Quantitative liquid chromatographic and tandem mass spectrometric determination of vitamin D 3 in human serum with derivatization: A comparison of in-tube LLE, 96-well plate LLE and in-tip SPME

Abstract

Sensitive and selective methods based on high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection were developed for the determination of vitamin D 3 in human serum. Derivatization of vitamin D 3 and its stable isotope labeled internal standard provided highly sensitive quantification and selective detection from endogenous compounds. Samples were prepared using the in-tube liquid–liquid extraction (LLE), 96-well plate LLE, and in-tip solid phase micro-extraction (SPME) in 96-well format. In all methods, the MS/MS detection was performed using Applied Biosystems-Sciex API 3000 tandem mass spectrometers interfaced with a heated nebulizer probe and operated in the positive ionization mode. Both tube and plate LLE methods achieved a lower limit of quantitation (LLOQ) of 0.5 ng/mL when 1.0 and 0.4 mL of human serum was processed, respectively, and were validated in the concentration range of 0.5–25 ng/mL; while for the in-tip SPME method, LLOQ was 5 ng/mL with only 0.1 mL of human serum required. Comparisons were made among three different methods, including precision and accuracy, sample throughput, recovery and matrix effects.