A candidate reference measurement procedure for quantifying serum concentrations of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 using isotope-dilution liquid chromatography-tandem mass spectrometry

Abstract

The inaccuracy of routine serum 25-hydroxyvitamin D measurements hampers the interpretation of data in patient care and public health research. We developed and validated a candidate reference measurement procedure (RMP) for highly accurate quantitation of two clinically important 25-hydroxyvitamin D metabolites in serum, 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3]. The two compounds of interest together with spiked deuterium-labeled internal standards [d3-25(OH)D2 and d6-25(OH)D3] were extracted from serum via liquid-liquid extraction. The featured isotope-dilution LC-MS/MS method used reversed-phase chromatography and atmospheric pressure chemical ionization in positive ion mode. A pentafluorophenylpropyl-packed UHPLC column together with isocratic elution allowed for complete baseline resolution of 25(OH)D2 and 25(OH)D3 from their structural C-3 isomers within 12 min. We evaluated method trueness, precision, potential interferences, matrix effects, limits of quantitation, and measurement uncertainty. Calibration materials were, or were traceable to, NIST Standard Reference Materials 2972. Within-day and total imprecision (CV) averaged 1.9% and 2.0% for 25(OH)D3, respectively, and 2.4% and 3.5% for 25(OH)D2, respectively. Mean trueness was 100.4% for 25(OH)D3 and 100.3% for 25(OH)D2. The limits of quantitation/limits of detection were 4.61/1.38 nmol/L for 25(OH)D3 and 1.46/0.13 nmol/L for 25(OH)D2. When we compared our RMP results to an established RMP using 40 serum samples, we found a nonsignificant mean bias of 0.2% for total 25(OH)D. This candidate RMP for 25(OH)D metabolites meets predefined method performance specifications (≤5% total CV and ≤1.7% bias) and provides sufficient sample throughput to meet the needs of the Centers for Disease Control and Prevention Vitamin D Standardization Certification Program.

Rapid and reliable 25-OH vitamin D2 and 25-OH vitamin D3 measurements by multitasker LC-MS/MS

“…The main function of Vitamin D in human metabolism is defined as regulating calcium and phosphorus levels in the bloodstream. There are an increasing amount of studies and views on the different metabolic functions of Vitamin D. Studies show that its` widespread impact and the need for its reliable and quick measurement is of paramount importance [1][2][3][4][5][6].…”

“…Due to 3-epi-25(OH)D3 being found only in trace amounts, it is not commonly studied in routine analyses carried out by clinical biochemists. Values below 12 ng/mL (30 nmol/mL) of 25-OH vitamin D are defined as a Vitamin D deficiency and point towards rickets in newly born babies and osteomalacia in adults [4][5][6][7][8][9]. A value of 12-20 ng/mL (20-50 nmol/mL) is considered inadequate for bone and overall health in healthy individuals.…”

“…The NIST provided service of SRM 972 to clinical laboratories in 2009 [10] and SRM 972a in 2013 [11] while UME CRM 1308 was provided by TUBITAK UME in 2016 [12]. While the various metrology institutes over the world report Vitamin D measurements with low uncertainties in the CCQM level, 2.3% to 6.8% for Vitamin D2 and 2.1% to 12.6% for Vitamin D3, the uncertainty values are generally not only not reported by second tier laboratories but also often report CV values between 10-30% [1][2][3][4][5][6][7][8][9].…”

“…Therefore the ID-LC-MS/MS is accepted as the primary method of measuring Vitamin D and because of its accurate and precise measurements it is becoming increasingly popular in clinical laboratories [4][5][6][7].…”

Various calibration procedures result in optimal standardization of routinely used 25(OH)D ID-LC-MS/MS methods

“…In a joined effort to implement standardized measurements for 25(OH)D, the National Institute of Health (NIH), the US Center for Disease Control and Prevention (CDC), the US National Institute of Standards and Technology (NIST) and the Belgian Laboratory for Analytical Chemistry in Ghent (UGhent), in collaboration with other researchers and organizations, established the Vitamin D Standardization Program (VDSP) [9]. The goal of this collaboration is to make 25(OH)D measurements traceable to the highest order reference, the NIST Standard Reference Material 2972a, by using recognized reference measurement procedures (RMP) operated at NIST, CDC, and UGhent and high quality serum based reference materials with values assigned by these RMPs [8, 10,11]. Although several immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays have been standardized by CDC’s Vitamin Standardization Certification Program [1], information about the accuracy of routinely used liquid chromatography-tandem mass spectrometry (LC-MS/MS) 25(OH)D methods is very limited.…”