Abstract

Determination of 25-OH-D₃ in plasma is used in the diagnosis of disorders in calcium metabolism, such as neonatal hypocalcemia, rickets and postmenopausal osteoporosis. Current available methods are radioimmunoassay ¹²⁵I-RIA), radioreceptorassay and HPLC. In contrast to immunological methods, which determine the total amount of 25-OH-D₃ and 25-OH-D₂ (depending on the specificity of the antibody), HPLC methods allow the analysis of both analytes. The Bio-Rad HPLC assay measures the 25-OH-D₃, using a novel simple extraction procedure followed by fast reverse phase chromatography. Method evaluation was performed for intra- and interassay precision, linearity, recovery, and a patient specimen comparison with the INCSTAR ¹²⁵I-RIA. For analysis acetonitrile containing the internal standard is added to 500 µl sample and mixed. Subsequently two different buffer solutions are added to stabilize the sample and to force phase separation between acetonitrile and the aequeous part. After centrifugation 50 µl of the upper acetonitrile layer is injected into a reverse phase column (3.2 mm ID, 90 mm) at 40°C and eluted isocratically within 12 minutes. Quantitation is performed by UV detection λ = 265 nm). A single point calibrator is used for calculating unknowns. The average intraassay precision (n = 25) was 3.72% (range 2.07 - 5.69%), the interassay precision (n = 25) was 4.34% (range 3.59 - 5.15%) at concentrations between 15 - 200 ng/ml. The method was found to be linear up to 250 ng/ml (determined according to the NCCLS document EP6-P), the detection limit was 2 - 5 ng/ml (depending on the performance of the UV detector). The mean recovery of added 25-OH-D₃ was 100% (range 92 - 105%) for concentrations between 20 - 200 ng/ml. The method comparison study included 197 patient with concentrations between 5 - 105 ng/ml. Regression analysis was performed by the method of Bablok and Passing using the Evapak 3.0 software package (Boehringer Mannheim). The equation of the regression analysis of the method comparison was HPLC = 0.93 [INCSTAR RIA] + 6.2, r = 0.873. These results confirm that the Bio-Rad 25-OH-D₃ HPLC assay provides a convenient and accurate method for the determination of 25-OH-D₃ levels in all patient categories.

DOI: CIin. Lab. 1999;45:657-659