Development of a mass spectrometry method for 1,25-dihydroxy vitamin D3 using immunoextraction sample preparation
| Autor: | Fiona Ivison, Neil Howarth, Lesley Tetlow, Mandy Pickersgill, Edward Hinchliffe |
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| Rok vydání: | 2019 |
| Předmět: |
Vitamin
Clinical Biochemistry 030209 endocrinology & metabolism Chemical Fractionation Mass spectrometry 01 natural sciences Mass Spectrometry 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Limit of Detection Liquid chromatography–mass spectrometry Vitamin D and neurology Blood calcium Humans Sample preparation Vitamin D Active metabolite Detection limit Chromatography 010401 analytical chemistry Analytic Sample Preparation Methods General Medicine 0104 chemical sciences chemistry Calibration |
| Zdroj: | Annals of Clinical Biochemistry: International Journal of Laboratory Medicine. 56:646-653 |
| ISSN: | 1758-1001 0004-5632 |
| Popis: | Background 1,25-Dihydroxy vitamin D3 (DHVD) is the active metabolite of vitamin D, required to maintain blood calcium concentrations. Measurement has proved challenging as it circulates in picomolar concentrations and must be differentiated from other dihydroxyvitamin D species. Clinically, it is essential to be able to determine the cause of hypercalcaemia, which may be due to DHVD excess. Methods The liquid chromatography-mass spectrometry (LCMS) assay which has been developed uses immunoextraction of 0.5 mL serum followed by Amplifex™ derivatization of the dried eluent, with the analysis using the SCIEX 6500+ instrument taking a run time of 11 min. Results The limit of quantitation was determined (15 pmol/L) and the method is linear up to at least 600 pmol/L. Repeatability ranged from 6.1% at 23 pmol/L to 2.5% at 172 pmol/L and intermediate imprecision was 15.6% at 26 pmol/L to 8.3% at 173 pmol/L. The method is unaffected by icterus, haemolysis or lipaemia. Good performance was achieved with the samples from the vitamin D external quality assessment scheme, demonstrating a negative bias compared with the all lab trimmed mean (average –13.8%) and the specific method group (average –7.75%). A negative bias was observed across the concentration range found in 78 patient samples in comparison to a commercial radioimmunoassay (mean –47.8%). This was not unexpected and is likely due to better specificity of the mass spectrometry assay and the lack of a commutable standard reference calibrator. Conclusions We have developed a sensitive and robust LCMS method for the analysis of DHVD in serum, utilizing immunoextraction and derivatization to provide specificity. |
| Databáze: | OpenAIRE |
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