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• W2900177426 abstract "Cerebrosides, including glucosylceramides (GlcCers) and galactosylceramides (GalCers), are important membrane components of animal cells with deficiencies resulting in devastating lysosomal storage disorders. Their quantification is essential for disease diagnosis and a better understanding of disease mechanisms. The simultaneous quantification of GlcCer and GalCer isomers is, however, particularly challenging due to their virtually identical structures. To address this challenge, we developed a new LC/MS-based method using differential ion mobility spectrometry (DMS) capable of rapidly and reproducibly separating and quantifying isomeric cerebrosides in a single run. We show that this LC/ESI/DMS/MS/MS method exhibits robust quantitative performance within an analyte concentration range of 2.8–355 nM. We further report the simultaneous quantification of nine GlcCers (16:0, 18:0, 20:0, 22:0, 23:0, 24:1, 24:0, 25:0, and 26:0) and five GalCers (16:0, 22:0, 23:0, 24:1, and 24:0) molecular species in human plasma, as well as six GalCers (18:0, 22:0, 23:0, 24:1, 24:0 and 25:0) and two GlcCers (24:1 and 24:0) in human cerebrospinal fluid. Our method expands the potential of DMS technology in the field of glycosphingolipid analysis for both biomarker discovery and drug screening by enabling the unambiguous assignment and quantification of cerebroside lipid species in biological samples. Cerebrosides, including glucosylceramides (GlcCers) and galactosylceramides (GalCers), are important membrane components of animal cells with deficiencies resulting in devastating lysosomal storage disorders. Their quantification is essential for disease diagnosis and a better understanding of disease mechanisms. The simultaneous quantification of GlcCer and GalCer isomers is, however, particularly challenging due to their virtually identical structures. To address this challenge, we developed a new LC/MS-based method using differential ion mobility spectrometry (DMS) capable of rapidly and reproducibly separating and quantifying isomeric cerebrosides in a single run. We show that this LC/ESI/DMS/MS/MS method exhibits robust quantitative performance within an analyte concentration range of 2.8–355 nM. We further report the simultaneous quantification of nine GlcCers (16:0, 18:0, 20:0, 22:0, 23:0, 24:1, 24:0, 25:0, and 26:0) and five GalCers (16:0, 22:0, 23:0, 24:1, and 24:0) molecular species in human plasma, as well as six GalCers (18:0, 22:0, 23:0, 24:1, 24:0 and 25:0) and two GlcCers (24:1 and 24:0) in human cerebrospinal fluid. Our method expands the potential of DMS technology in the field of glycosphingolipid analysis for both biomarker discovery and drug screening by enabling the unambiguous assignment and quantification of cerebroside lipid species in biological samples. Glycosphingolipids (GSLs) are essential plasma membrane lipid components of eukaryotes. Together with cholesterol and sphingomyelin, GSLs form microdomains on plasma membranes that play important roles in various cellular activities such as adhesion, growth, and differentiation (1Regina Todeschini A. Hakomori S.I. Functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains.Biochim. Biophys. Acta. 2008; 1780: 421-433Crossref PubMed Scopus (338) Google Scholar). The simplest GSLs are monohexosylceramides (also known as cerebrosides). Cerebrosides are composed of a lipid, ceramide, that is embedded in the outer leaflet of the plasma membrane, and a sugar moiety, either glucose or galactose, that is attached to the ceramide by a #x03BC;-glycosidic linkage. Galactosylceramide (GalCer) is the principal GSL in the brain and, together with its sulfated or sialylated derivatives, is an essential structural component of myelin. Glucosylceramide (GlcCer) is found in all mammalian membranes and is a major lipid component of skin and neurons. More complex GSLs can be further produced from GlcCer, to which additional sugar moieties and functional groups may be attached. GlcCer and GalCer are synthesized by their respective synthases, the GlcCer synthase [UDP-glucose:ceramide #x03BC;-1,1-glucosyltransferase (GlcT); EC 2.4.1.80] (2Ichikawa S. Sakiyama H. Suzuki G. Hidari K.I. Hirabayashi Y. Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.Proc. Natl. Acad. Sci. USA. 1996; 93: 4638-4643Crossref PubMed Scopus (220) Google Scholar) and GalCer synthase [UDP-galactose:ceramide galactosyltransferase (GalT); EC 2.4.1.45] (3Schulte S. Stoffel W. Ceramide UDPgalactosyltransferase from myelinating rat brain: purification, cloning, and expression.Proc. Natl. Acad. Sci. USA. 1993; 90: 10265-10269Crossref PubMed Scopus (164) Google Scholar, 4Kapitonov D. Yu R.K. Cloning, characterization, and expression of human ceramide galactosyltransferase cDNA.Biochem. Biophys. Res. Commun. 1997; 232: 449-453Crossref PubMed Scopus (24) Google Scholar, 5Bosio A. Binczek E. Le Beau M.M. Fernald A.A. Stoffel W. The human gene CGT encoding the UDP-galactose ceramide galactosyl transferase (cerebroside synthase): cloning, characterization, and assignment to human chromosome 4, band q26.Genomics. 1996; 34: 69-75Crossref PubMed Scopus (75) Google Scholar, 6Sprong H. Kruithof B. Leijendekker R. Slot J.W. van Meer G. van der Sluijs P. UDP-galactose:ceramide galactosyltransferase is a class I integral membrane protein of the endoplasmic reticulum.J. Biol. Chem. 1998; 273: 25880-25888Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The knockout and knockdown of these synthases have revealed critical biological relevancies (7Bosio A. Binczek E. Stoffel W. Functional breakdown of the lipid bilayer of the myelin membrane in central and peripheral nervous system by disrupted galactocerebroside synthesis.Proc. Natl. Acad. Sci. USA. 1996; 93: 13280-13285Crossref PubMed Scopus (282) Google Scholar, 8Coetzee T. Fujita N. Dupree J. Shi R. Blight A. Suzuki K. Suzuki K. Popko B. Myelination in the absence of galactocerebroside and sulfatide: normal structure with abnormal function and regional instability.Cell. 1996; 86: 209-219Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 9Yamashita T. Wada R. Sasaki T. Deng C. Bierfreund U. Sandhoff K. Proia R.L. A vital role for glycosphingolipid synthesis during development and differentiation.Proc. Natl. Acad. Sci. USA. 1999; 96: 9142-9147Crossref PubMed Scopus (404) Google Scholar, 10Jennemann R. Sandhoff R. Langbein L. Kaden S. Rothermel U. Gallala H. Sandhoff K. Wiegandt H. Grone H.J. Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis.J. Biol. Chem. 2007; 282: 3083-3094Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 11Jennemann R. Sandhoff R. Wang S. Kiss E. Gretz N. Zuliani C. Martin-Villalba A. Jager R. Schorle H. Kenzelmann M. et al.Cell-specific deletion of glucosylceramide synthase in brain leads to severe neural defects after birth.Proc. Natl. Acad. Sci. USA. 2005; 102: 12459-12464Crossref PubMed Scopus (152) Google Scholar). Both GlcCer and GalCer are essential to neural functions. GlcCer is involved in the axon growth of neural cells (12Schwarz A. Futerman A.H. Distinct roles for ceramide and glucosylceramide at different stages of neuronal growth.J. Neurosci. 