Quantitation of Sphingolipids in Tissue Extracts by LC/MS/MS
|
|
- Eugene Freeman
- 5 years ago
- Views:
Transcription
1 Quantitation of Sphingolipids in Tissue Extracts by LC/MS/MS Alexei Belenky CASSS, Meritage Resort, Napa 2008
2 Outline Role of lipid analysis in drug discovery and disease diagnostics Analytical tools and methods for lipid analysis and extraction from tissues Quantitation and normalization of the results Multidimensional approaches for Lipidomics
3 Lipids a free fatty acid a triglyceride cholesterol a phospholipid Lipids are broadly defined as any fat-soluble (lipophilic), naturally-occurring molecule, such as fats, oils, cholesterol, sterols, mono-, di-, tri- glycerides, phospholipids, sphingolipids and others. (From Wikipedia) The main biological functions of lipids include energy storage, acting as structural components of cell membranes, and participating as important signaling molecules. Because of the combinatorial nature of sphingolipids biosynthesis, the sphingolipidome is comprised of thousands of related and biologically connected species
4 Sphingolipid Pathway Map Sphingolipidomics similarly to genomics or proteomics helps to understand the disease progression, identify targets, mechanism of drug action and toxicity
5 Gaucher Disease Lysosomal storage disorder caused by deficiency of the enzyme β-glucosidase, leading to an accumulation of its substrate glucosylceramide. Fatty material can collect in the spleen, liver, kidney, braine and bone marrow. occurring in approximately 1 in 50,000 live births Structure of a Glucosylceramide Girl with Gaucher Disease..
6 Glycosphingolipid Pathways and Target Diseases Gangliosides Globosides G M1 G B4 Tay-Sachs Disease G M2 Type 2 Diabetes G M3 LacCer G B3 Fabry Disease Sandhoff Disease Enzyme Replacement Therapy glucosylceramide Gaucher Disease ceramide + glucose GluCer synthase Substrate Reduction Therapy
7 Analytical Challenges Separation analytes are a small part of a huge family of endogenous lipids Absence of pure synthetic standards Analyte concentration is ng/ml Solution to the problem: SELECTIVITY, SELECTIVITY, SELECTIVITY!
8 Work Flow for Sphingolipids Analysis Cells/Tissue/plasma/serum Cells/Tissue/plasma/serum 96/384-well plate format QQQ-MS Extraction HPLC UPLC API 4000 API 5000 LC separation MRM Int. (arb.) 0.0 DHSPH SPH min 8.01 Higher Cer throughput GluCer SM 9.35 S1P MRM-TIC min
9 Extraction and Analysis of Tissue Samples Kidney samples ( 100 mg/ml) are homogenized and extracted with a modified Folch method: Extraction buffer : 0.47/ 1.7/1.7 of water / methanol / chloroform; ~ 5mg/ml tissue HPLC-MS/MS of glucosylceramide isoforms, GL1 std 0.5ug/ml Intensity, cps 4.4e5 4.0e5 3.6e5 3.2e5 2.8e5 2.4e5 2.0e5 1.6e5 1.2e5 8.0e4 4.0e4 0.0 XIC of +MRM (6 pairs) C16 C18 C Time, min C22 C 24 C 23 HPLC (UPLC) Column: Waters Xbridge Phenyl 3.0 x 100 mm 3.5 um column. (Alternative: Phenomenex Luna C8,) The mobile phases : 0.2% (v/v) FA and 5 mm AmF in water (A) 0.2% FA (v/v) and 5 mm AmF in 1:1 meth/acetonitrile (B) The flow rate was 0.6 ml/min. The column was heated to 60 C. The gradient: 70% B to 100% B in 5 min, hold at 100% B for 3 min, reset ESI- MS, API-4000 (API-5000) ESI-MS conditions were: needle voltage, 3.5 kv. drying gas temperature, 400 C, collision gas density (CAD), 7. All MRM transitions included m/z or 184 for SM as the product ions.
10 MRM Transitions for Analysis of Sphingolipids in Tissues Intensity, cps Intensity, cps GalCer std Time, min Sphingoid backbone -H2O Gal m/z, amu 800 fatty acid chain O Ceramide R=H Glycosphingolipids R= carbohydrate O + Sphyngomyelin R = O-P-OCH 2 CH 2 N(CH 3 ) 3 OH m/z 184 Alfred H.Merrill Jr, et.al. Methods 36(2005)
11 LC-MRM Analysis of GL1 Isoforms Extracted from Different Amount of Tissue Tissues are extracted and reconstituted in the same volume MRM signal peak area 3.50E E E E E E E E Tissue, mg GL1 C16 GL1 C24.1 GL1 C22 GL1 C24 GL1 C23 GL1 C18 C16 C 24 1 C18 Time, min C22 C 24 C 23? Extraction or Matrix?
12 When is LC QQQ Analysis Quantitative? when matrix effect does not exist when isotopically labeled IS is available.. and if not? The alternative is - method of standard additions, which requires a well characterized standard.
13 LC-CAD/TOF Characterization Analysis of Standards of a Sphingomyelin using CAD-TOF Standard Method: e4 6.0e4 4.0e4 HPLC HPLC Column Charged Aerosol Detector, CAD 9 1 Eluent Split Mass Spectrometer TIC of +TOF MS: 600 to 850 amu from SM 214ug/m stnd Luna C8 Max. 9.2e4 cps. CAD Time, min Since the abundance of isoforms in the standard is not known, a universal detector (CAD) was used to quantitate all the isophorms present in the standard. 2.0e4 CAD, mv CAD of SM 214 ug/m stnd, Luna C8 MW SM C16:1 MW , C14 MW ,C16 MW SM C15 MW , C17 MW ,C18 MW ,C20:1 MW , C24:2 Intensity, mv Intensity, cps Sphingomyelin standard, 214ug/ml Time, min MW , C20 MS - Q STAR Time, min Time, min MW , C23:1 MW , C22 MW , C24:1 MW , C23 MW , C24 MW 84187, C26:1 MS - Q STAR CAD
14 Slopes for Different Mouse Models Kidney Samples, GL1 C24:1 Isoform Analysis Peak area 3.50E E E E+05 y = x R 2 = y = 9293x y = x R 2 = y = 6167x y = 6990x WT 45d Jck 45d Jck 64d WT 45d Jck 45d Jck 64d 1.50E E E+04 y = x R 2 = GL1 conc., ng/ml
15 Normalization of Results Not all the tissues are equal
16 Difference in Tissue Morphology Makes Normalization by Weight Inadequate normal adult kidney cystic adult kidney Conventional method of normalization is using total phosphate. We have developed a fast, sensitive and reliable method for total phosphate analysis based on inductively coupled plasma spectrometry (ICP).
