BIOO RESEARCH PRODUCTS Ascorbic Acid Assay Kit Manual Catalog # 1302-01 This kit is manufactured to the international quality standard ISO 9001:2008. ISO CI#: SARA-2009-CA-0114-01-B BIOO Scientific Corp.2011 Updated: August 24 th, 2010
TABLE OF CONTENTS GENERAL INFORMATION... 1 Product Description... 1 Procedure Overview... 1 Kit Contents, Storage and Shelf Life... 1 Required Materials Not Provided With the Kit... 2 Sensitivity (Detection Limit)... 2 Warnings and Precautions... 2 SAMPLE PREPARATION... 3 General information on sample preparation... 3 Beer... 3 Wine... 3 Fruit Juices and Vegetable Juices... 3 Potato-based Foods, Fruits and Vegetables... 3 Milk... 3 Flour... 4 ASCORBIC ACID DETECTION PROTOCOL... 4 Reagent Preparation... 4 Assay Protocol... 4 DATA ANALYSIS... 5 Determination of Ascorbic Acid in Food Samples... 5 ALTERNATIVE PROTOCOL USING A 96-WELL MICROPLATE... 5 MaxSignal Ascorbic Acid Assay Kit is intended for laboratory use only, unless otherwise indicated. This product is NOT for clinical diagnostic use. MaxSignal is a trademark of Bioo Scientific Corporation (BIOO).
GENERAL INFORMATION GENERAL INFORMATION Product Description MaxSignal Ascorbic Acid Assay Manual 1302-01 The MaxSignal Ascorbic Acid Assay Kit is a chemical assay for the determination of the ascorbic acid across a variety of food samples. The kit uses a direct spectrophotometric assay to detect ascorbic acid directly from food samples. The unique features of the kit are: High sensitivity and low detection limit (0.6 ppm ascorbic acid) A rapid (25 minutes) robust chemical assay High reproducibility Procedure Overview The MaxSignal Ascorbic Acid Assay Kit uses a specific visible, colorimetric reaction to detect ascorbic acid in food samples: ascorbic acid first reduces an electron carrier (PMS). This compound then reduces MTT to produce a colored MTT formazan product. This conversion, measured at 578 nm, is proportional to the level of ascorbic acids in the sample. The absorbance of each sample at 578 nm is measured using a spectrophotometer in a cuvette (or plate reader). The concentration of ascorbic acid in each sample is then directly determined from the change in absorbance at 578 nm within 30 minutes time. Kit Contents, Storage and Shelf Life The MaxSignal Ascorbic Acid Assay Kit has the capacity for 48 determinations. Store the kit at 4 C. The shelf life of the kit is 12 months, after receipt, when the kit is properly stored. Kit Contents Amount Storage MTT Solution 10 ml 4ºC PMS Reagent 2 ml 4ºC Ascorbate Oxidase 0.24 ml 4ºC Dilution Buffer 20 ml 4ºC BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 1
Required Materials Not Provided With the Kit MaxSignal Ascorbic Acid Assay Manual 1302-01 Spectrophotometer (578 nm) Low volume (0.3 ml) UV cuvettes, such as Brand Scientific catalog #7592 00 Deionized or distilled water 50 ml plastic conical tubes Sensitivity (Detection Limit) Sample Volume Detection Limit (mg/l) 0.01 ml 12 0.2 ml 0.6 The linear range of the assay is 0.6 500 mg/l (ppm). Samples containing higher amounts of ascorbic acid should be diluted prior to analysis. Warnings and Precautions BIOO strongly recommends that you read the following warnings and precautions to ensure your full awareness of the techniques and other details you should pay close attention to when running the assays. Periodically, optimizations and revisions are made to the kit and manual. Therefore, it is important to follow the protocol included with the kit. If you need further assistance, you may contact your local distributor or BIOO at techsupport2@biooscientific.com. Do not use the kit past the expiration date. Try to maintain a laboratory temperature of (20 25 C/68 77 F). Avoid running assays under or near air vents, as this may cause excessive cooling, heating and/or evaporation. Also, do not run assays in direct sunlight, as this may cause excessive heat and evaporation. Cold bench tops should be avoided by placing several layers of paper towel or some other insulation material under the assay plates during incubation. Make sure you are using only distilled deionized water since water quality is very important. When pipetting samples or reagents into an empty microtiter plate, place the pipette tips in the lower corner of the well, making contact with the plastic. BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. There is no warranty of merchantability of this product, or of the fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or consequential damage, or expense arising directly or indirectly from the use of this product. BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 2
SAMPLE PREPARATION SAMPLE PREPARATION General information on sample preparation Beer MaxSignal Ascorbic Acid Assay Manual 1302-01 1. Grind or homogenize solid samples then extract with water and filter to clarify sample. 2. Adjust samples to ph 4, if necessary, using either meta-phosphoric acid (1% - 15%) or KOH (2M). Allow ph of carbonated samples to stabilize for 15 minutes before final ph adjustment. 3. Measure colored samples against a sample blank containing only buffer plus sample (using the same sample volume used for the assay). Strongly colored samples can be treated with polyvinylpolypyrrolodine (PVPP, final concentration about 1%) to decolorize: Stir 1 minute and filter to remove solids. 