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Article: Mise au point et validation d’une méthode de dosage de la 25-hydroxy-vitamine D3 par chromatographie liquide haute performance
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ABSTRACT: Vitamin D is a liposoluble molecule playing an essential role in the regulation of bone metabolism. Vitamin D status is usually assessed by measuring the plasma 25-hydroxy-vitamin D3 concentration. 25-hydroxy-vitamin D3 is stable, has a long half-life and is the main circulating form of vitamin D. Our aims were to determine and validate a simple, sensitive and rapid method for plasma 25-hydroxy-vitamin D3 monitoring by HPLC which could be applicable in routine. In 25-hydroxy-vitamin D3 plasma monitoring, the first step was a deproteinization. The elution was made on extraction columns. The eluent was evaporated, suspended in the mobile phase and injected into the HPLC system. Results were quantified by the calibration curve and calculated by integrating the peak areas. We selected the best chromatographic conditions and proceeded to our technique validation. The calibration curve was constructed for aqueous standards with concentrations varying from 10 to 150 ng.ml−1. The correlation coefficient was 0.996. The limit of quantification was 0.34 ng.ml-1 and the limit of detection was 0.11 ng.ml-1. The coefficient of variation (CV) was equal to 1.84% showing that the method provides a good repeatability. The reproducibility expressed by the CV did not exceed 1.08% in all concentrations. In conclusion, we presented a rapid, reliable and low cost method for plasma 25-hydroxy-vitamin D3 monitoring.Revue Francophone des Laboratoires 11/2011; 41(436):89-92.
