BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
2-8°C储存,避光,室温。低温运输 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
在食品中，亚硝酸盐可与肉品中的肌红素结合而更稳定，在食品加工业中作为保色剂，以维持肉制品的良好外观，并防止肉毒梭状芽孢杆菌的产生，提高食用肉制品的安全，但是人体长期摄入亚硝酸盐过量的食品，可诱发消化系统癌变。
检测原理：在酸性条件下，亚硝酸盐与对氨基苯磺酸反应生成重氮化合物，再与N-1-萘基乙二胺形成紫红色偶氮化合物，在540nm处有特征吸收峰。
检测范围：1.563-100 μM
灵敏度：1.563 μM
注意：正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
自备仪器和试剂
操作步骤
1、试剂准备
1. 试剂准备
| 试剂名称 | 试剂准备 | 备注 |
| 提取液 I | 即用型 | 常温保存，本试剂为饱和溶液，有少量析出为正常现象，使用前充分摇匀。 |
| 提取液 II | 即用型 | 常温保存 |
| 提取液 III | 即用型 | 常温保存 |
| 活性炭 | 即用型 | 常温保存 |
| 试剂 一 | 即用型，使用前平衡到室温 | 2-8℃避光保存 |
| 试剂 二 | 即用型，使用前平衡到室温 | 2-8℃避光保存 |
亚硝酸钠标准品 (1M) | 2-8℃保存 |
2、标准曲线设置 ：用去离子水将1M 亚硝酸钠标准品稀释成100 μM。按下表所示，用去离子水将100 μM 亚硝酸钠标准品稀释至50、25、12.5、6.25、3.125、1.563 µM。
序号 | 标准品体积 | 去离子水体积 (μL) | 标准品浓度 (μM) |
标品 1 | 取 200 μL 100 μM 亚硝酸钠标准工作液 | 0 | 100 |
标品 2 | 取 100 μL 标品 1 (浓度 100 μM) | 100 | 50 |
标品 3 | 取 100 μL 标品 2 (50 μM) | 100 | 25 |
标品 4 | 取 100 μL 标品 3 (25 μM) | 100 | 12.5 |
标品 5 | 取 100 μL 标品 4 (12.5 μM) | 100 | 6.25 |
标品 6 | 取 100 μL 标品 5 (6.25 μM) | 100 | 3.125 |
标品 7 | 取 100 μL 标品 6 (3.125 μM) | 100 | 1.563 |
标品 8 | 0 | 200 | 0 |
3. 样本制备
称取样品约 0.2 g 鲜重或 0.05 g 干重，充分破碎均质；加入 0.5 mL 提取液 I，加盖密封沸水浴 15 分钟，冷却至室温；加入 0.5 mL 提取液 II，涡旋混匀；再加入 0.5 mL 提取液 III，用镊子加入活性炭约 1 mg，充分混匀；常温避光静置 30 分钟；8000×g、25 ℃ 离心 15 分钟，小心吸取中层澄清上清液待测。
注 1: 沸水浴务必拧紧离心管管盖，防止加热过程中溶剂挥发造成提取体积偏差。
注 2: 加入提取液 II、III 后样品体系变粘稠属于正常现象，不影响脱色与离心分离；深色样品活性炭用量可增至 2-3 mg，请勿过量添加，防止吸附亚硝酸盐造成结果偏低；若离心后上清浑浊，同等条件二次离心 10 分钟后再取样。
4. 实验步骤
4.1 酶标仪或可见分光光度计预热 30 分钟，调节波长到 540 nm。可见分光光度计用去离子水调零。
4.2 操作表 (下述操作在96孔板或微量玻璃比色皿中操作）
试剂 (μL) | 测定孔 | 标准孔 | 空白孔 |
待测样本上清液 | 70 | 0 | 0 |
亚硝酸钠标准工作液 | 0 | 70 | 0 |
去离子水 | 0 | 0 | 70 |
试剂 一 | 65 | 65 | 65 |
试剂 二 | 65 | 65 | 65 |
混匀，25℃静置15min，于540nm处测定吸光值，记为A测定、A标准、A空白，计算ΔA测=A测定-A空白、ΔA标=A标准-A空白。
注意：空白孔和标准孔只需测定1次。实验之前建议选择2-3个预期差异大的样本做预实验。如果ΔA测小于0.001可适当加大样本量。如果ΔA测大于1.0，样本可用去离子水进一步稀释，计算结果乘以稀释倍数，或减少提取用样本量。
5、结果计算
注意：我们为您提供的计算公式，包括推导过程计算公式和简洁计算公式。两者完全相等。建议以加粗的简洁计算公式为最终计算公式。
5.1 标准曲线的绘制
以标准液浓度为y轴，ΔA标为x轴绘制标准曲线。将ΔA测代入标准曲线公式计算出y（μM）。
5.2 样本亚硝酸盐含量计算
NO₂⁻ (μg/g)=y×V样总×46÷1,000÷W×n=0.069×y÷W×n
参数说明：
V样总：样本总体积，1.5mL；
46：NO₂⁻相对分子质量，g/mol；
1,000：单位换算系数，1L=1,000mL；
W：样本质量，g；
n：样本的稀释倍数。
6、结果展示
典型标准曲线
实验实例：
取0.2g咸菜使用96孔板测得计算ΔA=0.103，代入方程得y=10.345，按样本质量计算含量得： NO₂⁻ (μg/g)=0.069×10.345÷0.2×1=3.569 μg/g
取0.2 g罐头肉使用96孔板测得计算ΔA=0.007，代入方程得y=0.75，按样本质量计算含量得： NO₂⁻ (μg/g)=0.069×0.75÷0.2×1=0.259 μg/g
注意事项
本产品仅供科学研究使用，不适用于临床诊断。为了您的安全和健康，请穿实验服并戴一次性手套操作。
