Colorimetric determination of total serum calcium by o-cresolphthalein complexone assay

Summary

The near-o-cresolphthalein complexone colorimetric assay for the determination of total serum calcium test can be used clinically to measure serum calcium ion levels.

Operation method

Colorimetric determination of total serum calcium by o-cresolphthalein complexone assay

Principle

o-Cresolphthalein complex ketone is all genus Lohan indicator, but also acid-base indicator, in alkaline solution with calcium and magnesium chelate, generate purple-red compound. For calcium determination, 8-hydroxyquinoline is added to the reagent to eliminate the interference of magnesium in the specimen.

Materials and Instruments

Serum
AMP Alkaline Buffer
o-Cresolphthalein complex ketone color developer

Move

I. Experimental reagents:

l. o-cresolphthalein complex ketone color developer: weigh 500mg of 8 a hydroxyquinoline, placed in a beaker. Add concentrated HCL 15ml, make it dissolve and transfer to 500ml volumetric flask, then add o-cresolphthalein complex ketone 25mg, to be completely dissolved, such as Tritonx-10Oml mixing, and then add deionized water to the scale to be placed in polyethylene bottles for storage.

2. lmol / L AMP alkaline buffer: Weigh a-amino-2-methyl-l -propanol 89.14g, placed in 1L Volumetric flask, add 500ml of deionized water to dissolve until completely dissolved and added to the scale, placed in a polyethylene bottle and stored at room temperature.

3. Color development application solution: when applying, according to the number of specimens on the day of the two liquids will be mixed in equal quantities.

4. Calcium standard solution (2.5mmol/L): accurately weighed by the drying of 110 ℃ for 12h of the 250mg of calcium carbonate, placed in 1L volumetric flasks, add dilute hydrochloric acid (1 portion of concentrated HCL plus 9 portions of deionized water), and then mix with a mixture of calcium carbonate and calcium carbonate. HCL plus 9 parts of deionized water) 7ml dissolved, add 900ml of deionized water, and then use 500g/L ammonium acetate solution to adjust the PH value to 7.0, and finally add deionized water to the scale, mix well.

Second, the experimental operation:

Warm and homogeneous, put 5min, spectrophotometer wavelength 575nm, 10mm aperture colorimetric cup with a blank tube to adjust zero, read the absorbance of each tube.

If the specimen is turbid, jaundice, blood bath, need to do the correction test, in the blank tube and measuring tube each add 5g/L EGTA solution 0.05ml and then measure the absorbance, again read the absorbance difference, that is, the corrected absorbance.

Calculation:

Serum Calcium mmol/L= { [ Absorbance of measurement tube (or absorbance of correction tube)] / Absorbance of standard tube }x2.5

Reference value: Adult: 2.03-2.45 mmol/L

Child: 2.25-2.67 mmol/L

Caveat

1. Anticoagulation of plasma specimens with serum or heparin should not be done with calcium chelator (EDTA).Calcium chelators (EDTA) are not allowed.2Calcium chelator (EDTA Na2) and oxalate should not be used as anticoagulants.

2. o-cresolphthalein complex ketone reagent sensitivity is very high, the use of test tubes and utensils, such as trace contamination will also cause errors in the determination, therefore, the determination of the best use of plastic tubes, the use of the feed should be stored in polyethylene bottles. If the conditions do not allow the use of glass test tubes and utensils must be cleaned by dilute hydrochloric acid, and then rinse with deionized water before use.

Common Problems

Through the observation on the stability of color development, linear relationship, repeat test and recovery test, as well as the comparison with other methods, it is proved that this method has the characteristics of high sensitivity, simple method and good stability, and can be popularized and applied.

This experiment is based on the experiment instruction of Mudanjiang Medical College, "Serum Calcium-o-cresolphthalein Complexone Colorimetric Method", which is a professional experiment for undergraduates of 5-year undergraduates of Mudanjiang Medical College.

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