Vitamin C Assay Kit. ( DNPH method )

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1 Product No.SML-ROIK2-EX Vitamin C Assay Kit ( DNPH method ) Vitamin C (L-Ascorbic acid) is water-soluble vitamin with strong reducing action and is an important coenzyme for internal hydroxylation reactions (e.g. collagen). Vitamin C is found in both reduced form (ascorbic acid (AsA)) and oxidized form (dehydroascorbic acid (DHAsA)). This Vitamin C Assay Kit measures total vitamin C (AsA + DHAsA). The method of this kit is a refinement of the colorimetric assay described by Daniel (973). REAGENTS [Sufficient for assay points] * SAMPLE PREPARATION ) Reagent() : vial (2 ml) All operation should be performed on ice. Oxidizing agent Tissue 2) Reagent(2) : vial ( ml) () Homogenize one volume of fresh tissue 3 % Metaphosphoric acid/2% SnCl 2 volumes of.4% metaphosphoric acid. (Final: %) 3) Reagent(3): vial(dissolve in 3 ml 44% sulfuric acid) (2) Centrifuge at, x G for minutes at 4C 2,4-Dinitrophenylhydrazine (DNPH) (3) Remove clear supernatant carefully from pellet. 4) Reagent(4) : vial ( ml) Store supernatant at -2C until ready for use. % Metaphosphoric acid Plasma ) Vitamin C Stock Solution : vial ( ml) ()Add equal amount of % metaphosphoric acid to plasma (Final: %), and stir this solution. PREPARATION OF REGENT (2) Centrifuge at, g for minutes at 4C Note: 44% Sulfuric Acid is used to prepare reagent(3) and 8% Sulfuric acid is added during the assay protocol. Please (3) Remove clear supernatant carefully from pellet. Store supernatant at -2C until ready for use. prepare appropriate dilutions from sulfuric acid stock solution. CAUTION: Sulfuric acid causes severe burns. Use extreme ASSAY PROTOCOL care when handling. Control Reagent(3): Dissolve DNPH in 3 ml of 44% sulfuric acid (Std) * This kit is sufficient for assay points ( samples, duplicate assay points). PRINCIPLE OF THE ASSAY 2 ul 2 ul Reagent() 2 ul 2 ul Incubate 3- minutes at room temperature Reagent(2) * 7 ul 7 ul Total volume is 282 ul. Dispense into tubes as follows. Solution B AsA in a given sample is converted to DHAsA by oxidizing agent. DHAsA is then derivatized with DNPH. Total vitamin C concentration (AsA + DHAsA) can be determined by the specific UV absorption of the DNPH derivative. MATERIALS NOT SUPPLIED Micropipets Thermostat bath(37c) Microplate reader (3 nm) or spectrophotometer * ul cuvette (for spectrophotometer) Metaphosphoric acid (WAKO 3-27 or Sigma M6288) Sulfuric acid (SA) (Sigma 33974) (S) Control (C) (Std) Solution B Reagent(3) 2 ul ul Incubate 3 hours at 37C * 2 8% SA * 3 ul ul ul Reagent(3) ul ---- Incubate 3 minutes at room temperature Dispense ul into each well of a 96 well plate. Read absorbance at 3 nm on microplate reader. Or, read samples with spectrophotometer (3 nm) and ul cuvette. Real absorbance is calculated as the absorbance value of the minus the absorbance value of control(c).

2 Correction of s (Std) is not necessary. * Reagent (2) will contain insoluble material. Just before use, shake reagent(2) well. Use as suspended solution. Insoluble material will not affect assay results. Note: Addition of Reagent () to sample will cause purple color. Following addition of Reagent (2) incubate until purple color fades. *2 Red insoluble material will settle. *3 Resuspend insoluble material by shaking. STANDARD CURVE PROTOCOL Dilute vitamin C standard stock solution in reagent(4) conforming to this protocol. Use this diluted solution as standard (std) conforming to assay protocol. OPERATION PRECAUTION Hemolysis causes assay errors. Anticoagulants such as the EDTA do not affect measurements. STORAGE Storage: store Frozen Expiration date: 2 years after production REFERENCES Daniel W.B., Gladys E, James E.M. : Clinica Chimica Acta, 44, 47-2 (973) No. Vitamin C standard stock Reagent(4) Concentration solution ul 2 ul 39 ul 2. ug/ml 3 ul 39 ul ug/ml 4 2 ul 3 ug/ml 4 ul 36 ul 2 ug/ml A typical standard curve following this protocol is shown below. curve BECKMAN DU R 3 Life Science UV/Vis Spectrometer Wavelength:3 nm Optical path length: mm 2 2 2