In food products, nitrite can bind with myoglobin in meat to form a more stable compound. In the food processing industry, it is used as a color‑fixing agent to maintain the appealing appearance of meat products, prevent the growth of Clostridium botulinum, and enhance the safety of edible meat products. However, long‑term consumption of foods containing excessive nitrite may induce digestive system cancers.
Detection Principle
Under acidic conditions, nitrite reacts with sulfanilic acid to form a diazo compound, which then couples with N‑(1‑naphthyl)ethylenediamine to produce a purple‑red azo dye with a characteristic absorption peak at 540 nm.
Detection Range: 1.563‑100 µM
Sensitivity: 1.563 µM
Note: Before formal testing, it is recommended to perform a preliminary experiment with 2‑3 samples expected to show significant differences.
Reagents, consumables and Equipments not provided
Microplate reader or visible spectrophotometer (capable of measuring absorbance at 540 nm)
96‑well plate or micro‑volume glass cuvette, adjustable pipettes and tips
Ice maker, centrifuge, water bath
Deionized water
Mortar
Procedure
1. Reagent Preparation
| Reagent Name | Preparation | Notes |
| Extraction Solution Ⅰ | Ready‑to‑use | Store at room temperature. This is a saturated solution; slight precipitation is normal. Shake well before use. |
| Extraction Solution Ⅱ | Ready‑to‑use | Store at room temperature. |
| Extraction Solution Ⅲ | Ready‑to‑use | Store at room temperature. |
| Activated Carbon | Ready‑to‑use | Store at room temperature. |
| Reagent Ⅰ | Ready‑to‑use; equilibrate to room temperature before use | Store at 4 °C protected from light. |
| Reagent Ⅱ | Ready‑to‑use; equilibrate to room temperature before use | Store at 4 °C protected from light. |
| NaNO₂ Standard (1M) | Store at 4 °C. |
Note: Reagent Ⅱ and NaNO₂ Standard (1 M) are somewhat toxic; perform experiments in a fume hood.
2. Standard Curve Setup
Dilute the 1 M NaNO₂ Standard to 100 µM using deionized water. Then, dilute the 100 µM NaNO₂ Standard as shown in the table below to obtain 50, 25, 12.5, 6.25, 3.125, and 1.563 µM standards.
| Tube | Standard Volume | Deionized Water Volume (µL) | Standard Concentration (µM) |
| Std.1 | 200µL of 100μM NaNO₂ Standard | 0 | 100 |
| Std.2 | 100µL of Std.1 (100μM) | 100 | 50 |
| Std.3 | 100µL of Std.2 (50μM) | 100 | 25 |
| Std.4 | 100µL of Std.3 (25μM) | 100 | 12.5 |
| Std.5 | 100µL of Std.4 (12.5μM) | 100 | 6.25 |
| Std.6 | 100µL of Std.5 (6.25μM) | 100 | 3.125 |
| Std.7 | 100µL of Std.6 (3.125μM) | 100 | 1.563 |
Note: A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.
