Improving The Vitamin D Assay Using Ion Mobility Coupled With Liquid .

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IMPROVING THE VITAMIN D ASSAY USING ION MOBILITY COUPLED WITH LIQUIDCHROMATOGRAPHY/MASS SPECTROMETRYByNICHOLAS ROBERT ORANZIA DISSERTATION PRESENTED TO THE GRADUATE SCHOOLOF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYUNIVERSITY OF FLORIDA2019

2019 Nicholas Robert Oranzi

To Team NNN

ACKNOWLEDGMENTSFirst, with much specificity, I would like to give massive thanks to my family:Mom, Dad, Khun Yai, and especially my wife Nui, charged with running our family,raising an awesome little boy, giving me patience and space when I needed it, keepingan eye on me, refocusing me if I drifted too far, and giving me the resolve and power tofollow through. I want to thank my son, Neil, for keeping me smiling. I hope he canreflect on this experience when his turn comes. This work is theirs as much as it ismine.I would like to thank those that helped with my projects either in the lab, ascollaborators, providing samples, or providing advice and guidance. These includeTimothy Garrett, Nicolas Polfer, Nicole Horenstein, Kenneth Wagener, Ben Smith,Jiajun Lei, Allison Levy, Robin Kemperman, Michael Wei, Alan Rockwood, Aurelio LaRotta, Julia Kaszycki, Scott Granato, Ben Rochon, Kevin Dit Fouque, FranciscoFernandez-Lima, George Stafford, Vinicius Cruzeiro, Adrian Roitberg, Russell Grant,Brett Holmquist, and many others.I would like to thank Dr. Yost along with the rest of my graduate committee fortheir guidance throughout the years ensuring I reach my peak capacity and VioletaPetkovska for realizing my potential, forcing me to return to school. Finally, I would liketo thank my funding sources, including Agilent, Wellspring Clinical Lab, Partnership forClean Competition, and National Institute of Health grant #U24DK097209 for helping myresearch meet its resolution.4

TABLE OF CONTENTSpageACKNOWLEDGMENTS . 4LIST OF TABLES . 8LIST OF FIGURES . 9LIST OF ABBREVIATIONS . 12ABSTRACT . 17CHAPTER1INTRODUCTION . 19Ion Mobility-Mass Spectrometry Analysis . 19Analysis of 25-Hydroxyvitamin D . 21Vitamin D Metabolism . 21Current Vitamin D Assays . 23Ion Mobility Improves Analysis of 25-Hydroxyvitamin D . 24Instrumental Details . 26Ion Mobility Quadrupole Time-of-Flight Mass Spectrometer. 26Ionization . 27Electrodynamic ion guides. 30Time-of-Flight Mass Analysis . 31Internal Energy and Thermometer Ions . 32Implementations of Ion Mobility . 34Drift Tube Ion Mobility . 34Travelling Wave Ion Mobility. 35Trapped Ion Mobility . 36High-Field Asymmetric Ion Mobility . 37Dissertation Overview . 382INFLUENCE OF EXPERIMENTAL CONDITIONS ON THE RATIO OF 25HYDROXYVITAMIN D3 CONFORMERS FOR VALIDATING A LIQUIDCHROMATOGRAPHY-ION MOBILITY-MASS SPECTROMETRY METHODFOR ROUTINE QUANTITATION . 48Integrity of sodiated 25-hydroxyvitamin D3 conformers . 48Experimental . 51Chemicals. 51Instrumentation . 51Data Analysis . 52Infusion Experiments . 52Component optimization design of experiment. 535