1997; 17: 2929-2938Crossref PubMed Google Scholar), and GalCer is indispensable for multiple myelin functions (8Coetzee T. Fujita N. Dupree J. Shi R. Blight A. Suzuki K. Suzuki K. Popko B. Myelination in the absence of galactocerebroside and sulfatide: normal structure with abnormal function and regional instability.Cell. 1996; 86: 209-219Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Surprisingly, GalT-null mutants are still capable of myelination, albeit with reduced insulating capacity. While GalT knockout mice do not express any GalCers or their derivatives, they remain capable of synthesizing GlcCer using hydroxylated fatty acids, and they compensate for GalCer deficiency by incorporating GlcCer de novo into myelin (7Bosio A. Binczek E. Stoffel W. Functional breakdown of the lipid bilayer of the myelin membrane in central and peripheral nervous system by disrupted galactocerebroside synthesis.Proc. Natl. Acad. Sci. USA. 1996; 93: 13280-13285Crossref PubMed Scopus (282) Google Scholar, 8Coetzee T. Fujita N. Dupree J. Shi R. Blight A. Suzuki K. Suzuki K. Popko B. Myelination in the absence of galactocerebroside and sulfatide: normal structure with abnormal function and regional instability.Cell. 1996; 86: 209-219Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). By contrast, GalCer cannot compensate for neuronal GlcCer deficiencies. Brain-specific conditional knockout of GlcT is perinatal lethal with pups exhibiting structural and functional dysfunction in the cerebellum and peripheral nerves (11Jennemann R. Sandhoff R. Wang S. Kiss E. Gretz N. Zuliani C. Martin-Villalba A. Jager R. Schorle H. Kenzelmann M. et al.Cell-specific deletion of glucosylceramide synthase in brain leads to severe neural defects after birth.Proc. Natl. Acad. Sci. USA. 2005; 102: 12459-12464Crossref PubMed Scopus (152) Google Scholar). Constitutive GlcT knockout is embryonic lethal, consistent with the essential role of GlcCer in the synthesis of complex GSLs throughout the body (9Yamashita T. Wada R. Sasaki T. Deng C. Bierfreund U. Sandhoff K. Proia R.L. A vital role for glycosphingolipid synthesis during development and differentiation.Proc. Natl. Acad. Sci. USA. 1999; 96: 9142-9147Crossref PubMed Scopus (404) Google Scholar). New methods that can quickly profile and reliably quantify cerebrosides are necessary for both diagnosing and understanding GSL disorders, as well as for drug screening. Cerebrosides are the natural substrates of #x03BC;-glucocerebrosidase (GCase; EC 3.2.1.45) and #x03BC;-galactocerebrosidase (EC 3.2.1.46). Humans with mutations in the GBA gene that encodes GCase, and thus with deficient degradation of GlcCer, may develop Gaucher disease. Those with deficient GalCer degradation develop Krabbe disease (13Suzuki K. Suzuki Y. Globoid cell leucodystrophy (Krabbe's disease): deficiency of galactocerebroside beta-galactosidase.Proc. Natl. Acad. Sci. USA. 1970; 66: 302-309Crossref PubMed Scopus (402) Google Scholar, 14Zhao H. Grabowski G.A. Gaucher disease: perspectives on a prototype lysosomal disease.Cell. Mol. Life Sci. 2002; 59: 694-707Crossref PubMed Scopus (83) Google Scholar). The molecular mechanisms underlying how GSL accumulation mediates these disorders are complex and not yet fully understood (15Jmoudiak M. Futerman A.H. Gaucher disease: pathological mechanisms and modern management.Br. J. Haematol. 2005; 129: 178-188Crossref PubMed Scopus (229) Google Scholar). For example, mutations in the human GBA gene that moderately decrease GCase activity significantly increase the risk of Parkinson's disease (16Elstein D. Alcalay R. Zimran A. The emergence of Parkinson disease among patients with Gaucher disease.Best Pract. Res. Clin. Endocrinol. Metab. 2015; 29: 249-259Crossref PubMed Scopus (12) Google Scholar, 17Schapira A.H. Glucocerebrosidase and Parkinson disease: recent advances.Mol. Cell. Neurosci. 2015; 66: 37-42Crossref PubMed Scopus (158) Google Scholar, 18Li Y. Li P. Liang H. Zhao Z. Hashimoto M. Wei J. Gaucher-associated Parkinsonism.Cell. Mol. Neurobiol. 