3. Sample Preparation
Weigh approximately 0.2 g fresh weight or 0.05 g dry weight of sample, homogenize, add 0.5 mL of Extraction Solution Ⅰ, incubate in a boiling water bath for 15 minutes, cool to room temperature, add 0.5 mL of Extraction Solution Ⅱ, vortex to mix, add 0.5 mL of Extraction Solution Ⅲ, add a small amount of Activated Carbon (about 1 mg) using forceps, mix well, let stand for 30 minutes, centrifuge at 8,000 g, 25 °C for 15 minutes, and collect the supernatant for testing.
Note: After adding Extraction Solutions Ⅱ and Ⅲ, the sample may become viscous, which is normal.
4. Experimental Steps
4.1 Preheat the microplate reader or visible spectrophotometer for 30 minutes and set the wavelength to 540 nm. Zero the visible spectrophotometer with deionized water.
4.2 Operation table (perform the following steps in a 96‑well plate or micro‑volume glass cuvette):
| Reagent (µL) | Test Well | Standard Well | Blank Well |
| Sample | 70 | 0 | 0 |
| Standard | 0 | 70 | 0 |
| Deionized water | 0 | 0 | 70 |
| Reagent Ⅰ | 65 | 65 | 65 |
| Reagent Ⅱ | 65 | 65 | 65 |
Mix well, incubate at 25 °C for 15 minutes, measure the absorbance at 540 nm, recorded as ATest, AStandard, and ABlank. Calculate ΔATest = ATest – ABlank and ΔAStandard = AStandard – ABlank.
Note: The blank and standard wells need to be measured only once. It is recommended to perform a preliminary experiment with 2‑3 samples showing expected large differences before formal testing. If ΔATest is less than 0.001, appropriately increase the sample amount. If ΔATest is greater than 1.0, further dilute the sample with deionized water and multiply the result by the dilution factor, or reduce the amount of sample used for extraction.
5. Calculation of Results
Note: We provide two formula sets for your convenience: the derived calculation formulas and the simplified formulas. They are completely equivalent. The simplified formulas are recommended as the final calculation formulas.
5.1 Plotting the Standard Curve
Plot the standard curve with the standard concentration as the y‑axis and ΔAStandard as the x‑axis. Substitute ΔATest into the standard curve equation to obtain y (µM).
5.2 Calculation of Nitrite Content in Samples
NO₂⁻ (µg/g) = y × VTotal × 46 ÷ 1,000 ÷ W × n = 0.069 × y ÷ W × n
Parameter Description:
VTotal: Total volume of the sample extract, 1.5 mL;
46: Molecular weight of NO₂⁻, g/mol;
1,000: Unit conversion factor, 1 L = 1,000 mL;
W: Sample mass, g;
n: Dilution factor of the sample.
6. Example Results
Typical Standard Curve:
Experimental Examples:
For 0.2 g of pickled vegetable measured using a 96‑well plate: ΔA = 0.103. Substituting into the equation yields y = 10.345 µM. The calculated nitrite content is: NO₂⁻ (µg/g) = 0.069 × 10.345 ÷ 0.2 × 1 = 3.569 µg/g.
For 0.2 g of canned meat measured using a 96‑well plate: ΔA = 0.007. Substituting into the equation yields y = 0.75 µM. The calculated nitrite content is: NO₂⁻ (µg/g) = 0.069 × 0.75 ÷ 0.2 × 1 = 0.259 µg/g.
Notes
This product is for scientific research use only and is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.
储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 96T | 储存 | 单次测试使用量 |
N1507975NT | 食品中亚硝酸盐含量检测试剂盒 (盐酸萘乙二胺, 微量法) (常温组分) | N1507975A | 提取液 I | 无色透明液体 | 70 mL | Room temperature. | 按需 |
| | N1507975B | 提取液 II | 黄色液体 | 70 mL | Room temperature. | 按需 |
| | N1507975C | 提取液 III | 无色透明液体 | 70 mL | Room temperature | 按需 |
| | N1507975D | 活性炭 | 黑色固体 | 0.12 g | Room temperature. | 按需 |
N1507975WT | 食品中亚硝酸盐含量检测试剂盒 (盐酸萘乙二胺, 微量法) (2-8℃组分) | N1507975E | 试剂 一 | 无色透明液体 | 10 mL | 2-8℃. Protect from light | 65 µL |
| | N1507975F | 试剂 二 | 无色透明液体 | 10 mL | 2-8℃. Protect from light | 65 µL |
| | N1507975G | 亚硝酸钠标准品(1M) | 无色透明液体 | 500 µL | 2-8℃. | 70 µL |
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939411 |
分析证书 | N1507975 |
| ZJ26F0939412 |
分析证书 | N1507975 |
| G2602262 |
分析证书 | N1507975 |
| E2623009 |
分析证书 | N1507975 |
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