Ion accumulation trap funnel simulations . 53Quantitation of 25OHD3 . 54Results and Discussion. 55Conclusion . 623MEASURING THE INTEGRITY OF GAS-PHASE CONFORMERS OF 25HYDROXYVITAMIN D3 BY DRIFT TUBE, TRAVELLING WAVE, TRAPPED,AND HIGH-FIELD ASYMMETRIC ION MOBILITY . 77Expanding the instrument space for LC/IM-MS quantitation . 77Materials and Methods. 79Chemicals. 79Instrumentation . 80Calculating resolving power and resolution . 84Results and Discussion:. 85Internal energy-driven interconversion . 8525-hydroxyvitamin D3 across IMS platforms . 88Atmospheric-Pressure Drift Tube IMS . 90Travelling Wave IMS . 91Trapped IMS. 93High-Field Asymmetric IMS . 96Conclusions . 974RAPID QUANTITATION OF 25-HYDROXYVITAMIN D2 AND D3 IN HUMANSERUM USING LIQUID CHROMATOGRAPHY-DRIFT TUBE ION MOBILITYMASS SPECTROMETRY . 109Harmonizing Ion Mobility and Traditional LC/MS . 109Liquid Chromatography Ion Mobility Mass Spectrometry . 109Sample Preparation Considerations . 110Clinical Validation Protocols . 111Methods . 112Chemicals. 112Sample Preparation . 112Instrumentation . 113Data Processing . 114Method Validation . 115Method Comparison . 116Results . 116Discussion . 118Quantitation Software Comparison . 119Relative Informing Power of LC/IM-MS . 122Method Validation Figures of Merit . 124Impact of IMS on internal standard response . 126Conclusion . 1275CONCLUSION AND FUTURE DIRECTIONS . 1386

Rapid Quantitation of Epi25OHD3 using LC/IM-HRMS . 138Sample Analysis . 138Data Processing . 139Results . 140Datafile Preprocessing . 141Calculating Contributions from 25OHD3. 142Quantifying Against 25OHD3 Calibration Curve . 143Designing a Better Method for Epimer Quantitation . 144Formation of Sodiated 25-hydroxyvitamin D Conformers . 144Conclusion . 146LIST OF REFERENCES . 152BIOGRAPHICAL SKETCH . 1667

LIST OF TABLESTablepage2-1Effect-screening design of experiment (DOE) summary. . 643-1Nitrogen collision cross section (CCS) calibration ions for travelling wave ionmobility . 993-2Collision cross sections (CCS) for low-field ion mobility methods compared tostepped-field drift tube ion mobility measurements . 994-1Results for bias, precision and method efficiency for 25OHD2 and 25OHD3 . 1284-2Comparison of results from the four-day bias and precision study usingMassHunter and Skyline . 1285-1Calculated concentration of epi25OHD3 . 1478

LIST OF FIGURESFigurepage1-1Schematic of vitamin D metabolism. 391-2Structures of 25-hydroxyvitamin D2 and D3 with their corresponding 3epimer . 401-3Extracted drift spectrum of 25-hydroxyvitamin D3 and 3-epi-25hydroxyvitamin D3 with calculated structures . 411-4Schematic of Agilent 6560 ion mobility quadrupole time of flight massspectrometer. 421-5Cartoon of stacked ring electrode ion trap . 421-6Simulations of ion kinetic energy with respect to position within a stacked-ringion accumulation trap . 431-7Generalized structures of para-substituted benzylpyridinium parent andfragment thermometer ions. 431-8Cartoon depicting drift tube ion mobility . 441-9Cartoon depicting travelling wave ion mobility . 451-10 Cartoon depicting trapped ion mobility . 461-11 Cartoon depicting high-field asymmetric ion mobility . 472-1Simplified scheme of the LC/IM-MS workflow. 652-2Trap schematic, voltage gradient diagram for ion trap simulations, timingdiagram for IM transient . 652-3Percent of sodiated d6-25OHD3 in the open conformer as concentration andsample infusion flow rate increase. 662-4Percent of sodiated d6-25OHD3 in the open conformation for experiments atvarious concentration and flow rate combinations . 672-5Reaction scheme for the open-to-closed transition. 682-6Open and closed peak areas of sodiated d6-25OHD3 normalized to thesodiated epimer peak area . 692-7Using sodium acetate to control TIC . 709

