A micro-method for serum calcium and serum magnesium.

A simple, rapid, accurate method for determining calcium and magnesium separately in 0.1 ml. volumes of serum (or heparinized plasma) was required for the routine chemical pathology service of a children's hospital (Wilkinson, 1955-6). The apparatus used had to be generally available, the minimum of pipetting and transfer was desirable for the sake of accuracy, and the method had to be specific. Sobel and Hanok (1951) determined the sum of serum calcium and magnesium by direct titration with the disodium salt of ethylene-diamine tetra-acetic acid (E.D.T.A.) using eriochrome black T as the metal indicator. This indicator has a relatively sharp visual end-point. If serum calcium could be determined in a similar way then magnesium could be found by difference. Murexide (ammonium purpurate) in alkaline solution is a specific indicator for calcium in serum. The colour change of the calcium murexide complex to the free dye is gradual and the visual end-point of a titration with E.D.T.A. is not sharp (Davis and Smith, 1953). The colour change of the alkaline dye solution with calcium has been used as a colorimetric method (Williams and Moser, 1953; Gorsuch and Posner, 1955), but the fading is rapid. Diggins (1955) has reported a method of stabilizing the colour. An alternative procedure is to titrate serum calcium with E.D.T.A. using murexide as an indicator and to determine the end-point photometrically. Schwarzenbach (1955) has reviewed the whole subject of complexometric titrations. He showed the curves of colour change of indicator versus the volume of titrant added, for murexide and eriochrome black T in the presence of calcium and magnesium at different pH levels. The theory of spectrophotometric titrations with complexones has been discussed by Fortuin, Karsten, and Kies (1954) and Ringbom and Vanninen (1954). Methods which use these principles for the photometric titration of calcium have been described by Holtz and Seekles (1952), Kibrick, Ross, and Rogers (1952), Lehmann (1953), Fales (1953), Chalmers (1954), Poulie (1954), Karsten, Kies, van Engelen, and de Hoog (1955), Eldjarn, Nygaard, and Sveinsson (1955), and Horner (1955). An investigation of this type of analysis is described below and a simple method has been evolved for analysing 0.1 ml. quantities of serum for calcium and calcium plus magnesium.