2015; 35: 755-761Crossref PubMed Scopus (19) Google Scholar). Yet it is not clear how this impairment enhances Parkinson's disease risk. Elucidating the underlying mechanism is complicated by the technical challenges associated with discriminating between isomeric GlcCer and GalCer isoforms in biological samples. Both GlcCer and GalCer cerebrosides have virtually identical structures, with the only difference being the stereochemistry of the 4′-hydroxyl group as either axial (GalCer) or equatorial (GlcCer) (Fig. 1). GlcCer and GalCer isoforms also produce identical product ion spectra and possess similar physical properties, making their distinction very difficult by traditional LC/MS analysis. To address this issue, we exploited recent advances in differential ion mobility spectrometry (DMS) to develop a simple, rapid, reliable method for profiling and quantifying GlcCer and GalCer isoforms in biological samples. DMS has emerged over the past decade as a viable addition to chromatography as an orthogonal separation technique that achieves highly selective multidimensional separations (19Beach D.G. Melanson J.E. Purves R.W. Analysis of paralytic shellfish toxins using high-field asymmetric waveform ion mobility spectrometry with liquid chromatography-mass spectrometry.Anal. Bioanal. Chem. 2015; 407: 2473-2484Crossref PubMed Scopus (26) Google Scholar, 20Ray J.A. Kushnir M.M. Yost R.A. Rockwood A.L. Wayne Meikle A. Performance enhancement in the measurement of 5 endogenous steroids by LC-MS/MS combined with differential ion mobility spectrometry.Clin. Chim. Acta. 2015; 438: 330-336Crossref PubMed Scopus (67) Google Scholar, 21Varesio E. Le Blanc J.C.Y. Hopfgartner G. Real-time 2D separation by LC × differential ion mobility hyphenated to mass spectrometry.Anal. Bioanal. Chem. 2012; 402: 2555-2564Crossref PubMed Scopus (38) Google Scholar). This technique can be particularly useful in resolving isobaric and isomeric compounds in complex samples (22Parson W.B. Schneider B.B. Kertesz V. Corr J.J. Covey T.R. Van Berkel G.J. Rapid analysis of isomeric exogenous metabolites by differential mobility spectrometry-mass spectrometry.Rapid Commun Mass Spectrom. 2011; 25: 3382-3386Crossref PubMed Scopus (29) Google Scholar, 23Porta T. Varesio E. Hopfgartner G. Gas-phase separation of drugs and metabolites using modifier-assisted differential ion mobility spectrometry hyphenated to liquid extraction surface analysis and mass spectrometry.Anal. Chem. 2013; 85: 11771-11779Crossref PubMed Scopus (53) Google Scholar, 24Li H. Giles K. Bendiak B. Kaplan K. Siems W.F. Hill Jr., H.H. Resolving structural isomers of monosaccharide methyl glycosides using drift tube and traveling wave ion mobility mass spectrometry.Anal. Chem. 2012; 84: 3231-3239Crossref PubMed Scopus (84) Google Scholar, 25Kyle J.E. Zhang X. Weitz K.K. Monroe M.E. Ibrahim Y.M. Moore R.J. Cha J. Sun X. Lovelace E.S. Wagoner J. et al.Uncovering biologically significant lipid isomers with liquid chromatography, ion mobility spectrometry and mass spectrometry.Analyst. 2016; 141: 1649-1659Crossref PubMed Google Scholar). As such, DMS has recently evolved as a powerful tool in the field of lipidomics (26Baker P.R. Armando A.M. Campbell J.L. Quehenberger O. Dennis E.A. Three-dimensional enhanced lipidomics analysis combining UPLC, differential ion mobility spectrometry, and mass spectrometric separation strategies.J. Lipid Res. 2014; 55: 2432-2442Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 27Lintonen T.P. Baker P.R. Suoniemi M. Ubhi B.K. Koistinen K.M. Duchoslav E. Campbell J.L. Ekroos K. Differential mobility spectrometry-driven shotgun lipidomics.Anal. Chem. 2014; 86: 9662-9669Crossref PubMed Scopus (116) Google Scholar), yet its application in GSL analysis remains limited. Here, we explored the capabilities of DMS coupled with LC in