|Automated Analysis of Vitamin D on the PAL-RTC Autosampler, using MagSiMUS-D PREP Sample Preparation for LC-MS/MS
|Automating Complex Sample Workflows
|Dr Nina Meszaros
|Dr Leon de Bruin
Dr Stefan Cretnik
|Axel Semrau GmbH
Abstract Information :
The advantage of LC-MS/MS is the simultaneous measurement of multiple analytes (multiplexing) and its high sensitivity. But this also makes it susceptible for impurities and matrix effects, emphasizing the need for a good sample preparation process. Oftentimes, LC-MS/MS sample preparation consists of a laborious manual procedure, including protein precipitation with either organics or aqueous solutions containing acid, base or salts, after which the protein precipitate is removed by centrifugation. The MagSiMUS-D PREP (25-OH-Vitamin D2/D3 for LC-MS) now automates the combined LC-MS/MS measurement of the 2 most important Vitamin D metabolites from clinical serum samples. This was realized on the PAL RTC, equipped with FlipTube® and PAL Tools for opening, closing, transfer and vortexing, and easy control by the CHRONOS software (Axel Semrau).
The PAL-RTC automated MagSiMUS sample preparation was combined with LC-MS/MS to determine the levels of 25-OH-Vitamin D2 and 25-OH-Vitamin D3 in serum samples. Samples were completely free of any particles and protein removal was >98%. Detection limits and calibration curves covered the diagnostic range of the two Vitamin D3 metabolites with CVs <6.5% and accuracies between 90-110% when compared to conventional protein crash protocols. Automated MagSiMUS sample preparation on the PAL-RTC system reduced the total hands-on time while a batch of 64 serum samples in FlipTubes® was processed unattended and ready for direct injection within 75 minutes. Overall accuracy was improved by the automated processing.
MagSiMUS-D PREP (25-OH-Vitamin D2/D3) is a reliable sample preparation method which can be applied in an automated, medium-throughput work-flow on the PAL-RTC autosampler. The absence of a centrifugation step, which can take up to 10 minutes and limits the number of samples that can be treated simultaneously, improves the total sample throughput and reduces the preparation time. Furthermore, the PAL-RTC automated MagSiMUS method is very easy to implement in existing work-flows (direct connection to LC-MS/MS or (U)HPLC).