Development of novel measurement assay for calcium in serum by the chlorophosphonazo-III vanadate method

Background: Chlorophosphonazo-III (2,7-bis[4-chloro-2-phosphonophenylazo]- 1,8-dihydroxy-3,6-naphthalenedisulphonic acid, disodium salt; CPZ-III) reacts with calcium and magnesium in a sample under acidic to neutral conditions. However, the specific method of measuring calcium in serum using CPZ-III has not been established because of the difficulty of avoiding the interaction between CPZ-III and albumin. Methods: In this study, we found that the non-specific reaction between CPZ-III and albumin could be controlled and calcium in serum could be specifically detected using CPZ-III combined with vanadate. On the basis of this finding, we evaluated a novel method of serum calcium determination using CPZ-III. Results: This CPZ-III vanadate method gave linear results from 0 to 7.0 mmol/L. The coefficient of variation was 0.63-0.76%. There was no interference except with Omniscan. There was no change in control performance during 60 d under open-air conditions. The assay results correlated well with those of the Arsenazo-III (2,7-bis(2-arsonophenylazo)-1,8-dihydroxy-3, 6-naphthalenedisulphonic acid) method (slope = 1.067; intercept = 20.120; r = 0.989; Sy/x = 0.036 mmol/L), o-cresolphthalein complexone method (slope = 0.911; intercept = 0.186; r = 0.988; Sy/x = 0.035 mmol/L), amylase enzymatic method (slope = 0.981; intercept = 0.072; r = 0.989; Sy/x = 0.036 mmol/L) and inductively coupled plasma emission spectroscopy method (slope = 0.955; intercept = 20.001; r = 0.979; Sy/x = 0.048 mmol/L). Conclusions: These results suggested that the present method has great clinical potential for measuring calcium.