Instructions for Ascorbic Acid (Vc) Test Kit
First, understand the kit composition and configuration (50T/48 samples)
Reagent 1: 5 ml of stock solution in a bottle. Store at 4°C for 6 months.
Reagent 1 application solution preparation: When using, mix reagent with double distilled water in a ratio of 1:14 and prepare it accordingly.
Reagent 2: 1 powder bottle. Store at 4°C for 6 months.
Reagent 2 application solution: Dissolve the powder by adding 0.36 ml of glacial acetic acid and then dilute to 40 ml with water. Store at 4°C.
Reagent 3: 1 white crystal powder. Store at 4°C for 6 months.
Reagent 3 application solution: Dissolve in Jiaonan (a specific solvent), fully dissolve within 2-4 hours, then add 60 ml of 95% ethanol or anhydrous ethanol. Prepare and store at 4°C.
Reagent 4: 0.2 ml of yellow stock solution in a bottle. Store in a refrigerator at 4–8°C, away from light, for 6 months.
Reagent 4 application solution: Take 0.15 ml of the stock solution and dilute to 25 ml with water. Use immediately after preparation.
Reagent 5: 6 ml of liquid in a bottle. Store at 4°C for 6 months.
Reagent 6: Vc standard, 6 mg of powder in 3 bottles. Each contains vitamin C. Store at 4°C for 6 months.
Preparation of 60 µg/ml Vc standard solution: Dissolve the standard powder in 10 ml of the application solution.
Preparation of 6 µg/ml Vc standard solution: Dilute the 60 µg/ml solution 10 times using the application solution.
Note: Vitamin C standards are prone to oxidation. It is best to use the standard after preparing the sample supernatant and testing within 10 minutes.
Second, procedure:
Step 1: Preparation of supernatant
Take 0.15 ml of sample and mix with 0.45 ml of Reagent 1. Vortex and incubate for 15 minutes. Centrifuge at 3500–4000 rpm for 10 minutes. The supernatant is ready for testing.
Step 2: Determination of Vc in the supernatant
| Blank tube | Standard tube | Measuring tube |
| Reagent 1 (mL): 0.4 | 6 µg/ml Vc standard (mL): 0.4 | Supernatant (mL): 0.4 |
| Reagent 2 (mL): 0.5 | Reagent 2 (mL): 0.5 | Reagent 2 (mL): 0.5 |
| Reagent 3 (mL): 1 | Reagent 3 (mL): 1 | Reagent 3 (mL): 1 |
| Reagent 4 (mL): 0.25 | Reagent 4 (mL): 0.25 | Reagent 4 (mL): 0.25 |
| Mix well, incubate at 37°C for 30 minutes | ||
| Reagent 5 (mL): 0.1 | Reagent 5 (mL): 0.1 | Reagent 5 (mL): 0.1 |
Mix thoroughly, let stand for 10 minutes. Measure absorbance at 636 nm, using a 1 cm or 0.5 cm path length. Use double distilled water as blank reference.
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