Peroxide Value Assay Kit (Micro Method)

Cat. No.: P1522268

GRADE & PURITY BioReagent

Overview

Peroxide value is an important indicator of the degree of oxidation of oils, fats, and fatty acids, usually expressed as millimoles of active oxygen per kilogram of sample. It is used to determine whether food has deteriorated due to oxidation and is a key parameter for evaluating food hygiene quality. The higher the peroxide value, the more severe the rancidity of the oil, and the lower the quality and safety of the food.

Principle: Peroxides in the sample oxidize ferrous ions (Fe²⁺) to ferric ions (Fe³⁺). Ferric ions react with thiocyanate to form an orange-red ferric thiocyanate complex. The peroxide value can be determined by measuring the absorbance of this complex at 500 nm.

Contents & Storage:

Component No.	Component Name	Appearance	96T	Storage
P1522268A	Reagent I	Liquid	1EA	2-8℃. Store in the dark
P1522268B	Reagent II	Powder	1EA	2-8℃. Store in the dark
P1522268C	Standard	Liquid	2mL	2-8℃. Store in the dark

Precautions for Reagent Use:

1.Before opening Reagent II, ensure the powder has settled to the bottom (shake manually). When using, dissolve in 1.2 mL of pure water. The storage period is the same as the kit's shelf life.

2.Extraction solution must be prepared by the user. Pure methanol (analytical grade or higher, purity ≥98%) is recommended.

Experimental Equipment:

Mortar (homogenizer), ice box (ice machine), benchtop centrifuge, adjustable pipette, 96-well plate, centrifuge tubes, microplate reader, pure methanol, distilled water (deionized water or ultrapure water are all acceptable).

Measurement Procedure:

It is recommended to first select 1–3 samples with large differences (e.g., different types or groups) for a preliminary experiment to familiarize yourself with the procedure and determine or adjust the sample concentration based on the preliminary results, so as to avoid unnecessary waste of samples or reagents.

1. Sample Extraction:

① Animal and plant tissue samples:

Take approximately 0.1 g of tissue, add 1 mL of extraction solution, and homogenize in an ice bath. Centrifuge at 12,000 rpm at 4°C for 10 min. Collect the supernatant and keep it on ice until measurement.

② Liquid samples:

Measure directly. If turbid, centrifuge and use the supernatant for detection.

③ Bacterial/cell samples:

First collect the bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Take 5 million bacteria or cells, add 1 mL of extraction solution, and homogenize in an ice bath (various common electric homogenizers may be used); alternatively, use an ultrasonic disruptor to sonicate the bacteria or cells in an ice bath (power 200 W, sonication 3 s, interval 10 s, repeat 30 times). Centrifuge at 12,000 rpm at 4°C for 10 min and collect the supernatant for measurement.

[Note]: Cell samples may also be lysed with a lysis buffer for subsequent detection; the lysis method should follow the lysis buffer's operating instructions.

2. Detection Steps:

① Preheat the microplate reader for 30 min (waiting for the instrument to complete its self-check procedure is also acceptable), and set the wavelength to 500 nm.

② Bring all reagents to room temperature, and add them sequentially to EP tubes as shown in the table below:

Reagent (μL)	Assay Tube	Blank Tube (once only)
Sample	50	—
Pure methanol	900	950
Reagent I	10	10
Reagent II	10	10

Mix well and incubate at room temperature for 5 min. Transfer 200 μL to a 96-well plate and measure the absorbance (A) at 500 nm.ΔA = A_assay − A_blank.

[Note]: 

1. If the value of the assay tube (A) exceeds 1.8, the sample may be diluted before measurement; the dilution factor D should be substituted into the calculation formula.

2.If ΔA is less than 0.01, the sample mass W may be increased; the changed W must be substituted into the formula for recalculation.

Result Calculation:

Standard curve equation: y = 0.0055x − 0.0165, where x is the standard concentration (μg/mL) and y is ΔA.

1.Calculation based on mass:

Peroxide value (μg/g) = [(ΔA + 0.0165) ÷ 0.0055 × V₁] ÷ (W × V₁ ÷ V) × D= 181.82 × (ΔA + 0.0165) ÷ W × D

2.Calculation based on protein content:

Peroxide value (μg/mg prot) = [(ΔA + 0.0165) ÷ 0.0055 × V₁] ÷ (Cpr × V₁ ÷ V) × D= 181.82 × (ΔA + 0.0165) ÷ Cpr × D

3.Calculation based on liquid volume:

Peroxide value (μg/mL) = [(ΔA + 0.0165) ÷ 0.0055] × D = 181.82 × (ΔA + 0.0165) × D

4.Calculation based on bacterial/cell density:

Peroxide value (μg/10⁴ cells) = [(ΔA + 0.0165) ÷ 0.0055 × V₁] ÷ (V₁ ÷ V × 500) × D= 181.82 × (ΔA + 0.0165) ÷ 500 × D

W: sample mass, g;

V: extraction solution volume, 1 mL;

V₁: sample volume added, 0.05 mL;

500: total number of bacteria or cells, in units of 10⁴;

D: dilution factor (1 if undiluted);

Cpr: protein concentration of the supernatant, mg/mL.

Appendix: Standard Curve Preparation Procedure:

1.The standard curve is optional. Users may prepare a standard curve according to experimental needs, or directly use the calculation formula in the instructions for result calculation.

2.The standard stock solution concentration is 1000 μg/mL. Dilute the stock solution with pure methanol to prepare six concentration gradients of standards, for example: 0, 20, 40, 60, 80, 100 μg/mL. The standard concentrations may also be adjusted according to actual samples.

3.Refer to the following table for standard dilution:

Pipette 100 μL of the standard stock solution, add 900 μL of pure methanol, and mix well to obtain a 100 μg/mL standard diluent for later use.

Standard concentration (μg/mL)	0	20	40	60	80	100
Standard diluent (μL)	0	40	80	120	160	200
Pure methanol (μL)	200	160	120	80	40	0

Mix each standard tube well and set aside for use.

4.Operate according to the assay tube loading table. Based on the results, subtract the absorbance at 0 concentration from the absorbance at each concentration, and construct the standard curve through the zero point.

Reagent (μL)	Standard Tube (A)	Zero Concentration Tube A0 (once only)
Standard	50	—
Pure methanol	910	960
Reagent II	10	10

Mix well and incubate at room temperature for 5 min. Transfer 200 μL to a 96-well plate and measure the absorbance (A) at 500 nm. ΔA = A_{standard tube} − A_{0 concentration tube}.

Specifications

Specifications & Purity

BioReagent

Grade

BioReagent

Application

Cell Metabolism

Sample type

Animal and Plant tissues, Bacteria, Cell

Method Of Detection

Colorimetric Method

Quantitative

Quantitative

Detection instrument

Microplate reader

Detection wavelength

500nm

Sensitivity

Light-sensitive

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.

Contents & Storage

Component No.	Component Name	Appearance	96T	Storage
P1522268A	Reagent I	Liquid	1EA	2-8℃. Store in the dark
P1522268B	Reagent II	Powder	1EA	2-8℃. Store in the dark
P1522268C	Standard	Liquid	2mL	2-8℃. Store in the dark