Topic: Tox / TDM / Endocrine
Authors: Zhicheng Jin, PhD, Ashley N. Siverly, and Xin Yi, PhD
A 1α,25-dihydroxyvitamin D (DHVD) test is used to assess the level of the biologically active form of vitamin D in patients with chronic renal disease or for differential diagnosis of hypercalcemia. The DHVD assay is challenging because there are numerous isomers present in patient specimens, and the active form has very low circulating concentrations (picomolar). Our hospital is currently using a Cookson-type triazolinedione derivatization protocol followed by LC-MS/MS quantification to determine 1α,25-dihydroxyvitamin D2 and 1α,25-dihydroxyvitamin D3 levels in serum specimens (1). While investigating a positive comparison bias for DHVD assay results, we discovered that the reference laboratories had adopted a new methodology. We thus modified our methodology to address these discrepancies.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the “gold standard” for quantification of vitamin D metabolites in complex matrices. Our original method started with solid-phase extraction and derivatization using 4-phenyl-1,2,4-triazoline-3,5-dione. The derivatized complex mixtures were chromatographically separated on Acuity BEH C18 columns (1.7 µm, 2.1x100 mm) using a Shimadzu UHPLC system coupled to a Sciex Qtrap 5500 instrument operating in triple quadrupole mode. Recent studies showed that immunoaffinity purification may reduce an interference metabolite that was co-eluting with DHVD peaks. However, we optimized liquid chromatographic separation to resolve interference peaks that may lead to positive bias of the DHVD results.
Our recent in-house alternative proficiency tests showed a positive bias in comparison to DHVD send-out test. Specifically, the results of our calibrators were acceptable, while the majority of patient specimens showed positive bias. We performed a thorough investigation, ruled out assay issues, and finally determined that the change was likely not within our lab. Two months later, the reference laboratories reported a modified sample preparation method that incorporated immunoaffinity isolation steps. Their decreased DHVD levels in patient specimens were consistent with the protocol change. These results indicated that our method was likely overestimating DHVD if there was an interference that may be co-eluting with the targeted analyte. Recent studies showed that 4β,25-dihydroxyvitamin D and 1α,25-dihydroxy-3-epi-vitamin D have the same mass-to-charge ratio as 1α,25-dihydroxyvitamin D and may cause bias in the quantification of DHVD. Based on the retention time, we suspected that an interference peak was possible due to the presence of 4β,25-dihydroxyvitamin D (2). By using a phenyl-hexyl LC column and slightly extending the LC gradient, we were able to separate an interference peak from the DHVD peak. The assay results using our updated LC-MS/MS method were consistent with results from the reference laboratory.
We identified the root cause of positive bias in our DHVD assay. Optimizing chromatographic separation allowed us to address the deviation in a timely manner. Our quantification assay will be revalidated after the method modification.
1. Laha TJ, etc. Characterizing and harnessing antibody cross-reactivity for the immunoaffinity purification of analytes prior to multiplexed liquid chromatography-tandem mass spectrometry. Clin Chem. 2012, 58(2):1711-1716.
2. Wang Z, etc. Simultaneous measurement of plasma vitamin D3 metabolites including 4β,25-dihydroxyvitamin D3 using liquid chromatography-tandem mass spectrometry. Anal Biochem. 2011, 418(1):126-133.
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