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).