Measurement of Calcium
in Serum & Urine 1
This method reduces or removes potential interference from sodium, potassium and phosphates normally present in serum and urine by diluting the samples with a diluent containing ethylenediaminetetraacetic acid (EDTA).
An EDTA solution is prepared to negate the interference of competing ionic species during the determination of Calcium / Sodium / Potassium via flame photometry. To negate the formation of Calcium Phosphate in the samples which impedes the emission of calcium ions in solution, 1000mg/L solution is added due to its higher binding potential with phosphates present in serum and urine.
An EDTA Stock solution was prepared with 58.4grams of EDTA being diluted into 400mls of DI water. To this solution small aliquots of Ammonium Hydroxide were added, and the pH of the solution was tested with a pH probe until additions brought the pH of the solution to 8.0. This solution was then filtered into a 1L volumetric flask through Whatman N.5 Filter paper and diluted to 1L with DI water.
A range of standards were then formed in 5 volumetric flasks with the following ionic concentrations:
Na(mMol/L) 0, 50, 100, 150, 200
K(mMol/L) 0, 10, 20, 30, 40, 50
Ca(mMol/L) 0, 0.2, 0.4, 0.6, 0.8, 1.0
In addition to these calibration ions, 0.75mMol/L Mg was maintained throughout each standard if measuring blood and 0.125mg/L Mg if measuring urine, with 1ml of 1000mg/L Lanthanum Chloride solution being added to account for its addition to each sample.
2ml of the sample be that of serum or urine was placed into a polyethylene volumetric flask. To this volumetric flask 1ml of 1000mg/L Lanthanum Chloride solution was added then the solution was diluted to 100ml with the EDTA solution to dilute the sample fiftyfold. The volumetric flask was then mixed via inversion.
The solution was then labelled and placed aside whilst the flame photometer is calibrated.
The flame photometer was switched on and allowed to stabilise for 45 minutes prior to calibration.
The standard solutions prepared were then aspirated in accordance with the guidelines set out on the MultiPoint/Multi Ion guidelines set out in the BWB-Tech Installation and Operation manual. To avoid high concentration residue affecting the calibration series, all of the lowest concentration ions were calibrated initially (eg Calibrating all of the low concentration standards ions before moving up to the next calibration solution).
The flame photometer was then set to read mode and the sample was aspirated; the result of this determination was multiplied by 50 to account for the 50-fold dilution carried out in the sample preparation step of this methodology.
Preparation of Standard Graph
Set the flame photometer in accordance to MultiPoint/Single Ion Calibration found on page 24 of the BWB Technologies Installation and Operation Manual, to measure potassium emission. Nebulise the lithium working standard solutions and adjust the controls until steady zero and maximum readings are obtained. Nebulise the intermediate working standard solutions and construct a graph relating raw emission data (known as RAW in BWB the flame photometer) to concentration of all the standard solutions.
1 Thiers, R. and Hviid, K., ‘Interference-free Photometry of Calcium in Serum and Urine’, Clin.Chem., 8, (1962), p. 35-46.