Absorptive chemistry based extraction for LC-MS/MS analysis of small molecule analytes from biological fluids – an application for 25-hydroxyvitamin D

Abstract

Abstract Background: Already available sample preparation technologies for liquid chromatography-tandem mass spectrometry have substantial shortcomings with respect to automation. A novel approach is based on gel-like polymeric material with defined absorption chemistry, which is immobilized in micro-plate wells. It is referred to as Tecan Immobilized Coating Extraction ™ (TICE ™) technology and it enables easy automation on liquid handling systems. We aimed to study the performance of Tecan AC Extraction Plate ™ based on this principle by addressing 25-hydroxyvitamin D (25OHD) as an exemplary analyte. Methods: A protocol for extraction of 25OHD from serum samples based on TICE ™ technology was implemented on a robotic liquid handling system Freedom EVO ® (Tecan). An isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry method was used for quantification. Performance was tested according to a comprehensive protocol. Results: Linearity was found over a range from 4.3 to 65.8 ng/mL for 25OHD3. The coefficients of variation for the intra-day and inter-day precision were <6% and accuracy ranged between 96.9% and 99.8% for 25OHD3. Recovery was 84% and efficient control of matrix effects was verified. High sample throughput could be observed with 96 samples prepared in <60 min. Close agreement of results was found for clinical samples analyzed with a second tandem mass spectrometry method based on protein precipitation and two-dimensional ultra-performance liquid chromatography for sample preparation (r=0.988, n=73). Conclusions: The new TICE ™ technology was found to be a useful process for sample preparation in clinical mass spectrometry. Full automation suited for routine analysis was achieved.