Although immunoassays in measuring 25-hydroxyvitamin D [25(OH)D] have been improved recently, relatively large differences are still seen between results of 25(OH)D measured by immunoassays and by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the present studies, we compared two immunoassays with LC-MS/MS for measuring 25(OH)D concentrations. Concentrations of 25-hydroxyvitamin D
2 [25(OH)D
2] and 25-hydroxyvitamin D
3 [25(OH)D
3] in serum samples from 59 healthy subjects were measured by two immunoassays including Siemens ADVIA Centaur Vitamin D Total (Centaur) and Roche Elecsys Vitamin D Total (Elecsys) and LC-MS/MS. To determine the cross reactivity of Elecsys and Centaur toward 25(OH)D
2, a dosage of 200,000 IU vitamin D
2 was given after first sampling. Serum samples were obtained 30 days later and concentrations of 25(OH)D
2 and 25(OH)D
3 were measured again. The results showed poor agreement between the immunoassays and LC-MS/MS in 25(OH)D
2 and 25(OH)D
3 measurements. The percentage of 25(OH)D
2 cross-reactivity was 45.3% for Centaur and 41.2% for Elecsys and there was no significant difference between Centaur and Elecsys. In conclusion, Centaur and Elecsys perform unsatisfactorily in measuring 25(OH)D levels, especially for 25(OH)D
2 cross-reactivity. Therefore, clinicians need to be aware of the underestimation of vitamin D status when using these immunoassays for measuring individuals supplemented with vitamin D
2.
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