CALCIUM
(Arsenazo III method)
For the determination of Calcium in serum or plasma
(For In vitro Diagnostic Use Only)
SUMMARY:
Calcium, in the body, is found mainly in the bones (approximately 99%). In serum calcium exists equally in a free
ionized form and in a bound form (with albumin). Hence a decrease in Albumin causes lower Calcium levels and
vice versa. The levels of Calcium in serum depends on the parathyroid hormone. Increased Calcium levels are
found in bone tumours, hyperparathyroidism. Decreased levels are found in hypoparathyroidism, renal failure,
rickets, Vitamin D deficiency and pancreatitis.
PRINCIPLE:
Calcium combines specifically with Arsenazo III at neutral pH to form a blue purple colored complex. Intensity of
the color formed is directly proportional to the amount of calcium present in the sample.
Calcium + Arsenazo III Blue Purple coloured Complex
CONTENTS:
PACK SIZE CALCIUM REAGENT(A1) STANDARD (S)
1x50ml 1x50ml 5ml
STORAGE & STABILITY:
Reagents are stable at RT till the expiry mentioned on the labels.
REAGENT PREPARATION:
Reagents are ready to use. Protect from bright light.
SAMPLE MATERIAL:
Serum/ Heparinized plasma. Calcium is reported to be stable in serum for 7 days at 2-8°C.
GENERAL SYSTEM PARAMETERS:
Reaction Mode End Point Sample Volume 10 µl
Wavelength 630 nm Reagent volume 1000 µl
Blank Reagent Blank Standard 10mg/dl
Incubation 3 min at R.T Reaction Slope Increasing
Delay Time 5 Sec Linearity 20 mg/dl
Read Time 5Sec Units mg/dl
ASSAY PROCEDURE:
Wavelength/ Filter : 630nm
Temperature : R.T
Light Path : 1 cm
Pipette into clean dry test tubes labelled as Blank (B), Standard (S), and Test (T):
Addition Sequence B(µl) S(µl) T(µl)
Calcium Reagent(A1) 1000 1000 1000
Standard(S) - 10 -
Sample - - 10
Mix well and incubate at R.T. for 3 min. Measure the absorbance of the Standard (Abs. S), and
Test Sample (Abs. T) against Blank.
CALCULATIONS:
Abs.T
Calcium in mg / dl = ———— x 10
Abs.S
NORMAL REFERENCE VALUES:
Serum/Plasma : 8.7 - 11.0 mg/dl
Urine : 100 - 400 mg/24 hours
LINEARITY:
This procedure is linear up to 20 mg/dl. If values exceed this limit dilute the sample with distilled water and
repeat the assay. Calculate the value using the proper dilution factor.
NOTE:
As calcium is a very widely distributed ion, care should be taken to avoid any contamination. All glassware being
used for the test should first be rinsed with 1% or 0.1 N HCL and then with good quality deionised water before
use. It is suggested that after the rinsing of the tubes with HCL the reagent be pipetted in their respective tubes and
the tubes be rinsed with the reagent. The reagent then should be pooled together in the ‘blank’ tube and repipetted
out into the ‘standard’ and ‘test’ test tubes. This will ensure that any remaining contamination will be carried over
equally in all the tubes. For flow cell cuvettes it is suggested that some reagent be aspirated before the blank to
take away any contamination in the flow through tubing or cuvette which may cause a higher than the actual blank
of the reagent. Chelating agents such as EDTA, present even in traces, prevent the formation of the colour complex,
hence necessary care should be taken during the assay.
REFERENCE :
1. Smith, H.G. [Link] al, (1979) Biochem 18:5067.
