LC-MS/MS reduces interference by high levels of 25(OH)D and its metabolites on measured 1,25(OH)₂D

High-dose vitamin D supplementation has been recommended as treatment to several conditions; however, potential side effects such as hypercalcemia should be considered, plus the fact that high levels of 25(OH)D may interfere with potential 1,25(OH)₂D measurements. Our study compared two methods of measuring 1,25-dihydroxyvitamin D [1,25(OH)₂D] in samples with 25-hydroxyvitamin D [25(OH)D] levels above 150 ng/mL (375 nmol/L).

We studied serum samples referred to 25(OH)D and 1,25(OH)₂D quantification. The concentrations of 25(OH)D and 1,25(OH)₂D were measured using DiaSorin chemiluminescent assays (CLIA) in 213 samples (CLIA group), whereas in 357 samples 25(OH)D and 1,25(OH)₂D were measured by DiaSorin CLIA and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively (CLIA + MS group).

Median concentrations of 25(OH)D and 1,25(OH)₂D in the CLIA group were 371 ng/mL (928 nmol/L, range 154-856 ng/mL) and 350 pg/mL (875 pmol/L, range 41-1280 pg/mL), respectively, and correlated significantly (Spearman correlation coefficient r_s = 0.8469, P < 0.001). In the CLIA + MS group, the median concentrations of 25(OH)D and 1,25(OH)₂D were, respectively, 344 ng/mL (860 nmol/L, range 152-756 ng/mL) and 56 pg/mL (140 pmol/L, range 17-151 pg/mL), and were not correlated (r_s = 0.0218, P = 0.6811). No significant difference was found in calcium, creatinine, and PTH serum values between the groups.

Methods for measuring 1,25(OH)₂D in patients with high levels of 25(OH)D may be susceptible to interference by 25(OH)D and its metabolites and should be validated carefully. In such cases, measurement of 1,25(OH)₂D using LC-MS/MS is preferred.