17 Total Phosphate Levels in Tissues are Associated with Disease State healthy ICP count cystic mouse,# Normal Kidney ADPKD Kidney
18 Normalization by Total P Revealed Increased GL1 Levels in Cystic Mice: Distribution of GL1 isoforms in kidney tissues for cystic and normal mice 1.80E E E E E E E E E+04 Control Mice Cystic Mice GL1 C16 GL1 C18 GL1 C22 GL1 C24 1 GL1 C E E+02 Same data after normalization Control Mice Cystic Mice 1.50E E E E+00 GL1C16 GL1 C18 GL1 C22 GL1 C24 1 GL1 24 0
19 Correlation of Endogenous SM C16 and Total P Has Been Identified Comparison of Total Phosphate and SM C16 levels; data normalized to the same scale Phosphate SM C16/100 Correlation between Total P and levels of endogenous SM C16 in mice kidney 2.00E E+05 y = x Animal #184 Animal #183 Animal #182 Animal #2274 Animal #5780 Animal #1222 Total phosphate by ICP 1.20E E E E+00 R2= E E E E E E+03 SM C16 level by LC-MS/MS Alternative approach: normalization using selected endogenous markers
20 Why Phospholipids can serve as relevant normalization markers? Napa Phospholipids are cell building materials and can be used for estimation of the number and the nature of the extracted cells
21 Phospholipids palmitate Separate by HILIC and detect using precursor scan:+184 & neutral loss: 141
22 HILIC Mode of Separation Water Acetonitrile/Water (90:10) Separation is based on partitioning between adsorbed polar component of mobile phase (water) and hydrophobic mobile phase (acetonitrile). This mode can be used to separate sphingolipids by their polar groups.
23 A Standard and a Mouse Kidney Homogenate Sample, Analyzed by HILIC Intensity, cps 4.8e6 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e6 1.0e6 5.0e PC PM SM PC 2.83 standard 3.23 PE PE Time, min Intensity, cps 6.5e6 6.0e6 5.0e6 4.0e6 3.0e6 2.0e6 1.0e PC PC SM SM kidney sample PE PE Time, min m/z, amu +184 precursors of PC from standard m/z, amu +184 precursors of PC from sample Mobil Phase A: 96:2:2 (ACN:MeOH:Acetic acid:water, 5mM Ammonium Acetate) Mobil Phase B: 98:1:1 (MeOH:Acetic Acid:Water, 5mM Ammonium Acetate) Column: Phenomenex Luna 50 x 2mm, 3 micron HILIC; gradient : 0 to 15%B
24 Correlation Between PC Level Measured by LC-MS/MS and Total Phosphate Measured by ICP Acute jck Mice Model Study PC 10level by MS/MS P level by ICP ug/ml sample# Treated Untreated
25 Correlation Between PC Level Measured by LC-MS/MS and Total Phosphate Measured by ICP Acute jck mice kidney study Time Course y = 2.087x R 2 = ICP, total P, mm LC-MS/MS, PC mm
26 GL1 Analysis from Cell Pellets Extracts in Triplicate Data normalized by cell count control 100nM-C9 300nM-C9 1uM-C9 Normalization by cell count is hard because of cell clamping Data normalized by PC level hr 24hr 48hr 0.00 control 100nM-C9 300nM-C9 1uM-C9 Normalization by PC level helps because it reflects true cell count. As a result RSD is much improved and effect of treatment can be clearly seen.
27 Comments PC levels correlate well with the total phosphate levels Normalization by PC significantly reduces sample preparation work required by conventional total phosphate analysis
28 What have we accomplished so far? We have a selective LC MS/MS method to analyze Sphingolipids We are able to quantitate multiple lipids and their isoforms We know how to normalize data
29 Characterization of 7 Animal Models by Sphingplipids Analysis MRM raw data data after quantitation and normalization SM C18 GL1 C16 GL1 C57Blk6 (wt) 45 days C57Blk6 (wt) 64 days Jck (disease model ) 45 days Jck (disease model ) 64 days CD1(wt) Pcy (disease model ) 4-15 wk Pcy (disease model )15-30 wk
30 Quantitation of Glycosphingolipids Using 2D-LC-MS/MS Method
31 Time, min Reversed Phase Chromatography Separates GLs According to the Lengths of Their Fatty Acid Chains and Not By the Sugar Moieties Luna C8, 3u, 2x100mm Mobile Phase A= 95%ACN, 5%MeOH, 5mM NH4OOC, 0.2% FA Mobile Phase B= MeOH, 5 mm NH4OOC, 0.2% FA 250ul/min 50% B isocratic,. Q-Star TOF, EIC 1.91 Max cps GM 3 20ug/ml : C16, C22, C Max cps GL 2 2ug/ml: C16, C20, C22, C Max cps. Max cps GL1 1ug/ml: C18, C20, C22, C Max cps CER 1ug/ml : C18, C22, C
32 HILIC Chromatography Separates GLs According to their Sugar Moieties and Not by the Fatty Acid Chains Atlantis HILIC Silica, 2.1 x 150mm, 3um GL1 Mobile Phase A = 95%ACN, 4.8%MeOH, 5mM NH 4 F, 0.2% FA Mobile Phase B = MeOH, 4.8 mm NH 4 F, 0.2% FA 250 ul/min, 55C Q-Star TOF, EIC 2% B to 10%B in 2 min,10%b to 40%B in 0.1min ; hold 2 min, re-equilibrate 6min at 2%B, 250 ul/min. Max cps. CER GM3 GL Time, min
33 Analysis of GM3 - GL1 Mixture Using both Columns Connected in Sequence Atlantis HILIC Silica, 2.1 x 150mm, 3um Luna C8, 3u, 2x50mm Summed EIC of +TOF Sample 3 (GL1 & GM3 at 20ug/ml ) Max. 1.4e4 cps Time, min GL1 C16, C22, C Max cps Time, min GM3 C16, C22,C24 Gradient used in the 1st dimension works as a 100% organic isocratic mode in the 2nd (RP) dimension.
