Variability in the analysis of 25-hydroxyvitamin D by liquid chromatography-tandem mass spectrometry: The devil is in the detail

Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly used in clinical laboratories for the analysis of 25-hydroxyvitamin D (25OHD), but measurement is not straightforward. Importantly. LC-MS/MS is not a single technique: variables in sample preparation, chromatography and ionisation/fragmentation should each be considered. Methods: We analysed results from a survey organised by the international Vitamin D External Quality Assessment Scheme (DEQAS), to determine the influence of such variables on the results for two DEQAS distributions. Results: 65 laboratories returned questionnaires. 346 (57%) individual results were from laboratories using electrospray ionisation (ESI), and 259 (43%) from laboratories using atmospheric pressure chemical ionisation (APCI). Although the mean ratio of results was not significantly different between ESI and APCI (P = 0.5828), there was greater variation (P<0.0001) in results obtained by laboratories using ESI. Greater variation (P<0.05) was also observed between results from laboratories monitoring non-specific water-loss transitions. Only 3 laboratories (5%) could resolve the isobaric metabolite 3-epi-25OHD(3) from 25OHD(3). Conclusions: There are many variables to consider when using LC-MS/MS, including assay standardisation/calibration, chromatography and MS conditions. MS/MS alone cannot distinguish isobaric metabolites such as 3-epi-25OHD(3). Interference can also occur if non-specific transitions are used. Laboratories should always subscribe to an EQA scheme for 25OHD analysis. (C) 2012 Elsevier B.V. All rights reserved.