Inorganic phosphorus in serum primarily consists of two phosphate anions, H₂PO₄⁻ and HPO₄²⁻, which rapidly interconvert depending on the pH environment. In serum at pH 7.4, the concentration ratio is approximately 1:4. This 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 WHO recommends the colorimetric method for routine detection. 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 Under acidic conditions, inorganic phosphorus reacts with ammonium molybdate to form ammonium phosphomolybdate. This compound is then reduced by metol to form a blue-purple complex. The absorbance is measured at 650 nm using a spectrophotometer, and the inorganic phosphorus content is calculated based on a formula. This kit is for research use only and is not suitable for clinical diagnosis or other purposes.
I1506789
| Component
| 100T | Storage
| I1506789A
| Phosphorus Standard (1 mg/mL)
| 1 mL | 2-8℃
| I1506789B
| Standard Diluent
| 5 mL | RT. Store in the dark.
| I1506789C
| Ammonium Molybdate Powder
| 0.3 g | RT
| I1506789D
| Ammonium Molybdate Diluent
| 50 mL | RT
| I1506789E
| Metol
| 50 mg | RT. Store in the dark.
| I1506789F
| Metol Diluent
| 50 mL
| RT
| I1506789G
| Pi Deproteinization Reagent
| 100 mL
| RT. Store in the dark. |
Materials Required but Not Provided Deionized water Electronic balance, centrifuge tubes or test tubes, water bath or incubator or metal bath, cuvettes, spectrophotometer
Experimental Procedure 1. Sample Preparation 1.1 Plasma/Serum Samples Prepare according to standard methods. Can be used directly for assay with this kit. Store at -20°C for Pi detection. 1.2 Cell or Tissue Samples Homogenize appropriate amount of cells or tissue. Centrifuge at low speed to collect supernatant. Store at -20°C for Pi detection. 1.3 High Concentration Samples If sample contains high Pi concentration, dilute with distilled water. Note: After preparation, determine protein concentration using a BCA protein assay kit to calculate Pi content per unit protein weight in tissues/cells. 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 Phosphorus Standard (1 mg/mL) with Standard Diluent at a 1:99 ratio to obtain Phosphorus Standard Working Solution (10 μg/mL). Store at -20°C for Pi detection.
3. Preparation of Ammonium Molybdate Solution Mix 0.3 g Ammonium Molybdate Powder with 50 mL Ammonium Molybdate Diluent. Dissolve completely (use ultrasonication if necessary). Store prepared solution at 4°C protected from light; stable for 1 month.
4. Preparation of Metol Solution Dissolve 50 mg Metol in 25 mL deionized water. Mix this Metol aqueous solution with Metol Diluent at a 1:9 ratio. Store prepared solution at 4°C protected from light; stable for 1 month.
5. Pi Assay Setup Set up Blank, Standard, and Test tubes as below. Add reagents in order, avoid bubbles, mix gently. For high Pi concentrations, reduce sample volume or dilute appropriately. Run duplicates for samples and average the results. Reagent (mL)
| Blank Tube
| Standard Tube
| Test Tube
| ddH₂O
| 0.5
| —
| —
| Phosphorus Std Working Solition (10μg/mL)
| —
| 0.5
| —
| Test Sample
| —
| —
| 0.5
| Ammonium Molybdate Solution
| 0.5
| 0.5
| 0.5
| Metol Solution
| 0.5
| 0.5
| 0.5 |
Place tubes in boiling water bath for 30 min. Add 1 mL deionized water to each tube. Cool in water to room temperature. 6. Pi Measurement Using a 1.0 cm pathlength cuvette and zeroing with the Blank, measure absorbance of all tubes at 650 nm (record as A<sub>standard</sub> and A<sub>test</sub>).
7. Calculation of Results Serum/Plasma Inorganic Phosphorus: Phosphorus (mmol/L) = (Atest / Astandard) × 0.323 × N Phosphorus (mg/L) = (Atest / Astandard) × 10 × N Tissue Inorganic Phosphorus: Phosphorus (mmol/mg prot) = (Atest / Astandard) × 0.323 × N / c
Parameter Description: Atest: Absorbance of test tube Astandard: Absorbance of standard tube N: Sample dilution factor c: Protein concentration of test sample (mg/L) Unit Conversion: 1 mg/dL = 10 mg/L = 10 μg/mL = 0.323 mmol/L
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) |
Appendix 1: Deproteinization for Turbid Samples Samples with inverted serum albumin ratios may cause turbidity. Deproteinize as follows: Add 0.1 mL test serum to 0.9 mL Pi Deproteinization Reagent. Mix well, centrifuge at low speed. Take 0.05 mL supernatant. Treat Phosphorus Standard Working Solution (10 μg/mL) similarly. Proceed with remaining steps.
Appendix 2: Standard Curve Reference Range Absorbance of the 10 μg/mL Phosphorus Standard at 650 nm (blank zeroed) typically ranges 0.3~0.6. The standard curve obtained by measuring absorbances of phosphorus standards at 0, 1, 5, 10, 15, 20, 25, 30 μg/mL (following instructions) is shown below: 
Precautions Prepared Ammonium Molybdate and Metol solutions should be used promptly. Prolonged storage may cause slight turbidity in normal serum. Do not use if reagents turn blue. Dilute high-concentration samples with distilled water and remeasure, multiplying result by dilution factor. Color development intensity is highly dependent on pH, time, and temperature. Control reaction conditions carefully. The linear range of this method is 1~35 μg/mL. For your safety and health, wear a lab coat and disposable gloves during operation.
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