MSACL 2018 US Abstract

Topic: Endocrinology

Using Ion Mobility to Reduce Chromatography Time for Liquid Chromatography-Ion Mobility-Mass Spectrometry Quantitation of 25-Hydroxyvitamin D in Human Serum

Nicholas Oranzi (Presenter)
University of Florida

Bio: I am currently a Ph.D. candidate at the University of Florida studying the application of ion mobility-mass spectrometry for small molecule quantitation under the direction of Dr. Rick Yost. Current research focuses on the separation of 25-hydroxyvitamin D epimers using ion mobility to improve vitamin D quantitation.

Authorship: Nicholas Oranzi, Jiajun Lei, Nicolas C. Polfer, Timothy J. Garrett, Richard A. Yost
University of Florida

Short Abstract

The evidence of links between vitamin D deficiency and many diseases has led to a dramatic increase in the demand for vitamin D testing. Quantitation of the vitamin D metabolite 25-hydroxyvitamin D (25OHD) by liquid chromatography-mass spectrometry (LC-MS) provides high accuracy but suffers from long analysis times needed to resolve 25OHD from its epimer 3-epi-25-hydroxyvitamin D. Ion mobility is coupled with LC-MS, to rapidly separate the epimer based on differences in their gas-phase conformations, and significantly reduce liquid chromatography time to two minutes. Quantitation was demonstrated for 25-hydroxyvitamin D2 and D3 in human serum without interference from the epimer. Experimental conditions were investigated to ensure ion mobility did not bias quantitation. At high ion density, RF-heating reduces the unique conformer of 25OHD, potentially impacting quantitation.

Long Abstract


Vitamin D deficiency has been linked to many diseases beyond bone development disorders leading to an increase in demand for quantitation 25-hydroxyvitamin D (25OHD). Analysis by liquid chromatography-mass spectrometry (LC-MS) requires long chromatography times to resolve interference from the stereoisomer, 3-epi-25-hydroxyvitamin D (epi25OHD). Assays which fail to resolve this stereoisomer risk overreporting vitamin D levels. 25OHD can be resolved from epi25OHD using ion mobility because 25OHD can adopt a gas-phase conformation inaccessible to epi25OHD [1]. By incorporating ion mobility into an LC-MS method (LC-IM-MS), sample analysis time is reduced from over 10 minutes to 2 minutes. In this study, progress is made towards validating a clinically relevant LC-IM-MS method for quantitation of 25-hydroxyvitamin D in human serum. The dependence of experimental conditions associated with the use of ion mobility for quantitation of individual conformers are also investigated.


All experiments were performed on an Agilent 6560 ion mobility QTOF instrument with electrospray ionization source. Separation of the sodiated adducts of 25OHD and epi25OHD was conducted with a 78 cm drift tube using a 18 V/cm uniform electric field and nitrogen buffer gas. For quantitation, a two-minute liquid chromatography method was employed to separate 25OHD from potential matrix interferents. Matrix, composed of 4% bovine serum albumin in phosphate buffered saline (BSA/PBS), was fortified with 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) with isotopically labeled internal standards 25OHD2-[2H3] and 25OHD3-[2H6]. Fortified samples and commercial quality control human serum samples were extracted by solid phase extraction prior to analysis. To assess the stability of the 25OHD conformer ratios at different experimental conditions, methanol solutions containing 0.01 to 1 ppm of 25OHD3 were infused with a syringe pump. Data was acquired and processed with MassHunter B.8.0.0 and data analysis performed using JMP 13.


Two peaks were detected in the drift time spectrum for the sodiated adducts of 25OHD2 (m/z 435.364) and 25OHD3 (m/z 423.324) at 25 and 29 ms corresponding to a compact and extended conformer, respectively [1]. For epi25OHD2 and epi25OHD3, only one peak was detected at 25 ms. The unique drift peak at 29 ms was integrated for 25OHD2 and 25OHD3 in each sample for quantitation. Calibration curves showed good linearity (r2>0.990) between 2 and 500 ng/mL for 25OHD2 and 25OHD3. Commercial quality control samples quantified within 10% of the target value. Quality control sample composed of BSA/PBS fortified with 25OHD and epi25OHD at 100 ng/mL quantified within 10% of the target showing that the presence of the epimer did not interfere with quantitation.

Since 25OHD forms two conformers, the ratio must remain constant so that only the unique conformer can be used for quantitation. Experimental conditions were varied to determine the effect on the observed conformer ratio. Methanol solutions with five concentrations of 25OHD3 were infused between 2 and 300 ┬ÁL/min. The percentage of the open conformer with respect to total 25OHD3 decreases from 87% at the lowest flow rate and concentration to 20% at the highest concentration and flow rate. Both factors were significant based on linear least squares fit; however, when the total ion counts (all ions at all detected m/z) is included as a factor, concentration and flow rate are no longer significant. Interconversion between conformers at high ion counts is attributed to RF-heating in the ion transfer optics. When ion density is low, RF-heating is minimized and conformer ratios are constant, so that the open conformer is proportional to the concentration in solution and calibration curve is linear. For the quantitation experiment, the threshold for RF-heating was 2.6 million counts per second, while the average total ion counts were 1.8 million counts per second. Including two standard deviations (2σ = 700,000 counts per second), all samples remain below the RF-heating threshold, and conformer ratios are unaffected by RF-heating.

Conclusions & Discussion

A two-minute LC-IM-MS method for the quantitation of 25OHD demonstrates the advantages of utilizing ion mobility to increase throughput of sample analysis; however, experimental conditions must be controlled to ensure interconversion does not introduce bias into quantitation.

References & Acknowledgements:

[1] Chouinard, C.D., Cruzeiro, V.W.D., Beekman, C.R., Roitberg, A.E., Yost, R.A.: Investigating Differences in Gas-Phase Conformations of 25-Hydroxyvitamin D3 Sodiated Epimers using Ion Mobility-Mass Spectrometry and Theoretical Modeling. Journal of the American Society for Mass Spectrometry. 28, 1497-1505 (2017)

Financial Disclosure

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