2-8Percent of sodiated d6-25OHD3 in the open conformation in response tochanges in the ion optics voltages . 712-9Representative equilibrium position of m/z 423 ion during simulation of iontrapping region at low and high exit grid 1 voltage. 712-10 Percent of sodiated d6-25OHD3 in the open conformation in response to traprelease time . 722-11 Percent of sodiated d6-25OHD3 in the open conformation in response to IMtransient time . 732-12 Conformation dependence of co-infused sodiated 25OHD3 and d6-25OHD3 . 742-13 Total ion chromatogram, extracted ion chromatogram (m/z 423.324), and ionmobility spectrum. 752-14 Calibration curve for quantitation of 25OHD3 including quality controlsamples . 763-2Survival yield of para-substituted benzylpyridinium ions and percent openconformer of sodiated d6-25OHD3 in response to trapping voltages withinthe ion accumulation region of the LP-DTIMS instrument . 1003-3The internal energy distribution at selected trapping grid voltages based onbenzylpyridinium survival yield . 1013-4Internal energy distributions calculated from benzylpyridinium survival yieldsand percentage of open conformers using nanoESI and conventional ESIsources at low and high heating conditions in the trapping funne. 1023-5Collision cross section and dispersion field spectra of sodiated d6-25OHD3(429.3600) and epi25OHD3 (m/z 423.3239) for five commercial IMSimplementations . 1033-6The percent open conformer of sodiated d6-25OHD3 with respect to thedesolvation gas temperature in the AP-DTIMS instrument . 1043-7Percent open conformer of sodiated d6-25OHD3 analyzed by TWIMS . 1053-8Trapped ion mobility spectrum (top) and atmospheric pressure drift-tube ionmobility spectrum (bottom) for sodiated 3-epi-25-hydroxyvitamin D3 (m/z423.3239) with Gaussian peaks fitted . 1063-9Ion heating of sodiated d6-25OHD using TIMS . 1073-10 Survival yields of para-substituted benzylpyridinium ions analyzed by FAIMS . 1084-1Total ion chromatogram for high level serum quality control sample . 12910

4-2Eight-point calibration curves (n 5/level) for 25OHD2 and 25OHD3 . 1304-3Extracted ion chromatogram (m/z 423.3239) of 75 ng/mL quality control withepi25OHD3 and without . 1314-4Extracted ion drift time spectrum (m/z 423.3239 and RT 0.75-0.85 min) of thechromatographic peak containing 25OHD3 and epi25OHD3 . 1324-5Extracted drift time and ion chromatogram for 75 ng/mL quality control withand without 50 ng/mL epi25OHD3. 1334-6Correlation plot of patient samples between MassHunter and Skyline withorthogonal, equal variance regression . 1344-7Difference plots for the patient sample validation run compared to theLC/MS/MS reference laboratory results using both software packages . 1354-8Difference plot for 30 patient samples analyzed by LC/IM-MS and LC/MS/MS 1364-9Box and whisker plot of the internal standard peak area response, total ioncounts (TIC) and percent of d3-25OHD2 in the open conformation. 1375-1Correlation of reference and calculated concentrations of epi25OHD3 for 22patient samples . 1485-2Flowchart describing the data transformations to calculate the concentrationof epi25OHD3 . 1495-3Two-dimensional heatmap of m/z and drift time of patient sample . 1505-4Ion mobility spectrum from a patient sample for m/z 423.3239 showing thedrift-time filter window . 15111

LIST OF ABBREVIATIONSµReduced mass1,25(OH)2D1,25-dihydroxyvitamin D24,25(OH)2D25,25-dihydroxyvitamin D25OHD25-hydroxyvitamin D25OHD225-hydroxyvitamin D225OHD325-hydroxyvitamin D3AclosedAbundance of closed conformerANOVAAnalysis of varianceAopenAbundance of open conformerAPCIAtmospheric pressure chemical ionizationAP-DTIMSAtmospheric pressure drift tube ion mobilityspectrometryBPBenzylpyridiniumBSABovine serum albumincInstrument electronic delay coefficientCCSCollision cross section, ŲCCS'Mass normalized collision cross sectionCFCompensation field, TdCRMCharged residue modeld3-25OHD2[2H3]-25-hydroxyvitamin D2d6-25OHD3[2H6]-25-hydroxyvitamin D3DaDaltonDCDirect currentDFDispersion field, Td12

DMADynamic mobility analyzerDMSDifferential mobility spectrometryDOEDesign of experimentDTIMSDrift tube ion mobility spectrometrydV/dtFlow rate, µL/minEElectric field strength, V/cmE0Critical energyEICExtracted ion chromatogramepi25OHD3-epi-25-hydroxyvitamin Depi25OHD23-epi-25-hydroxyvitamin D2epi25OHD33-epi-25-hydroxyvitamin D3ErfRF potentialESIElectrospray ionizationFAIMSHigh-field asymmetric ion mobility spectrometryFDAUnited States Food and Drug AdministrationIEInternal energyIEMIon ejection modelIM-MSIon mobility-mass spectrometryIMSIon mobility spectrometryKMobility,K0Reduced mobilitykBBoltzmann constantKEKinetic energyLLengthLCLiquid chromatography13

