2.3. Urea determination
Urea was analyzed by the diacetyl monoxime method (Wang,
Feng, Wu, Zhang, & Pan, 2010). In brief, 10.0 mL of the yellow rice
wine was eluted using the Carb solid-phase micro-extraction column
(200 mg/6 mL). The primary 1 mL elution fraction was discarded
and the following 2 mL eluate was collected for further
analysis. Subsequently, 2-mL water, 0.5-mL 2% diacetyl monoxime
water solution, and 3-mL acid reagents (see below) were added to
2 mL of the yellow rice wine eluate. The fraction was allowed to
boil in water for 15 min, and the reaction mixture was cooled in
ice water and analyzed using an ultraviolet–visible spectrophotometer
at 525 nm. Water (2 mL) was used as a control instead
of the yellow rice wine. The aforementioned acid reagent was prepared
by adding 1 mL of 98% sulfuric acid and 16.5 mL of 85% phosphoric
acid to 50 mL of water and further cooled to room
temperature, to which 0.5 g cadmium sulfate and 12.5 mg thiosemicarbazide
were added to a final volume of 250 mL with water.
Urea concentration was calculated by using a standard curve.