Nitrate/Nitrite Assay Kit Manual Catalog #

BIOO RESEARCH PRODUCTS Nitrate/Nitrite Assay Kit Manual Catalog # 1305-01 This kit is manufactured to the international quality standard ISO 9001:2008. ISO CI#: SARA-2009-CA-0114-01-B BIOO Scientific Corp.2011 v.511

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... 2 Water, waste water, drinking water and mineral water... 3 Fruit Juices... 3 Dairy Products... 3 Meat Products... 3 Potato-based Foods, Fruits and Vegetables... 3 Cheese and Processed Cheese Products... 4 Baby Food Products... 4 NITRATE DETECTION PROTOCOL... 4 Reagent Preparation... 4 Assay Protocol... 5 DATA ANALYSIS... 5 Determination of Nitrate and Nitrite in Food Samples... 5 RELATED PRODUCTS... 6 The MaxSignal Nitrate/Nitrite Assay Kit is intended for laboratory use only, unless otherwise indicated. This product is NOT for clinical diagnostic use. MaxSignal is a registered trademark of Bioo Scientific Corporation (BIOO).

Product Description GENERAL INFORMATION The MaxSignal Nitrate/Nitrite Assay Kit is a cuvette-based enzymatic assay for the determination of nitrate and/or nitrite across a variety of food samples. The kit uses a spectrophotometric assay to detect the enzymatic degradation of nitrate and nitrite directly from food samples. The unique features of the kit are: High sensitivity and low detection limit (0.02 ppm) A rapid (45 minute), robust enzyme-based assay High reproducibility Flexible format Procedure Overview The MaxSignal Nitrate/Nitrite Assay Kit uses a coupled enzymatic reaction scheme to detect nitrate and/or nitrite in food samples: nitrate in the sample is converted by nitrate reductase enzyme to make nitrite. The nitrite then reacts with sulfanilamide and N-(1-naphthyl)-ethylene-diamine to form a red diazo dye. This dye, measured at 540 nm, is proportional to the level of nitrate and/or nitrite in the sample. The absorbance of each sample at 540 nm is measured using a spectrophotometer. The concentration of nitrate /nitrite in each sample is then directly determined from the diazo dye absorbance at 540 nm within 45 minutes. Kit Contents, Storage and Shelf Life The MaxSignal Nitrate/Nitrite Assay Kit has the capacity for 48 determinations or 24 determinations in duplicate. Store the kit at 4 C. The shelf life of the kit is 6 months, after receipt, when the kit is properly stored. Kit Contents Amount Storage Buffer Solution 4 ml 4 C Reagent Mix 1 vials 4 C Nitrate Reductase 1 vial 4 C Color Reagent 1 3 ml 4 C Color Reagent 2 3 ml 4 C Microtiter Plate One Room temp or 4 C BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 1

Required Materials Not Provided With the Kit MaxSignal Nitrate/Nitrite Assay Kit Manual 1305-01 Spectrophotometer (540 nm) Low volume (0.3 ml) UV cuvettes, such as Brand Scientific catalog #7592 00 Deionized or distilled water 100 ml volumetric flask 50 ml plastic conical tubes Carrez buffer I and Carrez buffer II (recipes included in protocol) Sensitivity (Detection Limit) Sample Volume Detection Limit (mg/l) 0.1 ml 0.02 The linear range of the assay is 0.02 40 mg/l (ppm). 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 techsupport@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. SAMPLE PREPARATION General information on sample preparation 1. Use clear, colorless or faintly colored solutions directly of dilute to a concentration of <5 mg/l nitrite or nitrate before using in test 2. Filter turbid solutions or clarify using Carrez solutions and dilute such that the nitrite or nitrate concentration is below 5 mg/l. Diluted solution can be directly used in the test. 3. After adding Carrez solutions I and II it is important to adjust ph to 8.0 ± 0.2. BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 2

