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The Link from HPLC to Isotope RatioMass Spectrometry
Thermo Scientific LC IsoLink™
Part of Thermo Fisher Scientific
m a s s s p e c t r o m e t r y
Carbohydrates
Amino Acids, Nucleotides
Organic Acids
Pharmaceuticals
ThermoScientific
LCIsoLink
The Thermo Scientific LC IsoLink is the first highsensitivity interface connecting HPLC with IsotopeRatio MS (IRMS) for reproducible and accurateon-line determination of 13C/12C isotope ratios. Allorganic compounds eluting from an HPLC columnare analyzed while maintaining the chromato-graphic resolution, opening up a new frontier in theapplication of isotope ratio mass spectrometry.
Unique Features of the LC IsoLink:
• On-line coupling of HPLC and Isotope Ratio MS
• Isotope ratio data acquired on an HPLC time scale
• Preserves the integrity of chromatographic resolution
• Sample analysis in the low nanomole range
• Accurate and reproducible results
• Direct injection of reference materials
• Maintenance-free operation
• High throughput
• Capability for full automation
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The Link from HPLC toIsotope Ratio MS…
Carbohydrates are one of the largest andmost common families of molecules in life.They serve as energy resource, backbone ofDNA and RNA, carrier of biologically activecompounds, and as structure buildingmaterial in plants. Sugars can carryinformation of their origin and processing.If isotopically labeled, they tell us abouttheir pathways and use.
Because pharmaceuticals are producedat various sites with different prices anddifferent formulations, it is essential forboth the producers and consumers to beable to distinguish between originals andgenerics. Aspirin® and Paracetamol arerepresentatives of mass produced drugswith a wide range of origin and price.
The Thermo Scientific LC IsoLink is built todirectly address the analysis of polar andthermolabile compounds. Most of thesecompounds (such as amino acids, peptides,carbohydrates, nucleotides, alcohols,organic acids, sulfa drugs, and many more)can be chromatographically separated inaqueous phase using inorganic buffers onion exchange, size exclusion, or specificreversed phase HPLC columns. The LCIsoLink enables the on-line high precisiondetermination of the 13C/12C isotope ratioof each separated component.
• Metabolism Studies
– Tracer experiments withamino acids, lipids,carbohydrates
– Nucleotide analysis forDNA synthesis rates
• Food and Food Ingredients
– Authentication of honey andfruit juices
– Determining origin of flavorsand nutrients
– Analysis of phospholipids,carbohydrates, amino acidsand vitamins
• Pharmaceuticals
– Authenticity control of drugs
– Investigation of agentsand additives
• Doping control
– Analysis of steroidsand hormones
• Biogeochemistry
– History of biomolecules insediments and soils
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…for a wide Range ofApplications...
Sugars on a cation exchange column. Drugs on a HyPURITY AQUASTAR™ HPLC column.
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Until now, the analysis of organic samplesfor 13C/12C isotope ratios was restricted toeither on-line GC separation followed bycombustion (isotope ratio monitoringGC/MS, irm-GC/MS) or bulk combustion ofsamples in an organic elemental analyzer.Biologically or pharmaceutically importantcompounds are often not GC-volatile or toothermolabile to be separated by gaschromatography. 13C/12C isotope ratiodetermination of these compounds in realanalytical applications, typically withincomplex matrices, was so far eitherimpossible or extremely labor intensive.
*GB2388800 B, US7213443 B2
The patented LC IsoLink* leapfrogs thehurdle that, until now, has preventedsuccessful isotope ratio monitoring LC/MS,namely the quantitative, fractionation-freeelimination of the liquid phase prior to theoxidative conversion of the sample into CO2.
In the LC IsoLink, the liquid phase is notremoved from the sample prior to oxidationbut the sample is oxidized while still withinthe aqueous solution, and only afterwardsthe CO2 is separated from the liquid phasefor isotopic analysis. This process isquantitative and fractionation-free in theLC IsoLink.
The oxidation reagent consists of twosolutions, the oxidizing agent and phos-phoric acid. Both are pumped separatelyand added to the mobile LC phase.