34 Analysis of CER GM3 Mixture Using Two Columns Connected in Sequence Atlantis HILIC Silica, 2.1 x 150mm, 3um Luna C8, 3u, 2x50mm Summed EIC of +TOF Sample 3 (CER & GM3 at 20ug/ml) Max. 1.2e4 cps CER C16, 18, 22, Max cps GM3 C16, 22, 24 Time, min
35 Comments: Direct hyphenation of normal and reversed phase separations on a single HPLC system was demonstrated for the 2D separation of GLs The presented on-line 2D separation system is fully compatible with ESI- MS/MS analysis Dramatic increase of the separation window for gangliosides was achieved in this 2D setup, which allowed for monitoring of GM3, GL2, GL1 and CER transitions in the same LC-MS/MS run.
36 Conclusions: We have demonstrated a new set of tools for quantitative targeted lipidomics These tools were successfully applied for analysis of many tissue types, including kidney, liver, muscle, adipose and blood serum New normalization approach simplified comparison of different animal models and disease states
37 Acknowledgments: Analytical R&D Aharon Cohen Bing Wang Eva Budman Clifford Phaneuf Alla Kloss Lingyun Li Kim Alving John Bailey Biology: Tom Natoli
38 Distribution of Glucosylceramide Isoforms for a Mice Kidney Sample and a Matreya Standard 3.00E E+06 Sample mice kidney Standard 2.00E+06 MRM, Are 1.50E E E E+00 C16 GL1 C18 GL1 C22 GL1 C23 GL1 C24:1GL1 C24 GL1
39 In this sequential setup GLs are separated first by their sugar moieties on the HILIC column (1 st dimension = normal phase separation), and then additional separation by the length of the fatty acid chain is achieved on the Luna column (2 nd dimension = reversed phase separation). Gradient used in the 1 st dimension works as a 100% organic isocratic mode in the 2 nd (RP) dimension. The last feature allows to use this 2D methodology in on-line flow through mode. Dramatic increase of the separation window for gangliosides was achieved in this 2D setup, which allowed for monitoring of GM3, GL2, GL1 and CER transitions in the same LC-MS/MS run.
40 Fragmentation of GM3 in the ESI-MS Source +TOF MS: to min from Sample 1 (GL1 and GM3 at 20 ug/ml) Cer LC - QSTAR Results Max counts. Cer Cer Cer GL1 GL2 GM m/z, amu Multiple GLs with shorter sugar chains are produced as result of fragmentation in ESI, which reduces selectivity of the MRM detection. Therefore separation of different GLs prior to MS/MS analysis is crucial.
41 Method for Phospholipid Analysis using HILIC Chromatography: Instrument UPLC-API 4000 LC method: Mobil Phase A: 96:2:1:1 (ACN:MeOH:Acetic acid:water, 5mM Ammonium Acetate) Mobil Phase B: 98:1:1 (MeOH:Acetic Acid:Water, 5mM Ammonium Acetate) Column: Phenomenex Luna 50 x 2mm 3 micron HILIC MS/MS method: Period 1 Scan type: precursor Polarity: positive Precursor of: 184 amu Period 2 Scan type: neutral loss Polarity: positive Neutral loss of: 141 amu Gradient profile corrected for system volume 20 % B PC SM PE Phosphatidylcholine Sphingomyelin Phosphatidylethanolamine PC SM PE minutes
The use of mass spectrometry in lipidomics. Outlines
The use of mass spectrometry in lipidomics Jeevan Prasain jprasain@uab.edu 6-2612 utlines Brief introduction to lipidomics Analytical methodology: MS/MS structure elucidation of phospholipids Phospholipid
More informationUsing Hydrophilic Interaction Chromatography (HILIC) for the Retention of Highly Polar Analytes
Using Hydrophilic Interaction Chromatography (HILIC) for the Retention of Highly Polar Analytes Eric S. Grumbach,, Diane M. Diehl and Jeffrey R. Mazzeo 2003 Waters Corp. Outline Introduction HILIC Definition
More informationPhospholipid characterization by a TQ-MS data based identification scheme
P-CN1716E Phospholipid characterization by a TQ-MS data based identification scheme ASMS 2017 MP-406 Tsuyoshi Nakanishi 1, Masaki Yamada 1, Ningombam Sanjib Meitei 2, 3 1 Shimadzu Corporation, Kyoto, Japan,
More informationMass Spectrometry based metabolomics
Mass Spectrometry based metabolomics Metabolomics- A realm of small molecules (
More informationA Definitive Lipidomics Workflow for Human Plasma Utilizing Off-line Enrichment and Class Specific Separation of Phospholipids
A Definitive Lipidomics Workflow for Human Plasma Utilizing Off-line Enrichment and Class Specific Separation of Phospholipids Jeremy Netto, 1 Stephen Wong, 1 Federico Torta, 2 Pradeep Narayanaswamy, 2
More informationLipid Class Separation Using UPC 2 /MS
Michael D. Jones, 1,3 Giorgis Isaac, 1 Giuseppe Astarita, 1 Andrew Aubin, 1 John Shockcor, 1 Vladimir Shulaev, 2 Cristina Legido-Quigley, 3 and Norman Smith 3 1 Waters Corporation, Milford, MA, USA 2 Department
More informationRapid Lipid Profiling of Serum by Reverse Phase UPLC-Tandem Quadrupole MS
Rapid Lipid Profiling of Serum by Reverse Phase UPLC-Tandem Quadrupole MS Mark Ritchie and Evelyn Goh Waters Pacific Pte Ltd., Singapore A P P L I C AT ION B E N E F I T S Delivers a rapid 10-min MRM method
More informationComprehensive Two-Dimensional HPLC and Informative Data Processing for Pharmaceuticals and Lipids
PO-CON1576E Comprehensive Two-Dimensional HPLC and Informative Data Processing for Pharmaceuticals and Lipids HPLC 2015 PSB-MULTI-06 Yoshiyuki WATABE, Tetsuo IIDA, Daisuke NAKAYAMA, Kanya TSUJII, Saki
More informationMass-Spectrometric Analysis of Lipids (Lipidomics)
