Method and apparatus for the extraction of vitamin D metabolites from human plasma

摘要

This invention relates to a method and apparatus or kit for extracting major metabolites of vitamin D from human plasma or serum. More particularly, the invention provides for the extraction from human plasma or serum samples comprising vitamin D metabolites such as 1,25-dihydroxy vitamin D3, 25-hydroxy vitamin D2, and D3 from protein binding, removal of protein and phospholipids, and isolation of the metabolites using a combination of ion-exchange and Lewis acid mechanisms without the requirement to acidify the samples. The method and apparatus of the invention comprise a cartridge or plurality of cartridges comprising at least one protein crash frit, a strong cation exchanged sorbent, and an acidified alumina sorbent to provide higher recoveries of vitamin D metabolites than existing phospholipid depletion plate techniques. Accurately quantifying 1,25-dihydroxy vitamin D3 is useful in differential diagnosis of vitamin D-related diseases and for monitoring vitamin D therapy in patients with chronic renal disease.

主权项

1. An apparatus for isolating metabolites of vitamin D or small molecules from a non-acidized serum or plasma sample comprising proteins, metabolites of vitamin D, small molecules, phospholipids and interfering compounds in an aqueous media, said apparatus comprising:
at least one well having a generally tubular side wall defining an interior well space having an upper inlet and a lower outlet; a first protein crash frit disposed in the interior well space between the upper inlet and the lower outlet defining an introduction zone above the first protein crash frit and between the upper inlet and the first protein crash frit; a first sorbent zone comprising a cation exchanged silica sorbent disposed adjacent to and below the first protein crash frit; a second sorbent zone disposed below said first adsorbent zone comprising an acidized alumina sorbent; a second protein crash frit disposed in the interior well space between the first sorbent zone and the second sorbent zone; and, a lower membrane zone being a hydrophobic protein crash plate disposed adjacent to and below the second sorbent zone, wherein the hydrophobic protein crash plate is a multi-layer protein crash plate constructed of at least three layers selected from the group consisting of polypropylene, polyethylene (PE), polyvinylidene difluoride (PVDF), polytetrafluoroethylene (PTFE), glass, and combinations thereof.