Abstract
The current analytical high-performance liquid chromatography (HPLC) methods by which the various forms of cobalamin can be separated and quantified are limited to tedious chromatographic gradients with run times of 20―30 min and limits of detection (LOD) of 2 nM (2.7 ng/mL). This LOD is far above the physiological range of 148―443 pM (200―600 pg/mL) that is the normal total cobalamin level in human plasma. In this manuscript, benefits of ultra-performance liquid chromatography (UPLC) in which the stationary phase particle size may be reduced from 3.5 μm with a mobile-phase backpressure of 6000 psi in traditional analytical HPLC to a stationary phase particle size of 1.7 μm and a mobile phase backpressure of 15,000 psi in UPLC are reported. UPLC can more than double the chromatographic resolution and reduce each chromatographic run time by 10-fold, such that a complete analysis takes only 3 min per sample.
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