4. Protein-rich samples can be deproteinized before performing the assay using acid such as 15% meta-phosphoric acid. After this treatment, adjust the sample to ph 4 using 2M KOH. To remove the carbonation (bubbles), stir 10 ml of beer in a beaker for 30 40 seconds with a glass rod or filter using filter paper. Wine Adjust sample to ph 4, if necessary using dilute meta-phosphoric acid. Wine containing sulfites must be treated before performing the assay: Add 25 µl 5% formaldehyde to 5 ml of wine, mix and incubate for 5 minutes at room temperature, then adjust the sample to ph 4 using dilute meta-phosphoric acid. Fruit Juices and Vegetable Juices Filter turbid juices to clarify. Moderately colored fruit juices can usually be diluted for direct use in the assay. Strongly colored juice samples can be treated with polyvinylpolypyrrolodine (PVPP, final concentration about 1%) to decolorize: Stir 1 minute and filter to remove solids. Adjust sample to ph 4, if necessary using dilute meta-phosphoric acid. Potato-based Foods, Fruits and Vegetables Milk Homogenize > 25 g sample (using a blender or other homogenizer).transfer 5 g to a 50 ml tube and add 5 ml 15% meta-phosphoric acid + 10 µl n-butanol. Vortex mix or shake for 1 minute. Adjust sample to ph 4 with 2M KOH. Increase volume to 50 ml with dh2o, mix and filter sample to clarify. Carefully adjust the sample to ph 4 using citric acid (monohydrate) powder. Filter and discard the first portion of filtrate. The remaining filtrate is ready for the assay. BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 3
MaxSignal Ascorbic Acid Assay Manual 1302-01 Flour Mix 8 g of flour with 40 ml 15% metaphosphoric acid in a 50 ml flask or tube. Shake to obtain a homogenous suspension. Adjust sample to ph 4 with 2M KOH. Filter the sample to remove solids. ASCORBIC ASCORBIC ACID DETECTION ACID DETECTION PROTOCOL PROTOCOL Reagent Preparation IMPORTANT: Make sure you read Warnings and Precautions section on page 2. ALL REAGENTS SHOULD BE BROUGHT UP TO ROOM TEMPERATURE BEFORE USE (30 MIN - 1 HOUR AT 20 25 C/68 77 F). Gently tap the Ascorbate Oxidase tube several times to evenly disperse the suspension. Assay Protocol Note: The determinations are performed in 0.3 ml microcuvettes. To ensure that the color change in the assay is specifically produced by ascorbic acid, each determination is performed, in parallel, using samples treated with enzyme (ascorbate oxidase) to degrade ascorbic acid (Sample blank). The color change in the ascorbic acid depleted reaction (Sample blank) is then subtracted from the color change of corresponding untreated reactions (Sample reaction) to correct for individual sample background signals. Pipet into low-volume cuvettes Sample Dilution Buffer Note: Sample volume + Buffer volume should equal 200 ul. MTT Solution Ascorbate Oxidase Sample blank 10 µl 190 µl 100 µl 4 µl Sample reaction 10 µl 190 µl 100 µl ---- Mix and incubate 10 min at 37 C or 15 min at room temperature. Measure the absorbance at 578 nm (A1) and then add the PMS reagent: PMS Reagent 20 µl 20 µl Mix and incubate 15 min at 37 C or 30 min at room temperature. Measure the absorbance at 578 nm (A2). Note: If samples are not strongly colored, then sample volumes greater than 10 µl can be used; however, in such cases, the complementary volume of the Dilution Buffer must be lowered so that the total volume (sample + buffer) equals 200 µl. BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 4
MaxSignal Ascorbic Acid Assay Manual 1302-01 DATA ANALYSIS DATA ANALYSIS Determination of Ascorbic Acid in Food Samples To calculate the ascorbic acid concentration, use the following general equation: c = V x MW x ΔA (mg / L) ε x d x v V = final volume (0.322 ml) v = sample volume (0.01 0.1 ml) MW = molecular weight of ascorbic acid (176.13 g/mol) d = light path (1 cm) ε = extinction coefficient of MTT formazan (16900 at 578 nm) ΔA = (A2 A1)sample reaction (A2 A1)sample blank where sample blank is the sample treated with ascorbate oxidase. For example, if a 10 µl (0.01 ml) sample volume is used: c = 0.322 x 176.13 x ΔA (g/l) =0.334 x ΔA (g ascorbic acid / L sample solution) 16900 x 1 x 0.01 Note: If the sample has been diluted during the sample preparation protocol, the result must be multiplied by the dilution factor, F, to correct for this dilution. For calculating the recovery of ascorbic acid from a spiked solution, the dilution factor (F) will vary based on the standard material. ALTERNATIVE ALTERNATIVE PROTOCOL PROTOCOL USING USING A 96-WELL 96-WELL MICROPLATE MICROPLATE Note: The microplate protocol uses a reaction volume (300 μl). 1. Add 10 µl to 180 µl of sample solution (in duplicate) into microtiter plate wells, depending on the sample type. 2. Add Dilution Buffer into the cuvette for a total of 180 μl in cuvette. 3. Add 100 µl MTT Solution to each cuvette. 4. Add 2 µl of Ascorbate Oxidase to one well from each duplicate set (Sample Blank). Mix. Add 2 µl dh2o to the other duplicate (Sample Reaction), mix and incubate for 15 minutes at room temperature then measure the absorbance of each sample at 570 nm (A1). 5. Add 20 µl of PMS Solution. Mix and incubate for 30 minutes at room temperature and then measure the absorbance of each sample at 570 nm (A2). BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 5
MaxSignal Ascorbic Acid Assay Manual 1302-01 Data analysis is performed in the manner described in the previous section, except use a value of 17000 (M -1 cm -1 ) for the extinction coefficient at 570 nm. Bioo Scientific Corporation 3913 Todd Lane Suite 312 Austin, TX 78744 USA Tel: 1.888.208.2246 Fax: (512) 707-8122 Made in USA BIOO Research Products Group info@biooscientific.com techsupport2@biooscientific.com www.biooscientific.com BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 6