Mass-Spectrometric Analysis of Lipids (Lipidomics) 1. Identification 2. Quantification 3. Metabolism Why to do lipidomics? Biology: Functions of different lipids? Medicine: Diagnostics and Therapy Industry:
More informationLC/MS Method for Comprehensive Analysis of Plasma Lipids
Application Note omics LC/MS Method for Comprehensive Analysis of Plasma s Authors Tomas Cajka and Oliver Fiehn West Coast Metabolomics Center, University of California Davis, 451 Health Sciences Drive,
More informationDr. Erin E. Chambers Waters Corporation. Presented by Dr. Diego Rodriguez Cabaleiro Waters Europe Waters Corporation 1
Development of an SPE-LC/MS/MS Assay for the Simultaneous Quantification of Amyloid Beta Peptides in Cerebrospinal Fluid in Support of Alzheimer s Research Dr. Erin E. Chambers Waters Corporation Presented
More informationSupporting Information
Supporting Information Development of a High Coverage Pseudotargeted Lipidomics Method Based on Ultra-High Performance Liquid Chromatography-Mass Spectrometry Qiuhui Xuan 1,2#, Chunxiu Hu 1#, Di Yu 1,2,
More informationDevelopment of a Bioanalytical Method for Quantification of Amyloid Beta Peptides in Cerebrospinal Fluid
Development of a Bioanalytical Method for Quantification of Amyloid Beta Peptides in Cerebrospinal Fluid Joanne ( 乔安妮 ) Mather Senior Scientist Waters Corporation Data courtesy of Erin Chambers and Mary
More informationSupporting information
Supporting information Figure legends Supplementary Table 1. Specific product ions obtained from fragmentation of lithium adducts in the positive ion mode comparing the different positional isomers of
More informationLC-Based Lipidomics Analysis on QTRAP Instruments
LC-Based Lipidomics Analysis on QTRAP Instruments Junhua Wang and Paul RS Baker SCIEX LC-Based Lipidomics Analysis Topics Covered Lipid extraction techniques Hydrophilic Interaction Chromatography (HILIC)
More informationImpact of Chromatography on Lipid Profiling of Liver Tissue Extracts
Impact of Chromatography on Lipid Profiling of Liver Tissue Extracts Application Note Clinical Research Authors Mark Sartain and Theodore Sana Agilent Technologies, Inc. Santa Clara, California, USA Introduction
More informationWhat You Can t See Can Hurt You. How MS/MS Specificity Can Bite Your Backside
What You Can t See Can Hurt You How MS/MS Specificity Can Bite Your Backside Johan van den Heever, Tom Thompson, and Don Noot Agri-Food Laboratories Branch Advances in Trace rganic Residue Analysis early
More informationQuantification of PtdInsP 3 molecular species in cells and tissues by mass spectrometry
Nature Methods Quantification of PtdInsP 3 molecular species in cells and tissues by mass spectrometry Jonathan Clark, Karen E Anderson, Veronique Juvin, Trevor S Smith, Fredrik Karpe, Michael J Wakelam,
More informationPerformance of an ultra low elution volume 96-well plate
Performance of an ultra low elution volume 96-well plate Claude R. Mallet, Ziling Lu, Jeff R. Mazzeo, Uwe D. Neue Waters Corporation PittCon 2003 March 10-14 2003 Orlando, Florida Today s Challenges Faced
More informationSelexION Technology for Lipid Analysis: Pushing the Boundaries of Lipidomics
ANSWERS FOR SCIENCE. KNOWLEDGE FOR LIFE. SelexION Technology for Lipid Analysis: Pushing the Boundaries of Lipidomics Baljit Ubhi, Ph.D ASMS Baltimore, June 2014 Lipidomics Profiling Needs and Deliverables
More informationA Novel Solution for Vitamin K₁ and K₂ Analysis in Human Plasma by LC-MS/MS
A Novel Solution for Vitamin K₁ and K₂ Analysis in Human Plasma by LC-MS/MS By Shun-Hsin Liang and Frances Carroll Abstract Vitamin K₁ and K₂ analysis is typically complex and time-consuming because these
More informationThe Raptor HILIC-Si Column
The Raptor HILIC-Si Column With Raptor LC columns, Restek chemists became the first to combine the speed of superficially porous particles (also known as SPP or core-shell particles) with the resolution
More informationSupplementary Information
Supplementary Information Molecular imaging of brain localization of liposomes in mice using MALDI mass spectrometry Annabelle Fülöp 1,2, Denis A. Sammour 1,2, Katrin Erich 1,2, Johanna von Gerichten 4,
More informationCore E Analysis of Neutral Lipids from Human Plasma June 4, 2010 Thomas J. Leiker and Robert M. Barkley
Core E Analysis of Neutral Lipids from Human Plasma June 4, 2010 Thomas J. Leiker and Robert M. Barkley This protocol describes the extraction and direct measurement of cholesterol esters (CEs) and triacylglycerols
More informationUPLC/MS Monitoring of Water-Soluble Vitamin Bs in Cell Culture Media in Minutes
UPLC/MS Monitoring of Water-Soluble Vitamin Bs in Cell Culture Media in Minutes Catalin E. Doneanu, Weibin Chen, and Jeffrey R. Mazzeo Waters Corporation, Milford, MA, U.S. A P P L I C AT ION B E N E F
More informationLipids ON HPLC COLUMNS
Lipids N HPLC CLUMNS No.TI17E cis - trans Isomers of 9-octadecenoic acid 2 1 1 2 mau 5 1 15 2 min Cadenza CD-C18, 15 x 4.6 mm ACN / water / formic acid = 9 / 1 /.5.8 ml/min, 37 C UV at 215 nm, 3.7 MPa
More informationSimple Method (IS-MRM) to Monitor Lysophospholipids and Phospholipids During LC-MS Method Development via In-Source CID
Simple Method (IS-MRM) to Monitor Lysophospholipids and Phospholipids During LC-MS Method Development via In-Source CID James Little, Eastman Chemical Company, Kingsport, TN Overview Phospholipids and