Water, Waste Water, Drinking Water and Mineral Water If necessary, dilute clear, colorless water samples to a nitrite or nitrate concentration of <5 mg/l and use in the test. Filter turbid water samples or samples containing carbon dioxide (to remove the gas) before use in the test.. Fruit Juices Accurately weigh 2.5 g of homogenized sample into a 50 ml conical tube and add 10 ml dh20 and mix. Add 2.5 ml diluted Carrez solution (3.6 g potassium hexacyanoferrate[ii] trihydrate/100 ml), mix well. Add 2.5 ml Carrez solution II (7.2 g zinc sulfate heptahydrate/100 ml) and mix well again. Adjust ph to 8.0 with 1M sodium hydroxide solution. Bring solution up to 50 ml with dh20 and mix well. Filter if necessary or centrifuge to clarify for testing. Use the clear liquid for the test. Dairy Products Accurately weigh out 2 g sample and transfer to a 100 ml volumetric flask. Add 50 ml of boiling dh20 and mix well. Boil on a water bath for 15 min and then allow sample to cool to ~ 25 C. Add 3 ml each concentrated Carrez I (15 g potassium hexacyanoferrate[ii] trihydrate/100 ml) and Carrez II (30 g zinc sulfate heptahydrate/100 ml) solutions, mixing well. Adjust ph to 8.0 with 1M sodium hydroxide solution. Bring solution up to 100 ml with dh20 and mix well. Transfer an aliquot 10-20 ml to a tube and centrifuge at 3,000 x g for 15 minutes. Filter the supernatant with a membrane filter and use the clear liquid for the test. Animal Feed Accurately weigh out 5 g of well homogenized sample into a 100 ml beaker and add 50 ml of boiling dh20. Boil on a water bath for 15 min and then allow sample to cool to ~ 25 C. Add 3 ml each concentrated Carrez I (15 g potassium hexacyanoferrate [II] trihydrate/100 ml) and Carrez II (30 g zinc sulfate heptahydrate/100 ml) solutions, mixing well. Adjust ph to 8.0 with 1M sodium hydroxide solution. Bring solution up to 100 ml with dh20 and mix well. Transfer an aliquot 10-20 ml to a tube and centrifuge at 3,000 x g for 15 minutes. Filter the supernatant with a membrane filter and use the clear liquid for the test. Meat Products Accurately weigh out 5 g of well homogenized sample into a 100 ml beaker and add 50 ml of boiling dh20. Boil on a water bath for 15 min and then allow sample to cool to ~ 25 C. Add 3 ml each concentrated Carrez I (15 g potassium hexacyanoferrate[ii] trihydrate/100 ml) and Carrez II (30 g zinc sulfate heptahydrate/100 ml) solutions, mixing well. Adjust ph to 8.0 with 1M sodium hydroxide solution. Bring solution up to 100 ml with dh20 and mix well. Transfer an aliquot 10-20 ml to a tube and centrifuge at 3,000 x g for 15 minutes. Filter the supernatant with a membrane filter and use the clear liquid for the test. Potato-based Foods, Fruits and Vegetables Accurately weigh out 3 g of well homogenized sample into a 100 ml beaker and add 60 ml of hot dh20. Mix well or shake, and incubate for 15 min. on a water bath set at 70 C. Treat samples that are intensely colored or that have high starch content with Carrez solutions (same concentration as the fruit juice protocol listed above). Allow contents of the beaker to cool to ~ 25 C, and bring volume of solution up to 100 ml with dh20. Filter if necessary and BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 3

use the clear liquid for the test. Cheese and Processed Cheese Products Accurately weigh out 3 g of homogenized sample into a 100 ml volumetric flask, add 20 ml dh20 and heat in microwave oven until the cheese is melted (about 10-20 sec). Stir with a spatula and microwave again until the mixture boils gently. Add about 50 ml of boiling dh20 and boil for 15 min on a water bath. Cool solution to ~ 25 C and add 5 ml each of concentrated Carrez I (15 g potassium hexacyanoferrate[ii] trihydrate/100 ml) and Carrez II (30 g zinc sulfate heptahydrate/100 ml) solutions, mixing well. Adjust ph to 8.0 with 1M sodium hydroxide solution. Bring solution up to 100 ml with dh20 and mix well. Transfer a 10-20 ml aliquot to a tube and centrifuge at 3,000 x g for 15 minutes. Filter the supernatant with a membrane filter and use the clear liquid for the test. Baby Food Products Accurately weigh out 1.5 g of well homogenized sample into a 100 ml beaker and add 50 ml of boiling dh20. Boil on a water bath for 15 min and then allow sample to cool to ~ 25 C. Add 3 ml each concentrated Carrez I (15 g potassium hexacyanoferrate[ii] trihydrate/100 ml) and Carrez II (30 g zinc sulfate heptahydrate/100 ml) solutions, mixing well. Adjust ph to 8.0 with 1M sodium hydroxide solution. Bring solution up to 100 ml with dh20 and mix well. Transfer an aliquot (10-20 ml) to a tube and centrifuge at 3,000 x g for 15 minutes. Filter the supernatant with a membrane filter and use the clear liquid for the test. Reagent Preparation NITRATE DETECTION PROTOCOL 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). Prepare Nitrate Reductase: Reconstitute the lyophilized Nitrate Reductase by adding 350 µl dh20 and gently swirling to mix. After use, the reconstituted enzyme can be stored at 4 C for up to one month. Prepare Reagent Mix solution: Reconstitute the Reagent Mix by adding 3 ml Buffer Solution to the vial containing a Reagent Mix. Gently swirl or invert the vial to completely dissolve the tablet. After use, the Reagent Mix can be stored at 4 C for up to one month. Prepare 500 mg/l sodium nitrite standard: Weigh 75 mg sodium nitrite into a 100 ml volumetric flask, dissolve in dh2o and make up to the mark with the same solvent (stock solution = 500 mg nitrite/l). Prepare a nitrite calibration curve by serially diluting the stock solution with dh2o to give concentrations in the range from 5.00 mg and 0.05 mg nitrite/l. Prepare 500 mg/l potassium nitrate standard: Weigh 81.5 mg potassium nitrate into a 100 ml volumetric flask, dissolve in dh2o and make up to the mark with the same solvent (stock solution = 500 mg nitrite/l). Prepare a nitrate calibration curve by diluting the stock solution with dh2o to give concentrations in the range from 5.00 mg and 0.05 mg nitrate/l. BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 4

