title: On-line SPE sample treatment as a tool for method automatization and detection limits reduction: Quantification of 25-hydroxyvitamin D3/D2.
abstract: The development and approbation of new, automated UHPLC-DAD method for the quantification of 25-hydroxyvitamin D3/D2(25OH-D3/D2) metabolites in plasma/serum for the evaluation of patient's vitamin D status are presented. The method was developed on the Ultimate 3000 UHPLC dual gradient system supplied with the on-line SPE-concentration column coupled through six port switching valve to analytical column. This configuration and materials selected enable large volume sample injection (500μL) and on-line sample preconcentration, clean up and subsequent selective metabolites transfer onto the analytical column. The new method abrogates main conventional time consuming and error source off-line steps of analysis and thus simplifies analysis. The large volume injection increases the sensitivity of instrumental analysis by about ten-fold on-line pre-concentration of metabolites. The instrument response is linear (R>0.99) in the investigated concentration range 10-100ngmL-1which covers all the possible vitamin D status from serious deficiency (<12ngmL-1) to excess. The method detection limits (S/N=3) are LOD (25OH-D3)=0.94ngmL-1and LOD (25OH-D2)=2.4ngmL-1. The method performance was assessed with the use of certified reference samples and perfect agreement between certified and measured values is demonstrated. The method was applied to human samples previously analyzed for total vitamin D by Competitive Protein-binding assay and findings of the two methods are compared.
authors.affiliation: University of Thessaly, Faculty of Medicine, Laboratory of Biochemistry, Larisa, Greece.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Palaiogiannis
authors.affiliation: University of Thessaly, Faculty of Medicine, Laboratory of Biochemistry, Larisa, Greece; Aristotle University of Thessaloniki, Department of Chemistry, Thessaloniki, Greece.
authors.forename: Evangelia
authors.initials: E
authors.lastname: Bekou
authors.affiliation: Department of Endocrinology, Theagenio Cancer Hospital, Thessaloniki, Greece.
authors.forename: Kalliopi
authors.initials: K
authors.lastname: Pazaitou-Panayiotou
authors.affiliation: Aristotle University of Thessaloniki, Department of Chemistry, Thessaloniki, Greece.
authors.forename: Victoria
authors.initials: V
authors.lastname: Samanidou
authors.affiliation: University of Thessaly, Faculty of Medicine, Laboratory of Biochemistry, Larisa, Greece. Electronic address: atsakal@med.uth.gr.
authors.forename: Andreas
authors.initials: A
authors.lastname: Tsakalof
mesh_terms: D015652:25-Hydroxyvitamin D 2 / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D002112:Calcifediol / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods*
chemical_list: D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol
keywords: 25-Hydroxy vitamin D(3)/D(2); Large volume injection; On-line solid phase extraction; Vitamin D