More informationHiroya Hidaka *1), Masaki Takiwaki 2), Mine Yamashita 2), Shinya Otsuki 1), Kenji Kawasaki 3), Mitsutoshi Sugano 3) and Takayuki Honda 4)
Mild acid hydrolysis of sphingolipids yields lysosphingolipids: a matrix-assisted laser desorption and ionization time-of-flight mass spectrometry study Hiroya Hidaka *1), Masaki Takiwaki 2), Mine Yamashita
More informationSUPPLEMENTARY DATA. Materials and Methods
SUPPLEMENTARY DATA Materials and Methods HPLC-UV of phospholipid classes and HETE isomer determination. Fractionation of platelet lipid classes was undertaken on a Spherisorb S5W 150 x 4.6 mm column (Waters
More informationSPME-LC Fibers for a Variety of Applications
SPME-LC Fibers for a Variety of Applications Robert Shirey 1, Craig Aurand 1, Dajana Vuckovic 2, Katherine Stenerson 1, Yong Chen 1, Leonard Sidisky 1, and Janusz Pawliszyn 2 1 Supelco, Div. of Sigma-Aldrich,
More informationAutomated Purification and Analytical Reinjection of a Small Molecule Drug, Probenecid, on a Gilson LC/MS Dual Function System
Automated Purification and Analytical Reinjection of a Small Molecule Drug, Probenecid, on a Gilson LC/MS Dual Function System Keywords Introduction Application Note PHA0413 High Pressure Liquid Chromatography
More informationEnrichment of Phospholipids from Biological Matrices with Zirconium Oxide-Modified Silica Sorbents
Enrichment of Phospholipids from Biological Matrices with Zirconium Oxide-Modified Silica Sorbents Xiaoning Lu, Jennifer E. Claus, and David S. Bell Supelco, Div. of Sigma-Aldrich Bellefonte, PA 16823
More informationGlycerolipid Analysis. LC/MS/MS Analytical Services
Glycerolipid Analysis LC/MS/MS Analytical Services Molecular Characterization and Quantitation of Glycerophospholipids in Commercial Lecithins by High Performance Liquid Chromatography with Mass Spectrometric
More informationMASS SPECTROMETRY BASED METABOLOMICS. Pavel Aronov. ABRF2010 Metabolomics Research Group March 21, 2010
MASS SPECTROMETRY BASED METABOLOMICS Pavel Aronov ABRF2010 Metabolomics Research Group March 21, 2010 Types of Experiments in Metabolomics targeted non targeted Number of analyzed metabolites is limited
More informationChip-Based E-Tip SPE followed by Infusion MS. Copyright by Jack Henion, 2015 Lecture 1, Page 30
Chip-Based E-Tip SPE followed by Infusion MS Copyright by Jack Henion, 2015 Lecture 1, Page 30 Infusion -MS Analysis of Sample Mixtures HPLC Mass Spec Elution/spray solvent Pipette tip with micro SPE packing
More informationI. Structure and Properties of Lipids
I. Structure and Properties of Lipids Lipids: A diverse group of compounds characterized by their low solubility in water and a high solubility in organic solvents such as chloroform and methanol. Nonpolar
More informationMethod Development for the Analysis of Endogenous Steroids Using Convergence Chromatography with Mass Spectrometric Detection
Method Development for the Analysis of Endogenous Steroids Using Convergence Chromatography with Mass Spectrometric Detection Christopher J. Hudalla, Stuart Chadwick, Fiona Liddicoat, Andrew Peck, and
More informationHigh-throughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines
High-throughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines Darren S. Dumlao, Matthew W. Buczynski, Paul C. Norris, Richard Harkewicz and Edward A. Dennis. Biochimica et
More informationAbout bioassay of Oximes:? New isolation alternatives from biomatrices? Chromatographic separation issues? Reserve on using MS or MS/MS detection
State of the art: xime-type AChE Reactivators: Wide use as antidotes in organo phosphorous compounds poisoning; Synthesis of new congeners; Intense studies on their transfer through BBB; Intense studies
More informationIon Exchange and Reversed Phase interactions in selective Bio-SPME extractions of designer drugs
Ion Exchange and Reversed Phase interactions in selective Bio-SPME extractions of designer drugs Frank Michel, Craig R. Aurand, Robert E. Shirey, Yong Chen, Leonard M. Sidisky sigma-aldrich.com/analytical
More informationLipids Analysis. Lipids
Lipids Analysis Stephen Barnes 3 5 15 Lipids Lipids are mostly very hydrophobic Most are conjugates of fatty acids of a variety of chain lengths, which have different degrees of unsaturation, cis trans
More informationAnalysis of HMF by HPLC
COST Action 927 Training School Building Skills on the Analysis of Thermal Process Contaminants in Foods 22-26 October 2007, Ankara Analysis of HMF by HPLC Vural Gökmen O O OH Background O COOH O R 2 Carbonyl
More informationSample Preparation is Key
PLOS ONE DOI: 10.1371/journal.pone.0117232 February 6, 2015 Presented by Katie Gibbs Sample Preparation is Key Sample extraction and instrumental analysis methods are well documented in metabolomics. Understanding
More informationRapid Analysis of Water-Soluble Vitamins in Infant Formula by Standard-Addition
Rapid Analysis of Water-Soluble Vitamins in Infant Formula by Standard-Addition Evelyn Goh Waters Pacific, Singapore APPLICATION BENEFITS This method allows for the simultaneous analysis of 12 water-soluble
More informationMEMBRANE LIPIDS I and II: GLYCEROPHOSPHOLIPIDS AND SPHINGOLIPIDS
December 6, 2011 Lecturer: Eileen M. Lafer MEMBRANE LIPIDS I and II: GLYCEROPHOSPHOLIPIDS AND SPHINGOLIPIDS Reading: Stryer Edition 6: Chapter 26 Images: All images in these notes were taken from Lehninger,