Assay Protocol Note: Each determination is performed in 0.3 ml microcuvettes, in parallel, using samples diluted into water without enzyme. The color change of these no-enzyme controls (Sample blank) is then subtracted from the color change of corresponding enzyme reactions (Sample reaction) to correct for sample background signals. Pipet into low volume cuvettes: Sample or standard * dh20 ** Reagent Mix solution Nitrate Reductase Nitrite blank 1 Nitrite sample 10-100 μl Up to 1 Nitrite + nitrate blank 100 μl 45 μl 5 μl Nitrite + nitrate sample 10 100 μl Up to 100 μl 45 μl 5 μl Mix and incubate reactions for 30 minutes at room temperature, measure the absorbance at 540 nm (A1) and then add: Color reagent 1 Color reagent 2 Mix and incubate 15 min at room temperature (away from light).and measure the absorbance at 540 nm (A2). * Volume of sample used in assay can be between 10 to 100 µl. ** Volume of dh20 added to sample must bring total volume to 1 for nitrite samples and to 100 μl for nitrate samples. Note: The same procedure can be performed using the same volumes with a microplate and reading the results with a microplate reader. DATA ANALYSIS Determination of Nitrate and Nitrite in Food Samples To calculate the nitrate and nitrite concentrations, use the results calculated from the calibration curves constructed using the standard solutions. Plot the change in absorbance obtained for the sodium nitrite and potassium nitrate standard solutions on the y-axis against the corresponding nitrite or nitrate concentrations in mg/l on the x-axis. ΔAnitrite = (A2 A1)nitrite sample (A2 A1)nitrite blank ΔAnitrite+ nitrate = (A2 A1)nitrite + nitrate sample (A2 A1)nitrite + nitrate blank ΔAnitrate = ΔAnitrite+ nitrate - ΔAnitrite Determine the concentrations of nitrite and nitrate in the sample from the calibration curves using the change in absorbance measured. BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 5

Note: The calibration curves do not have to be plotted every time a determination is made. It is sufficient to check the calibration curves from time to time and to include a sodium nitrite or potassium nitrate standard solution as a control. If the sample has been diluted during the sample preparation protocol, the result must be multiplied by the dilution factor (F). When analyzing solid or semisolid samples that have to be weighed out; calculate the result with respect to the mass of sample. RELATED PRODUCTS Constituent Analysis Kits: Product Catalog Number MaxSignal Ammonia Assay 1304-01 MaxSignal Glucose Assay 1301-01 MaxSignal Urea Assay 1307-01 MaxSignal Fructose/Glucose Assay 1306-01 MaxSignal Ascorbic Acid Assay 1302-01 Bioo Scientific Corporation 7050 Burleson Road Austin, TX 78744 USA Tel: 1.888.208.2246 Fax: (512) 707-8122 Made in USA BIOO Food & Feed Safety Products info@biooscientific.com foodfeedsafety@biooscientific.com www.biooscientific.com BIOO FOOD AND FEED SAFETY WWW.BIOOSCIENTIFIC.COM 6