Automated Vitamin D Extraction from Serum Samples

96 samples in < 10 minutes
Automation Compatibility: Hamilton Robotics

Vitamin D is an essential prohormone that regulates calcium and phosphate homeostasis. Vitamin D deficiency is a growing problem, manifesting as calcium malabsorption, secondary hyperthyroidism, muscle weakness, and/or osteoporosis/osteomalacia. With an estimated 50% of the global population being deficient in vitamin D, the extent of clinical testing continues to rise, with an increased emphasis on accuracy. Vitamin D assessment is performed by quantitation of 25-hydroxyvitamin D in serum and/or plasma. 25-hydroxyvitamin D has two forms: 25-hydroxyvitamin D2 (25OHD2) from vitamin D2, a plant based supplement, and 25-hydroxy-vitamin D3 (25OHD3) from vitamin D3, which is produced in the skin as a result of photochemical reaction.

Both 25OHD2 and 25OHD3 can be accurately and efficiently analyzed using LC-MS/MS. However, removal of the sample matrix is required to ensure that interferences do not adversely affect quantitative analysis. Therefore, most applications require protein precipitation followed by solid-phase extraction (SPE) prior to chromatographic analysis. In the current study, a patent-pending automated Tip-on-Tip (ToT) filtration technology is coupled to dispersive pipette extraction technology on an automated liquid handling system (ALH) for rapid and efficient sample preparation.

DPX ToT Filtration technology automates the traditional mixing and centrifugation steps used to protein precipitate serum samples. Efficiency is enhanced as ToT Filtration may be performed in a 96-sample format. Following filtration, XTR tips with weak anion exchange sorbent and salt necessary for SALLE (salt-assisted liquid liquid extraction) are used to rapidly remove matrix interferences. This method processes up to 96 samples in less than ten minutes.

sample preparation for blood/ serum samples prior to LC-MS/MS analysis
1Load Platform
2Protein Crash
– Add ACN and mix
4Aspirate and Dispense Filtrate 3x

XTR Tips with WAX-S sorbent

5Transfer Upper Layer