Development and Validation of an HPLC Method for the Determination of Thiamine and Riboflavin in Green Leafy Vegetables Using Clara‐Diastase

  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).