More informationUMR 8612, Faculty of Pharmacy Chatenay-Malabry. Natura-Brasil. EA Laboratory of Dermatological Research,
Iuliana Popa 1, Noëlle Remoué 2 and Jacques Portoukalian 3 1 UMR 8612, Faculty of Pharmacy Chatenay-Malabry 2 Natura-Brasil 3 EA 41 69 Laboratory of Dermatological Research, University of Lyon I, Faculty
More informationPrinciples of Shotgun Lipidomics
Principles of Shotgun Lipidomics Xianlin Han Diabetes and Obesity Research Center Sanford-Burnham Medical Research Institute Lake Nona Orlando, FL 32827 What is shotgun lipidomics? Original definition
More informationAccurate Quantification of Lipid Species by Electrospray Ionization Mass Spectrometry Meets a Key Challenge in Lipidomics
Metabolites 2011, 1, 21-40; doi:10.3390/metabo1010021 Review OPEN ACCESS metabolites ISSN 2218-1989 www.mdpi.com/journal/metabolites/ Accurate Quantification of Lipid Species by Electrospray Ionization
More informationAdvancing your Forensic Toxicology Analyses; Adopting the Latest in Mass Spectrometry Innovations
Advancing your Forensic Toxicology Analyses; Adopting the Latest in Mass Spectrometry Innovations For Research Use Only. Not for use in diagnostic procedures. 1 2015 AB Sciex RUO-MKT-11-1018-A For research
More informationThe detergent-solubilized and gel filtration purified rhodopsin was partitioned against
Supplement Jastrzebska et al. Materials and Methods The detergent-solubilized and gel filtration purified rhodopsin was partitioned against H 2 O/MeOH/CHCl 3, and the bottom layer was removed, dried down,
More informationRelative Quantitation of Human Polymorphonuclear Leukocyte Cell Membrane GPEtn Lipids
Relative Quantitation of Human Polymorphonuclear Leukocyte Cell Membrane GPEtn Lipids Using the QTRAP System with mtraq Reagents Karin A. Zemski-Berry 1, John M. Hevko 2, and Robert C. Murphy 1 1 Department
More informationObtaining Answers to Biological Questions Sample Prep to Data Analysis. Jeremiah D. Tipton, Ph.D. SCIEX Advanced Workflow Specialist in OMICS
Obtaining Answers to Biological Questions Sample Prep to Data Analysis Jeremiah D. Tipton, Ph.D. SCIEX Advanced Workflow Specialist in OMICS OMICS Research Why Quantitative OMICS? 2 2015 AB Sciex SCIEX
More informationLipids. Lipids: a Diverse group of chemicals. Storage Lipids: derivatives of fatty acids. 11/21/10
1 Lipids Lehninger 3 rd ed. Chapter 11 (For biosynthesis see Chapter 21) 2 Lipids: a Diverse group of chemicals Insolubility in water. Fats and oils: energy stores. Phospholipids and sterols: structural
More informationO O H. Robert S. Plumb and Paul D. Rainville Waters Corporation, Milford, MA, U.S. INTRODUCTION EXPERIMENTAL. LC /MS conditions
Simplifying Qual/Quan Analysis in Discovery DMPK using UPLC and Xevo TQ MS Robert S. Plumb and Paul D. Rainville Waters Corporation, Milford, MA, U.S. INTRODUCTION The determination of the drug metabolism
More informationOverview on the identification of different classes of. lipids by HPTLC (High Performance Thin Layer. Chromatography) and ITLC (Immuno Thin Layer
Overview on the identification of different classes of lipids by HPTLC (High Performance Thin Layer Chromatography) and ITLC (Immuno Thin Layer Chromatography) Iuliana Popa 1, Marie-Jeanne David 2, Daniel
More informationThe Development of LC/MS Methods for Determination of Polar Drugs of Abuse in Biological Samples
WA20259 The Development of LC/MS Methods for Determination of Polar Drugs of Abuse in Biological Samples Michael S. Young and Kevin M. Jenkins Waters Corporation, 34 Maple Street, Milford, MA 01757 Introduction
More informationNew Solvent Grade Targeted for Trace Analysis by UHPLC-MS
New Solvent Grade Targeted for Trace Analysis by UHPLC-MS Subhra Bhattacharya, Deva H. Puranam, and Stephen C. Roemer Thermo Fisher Scientific Fisher Chemical, One Reagent Lane, Fair Lawn, NJ Material
More informationThe HPLC Preparative Scale-Up of Soybean Phospholipids Application
The HPLC Preparative Scale-Up of Soybean Phospholipids Application Food and Flavors Authors Cliff Woodward and Ron Majors Agilent Technologies, Inc. 285 Centerville Road Wilmington, DE 1988-161 USA Abstract
More informationNeonatal Screening for Lysosomal Storage Disorders (LSD) by Tandem Mass Spectrometry (MSMS)
Neonatal Screening for Lysosomal Storage isorders (LS) by Tandem Mass Spectrometry (MSMS) Enzo Ranieri and Samantha Stark 1 Head, Neonatal Screening Centre & Biochemical Genetics (G&MP) irectorate of Genetics
More informationAnalysis of Testosterone, Androstenedione, and Dehydroepiandrosterone Sulfate in Serum for Clinical Research
Analysis of Testosterone, Androstenedione, and Dehydroepiandrosterone Sulfate in Serum for Clinical Research Dominic Foley, Michelle Wills, and Lisa Calton Waters Corporation, Wilmslow, UK APPLICATION
More informationComprehensive Lipid Profiling of Human Liver Tissue Extracts of Non-Alcoholic Fatty Liver Disease
Comprehensive Lipid Profiling of Human Liver Tissue Extracts of Non-Alcoholic Fatty Liver Disease Multiplexed Precursor Ion Scanning and LipidView Software Brigitte Simons 1 and Bianca Arendt 2 1 AB SCIEX,
More informationLC-MS/MS analysis of Chlorates in Milk and Whey Powder using the Agilent 6470 QQQ
LC-MS/MS analysis of Chlorates in Milk and Whey Powder using the Agilent 6470 QQQ Anthony Sullivan, LC/MS Product Specialist Melanie Mülek and Christoph Müller LC-MS Applications Specialists Hewlett-Packard-Str.