All individual organic compoundseluting from the HPLC column are oxidizedquantitatively into CO2 within this mixturewhen passing through a heated reactor. Ina downstream degassing unit, the CO2 isremoved from the liquid phase andentrained into a stream of He.
The individual CO2 peaks in He aresubsequently dried in an on-line gasdrying unit (Nafion®) and then admittedto the Isotope Ratio MS via an open splitinterface.
Since the liquid phase is not vaporizedat all, inorganic buffers, e.g. phosphatebuffers, have no influence on the operationof the interface, and can not accumulateanywhere in the system. This is in markedcontrast to any other LC/MS interfaceand makes the LC IsoLink essentiallymaintenance free.
ThermoScientific
LCIsoLink
The Concept of HPLC Coupling to IRMS
Amino acids and peptides are the basic elements for buildingproteins. Their structure and metabolism are of major interest inmedicine and biology. Either naturally occurring isotope differencesor labelling experiments can be used to give insight into theirpathways.
Archaeological Bone CollagenThe reconstruction of ancient diets using body tissue stable isotopevalues is an important aspect of paleodietary studies which providearchaeological science with a quantitative understanding of whatpeople consumed in the past.
Isotopic studies of diet are based on the principle “You arewhat you eat”, and collagen, being a well-preserved protein foundin bone, has been most often used for these purposes.
Isotopic signatures of individual amino acids can differconsistently and widely from each other. More dietary informationis potentially available at the individual amino acid level.
Separation of up to 18 amino acids can be performed ona single HPLC column.
Short-chained peptides and amino acid on a C18 reversed phaseendcapped column.
irm-LC/MS chromatogram of archaeological bone collagen hydrolysate.
RNA Nucleotides: Molecular biologicalmethods based on detection, sequencing,and phylogenetic identification of ribosomalRNA (rRNA) and the genes encoding it(rDNA) allow description of naturalmicrobial communities without isolation andculturing of individual species. By carbonisotopic characterization of rRNA speciesidentity can be linked with carbon sourceutilization.
Organic acids play an important rolein the metabolism of plants. They areinvolved in various anabolic and catabolicprocesses of metabolic pathways, such ascitric acid cycle.
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Principle of HPLC Coupling to IRMS
m/z
44(m
V)
Time (s)
irm-LC/MS analysis of NaOH-hydrolyzed E. coli RNA, LB-grown. irm-LC/MS chromatogram (m/z 44 trace) of an organic acid mixture.
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ThermoScientific
LCIsoLink
Isodat Software Suite
The irm-LC/MS system is fully automatedand controlled by the Isodat software suite.The Thermo Scientific Surveyor™ LC iscompletely integrated, other HPLC systemsare synchronized by contact closure.
The LC IsoLink and the mass spectrom-eter are entirely under full computer control,resulting in analyses with no userinteraction. When coupled to anyautosampler, the system can be operatedfully unattended for high sample throughputand high productivity.
The irm-LC/MS does not require anymanual intervention during operation exceptfor refilling of the reagents. Data evaluationand report generation are fully automatedby Isodat software suite. Since the interfacehas only few variables, e.g. flow of oxidizingagent and He-flow, method developmentand operation are also exceedingly simple.
• Easy and fast method and sequencesetup for Isotope Ratio MS and LC IsoLink
• Complete control and automation of allinterface functions before and duringdata acquisition
• Automated peak and backgrounddetection
• Fully automated ion correction forisobaric ion contribution such as 12C17O16Oon 13C16O16O
• Full access to all raw and processed data
• Complete raw data integrity and securesample identity
• Software suite flexible export ofevaluated data to Excel, otherspreadsheet programs and databases(LIMS)
• Customized printouts and data export
• Full network compatibility with direct andfast access to Windows® tools
• Complete Isodat software suite systembackup using the Isodat Version Handler
• Based on the unique “Plug and Measure”concept of Thermo Scientific IsotopeRatio MS (Thermo Scientific DELTA VAdvantage, DELTA V Plus and MAT 253),the LC IsoLink is immediately recognizedand operational
Analysis of an Extract of GeraniumPratenseChanges in the isotopic signature oforganic acid are studied in the process ofontogenesis of plants. Investigations can beimportant to understand development ofplant metabolism and to estimate theimpact of environmental factors.The most dominant organic acids in anextract of Geranium Pratense are malic acidand citric acid. Malic acid is isotopicallyheavier than citric acid.
