The evaluation of using high performance liquid chromatography as a method of monitoring vitamin D2 and D3 concentrations in patients

Abstract

Vitamin D is actually a steroid hormone. It exists in two forms; Vitamin D2 and D3 and can be introduced into a person via dietary intake or by the conversion of cholesterol in the skin by UV radiation, which initiates a series of chemical reactions to eventually produce the active form of Vitamin D; 1-2Sdihydroxycholecalciferol. It is extremely influential in the metabolism of Calcium and Phosphate, therefore its concentrations have a direct influence over the concentrations of these two analytes in circulation Vitamin D deficiencies are renown worldwide as causes such disorders as rickets, osteomalacia and are believed to be linked with other conditions such as Bowel Cancer and Immune system disorders. Vitamin D toxicity can be just as detrimental, therefore it is very important that a person's Vitamin D concentrations are monitored. Currently in the Royal Gwent Hospital samples are sent away for analysis at the University Hospital, Wales. UHW use a Liquid Chromatography Method with Mass Spectrometry detection. The purpose of this project is to evaluate and implement a new High Performance Liquid Chromatography method into the laboratory of the RGH. Tests were performed to determine the validation of High Performance Liquid Chromatography as a method of Vitamin D analysis. A comparative study was performed with the current "Gold Standard" method – Liquid Chromatography with Mass Spectrometry. Within batch and Between batch Quality Controls were run to determine the precision of the method. Patient samples that had been received in different collection tubes were spiked with Vitamin D to determine whether different additives would interfere with the analysis of the sample. Sensitivity and Specificity was also investigated by looking at the Coefficient Variant. By comparing patient results as produced by LCMS and HPLC there was a correlation of 0.957 which suggests that they are comparable. Quality Controls were run to determine precision of the method. The results of both the low and the high were within the manufacturers Reference Ranges provided. 28.4 and 88.4µg/l (low and high respectively) for Vitamin D3 where targets were 29.1µg/l and 95.2µg/1. The high result was within the reference range and therefore deemed acceptable. Vitamin D2 produced results of 14 and 46.4µg/l where targets were 14.7 and 49µg/l respectively. SST was the only collection tube that produced significant interference that would mask Vitamin D results, therefore when requesting Vitamin D a clinician would not be able to produce an SST sample. As UV detection is used, it was necessary to determine whether the state of the blood sample eg. Haemolysis, lipaemia and icterus, would interfere with the UV detection. The CV for both Specificity and Selectivity were below 15% which means that the method is adequate. These results provide evidence that HPLC is a reliable method and can be implemented into the Laboratory as a replacement to the LCMS method currently in use. Samples should be requested in EDTA, Lithium Heparin or plain blood sample tubes as other additives can create excessive interference. It also suggests that Vitamin D screening should be performed more frequently.