Validation and comparison of a rapid liquid chromatography tandem mass spectrometry method for serum 25OHD with the efficiency of separating 3-epi 25OHD3

• A rapid and precise LC-MS/MS for 25OHD measurements with the efficiency to separate 3-epi 25OHD3 was developed.
• The performance of the newly built method was excellent and would be suitable for using in clinical laboratory.
• 3-epi 25OHD3 had little effects on routinely used NEPI-LC-MS/MS that could not separate 3-epi 25OHD3 from 25OHD3.
• 3-epi 25OHD3 status was firstly evaluated in China.

ObjectivesTo develop a rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method with ability to separate 3-epi 25OHD3 (EPI-LC-MS/MS) from 25OHD3, and evaluate the effects of 3-epi 25OHD3 on routine LC-MS/MS that cannot separate 3-epi 25OHD3 (NEPI-LC-MS/MS).Design and methodsPerformance of the newly built EPI-LC-MS/MS was validated, and 982 samples were analyzed and compared by the two methods.ResultsBoth methods showed a linearity coefficient correlation exceeding 0.999 in the 6.25–500 nmol/L range for 25OHD2 and 25OHD3. Moreover, they showed a between run coefficient variation (CV) and total CV of < 5% for 25OHD2 and 25OHD3. The results of the accuracy test showed that the bias was below 6.19% in the absence of 3-epi 25OHD3. Comparison of the 25OHD results obtained by the two methods for 982 patients (age 1‐100 years) revealed excellent clinical agreement (Cohen’s kappa = 0.875) and correlation (R2 = 0.973). Among the 982 patients, only 73 patients had 3-epi 25OHD3 (> 6.25 nmol/L); out of these 73 patients, the 3-epi 25OHD3 level in 58 patients was between 6.25 and 12.5 nmol/L. In patients with < 375 nmol/L 25OHD (25OHD2 + 25OHD3), only 8 had 3-epi 25OHD3 levels exceeding 12.5 nmol/L (range: 13.3 - 27.5 nmol/L). Among samples containing 3-epi 25OHD3, only three were separated into different 25OHD-deficiency groups using the above methods.ConclusionA rapid and precise EPI-LC-MS/MS method for measuring 25OHD with efficient separation of 3-epi 25OHD3 was developed. Our results showed that 3-epi 25OHD3 had little effect on the routinely used NEPI-LC-MS/MS.