LC/IM-MSLiquid chromatography/ion mobility-mass spectrometryLC/MSLiquid chromatography/mass spectrometryLLELiquid-liquid extractionLODLimit of detectionLOQLimit of quantitationLP-DTIMSLow-pressure drift tube ion mobility spectrometrymMass, ionMMass, buffer gasm/zMass-to-charge ratioMHzMegahertzMSMass SpectrometryNGas number densitynanoESINano electrospray ionizationng/mLNanograms/milliliterNIST SRM 972aNational Institute of Standards and TechnologyStandard Reference Material 972a, Vitamin Dmetabolites in human serumPBSPhosphate buffered salinePPEProtein precipitation extractionppmParts per millionPTHParathyroid hormoneqChargeQCQuality let radius14

RFRadio frequencyRpResolving powerRsPeak-to-peak resolutionSPESolid phase extractionSYSurvival yieldTTemperature, Kt'Normalized drift timet0Non-drift-tube flight timetdDrift time, sTdTownsend, 1 Td 10-21 V*m²texpExperimental drift timeTICTotal ion countsTIMSTrapped ion mobility spectrometryTOFTime-of-flight (mass analyzer)tTOFTime of flight (ion flight time)TWIMSTraveling wave ion mobility spectrometryUaApplied electrospray voltageUHPLCUltra-high pressure (performance) liquidchromatographyUTThreshold electrospray voltageUVUltravioletvVelocityV*Effective potentialWclosedBase peak width of closed conformerWopenBase peak width of open conformer15

zIon chargeγSolvent surface tensionΔCCSCollision cross section differenceϑTaylor cone angleρSolvent densityωAngular frequencyΩCollision cross section, Ų16

Abstract of Dissertation Presented to the Graduate Schoolof the University of Florida in Partial Fulfillment of theRequirements for the Degree of Doctor of PhilosophyIMPROVING THE VITAMIN D ASSAY USING ION MOBILITY COUPLED WITH LIQUIDCHROMATOGRAPHY/MASS SPECTROMETRYByNicholas Robert OranziAugust 2019Chair: Richard A. YostMajor: ChemistryThe quantitation of 25-hydroxyvitamin D by liquid chromatography/massspectrometry is a high-value clinical assay that has superior accuracy and precision tothe more common immunoassay, but suffers from low throughput due to the need toseparate the interference from its stereoisomer, 3-epi-25-hydroxyvitamin D. Whenanalyzed by drift tube ion mobility, sodiated 25-hydroxyvitamin D is shown to adopt bothan open and a closed gas-phase conformer, of which the closed conformer is sharedwith 3-epi-25-hydroxyvitamin D. This open conformer enables the separation of theisomer pair by ion mobility on the millisecond timescale, potentially reducing timerequired for chromatographic separation when coupled with liquid chromatography andmass spectrometry. Ion mobility has been rarely used in quantitation, so its impact onmethod validation has not been fully elucidated. This work focuses on understandingthe impact of coupling ion mobility to liquid chromatography/mass spectrometry byinvestigating the stability of the open conformer on various ion mobility platforms for theeventual validation of a liquid chromatography-ion mobility-mass spectrometry methodfor the quantification of 25-hydroxyvitamin D in human serum.17

The abundance of the unique open conformer is found to be sensitive toinstrumental conditions rather than sample composition. Ion heating within the massspectrometer can drive interconversion from the open to closed conformer; this canbecome an issue at high ion density in the trapping funnel in front of the ion mobility drifttube. To correct for bias associated with interconversion, an isotopically labeled internalstandard is required. Four different low-field ion mobility platforms are shown tosuccessfully detect the two conformers of 25-hydroxyvitamin D, suggesting thatquantitation is platform independent, expanding the potential instrument space for thisapplication. Note that high-field ion mobility is unable to resolve the two conformers.Finally, a liquid chromatography/ion mobility-mass spectrometry method for thequantitation of 25-hydroxyvitamin D in human serum is developed and validated toensure accuracy and precision within the recommendations of the Food and DrugAdministration, and the suitability of the method is validated with real patient samples.The two-minute per sample chromatography time represents a significant reduction insample analysis time versus existing liquid chromatography/mass spectrometrymethods.18