3 Vitamin C Assay Report (Glucose) Assay date:28/3/3 A:Glucose solution (Glucose conc. ~ mg/ml) B:Vitamin C + Glucose solution (Vitamin C conc. µg/ml + Glucose conc. ~ mg/ml) Used Kit:Vitamin C Assay Kit (SHIMA LABORATORIES) Methods Preparation of Glucose solution (Glucose conc. ~ mg/ml) -. g Glucose was dissolved in ml of % Metaphosphoric acid solution (MPA). (Glucose conc. mg/ml) -2. mg/ml Glucose solution was diluted in % MPA to ~ mg/ml Glucose solution. (See table..2) 2 Preparation of Vitamin C + Glucose solution (Vitamin C conc. µg/ml + Glucose conc. ~ mg/ml) 2-. g Vitamin C was dissolved in ml of % MPA. (Vitamin C conc. mg/ml) 2-2. Furthermore, mg/ml Vitamin C solution was diluted in % MPA to µg/ml Vitamin C solution g Glucose was dissolved in ml of µg/ml Vitamin C solution. (Vitamin C conc. µg/ml, Glucose conc. mg/ml) 2-4. µg/ml Vitamin C + mg/ml Glucose solution was diluted in µg/ml Vitamin C solution. (See table 2.2.4) 3 After preparation of samples, the measurement of Vitamin C was performed using Vitamin C Assay Kit according to the manufacture's instruction. VC conc. (µg/ml) Slope.39 Intercept VC y =.39x -.6 R 2 = Vitamin C conc. ( µg/ml) Table..2 A:Glucose solution Glucose conc. (mg/ml) Table B:Vitamin C + Glucose solution Glucose conc. Vitamin C conc. (mg/ml) (µg/ml) A:Glucose solution B:Vitamin C + Glucose solution (Glucose conc. ~ mg/ml) (Vitamin C conc. µg/ml Glucose conc. ~ mg/ml) Glucose Absorbanc Absorbanc Glucose Absorbanc VC conc. conc. e e conc. e (µg/ml) (Control) (mg/ml) () (Control) (mg/ml) () A:Glucose solution (Semi log plot) B:VC + Glucose solution (Semi log plot).7.3. Glucose conc. (mg/ml).7.3. Glucose conc., (mg/ml) VC µg/ml.3 In this report, the reaction of Vitamin C Assay Kit and Glucose was evaluated. The Glucose solution ( A) and Vitamin C + Glucose solution ( B) were used for the samples. The sample of Glucose concentration was to times higher than Vitamin C concentration. The Vitamin C Assay kit did not react with ~ mg/ml Glucose solution (Result of A). The Vitamin C Assay kit slightly react with mg/ml Glucose solution (Result of A), but did not react with Vitamin C + mg/ml Glucose solution (Result of B). In conclusion, The Vitamin C Assay kit can be measured Vitamin C including mg/ml Glucose.

4 Vitamin C Assay Report (Tomato) Homogenizing: 27/2/7 Vitamin C Assay: 27/2/ : Tomato (Made in Japan) Kit : Vitamin C Assay Kit (SHIMA Laboratories CO., LTD) Spectrophotometer : BECKMAN DUR3 Life Science UV/Vis Spectrometer Method. Homogenize one volume of fresh tomato with volumes of 6% metaphosphoric acid. (Final metaphosphoric acid conc.: %) No. Dilution ratio 2. Centrifuge at, rpm for minutes at 4 (Supernatant) (.) 3. Remove clear supernatant and dilute it according to the right chart Store solution at Vitamin C was measured according to the Kit protocol curve Vitamin C (µg/ml) y =.29 x -.33 R 2 = Vitamin C Control No. S - C (µg/ml) Tomato Tomato Approximation curve Slope.29 Y-intercept y = 23.3 x - 3 R 2 =.99 y = x -.27 R 2 =.998 Tomato Tomato2 Conclusion Both sample curves showed good linearity with y-intercept values near the origin. Therefore, this assay kit may be used to quantify Vitamin C in tomato...8. Dilution ratio

5 Vitamin C Assay Report (Lettuce) Homogenizing: 27/2/7 Vitamin C Assay: 27/2/ : Lettuce (Made in Japan) Kit : Vitamin C Assay Kit (SHIMA Laboratories CO., LTD) Spectrophotometer : BECKMAN DUR3 Life Science UV/Vis Spectrometer Method. Homogenize one volume of fresh lettuce with one volume of % metaphosphoric acid. (Final Mataphosphoric acid conc.: %) No. Dilution ratio 2. Centrifuge at, rpm for minutes at 4 (Supernatant) (.) 3. Remove clear supernatant and dilute it according to the right chart Store solution at Vitamin C was measured according to the kit protocol curve Vitamin C (µg/ml) y =.29 x -.33 R 2 = Vitamin C Control No. S - C (µg/ml) Lettuce Lettuce Approximation curve Slope.29 Y-intercept No.-3 were excluded from the result. Because those absorbance were more than y = x R 2 =.994 y = x R 2 =.997 Lettuce Lettuce2 Conclusion Both sample curves showed good linearity with y-intercept values near the origin. Therefore, this assay kit can be used to quantify Vitamin C in Lettuce...3 Dilution ratio

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