The utility of adding ion mobility (IM) to liquid chromatography/mass spectrometry (LC/MS) for quantitation of 25hydroxyvitamin D 3 (25OHD3) is evaluated. Sodiated 25OHD3 ions adopt both closed and open conformations, whereas the stereoisomer 3-epi-25-hydroxyvitamin D (epi25OHD), when sodiated, adopts only a closed gas-phase conformation. The unique open conformation for sodiated 25OHD3 permits unambiguous quantitation. Nonetheless, the ratio of open versus closed gas-phase conformations for sodiated 25OHD3 can vary with instrumental conditions; conversion from the open to the closed conformer is attributed to radio frequency (rf) heating within the ion accumulation trap. Ion heating becomes significant when space charge in the trap forces ions to larger radii where the rf field is higher. To avoid biasing quantitation, an isotopically labeled internal standard must be used to account for changes in conformer ratio. Thirty-three serum extract samples were evaluated by LC/IM-MS and were found to not be biased by changes in ion conformer ratios, permitting reliable quantitation of 25OHD3 without interference from the epimer.

Ion mobility was integrated with liquid chromatography/high resolution mass spectrometry (LC/IM-HRMS) to quantify 25-hydroxyvitamin D (25OHD) in human serum. It has previously been shown that 25OHD adopts two gas-phase conformations which are resolved using ion mobility; in contrast, the inactive epimer, 3-epi-25-hydroxyvitamin D (epi25OHD), only adopts one. Interference from epi25OHD was eliminated by filtering the chromatogram to retain the drift time that corresponds to the unique gas-phase conformation of 25OHD. Although ion mobility separates the epimers, some chromatography is required to separate compounds which interfere with ionization or fall at the same nominal m/z. Standards were prepared in 4% albumin solutions and compared against commercial serum quality controls. Standards and quality controls were analyzed and validated using a 2 min LC/IM-MS method. 25-Hydroxyvitamin D3 and D2 were quantified over the range between 2 and 500 ng/mL with bias and precision within 15%. When epi25OHD was spiked into quality control samples, no significant bias was introduced, and analysis of 30 patient samples shows good agreement between this LC/IM-MS and traditional LC/MS/MS methods. This work shows that ion mobility can be incorporated with liquid chromatography and mass spectrometry for rapid quantitation of 25OHD in human serum.

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<p>Ion mobility was integrated with liquid chromatography/high resolution mass spectrometry (LC/IM-HRMS) to quantify 25-hydroxyvitamin D (25OHD) in human serum. It has previously been shown that 25OHD adopts two gas-phase conformations which are resolved using ion mobility; in contrast, the inactive epimer, 3-epi-25-hydroxyvitamin D (epi25OHD) only adopts one. Interference from epi25OHD was eliminated by filtering the chromatogram to retain the drift time that corresponds to the unique gas-phase conformation of 25OHD. Although ion mobility separates the epimers, some chromatography is required to separate compounds which interfere with ionization or fall at the same nominal <i>m/z</i>. Standards were prepared in 4% albumin solutions and compared against commercial serum quality controls. Standards and quality controls were analyzed and validated using a two-minute LC/IM-MS method. 25-hydroxyvitamin D3 and D2 were quantified over the range between 2 and 500 ng/mL with bias and precision within 15%. When epi25OHD was spiked into quality control samples, no significant bias was introduced, and analysis of 30 patient samples shows good agreement between this LC/IM-MS and traditional LC/MS/MS methods. This work shows that ion mobility can be incorporated with liquid chromatography and mass spectrometry for rapid quantitation of 25OHD in human serum.</p>

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Building an accurate lipid inventory relies on coordinated information from orthogonal analytical capabilities. Integrating the familiar workflow of liquid chromatography (LC), high-resolution mass spectrometry (HRMS), and tandem mass spectrometry (MS/MS) with proton nuclear magnetic resonance spectroscopy (1H NMR) would be ideal for building that inventory. For absolute lipid structural elucidation, LC–HRMS/MS can provide lower-level structural information with superior sensitivity, while 1H NMR can provide invaluable higher-order structural information for the disambiguation of isomers with absolute chemical specificity. Digitization of the LC eluent followed by splitting the microfractions into two flow paths in a defined ratio for HRMS/MS and NMR would be the ideal strategy to permit correlation of the MS and NMR data as a function of chromatographic retention time. Here, we report an active segmentation platform to transform analytical flow rate LC eluent into parallel microliter segmented flow queues for high confidence correlation of the MS, MS/MS, and NMR data. The practical details in implementing this strategy to achieve an integrated LC–MS–NMR platform are presented, including the development of an active segmentation technology using a four-port two-way valve to transform the LC eluent into parallel segmented flows for online MS analysis followed by offline segment-specific 1H NMR and optimization of the detector response toward segmented flow. To demonstrate the practicality of this novel platform, it was tested using lipid mixture samples.