CHAPTER 1INTRODUCTIONIon Mobility-Mass Spectrometry AnalysisIon mobility spectrometry (IMS) describes a broad category of instruments thatseparate ions based on differences in gas-phase mobility [1], the velocity of an ionthrough a gas in an electric field. IMS has been applied to the detection of explosivesresidues and chemical warfare agents [2], illicit drug residues [3] and to environmentalanalysis [4]. Coupling IMS with mass spectrometry (IM-MS) has enabled researchers toinvestigate the properties of synthetic polymers [5], proteins [6], polynucleotides [7],carbohydrates [8], peptides [9], lipids [10], and metabolites [11]. Many custom andcommercially available IMS platforms are available utilizing different ion mobilityprinciples. The four most common types of IMS are travelling-wave ion mobility(TWIMS) [12], high-field asymmetric ion mobility (FAIMS) [13], trapped ion mobility(TIMS) [14], and drift-tube ion mobility spectrometry (DTIMS) [15]. While each methodoperates under different principles, they each allow separation of ions based on mass,charge and ion shape.Drift tube ion mobility is arguably the simplest and earliest of the four methods.Ions travel under the influence of a uniform electric field through an inert buffer gas.The electric field exerts an accelerating force while frequent, low-energy collisions withthe buffer gas exert a decelerating force, resulting in a terminal ion velocity. The ratio ofthe ion velocity (v) to electric field (E) is described in eq 1 as the mobility (K) of the ion[16]:K vE(1-1)19

For an ion travelling through a fixed drift-tube length and constant electric field,the mobility is inversely proportional to the drift time (td), the time for an ion to traversethe drift tube. In DTIMS experiments, ions are accumulated in an electrostatic trap andpulsed into the drift tube as a uniform ion packet. Ions of lower mobility separate fromthose of higher mobility, since they encounter more collisions with the buffer gas, andare detected at a later drift time [17]. An ion’s mobility is dependent on its mass,charge, and rotationally-averaged collision cross section (CCS). Coupling IMS with atime-of-flight (TOF) mass analyzer enables the rapid measurement of the mass-tocharge ratio (m/z) so that CCS can be calculated for each ion based on the drift timeand instrumental parameters according to the Mason-Schamp equation [1], asdiscussed later in this chapter. Of the four major types of ion mobility, only DTIMS candirectly measure CCS which has driven its popularity among structural biologists andchemists interested in an analyte’s gas-phase structure.The duty cycle of a DTIMS experiment can be completed in under 100milliseconds in most conditions. The short timescale relative to liquid chromatography(LC), which has run times of minutes to hours, means that ion mobility is well suited forcoupling with LC; for example, a ten-second chromatographic peak could contain overone hundred ion mobility scans. While liquid chromatography/mass spectrometry(LC/MS) has long been used for clinical quantitation [18], integration of ion mobility(LC/IM-MS) for routine clinical quantitation has been limited, with few publishedexamples. This is due in large part to the high cost and size of commercial ion mobilityinstruments, and also due to a lack of a “killer application” where IMS offers clearadvantages to justify the investment required. One potential application is the20

quantitation of 25-hydroxyvtiamin D (25OHD), the clinically relevant metabolite ofvitamin D to monitor vitamin D deficiency.Analysis of 25-Hydroxyvitamin DVitamin D is most recognized for its role in regulating bone growth by promotingthe expression of calcium absorbing proteins and regulating parathyroid hormone (PTH)levels [19]. Insufficient levels of vitamin D have been associated with bonedevelopment disorders such as osteomalacia and Ricketts [20], and increasingly,evidence shows that vitamin D deficiency can potentially contribute to cardiovasculardisease, diabetes, and cancer [21]. In pregnant women, low levels of vitamin D havebeen linked to adverse neonatal outcomes such as low birth weight, preterm birth andincreased risk of respiratory tract infections [22–24]. This evidence has led, in part, toan explosion in the demand for vitamin D testing since the early 2000s [25]. To meetthis demand, clinical laboratories have been seeking out faster and more accurateassays.Vitamin D MetabolismVitamin D enters the metabolism through dietary sources, supplementation, orbiosynthesis upon exposure to UV radiation [26]. Approximately 60% of circulatingvitamin D is produced endogenously through biosynthesis, with the remaining sourcedfrom the diet [27]. Regional and seasonal differences in vitamin D levels in the UnitedStates have been attributed

Dissertation Overview . 38 2 INFLUENCE OF EXPERIMENTAL CONDITIONS ON THE RATIO OF 25-HYDROXYVITAMIN D3 CONFORMERS FOR VALIDATING A LIQUID . LC/IM-MS Liquid chromatography/ion mobility-mass spectrometry LC/MS Liquid chromatography/mass spectrometry LLE Liquid-liquid extraction

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