A rubber transfer gasket to improve the throughput of liquid–liquid extraction in 96-well plates: Application to vitamin D testing

BackgroundThe unmitigated rise in demand for the assessment of vitamin D status has taxed the ability of clinical mass spectrometry laboratories to preserve turn-around times. We aimed to improve the throughput of liquid–liquid extraction of plasma/serum for the assay of 25-hydroxy vitamin D.MethodsWe designed and fabricated a flexible rubber gasket that seals two 96-well plates together to quantitatively transfer the contents of one plate to another. Using the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant, we developed a novel liquid–liquid extraction workflow in a 96-well plate format. We applied the technology to the mass spectrometric quantification of 25-hydroxy vitamin D.ResultsCross-contamination between wells was ≤0.13%. The interassay imprecision over 132 days of clinical implementation was less than 10%. The method compared favorably to a standard liquid–liquid extraction in glass tubes (Deming slope = 1.018, Sx|y = 0.022). The accuracy of the assay was 102–105% as assessed with the recently released control materials from NIST.ConclusionsThe development of a plate-sealing gasket permits the liquid–liquid extraction of clinical specimens in a moderate-throughput workflow and the reliable assay of vitamin D status. In the future, the gasket may also prove useful in other sample preparation techniques for HPLC or mass spectrometry.