8th Edition, revised in February, 2018

（FOR RESEARCH USE ONLY. DO NOT USE IT IN CLINICAL DIAGNOSIS !）

Vitamin C (VC) Colorimetric Assay Kit

Catalog No: E-BC-K034-S

Method: Colorimetric method
Specification: 100 assays (Can detect 96 samples without duplication)
Measuring instrument: Spectrophotometer
Sensitivity: 0.35 μg/mL
Detection range: 0.35-20 μg/mL

This manual must be read attentively and completely before using this product.

If you have any problem, please contact our Technical Service Center for help.

Phone: 240-252-7368(USA) Fax: 240-252-7376(USA)

Email: techsupport@elabscience.com
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Please kindly provide us the lot number (on the outside of the box) of the kit for more efficient service.

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8th Edition, revised in February, 2018
Application
This kit can be used for detection of VC content in serum, plasma, animal/plant tissue and other samples.

Detection significance
Vitamin C (VC) is involved in the synthesis of collagen in the body. The deficiency of VC will result in
impaired wound healing and increased vascular fragility. VC is a strong reducing agent, which can reduce
Fe3+ to Fe2+ and promote the iron absorption in intestinal. VC can be used as scavenger of free radical
and it can remove O2- and OH-. Therefore, VC can protect the body from the damage of endogenous
oxygen free radicals, and can reduce the α2-tocopherol free radicals to vitamin E. VC is the most effective
antioxidant in organism.

Detection principle
The most obvious chemical activity of VC is that reduce Fe3+ to Fe2+, then promote iron absorption in
the intestine, promote the storage and utilization of iron. Fe3+ react immediately with reducing ascorbic
acid to form Fe2+ .then Fe2+ react with phenanthroline and the color developing reaction occurs. The
content of vitamin C in plasma can be determined. Measure the OD value and calculate the VC content
indirectly.

Kit components
Components Specifications Storage
Reagent 1 Extracting Solution 10 mL × 1 vial 2-8℃, 6 months, shading light
Preparation of Reagent 1 application solution: Dilute the reagent 1 with double distilled water at a
ratio of 1:14 and mix fully. The prepared solution can be stored at 2-8℃ for 7 days.
Reagent 2 Buffer Solution 50 mL × 1 vial 2-8℃, 6 months
Reagent 3 Chromogenic Agent 12 mL ×1 vial 2-8℃, 6 months, shading light
Preparation of substrate application solution: Dilute the reagent 3 with absolute ethyl alcohol (self-
prepared) at a ratio of 1:9 and mix fully. The prepared solution can be stored at 2-8℃ for 7 days with
shading light.
Reagent 4 Ferrum Reagent 1 mL × 1 vial 2-8℃, 6 months, shading light
Preparation of Reagent 4 application solution: Dilute 0.15 mL of the reagent 4 with double distilled
water to a final volume of 25 mL. Prepare the fresh solution before use.
Reagent 5 Stop Solution 12 mL × 1 vial 2-8℃, 6 months
Reagent 6 VC Standard 6 mg × 3 vials 2-8℃, 6 months, shading light
Preparation of 6 μg/mL standard solution: Dissolve a vial of reagent 6 with 1 mL of Reagent 1
application solution to prepare 6 mg/mL standard solution. Then dilute 6 mg/mL standard solution
with Reagent 1 application solution for 1000 times to prepare 6 μg/mL standard solution.
Note: VC standard is easy to oxidized, it is best to use 6 μg/mL VC Standard solution within 10 min

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8th Edition, revised in February, 2018

Experimental instruments
Test tube, Micropipettor, Vortex mixer, Centrifuge, 37℃ water bath, Spectrophotometer (536 nm)

Sample preparation
1. Serum (plasma): Take 0.15 mL of sample, add 0.45 mL of Reagent 1 application solution, mix
fully and stand for 15 min, then centrifuge at 2000 g for 10 min. Take the upper clear liquid for test.

