form of clara‐diastase (≥ 35 U/mg; Fluka Product No. 86959) in the analysis of vitamins B 1 and B 2 . Consequently, a method for the determination of total riboflavin and total thiamine in green leafy vegetables by high‐pressure liquid chromatography (HPLC) following sample preparation by sequential acid hydrolysis, enzymatic hydrolysis with clara‐diastase ≥ 35 U/mg, and for B1, derivatization to thiochrome, was developed and validated. Prepared samples are run at 35 °C on a 250 × 4.6 mm column of 5 μm Kromasil 100 C 18 , using a 0.8 mL/min flow of a 72:28 mixture of 5 mmol/L ammonium acetate and methanol as mobile phase and fluorescence detection. The linear response ranges and limits of detection are, respectively, 3.0 to 60.0 and 0.15 ng/mL for B 1 , and 3.1 to 155.0 and 0.103 ng/mL for B 2 . Of the 4 green leafy vegetables examined, young turnip tops had the highest B 1 and B 2 levels (0.19 and 0.20 mg/100 g fresh weight), followed by spinach (0.12 and 0.09 mg/100 g), lettuce (0.11 and 0.05 mg/100 g), and chard (0.04 mg/100 g).