More informationDetection of Low Level of Chloramphenicol in Milk and Honey with MIP SPE and LC-MS-MS
Detection of Low Level of Chloramphenicol in Milk and Honey with MIP SPE and LC-MS-MS Olga Shimelis, An Trinh, and Michael Ye Supelco, Div. of Sigma-Aldrich, Bellefonte, PA T407125 Introduction Molecularly
More informationSUPPORTING INFORMATION:
1S SUPPORTING INFORMATION: The discovery of the once weekly glucagon like peptide 1 (GLP-1) analog semaglutide Jesper Lau*, Paw Bloch, Lauge Schäffer, Ingrid Pettersson, Jane Spetzler, Jacob Kofoed, Kjeld
More informationTest Bank for Lehninger Principles of Biochemistry 5th Edition by Nelson
Test Bank for Lehninger Principles of Biochemistry 5th Edition by Nelson Link download full: http://testbankair.com/download/test-bank-forlehninger-principles-of-biochemistry-5th-edition-by-nelson/ Chapter
More informationRobust extraction, separation, and quantitation of structural isomer steroids from human plasma by SPE-UHPLC-MS/MS
TECHNICAL NOTE 21882 Robust extraction, separation, and quantitation of structural isomer steroids human plasma by SPE-UHPLC-MS/MS Authors Jon Bardsley 1, Kean Woodmansey 1, and Stacy Tremintin 2 1 Thermo
More informationAgilent 6470 Triple Quadrupole LC/MS System and EMR-Lipid. The Combination for Confident Pesticide Quantitation. Christoph Mueller
Agilent 670 Triple Quadrupole LC/MS System and EMR-Lipid The Combination for Confident Pesticide Quantitation Christoph Mueller Agilent Technologies LCMS Application Scientist EMEAI Food Market Program
More informationMetabolomics Core Lab School of Medicine University of Utah
Implementation Standard Operating Procedure-Sample Preparation for LC-MS 1. Purpose To prepare and analyze samples for LC-MS and/or LC-MS-MS lipidomics. 2. Scope This SOP applies to all LC-MS samples submitted
More informationFinnur Freyr Eiríksson
Effects of protolichesterinic acid isolated from Cetraria islandica on lipid composition in cultured cancer cells evaluated using HPLC-MS/MS Finnur Freyr Eiríksson Båstad, 9. nov 2011 Outline Introduction
More informationSeparation of Vitamin D and Vitamin D Metabolites on FLARE C18 MM (Mixed Mode) HPLC Column
Diamond Analytics Technical Note: T131-1 Separation of Vitamin D and Vitamin D Metabolites on FLARE C1 MM (Mixed Mode) HPLC Column Introduction In this technical note, the versatility of the diamond-based
More informationVitamin D3 and related compounds by ESI and APCI
Liquid Chromatography Mass Spectrometry SSI-LCMS-9 Vitamin D and related compounds by ESI and APCI LCMS-8 Summary Vitamin D and related compounds were measured by LC-ESI/APCI-MS-MS. Background Accurate
More informationAnalysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS)
Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS) Cameron Sullards, Ying Liu, Yanfeng Chen and Alfred H. Merrill
More informationLOCALISATION, IDENTIFICATION AND SEPARATION OF MOLECULES. Gilles Frache Materials Characterization Day October 14 th 2016
LOCALISATION, IDENTIFICATION AND SEPARATION OF MOLECULES Gilles Frache Materials Characterization Day October 14 th 2016 1 MOLECULAR ANALYSES Which focus? LOCALIZATION of molecules by Mass Spectrometry
More informationSupporting information
Supporting information A novel lipidomics workflow for improved human plasma identification and quantification using RPLC-MSn methods and isotope dilution strategies Evelyn Rampler 1,2,3, Angela Criscuolo
More informationA Robustness Study for the Agilent 6470 LC-MS/MS Mass Spectrometer
A Robustness Study for the Agilent 7 LC-MS/MS Mass Spectrometer Application Note Clinical Research Authors Linda Côté, Siji Joseph, Sreelakshmy Menon, and Kevin McCann Agilent Technologies, Inc. Abstract
More informationLC-MS-based Metabolomics: Workflows, Strategies and Challenges
LC-MS-based Metabolomics: Workflows, Strategies and Challenges Presented by: Sponsored by: Dr. Clary Clish Director of the Metabolite Profiling Platform at the Broad Institute of MIT and Harvard LC-MS-based
More informationQuantitative analysis of sphingolipids for lipidomics using triple quadrupole and. quadrupole linear ion trap mass spectrometers
Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers Revised D8:00035-JLR (11-16-2008) Rebecca L. Shaner *, Jeremy C. Allegood
More informationMASS SPECTROMETRY IN METABOLOMICS
For personal use only. Please do not reuse or reproduce without the author s permission MASS SPECTRMETRY IN METABLMICS Pavel Aronov Stanford Mass Spectrometry Users Meeting August 21, 2008 rigin of Metabolomics
More informationFast quantitative Forensic Analysis of THC and its Metabolites in Biological Samples using Captiva EMR- Lipid and LC/MSMS
Fast quantitative Forensic Analysis of THC and its Metabolites in Biological Samples using Captiva EMR- Lipid and LC/MSMS Christophe Deckers, M.Sc. Sample prep Application Scientist Types of ``Interferences``
More informationUpdate on the Development of a Sensitive, Accurate, and User- Friendly Method for the Direct Determination of 3-MCPD 3
Update on the Development of a Sensitive, Accurate, and User- Friendly Method for the Direct Determination of 3-MCPD 3 Esters J. D. Pinkston, P.J. Stoffolano, P. Y. Lin The Procter & Gamble Company, Winton
More informationEssential Lipidomics Experiments using the LTQ Orbitrap Hybrid Mass Spectrometer
Application Note: 367 Essential Lipidomics Experiments using the LTQ rbitrap Hybrid Mass Spectrometer Thomas Moehring 1, Michaela Scigelova 2, Christer S. Ejsing 3, Dominik Schwudke 3, Andrej Shevchenko
More informationSunil Kulkarni Product Specialist Agilent Technologies