2. 10% tissue homogenate: Weigh the tissue accurately and add PBS (0.01M, PH7.4) at a ratio of
weight (g): volume (mL) =1: 9, homogenize the tissue in ice bath, centrifuge at 10000 g for 10 min.
Take 0.15 mL of supernatant, add 0.45 mL of Reagent 1 application solution and mix fully, and
stand for 15 min, then centrifuge at 2000 g for 10 min. Take the upper clear liquid for test. And detect
the concentration of protein at the same time.

Operation steps
1. Blank well: add 0.4 mL of Reagent 1 application solution to the 5 mL EP tube.
Standard well: add 0.4 mL of 6 μg/mL standard solution to the 5 mL EP tube.
Sample well: add 0.4 mL of the upper clear liquid in sample preparation step to the 5 mL EP tube.
2. Add 0.5 mL of Reagent 2, 1 mL of Reagent 3 application solution and 0.25 mL of Reagent 4
application solution.
3. Mix fully with a vortex mixer and incubate at 37℃ water bath for 30 min.
4. Add 0.1 mL of Reagent 5 and mix fully with a vortex mixer.
5. Stand for 10 min at room temperature. Set the spectrophotometer to zero with double-distilled water
and measure the OD values of each tube at 536 nm with 1 cm optical path cuvette.

Note: The following operating table could be as a reference.

Blank tube Standard tube Sample tube
Reagent 1 application solution (mL) 0.4
6 μg/mL standard solution (mL) 0.4
The upper clear liquid (mL) 0.4
Reagent 2 (mL) 0. 5 0.5 0.5
Reagent 3 application solution (mL) 1 1 1
Reagent 4 application solution (mL) 0.25 0.25 0.25
Mix fully and incubate at 37℃ water bath for 30 min.
Reagent 5 (mL) 0.1 0.1 0.1
Mix fully and stand for 10 min at room temperature. Set the spectrophotometer to zero with
double-distilled water and measure the OD values of each tube at 536 nm with 1 cm optical
path cuvette.

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8th Edition, revised in February, 2018
Calculation of results
1. Serum (plasma) sample:

VC content ΔA1
= × c× f ×4*
(μg/mL) ΔA2

2. Tissue sample:
VC content ΔA1
= × c× f ÷Cpr×4*
(μg/mgprot) ΔA2

[Note]:
ΔA1: ODSample – ODBlank
ΔA2: ODStandard – ODBlank
c: Concentration of standard, 6 μg/mL.
f: Dilution factor of sample before test.
Cpr: Concentration of protein in sample, mgprot/mL.
4*: Dilution factor of sample preparation, 4 times.

Technical parameter
1. The sensitivity of the kit is 0.35 μg/mL.
2. The intra-assay CV is 2.3% and the inter-assay CV is 3.3%.
3. The recovery of the kit is 104%.
4. The detection range of the kit is 0.35-20 μg/mL.

Notes
1. This kit is for research use only.
2. Instructions should be followed strictly, changes of operation may result in unreliable results.
3. The validity of kit is 6 months.
4. Do not use components from different batches of kit.

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8th Edition, revised in February, 2018

Appendix: Standard curve

(This is for reference only)

Dilution of standard
Dilute 600 μg/mL VC Standard solution with Reagent 1 working solution to a serial concentration. The
recommended dilution gradient is as follows: 20, 15. 12, 9, 6, 3, 0 μg/mL.

Operation steps
Standard tube
Standard solution with different concentration (mL) 0.4
Reagent 2 (mL) 0.5
Reagent 3 application solution (mL) 1
Reagent 4 application solution (mL) 0.25
Mix fully and incubate at 37℃ water bath for 30 min.
Reagent 5 (mL) 0.1
Mix fully and stand for 10 min at room temperature. Set the spectrophotometer
to zero with double-distilled water and measure the OD values of each tube at
536 nm with 1 cm optical path cuvette.

Standard curve

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