High-Throughput Serum 25-Hydroxy Vitamin D Testing with Automated Sample Preparation

Serum from bar-coded tubes, and then internal standard, are pipetted to 96-well plates with an 8-channel automated liquid handler (ALH). The first precipitation reagent (methanol:ZnSO4) is added and mixed with the 8-channel ALH. A second protein precipitating agent, 1 % formic acid in acetonitrile, is added and mixed with a 96-channel ALH. After a 4-min delay for larger precipitates to settle to the bottom of the plate, the upper 36 % of the precipitate/supernatant mix is transferred with the 96-channel ALH to a Sigma Hybrid SPE® plate and vacuumed through for removal of phospholipids and precipitated proteins. The filtrate is collected in a second 96-well plate (collection plate) which is foil-sealed, placed in the autosampler (ALS), and injected into a multiplexed LC-MS/MS system running AB Sciex Cliquid® and MPX® software. Two Shimadzu LC stacks, with multiplex timing controlled by MPX® software, inject alternately to one AB Sciex API-5000 MS/MS using positive atmospheric pressure chemical ionization (APCI) and a 1.87 min water/acetonitrile LC gradient with a 2.1 × 20 mm, 2.7 μm, C18 fused core particle column (Sigma Ascentis Express). LC-MS/MS through put is ~44 samples/h/LC-MS/MS system with dual-LC channel multiplexing. Plate maps are transferred electronically from the ALH and reformatted into LC-MS/MS sample table format using the Data Innovations LLC (DI) Instrument Manager middleware application. Before collection plates are lo...
Source: Springer protocols feed by Imaging/Radiology - Category: Radiology Source Type: news