Inorganic Phosphate Content Assay Kit (FSMB, Colorimetric Method) - BioReagent

Cat. No.: I1506776

GRADE & PURITY BioReagent

Synonyms

Inorganic Phosphate Content Assay Kit (Ferrous Sulfate Molybdenum Blue, Colorimetric Method) | Inorganic Phosphorus Content Assay Kit

Storage

Store at 2-8°C,Protected from light,Room temperature

Shipped In

Wet ice

Overview

Inorganic phosphorus in serum primarily consists of two phosphate anions, H₂PO₄⁻ and HPO₄²⁻, which can rapidly interconvert depending on the pH environment. In serum at pH 7.4, the concentration ratio of these two anions is approximately 1:4. This ratio shifts to about 1:1 in acidosis and 1:9 in alkalosis. In urine at pH 4.5, the ratio is approximately 100:1. The colorimetric method is the routine assay recommended by the WHO. The Chinese Ministry of Health Clinical Test Center recommends the ferrous sulfate molybdenum blue method and the metol molybdenum blue method as routine procedures.

Detection Principle

Proteins are first precipitated and purified using ferrous sulfate. Inorganic phosphorus reacts with ammonium molybdate to form ammonium phosphomolybdate, which is then reduced by ferrous sulfate to form a blue-purple complex. The absorbance of this complex is measured at 640 nm using a spectrophotometer, and the inorganic phosphorus content is calculated based on a formula. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.

I1506776	Component	50T	100T	Storage
I1506776A	Phosphorus Standard (1 mg/mL)	1 mL	2 mL	2-8℃
I1506776B	Standard Diluent	25 mL	50 mL	RT. Store in the dark.
I1506776C	Pi Assay Buffer	250 mL	500 mL	2-8℃. Store in the dark.
I1506776D	Ammonium Molybdate Powder	0.66 g	1.32 g	RT
I1506776E	Ammonium Molybdate Diluent	15 mL	30 mL	RT

Materials Required but Not Provided

1. Deionized water

2. Electronic balance, centrifuge tubes or test tubes, centrifuge, cuvettes, spectrophotometer

Experimental Procedure

1. Sample Preparation

1.1 Plasma/Serum Samples

Add 0.1 ml of the plasma or serum sample to 2.4 ml of Pi Assay Buffer. Mix thoroughly and let stand at room temperature for 10 minutes. Centrifuge at 3000 × g for 10 minutes. Collect the supernatant and store at -20°C for Pi detection.

1.2 Urine Samples

Adjust the pH of an appropriate amount of urine to 6.0 using 50% hydrochloric acid. Dilute 1:10 to 1:20 with distilled water. Add 0.1 ml of the diluted urine sample to 2.4 ml of Pi Assay Buffer. Mix thoroughly and let stand at room temperature for 10 minutes. Centrifuge at 3000 × g for 10 minutes. Collect the supernatant and store at -20°C for Pi detection.

1.3 Cell or Tissue Samples

Homogenize an appropriate amount of cells or tissue. Centrifuge at low speed to collect the supernatant. Add 0.1 ml of the homogenate supernatant to 2.4 ml of Pi Assay Buffer. Mix thoroughly and let stand at room temperature for 10 minutes. Centrifuge at 3000 × g for 10 minutes. Collect the supernatant and store at -20°C for Pi detection.

1.4 High Concentration Samples

If the sample contains a high concentration of Pi, it can be diluted with deionized water.

Note: After sample preparation, the protein concentration can be determined using a BCA protein assay kit to facilitate subsequent calculation of Pi content per unit protein weight in tissues or cells. The Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.

2. Preparation of Phosphorus Standard Working Solution

Dilute an appropriate amount of the Phosphorus Standard (1 mg/mL) with Standard Diluent at a ratio of 1:24 (Phosphorus Standard : Standard Diluent) to obtain a Phosphorus Standard (1.292 mmol/L). Add 0.1 ml of this Phosphorus Standard (1.292 mmol/L) to 2.4 ml of Pi Assay Buffer. Mix thoroughly, let stand at room temperature for 10 minutes, and centrifuge at 3000 × g for 10 minutes. Collect the supernatant; this is the Phosphorus Standard Working Solution. Store at -20°C for Pi detection.

3. Preparation of Ammonium Molybdate Chromogenic Solution

Dissolve the Ammonium Molybdate Powder in the Ammonium Molybdate Diluent at a ratio of 0.44 g powder per 10 ml diluent. Mix until dissolved. The prepared Ammonium Molybdate Chromogenic Solution should not be stored long-term; it is generally stable for 2 weeks when stored at 4°C after preparation.

4. Pi Assay Setup

Set up Blank, Standard, and Test tubes according to the table below. Add solutions in the order specified, avoiding bubble formation. If the Pi content in the sample is too high, reduce the sample volume or dilute appropriately before assay. It is best to set up duplicate tubes for samples and use the average value.

Reagent (ml)	Blank Tube	Standard Tube	Test Tube
Pi Assay Buffer	2	—	—
Treated Std Working Solition	—	2	—
Treated Sample Supernatant	—	—	2
Ammonium Molybdate Chromogenic Solution	0.25	0.25	0.25

5. Pi Measurement

Mix well and incubate at room temperature for 15 minutes. Using the Blank tube to zero the spectrophotometer, measure the absorbance of the Standard tube and Test tubes at 640 nm using a cuvette with a 1 cm light path. Record the values as A_standard and A_test.

6. Calculation of Results

• Serum/Plasma Inorganic Phosphorus:

  Phosphorus (mmol/L) = (A_test / A_standard) × 1.292

• Urine Inorganic Phosphorus:

  Phosphorus (mmol/d) = (A_test / A_standard) × 1.292 × N × 24-hour urine volume (L)

• Tissue Inorganic Phosphorus:

  Phosphorus (mmol/mg prot) = (A_test / A_standard) × 1.292 / Protein concentration of test sample (mg/L)

Parameter Description:

• A_test: Absorbance of the test tube

• A_standard: Absorbance of the standard tube

• N: Urine dilution factor

• Unit Conversion: 1 mg/dL = 10 mg/L = (1 mmol/L) × 0.323

Reference Interval:

Sample Type	Serum Phosphorus Concentration
Healthy Adults	0.96~1.62mmol/L (3~5mg/dl)
Children	1.45~2.1mmol/L (4.5~6.5mg/dl)

Precautions

1. Hemolyzed samples interfere with the assay; avoid using them if possible.

2. This method can be adapted for automated biochemical analyzers using an endpoint detection method.

3. If the sample concentration is too high, dilute with distilled water and re-assay, multiplying the result by the dilution factor.

4. The linear range of this assay is 0.01 ~ 0.2 mg/ml. The lower limit of detection is 0.005, and the upper limit is 1.

5. For your safety and health, please wear a lab coat and disposable gloves during operation.

Appendix: Standard Curve Preparation

When operated at room temperature according to the instructions, measure the absorbance of a series of phosphorus standards (1/100, 1/40, 1/25, 1/15, 1/10, 1/8 mg/ml) using a spectrophotometer. The resulting standard curve is shown below (for reference only):

Specifications

Synonyms

Inorganic Phosphate Content Assay Kit (Ferrous Sulfate Molybdenum Blue, Colorimetric Method) | Inorganic Phosphorus Content Assay Kit

Specifications & Purity

BioReagent

Storage

Store at 2-8°C, Protected from light, Room temperature

Shipped In

Wet ice

This product requires cold chain shipping. Ground and other economy services are not available.

Grade

BioReagent

Chemical and Physical Properties

• Sensitivity: Light-sensitive