Plant metabolism study of organic acids.
Confirmation of SyntheticTestosteroneThe administration of synthetic testosteroneis detected by the testosterone/epitestos-terone (T/E) ratio in urine using GC/MS.Confirmation is performed by irm-GC/MS.
The 13C/12C ratio of synthetictestosterone differs from the 13C/12C ratioof endogenous testosterone.
The range of applications for theLC IsoLink can be extended by high
temperature HPLC. At elevated temperatures, water acts like aweak polar organic solvent. Therefore, it’s also capable ofseparating mid polar and even non-polar analytes. Steroids can beseparated under 100% aqueous conditions, selectivity can be tunedby temperature.
Analysis of steroids in urine (LC fraction) by high temperature HPLCcoupled to IRMS.
Accuracy, Precision and SensitivityAccuracy and precision has been testedusing standards with certified isotoperatios. The δ13C value of 400 ng of benzoicacid analyzed by repetitive loop injectionhas been shown to be correct within0.08‰ with a reproducibility of 0.11‰.These values compare favorably withthe accuracy and precision achievablewith bulk analysis by Elemental AnalyzerIsotope Ratio MS – but require 100 timesless sample.
The sensitivity of the integratedirm-LC/MS system is sufficient to allow theisotope ratio determination of 13C/12C onsample amounts as low as 50 ng in directinjection mode. The minimum amount inHPLC mode depends on the backgroundcreated by the HPLC column bleed.
Acceptance SpecificationsAfter installation, the system performanceof the LC IsoLink will be demonstrated bydirect loop injection, to show an externalprecision of < 0.3‰ for δ13C (n=5). Thisrequires an organic-free HPLC pump.
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Analytical Performance
Mass Spectrometer DELTA Series and MAT 253
LC IsoLink Basic Performance
10 pulses of reference gas (amplitude 3V), δ notationPrecision (1σ) Linearity
CO213C 0.06‰ 0.02‰/nA
External precision using the LC IsoLink (n=5, 1σ), δ notation
Benzoic Acid (direct loop injection)400 ng in 10 µL of water
3 nmol Benzoic Acid
21 nmol (250 ng) C as CO2 < 0.3‰
BR30039_E 03/08C
Thermo Fisher Scientific (Bremen) GmbHis certified DIN EN ISO 2001:9000
©2005, 2008 Thermo Fisher Scientific Inc. All rightsreserved. Aspirin is a registered trademark of Bayer AG.Nafion is a registered trademark of Dupont and/or itsaffiliates. Windows is a registered trademark of MicrosoftCorporation. All other trademarks are the property ofThermo Fisher Scientific Inc. and its subsidiaries.
Specifications, terms and pricing are subject to change.Not all products are available in all countries. Pleaseconsult your local sales representative for details.
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Laboratory Solutions Backed by Worldwide Service and Support
Installation Requirements
Power230 V single phase, 8 A
Gases
• He, better 99.999%• CO2, better 99.995%
Supplies
• Water (HPLC grade)• Reagent• Catalyst
Recommended Supplies
• Thermo Scientific Surveyor HPLCsystem with MS pump
• Thermo Scientific columns
LiteratureKrummen, M., A. W. Hilkert,D. Juchelka, A. Duhr, H.-J. Schlüterand R. Pesch: A new concept forisotope ratio monitoring liquidchromatography/mass spectrometry.Rapid Commun. Mass Spectrom.2004; 18: 2260-2266. USA, 1991,21.-26.05.1994, 875
Analytical data is available in theLC IsoLink Application Notes.
Thermo Scientific LC IsoLink Dimensions
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