Determination of Water-soluble Vitamin by Microbiological Assay
B. Preparation of Sample Assay Solution
8.3 Oxidation of ascorbic acid in the extract
1) Dilute standards (1, 3, 5, 10 ml of solution E) corresponding to 1, 3, 5, 10 mg of ascorbic acid, respectively, are made up to the mark in a 100 ml vol flask with solution A. The final concentration of each standard is 10, 30, 50 and 100 μg per ml, respectively.
2) Transfer sample solution or standards to each 250 ml Erlenmeyer flask containing 2 g acid-washed Norit and swirl vigorously for two minutes.
Immediately filter through whatman #42 paper into a 125 ml Erlenmeyer flask, discarding the first few cloudy drops of filtrate. Note: If the filtrate is gray or cloudy, it must be refiltered through a 0.45 micron syringe tip filter before continuing with the procedure.
3) To the blank volumetrics (containing 5 ml of H3BO3 / NaOAc solution), add 5.0 ml sample or standard filtrate and allow to stand for 15 minutes with occasional swirling. It is important that all blanks sit at least 15 minutes.
8.4 Fluorimetry (Formation of Quinozaline)
1) Add 5.0 ml sample or standard filtrate to the other 100 ml volumetrics flask containing the 5.0 ml NaOAc solution. Dilute to volume with deionized water and mix well.
2) After 15 minutes has elapsed, dilute blanks to volume with deionized water and mix well. Transfer 2.0 ml from each 100 ml volumetric flask into two test tubes. Two sample tubes and two blank tubes for each sample or standard - four tubes in all).
Note: The rest of the procedure should be performed in subdued lighting.
3) Add 5.0 ml o-phenylenediamine solution to tubes and stir with vortex mixer.
4) Let tubes stand 35 minutes in the dark at room temperature. The 35 minute time period can be started after the first set of four tubes has been stirred with the vortex mixer, as the time elapse during tube reading is about the same as for o-phenylenediamine and stirring.
5) Allow 15 minutes for the spectrofluorometer and computer system to warm up.
6) The emission at 430 nm with a 350 nm excitation is then measured (main and blank values). Record the fluorescence of the blank, sample, standards.
9. CALCULATION
1) Standard test values for the ascorbic acid standard concentrations of ascorbic acid per ml can be plotted or calculated by linear regression.
2) The values of sample in micrograms of ascorbic acid per ml can be read off the standard calibration curve or calculated.
3) The ascorbic acid content in 100 g of the sample material investigated can be calculated.
mg ascorbic acid/g or mL = [(average X – average D)/(average C – average B)] x (20 x (20 x S x V/E)
Where, V = 100 = initial assay solution volume E = number of g or mL of sample
S = concentration of standard in mg/mL added to reading tube 10. ACCEPTANCE OF RESULTS
1) In-house control sample using orange juice powders (Tang).
2) The linearity of the calibration curve is in the range 5-150 μg ascorbic acid per ml of standard test solution.
3) A recovery of 100 + 5% is found in the supplementary test.
11. METHOD VALIDATION
An amount of 5 mg of ascorbic acid per 100 g of sample or 10 μg per ml of standard test solution can be quantitatively evaluated. The upper detection limit for this method is 150 mg per 100 g of sample.
12. APPENDIX
12.1 Charcoal activated (Sigma, C-4386) can be used instead of Norit.
12.2 Instrument Performance Checks 12.2.1 Sensitivity
Check frequency: When use
Perform an instrument sensitivity check with a 100 ug/mL standard solution and plot the signal on the control chart. The signal should be within the acceptable control limit.
12.2.2 Detection Limit Frequency: Yearly
Determine a reagent blank and a 0.25 ug/mL standard solution 10 consecutive times. The standard solution should give a minimum response of three times the standard deviation of the blank.
Compare the detection limit against previous results.
12.3 Recommended QA/QC protocols include:
12.3.1 An instrument sensitivity check using the 100 ug/mL standard solution and plotting the signal on the control chart. The signal should be within the acceptable control limit.
12.3.2 QC standard check of 100 ug/mL (sec. 8.2) per batch of 10 samples. A 15% acceptance criteria applies.
12.3.3 1 reagent blank per batch of 10 samples.
12.3.4 1 spike per batch of samples if batch is larger than 10 samples or per every 10 samples tested if batch is smaller than 10 samples.
12.3.5 1 duplicate sample per batch of samples if batch is larger than 10 samples or per every 10 samples if batch is smaller than 10 samples.
VITAMIN C ANALYSIS IN JUICES BY TITRATION METHOD 1. PURPOSE/SCOPE:
The method is applicable to determination of reduced ascorbic acid in food products, juices and vitamin preparation. The method is not applicable to juices with dark red, blue and violet color.
2. SAFETY:
2.1 Carry out sample preparation and analysis under subdued light 2.2 Wear gloves to reduce contact of corrosive substances with skin.
2.3 Use fume hood to reduce inhalation of acids.
2.4 If you do not know the hazards of the chemicals involved, consult MSDS.
3. REFERENCES:
Official Methods of Analysis of AOAC International. 1997, 16th ed., 3rd rev.
Association of Vitamin Chemists Inc. 1951 Methods of Vitamin Assay. 2nd ed.
on. Interscience Publishers, Inc., New York.
4. DEFINITION:
Vitamin C is the ascorbic acid determined by the reduction with 2, 6- dichloroindophenol solution to a colorless dye.
5. PRINCIPLE:
Ascorbic acid reduces oxidation-reduction indicator dye, 2, 6-dichloroindophenol to colorless solution. After the ascorbic acid is oxidized to dehydroascorbic acid, excess dye remains pink in acid solution. At end point, excess unreduced dye is rose pink in acid solution. Vitamin is extracted and titration performed in presence of HPO3-CH3COOH or HPO3-CH3COOH-H2SO4 solution to maintain proper acidity for reaction and to avoid autoxidation of ascorbic acid at high pH.
REACTION
O=C O=C
HO-C O=C
HO-C + O=C
C H -C
HO-C-H HO-C-H
CH2OH CH2OH
2,6 Dichloroindophenol Ascorbic acid colorless dye Dehydroascorbic acid
Cl Cl O
N
+
Cl O Cl
NH
OH OH