ESI/MS/MS for differentially quantifying isomeric cerebrosides and their glucosylsphingosine (GlcSph) and galactosylsphingosine (GalSph) lyso forms. We show that 32 cerebroside species (16 isomeric GalCer-GlcCer pairs) and 1 pair of their GlcSph-GalSph metabolites can be separated in the DMS cell using isopropanol (IPA) as the gas-phase chemical modifier under the optimized conditions described here. Our results demonstrate robust quantitative performance, showing reproducibility and precision with a linear detection range of 2.8–355 nM. Using this method, we describe the profiling and quantification of GlcCer(d18:1/16:0), GalCer(d18:1/16:0), GlcCer(d18:1/22:0), GalCer(d18:1/22:0), GlcCer(d18:1/23:0), GalCer(d18:1/23:0), GlcCer(d18:1/24:1), GalCer(d18:1/24:1), GlcCer(d18:1/24:0), and GalCer(d18:1/24:0) as well as GlcCer(d18:1/18:0), GlcCer(d18:1/20:0), GlcCer(d18:1/25:0), and GlcCer(d18:1/26:0), but in the absence of their isomeric GalCer species in human plasma. We further detected and quantified GalCer(d18:1/18:0), GalCer(d18:1/22:0), GalCer(d18:1/23:0), GalCer(d18:1/24:0), GalCer(d18:1/24:1), and GalCer(d18:1/25:0) but only two GlcCer species [GlcCer(d18:1/24:0) and GlcCer(d18:1/24:1)] in human CSF. This study demonstrates the value of LC/ESI/DMS/MS/MS in the unambiguous assignment and reliable quantification of closely related stereoisomeric molecular species of cerebrosides and their lyso forms. Methanol, chloroform, and IPA were purchased from Fisher Scientific Co. (Ottawa, Canada). Anhydrous ethanol was obtained from Commercial Alcohols (Brampton, Canada). Ammonium acetate, formic acid, HPLC-grade 1-propanol, and HPLC-grade 1-butanol were purchased from Sigma-Aldrich Canada Co. (Oakville, Canada). LC/MS-grade water and acetonitrile were purchased from Avantor Performance Materials (Central Valley, PA). Microwell plates were from Agilent Technologies Canada Inc. (Mississauga, Canada). Lipid standards, including GalSph(d18:1), GlcSph(d18:1), GalCer(d18:1/8:0), GlcCer(d18:1/8:0), GalCer(d18:1/16:0), GlcCer(d18:1/16:0), GalCer(d18:1/18:0), GlcCer(d18:1/18:0), GalCer(d18:1/24:1), and GlcCer(d18:1/24:1), were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). GlcCers and GalCers with 14:0, 16:1, 18:1, 20:0, 21:0, 22:0, 23:0, 24:0, 25:0, 26:0, 22:1, and 31:0 N-fatty acyl chains on the d18:1 sphingosine backbone were custom-synthesized by Avanti Polar Lipids, Inc. All lipid standards used in this study have a d18:1 sphingosine backbone with the sugar moieties attached via a #x03BC;-glycosidic linkage. Human blood from a 50-year-old Caucasian woman enrolled in the Ottawa Biobank Study was collected in K2EDTA-coated lavender BD Hemogard tubes. The subject was assessed as cognitively normal using the Montreal Cognitive Assessment and Mini-Mental State Exam and had no significant acute medical illness (e.g., severely disturbed liver, kidney, or lung function), major psychiatric condition (e.g., major depressive disorder, schizophrenia, bipolar disorder), history of hospitalization for clinical stroke, or contraindication to MRI (e.g., metal in body, pacemaker). Consent was obtained in strict accordance with the Ottawa Hospital Research Institute Research Ethics Committee. The CSF sample was a pooled reference standard used to ensure normalization and standardization across sites, platforms, and assays and was from the National Institute of Neurological Disorders and Stroke (NINDS) Parkinson's Disease Biomarkers Program (PDBP). It was obtained in agreement with the National Institutes of Health and NINDS and the Icahn School of Medicine at Mount Sinai Research Ethics Committee as part of a study to evaluate pathway biomarkers for Parkinson's disease. Each pair of isomeric GlcCers and GalCers was prepared by mixing the individual standard to a final concentration of 1 μM each in ethanol. Human plasma (100 #x03BC;l) and human CSF (200 #x03BC;l) were extracted using the modified method of Bligh and Dyer (28Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42828) Google Scholar, 29Granger, M. W., H. Liu, C. F. Fowler, A. P. Blanchard, M. Taylor, S. P. M. Sherman, H. Xu, W. Le, and S. A. L. Bennett, . 