Drug Residue Analysis by LC/MS methods Sunil Kulkarni Product Specialist Agilent Technologies Outline General Drug Residue information Veterinary drug residue Analysis by LC/MS QQQ and Q-TOF method Analytical
More informationHigh Throughput Extraction of Opiates from Urine and Analysis by GC/MS or LC/MS/MS)
High Throughput Extraction of Opiates from Urine and Analysis by GC/MS or LC/MS/MS) Michael Rummel, Matthew Trass, Michael Campognone, and Sky Countryman Phenomenex, Inc., 411 Madrid Avenue, Torrance,
More informationAnalytical Challenges in Veterinary Toxicology: Bromethalin
Analytical Challenges in Veterinary Toxicology: Bromethalin Mike Filigenzi, Birgit Puschner, Linda Aston, Robert Poppenga CAHFS-UC Davis School of Veterinary Medicine First things first: Acknowledgements
More informationSimultaneous Analysis of Intact Human Insulin and Five Analogs in Human Plasma Using μelution SPE and a CORTECS UPLC Column
Simultaneous Analysis of Intact Human Insulin and Five Analogs in Human Plasma Using μelution SPE and a CORTECS UPLC Column Erin E. Chambers and Kenneth J. Fountain Waters Corporation, Milford, MA, USA
More informationA RAPID AND SENSITIVE ANALYSIS METHOD OF SUDAN RED I, II, III & IV IN TOMATO SAUCE USING ULTRA PERFORMANCE LC MS/MS
A RAPID AD SESITIVE AALYSIS METD OF SUDA RED I, II, III & IV I TOMATO SAUCE USIG ULTRA PERFORMACE LC MS/MS Choon Keow G, aomi TAAKA, Michelle KIM, Swee Lee YAP Waters Asia, Regional Technology Center,
More informationProbing for Packaging Migrants in a Pharmaceutical Impurities Assay Using UHPLC with UV and Mass Detection INTRODUCTION
Probing for Packaging Migrants in a Pharmaceutical Impurities Assay Using UHPLC with UV and Mass Detection Michael Jones Waters Corporation, Wilmslow, UK APPLICATION BENEFITS The ACQUITY Arc System is
More informationRapid, Simple Impurity Characterization with the Xevo TQ Mass Spectrometer
Robert Plumb, Michael D. Jones, and Marian Twohig Waters Corporation, Milford, MA, USA INTRODUCTION The detection and characterization of impurities and degradation products of an active pharmaceutical
More informationMetabolomic and Lipidomic Research in Diabetes and Obesity 08/07/2013
Novel Tools for omics -based Analysis of Complex Mixtures; Application to a Investigation of the Effect of Prolonged Glucose Stimulation on the Lipid Profile of Mouse Heart Muscle John P. Shockcor, PhD,
More informationDetermination of 6-Chloropicolinic Acid (6-CPA) in Crops by Liquid Chromatography with Tandem Mass Spectrometry Detection. EPL-BAS Method No.
Page 1 of 10 Determination of 6-Chloropicolinic Acid (6-CPA) in Crops by Liquid Chromatography with Tandem Mass Spectrometry Detection EPL-BAS Method No. 205G881B Method Summary: Residues of 6-CPA are
More informationMetabolomic fingerprinting of serum samples by direct infusion mass spectrometry
Metabolomic fingerprinting of serum samples by direct infusion mass spectrometry Raúl González-Domínguez * Department of Chemistry, Faculty of Experimental Sciences. University of Huelva, Spain. * Corresponding
More informationBiomed Pap Med Fac Univ Palacky Olomouc Czech Repub
SUPPLEMENTAL MATERIAL Sistik P, Turjap M, Iordache AM, Saldanha H, Lemr K, Bednar P. Quantification of selected antidepressants and antipsychotics in clinical samples using chromatographic methods combined
More information2D-LC as an Automated Desalting Tool for MSD Analysis
2D-LC as an Automated Desalting Tool for MSD Analysis Direct Mass Selective Detection of a Pharmaceutical Peptide from an MS-Incompatible USP Method Application Note Biologics and Biosimilars Author Sonja
More informationThe Comparison of High Resolution MS with Triple Quadrupole MS for the Analysis of Oligonucleotides
The Comparison of High Resolution MS with Triple Quadrupole MS for the Analysis of Oligonucleotides Mohammed Abrar Unilabs York Bioanalytical Solutions Outline Introduction Why LC-MS/MS? Limitations of
More informationApplying a Novel Glycan Tagging Reagent, RapiFluor-MS, and an Integrated UPLC-FLR/QTof MS System for Low Abundant N-Glycan Analysis
Applying a Novel Glycan Tagging Reagent, RapiFluor-MS, and an Integrated UPLC-FLR/QTof MS System for Low Abundant N-Glycan Analysis Ying Qing Yu Waters Corporation, Milford, MA, USA APPLICATION BENEFITS
More informationSurviving Matrix Effects Experiments. Grace van der Gugten St. Paul s Hospital, Vancouver, BC, Canada
Surviving Matrix Effects Experiments Grace van der Gugten St. Paul s Hospital, Vancouver, BC, Canada gvandergugten@providencehealth.bc.ca Outline What are matrix effects Post Column Infusion Phospholipid
More informationHuman Complex Milk Lipids: Concentrations, benefits and the implications for Paediatric Nutrition
Human Complex Milk Lipids: Concentrations, benefits and the implications for Paediatric Nutrition Alastair MacGibbon, Bertram Fong, Angela Rowan and Paul McJarrow Fonterra Research and Development Centre,
More informationSPE-LC-MS/MS Method for the Determination of Nicotine, Cotinine, and Trans-3-hydroxycotinine in Urine
SPE-LC-MS/MS Method for the Determination of Nicotine, Cotinine, and Trans-3-hydroxycotinine in Urine J. Jones, Thermo Fisher Scientific, Runcorn, Cheshire, UK Application Note 709 Key Words SPE, SOLA
More informationJose Castro-Perez, Henry Shion, Kate Yu, John Shockcor, Emma Marsden-Edwards, Jeff Goshawk Waters Corporation, Milford, MA, U.S. and Manchester, UK
HIGH-THRUGHPUT REACTIVE METABLITE SCREEIG FR DICLFEAC BY UPLC AD XEV TQ MS WITH SCAWAVE Jose Castro-Perez, Henry Shion, Kate Yu, John Shockcor, Emma Marsden-Edwards, Jeff Goshawk Waters Corporation, Milford,
More information