A rubber transfer gasket to improve the throughput of liquid-liquid extraction in 96-well plates: application to vitamin D testing.
BACKGROUND
The 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.


METHODS
We 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.


RESULTS
Cross-contamination between wells was < or = 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, S(x|y)=0.022). The accuracy of the assay was 102-105% as assessed with the recently released control materials from NIST.


CONCLUSIONS
The 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.
Hoofnagle
Andrew N
AN
Department of Laboratory Medicine, University of Washington, Seattle, WA 98195-7110, USA. ahoof@u.washington.edu
Laha
Thomas J
TJ
Donaldson
Thomas F
TF
D005591:Chemical Fractionation / Q000295:instrumentation / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D006801:Humans; D012044:Regression Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000097:blood
D014807:Vitamin D; C104450:25-hydroxyvitamin D
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