2018. Distinct disruptions in Land's cycle remodeling of glycerophosphocholines in murine cortex mark symptomatic onset and progression in two Alzheimer's disease mouse models. J. Neurochem. Epub ahead of print. July 25 2018; .Google Scholar), with an equimolar mixture of GalCer(d18:1/8:0) and GlcCer(d18:1/8:0) standards spiked in at the time of extraction for normalization. Extracted lipids were dried under a gentle stream of nitrogen gas at room temperature and were redissolved in ethanol. These lipid samples were stored in amber glass vials, flushed with nitrogen gas, and kept at −80°C until use. Lipid samples were analyzed using a QTRAP 5500 triple quadrupole-linear ion trap mass spectrometer equipped with a Turbo V ion source (AB SCIEX, Concord, Canada). Source parameters (temperature, ionspray voltage, and gas flow) were optimized using a mixture of cerebroside standards that were T-infused with a syringe pump set at 5 #x03BC;l/min into the flow of 10 #x03BC;l/min of 100#x0025; solvent B (acetonitrile-IPA at a 5:2 v/v ratio containing 0.1#x0025; formic acid and 10 mM ammonium acetate) delivered by an Agilent Infinity II system (supplemental Table S1). A microfilter assembly (M-547; IDEX Health and Science, Oak Harbor, WA) was installed to prevent the potential blockage of the turbo-spray electrode (25 µm inner diameter) and contamination of the MS system. Nitrogen was used as curtain gas, collision gas, and ion source gas 1 and 2 unless otherwise stated. Compound parameters (declustering potential, entrance potential, collision energy, and collision cell exit potential) were individually optimized for each isomeric pair of cerebrosides and their lyso forms using the same method with the optimized source parameters. For these experiments, the DMS cell was physically installed but was set to “OFF” [DMS temperature (DT) set to #x201C;LOW#x201D; at 150°C by default)] during source and compound optimization. DMS separation was performed in a SelexION differential ion mobility device interfaced with a triple quadrupole-linear ion trap mass spectrometer (QTRAP 5500; AB SCIEX). The DMS cell was mounted in the atmospheric pressure region between the curtain and the orifice plate of the mass spectrometer, and nitrogen was used as a transport gas. As compensation voltage (CoV) is influenced by mobile-phase and source conditions, DMS optimization was performed by injecting analytes at a flow rate and mobile-phase composition comparable to the intended LC conditions via T-infusion (see above). DMS parameters were optimized following the manufacturer's instructions using T-infusion of each isomeric pair of standards, and common parameters were determined as follows: DMS cell temperature = 150°C (low), modifier concentration = low (1.5#x0025; v/v in the transport gas, corresponding to a 173.8 µl/min flow rate for IPA), DMS resolution enhancement = 30 psi (medium) and DMS offset = −3.0 V, and nitrogen resolving gas = 30 psi. Four solvents, isopropanol, 1-propanol, 1-butanol, and methanol, were tested for their suitability as the DMS chemical modifier. To determine the optimal separation voltage (SV) and CoV pair for each isomeric pair of standards, CoV was ramped for each SV value in 100 V steps in the range of 3,000–4,100 V. Multiple reaction monitoring (MRM) spectra were recorded during CoV ramping, and the obtained data were plotted in the form of ionograms (signal intensity vs. CoV). With the highest signal intensity, a characteristic CoV value ensuring baseline or near-baseline separation was selected for each target analyte ion. CoV values at the peak apex were determined after Gaussian smoothing (smoothing width: 0.5 points for IPA and 1.5 points for 1-propanol, methanol, and 1-butanol). Lipid standards at 1 #x03BC;M each in ethanol were used for DMS optimization. LC was performed with an Agilent Infinity II system operating at a flow rate of 10 #x03BC;l/min with 8 #x03BC;l (plasma) or 10 #x03BC;l (CSF) sample injections by an autosampler maintained at 4°C. A 100 mm × 250 #x03BC;m (inner diameter) capillary column packed with ReproSil-Pur 120 C8 (particle size of 3 µm and pore size of 120 Å) was used with a binary solvent gradient consisting of water with 0.1#x0025; formic acid and 10 mM ammonium acetate (solvent A) and acetonitrile-IPA (5:2; v/v) with 0.1#x0025; formic acid and 10 mM ammonium acetate (solvent B). The gradient started from 30#x0025; solvent B, reached 100#x0025; solvent B in 5 min, and was maintained for 30 min. The composition returned to 30#x0025; solvent B within 1 min and was maintained for 10 min to reequilibrate the column prior to the next sample injection. Data acquisition was performed in the positive ion mode using MRM with the optimized DMS parameters (see supplemental Table S2 for optimized DMS parameters and MRM transitions). Each duty cycle was 0.85 s, and the MRM data were acquired over the 45 min chromatography period. Instrument control and data acquisition were performed with Analyst software version 1.6.2 (AB SCIEX). PeakView software version 2.2.0 (AB SCIEX) was used for data processing and visualization. MultiQuant software version 3.0.2 (AB SCIEX) was used for processing quantitative MRM data. Source parameters were first optimized using a mixture of cerebroside standards that were T-infused with solvent B into the mass spectrometer (supplemental Table S1). Compound parameters were then optimized for each isomeric pair of GlcCer/GalCer and GlcSph/GalSph standards (supplemental Table S2). Collision energy was the only parameter that differed significantly between compounds, whereas declustering potential, entrance potential, and collision cell exit potential were all of very similar values (supplemental Tables S1, S2). For all standards, the precursor ion scan spectra showed the protonated [M+H]+ ion as the base peak, and the product ion spectra showed the ion with m/z 264.3 (corresponding to the didehydrated sphingosine) as the most intense fragment. Thus, the most intense MS/MS ion transition from [M+H]+ → m/z 264.3 was monitored for each compound for DMS optimization and subsequent quantitative analyses. With these optimized source and compound parameters, DMS was further optimized for the separation of isomeric GlcCer/GalCer and GlcSph/GalSph standards, with the intent of achieving the optimal separation with the best detection sensitivity. Experimental conditions were systematically varied by examining the influence of the main factors, including DT, transport gas type, SV, and the addition of gas-phase organic chemical modifiers, on DMS separation. DT influences DMS performance by affecting transport gas density and the clustering-declustering equilibrium of analytes according to the modifier (30Schneider B.B. Nazarov E.G. Londry F. Vouros P. Covey T.R. Differential mobility spectrometry/mass spectrometry history, theory, design optimization, simulations, and applications.Mass Spectrom. Rev. 2016; 35: 687-737Crossref PubMed Scopus (120) Google Scholar, 31Schneider B.B. Covey T.R. Coy S.L. Krylov E.V. Nazarov E.G. Planar differential mobility spectrometer as a pre-filter for atmospheric pressure ionization mass spectrometry.Int. J. Mass Spectrom. 2010; 298: 45-54Crossref PubMed Scopus (131) Google Scholar). We evaluated the three preset DTs on DMS performance mainly based on achieving the best sensitivity and found that the most intense and stable signals were obtained at the low setting of 150°C (supplemental Table S1). We next optimized SV and CoV simultaneously to find the SV and CoV combination that gave the best sensitivity and separation. The SV shifts the ion trajectories in the ion mobility cell, deviating ions from their central path entering the mass spectrometer. The CoV, a compound-specific parameter, compensates for the SV-causing shift of an ion trajectory, allowing specific ions to enter the mass spectrometer. We started with no chemical modifier added to the transport gas and noticed no separation of any of the isomeric analytes. It has been shown that the addition of gas-phase chemical modifiers drastically improves the separation power of the DMS (32Eiceman G.A. Krylov E.V. Krylova N.S. Nazarov E.G. Miller R.A. Separation of ions from explosives in differential mobility spectrometry by vapor-modified drift gas.Anal. Chem. 2004; 76: 4937-4944Crossref PubMed Scopus (177) Google Scholar, 33Schneider B.B. Covey T.R. Coy S.L. Krylov E.V. Nazarov E.G. Chemical effects in the separation process of a differential mobility/mass spectrometer system.Anal. Chem. 2010; 82: 1867-1880Crossref PubMed Scopus (144) Google Scholar, 34Levin D.S. Vouros P. Miller R.A. Nazarov E.G. Morris J.C. Characterization of gas-phase molecular interactions on differential mobility ion behavior utilizing an electrospray ionization-differential mobility-mass spectrometer system.Anal. Chem. 2006; 78: 96-106Crossref PubMed Scopus (70) Google Scholar, 35Rorrer Iii L.C. Yost R.A. Solvent vapor effects on planar high-field asymmetric waveform ion mobility spectrometry.Int. J. Mass Spectrom. 2011; 300: 173-181Crossref Scopus (63) Google Scholar, 36Tsai C-W. Yost R.A. Garrett T.J. High-field asymmetric waveform ion mobility spectrometry with solvent vapor addition: a potential greener bioanalytical technique.Bioanalysis. 2012; 4: 1363-1375Crossref PubMed Scopus (19) Google Scholar). Indeed, the addition of polar organic chemical modifiers to the DMS cell resulted in partial or complete separation of isomeric compounds (Fig. 2A–D). Among the modifiers examined, IPA performed the best in terms of peak separation and sensitivity (Fig. 2D), followed by 1-propanol (Fig. 2C), 1-butanol (Fig. 2B), and methanol (Fig. 2A). However, with 1-butanol or methanol as modifiers, peaks were #x201C;noisier#x201D; and sometimes split. In addition, the isomeric hexosylceramide (HexCer) (d18:1/31:0) could not be effectively separated into GlcCer(d18:1/31:0) and GalCer(d18:1/31:0) isomers with methanol as a modifier. The effects of the modifier concentration were also investigated at the low and high settings. The use of a high concentration of modifiers resulted in an almost complete loss of signal. Thus, with IPA selected as a gas-phase chemical modifier at the low concentration setting, optimal SV and CoV values were determined for each pair of isomeric analytes by sca" @default.
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• W2900177426 cites W2030480923 @default.
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• W2900177426 cites W2039082895 @default.
• W2900177426 cites W2039736612 @default.
• W2900177426 cites W2039772623 @default.
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• W2900177426 cites W2085730038 @default.
• W2900177426 cites W2090515621 @default.
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• W2900177426 cites W2124717330 @default.
• W2900177426 cites W2128517041 @default.
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