Finnigan™ LC IsoLink

Analyze • Detect • Measure • Control™

Isotope Ratio

Mass Spectrometry

m a s s s p e c t r o m e t r y

The Finnigan LC IsoLink is the first high sensitivity interface connecting HPLC with Isotope Ratio MS forreproducible and accurate on-line determination of 13C/12C isotope ratios. All organic compounds elutingfrom an HPLC column are analyzed while maintaining the chromatographic resolution, opening up a newfrontier in the application of isotope ratio mass spectrometry.

• 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

Finnigan LC IsoLinkThe Link from

HPLC to Isotope Ratio M

S

Finnigan™ LC IsoLinkThe Link from HPLC to Isotope Ratio MS

The Linkfrom HPLC to

Isotope Ratio MS

A wide range ofapplicationsThe Finnigan LC IsoLink is builtto directly address the analysisof polar and thermo-labilecompounds. Most of thesecompounds (such as amino acids,peptides, carbohydrates,nucleotides, alcohols, organicacids, sulfa drugs, and manymore) can be chromato-graphically separated in aqueousphase using inorganic buffers onion exchange, size exclusion orspecific reversed phase HPLCcolumns. The Finnigan LC IsoLinkenables the on-line highprecision determination of the13C/12C isotope ratio of eachseparated component.

• Metabolism studies– Tracer experiments with

amino acids, lipids,carbohydrates

– Nucleotide analysis for DNAsynthesis rates

• Food and food ingredients– Adulteration control of

honey and fruit juices– Determinating origin of

flavors and nutrients– Analysis of phospholipids,

carbohydrates, amino acidsand vitamins

• Pharmaceuticals– Authenticity control of drugs– Investigation of agents and

additives• Doping control

– Analysis of steroids andhormones

• Biogeochemistry– History of biomolecules in

sediments and soils

A completely new conceptUp until now, the analysis of organicsamples for 13C/12C isotope ratios wasrestricted to either on-line GC separationfollowed by combustion (isotope ratiomonitoring GC/MS, irm-GC/MS) or bulkcombustion of samples in an organicelemental analyzer. Because biologically orpharmaceutically important compounds aremostly not GC-volatile or are too thermo-labile to be separated by gaschromatography, 13C/12C isotope ratiodetermination of these compounds in realanalytical applications, typically withincomplex mixtures, was either impossible orextremely labor intensive.

The Finnigan LC IsoLink (patentpending) leapfrogs the hurdle that, up until

now, has prevented successful isotope ratiomonitoring LC/MS, namely the quantitative,fractionation-free elimination of the liquidphase prior to the oxidative conversion ofthe sample into CO2 (1-3). In the Finnigan LCIsoLink, the liquid phase is not removedfrom the sample prior to oxidation but thesample is oxidized while still within theaqueous solution, and only afterwards theCO2 is separated from the liquid phase forisotopic analysis. This process isquantitative and fractionation-free in theFinnigan LC IsoLink.

The oxidation reagent consists of twosolutions, the oxidizing agent and a catalyst.Both are pumped separately and added tothe mobile LC phase. All individual organiccompounds eluting from the HPLC column

are oxidized quantitatively into CO2 withinthis mixture when passing through a heatedreactor. In a downstream degassing unit,the CO2 is removed from the liquid phaseand entrained into a stream of He.

The individual CO2 peaks in He aresubsequently dried in an on-line gas dryingunit (Nafion®) and then admitted to theIsotope 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 interface andmakes the Finnigan LC IsoLink essentiallymaintenance free.

The InterfaceFinnigan LC IsoLinkThe Link from

HPLC to Isotope Ratio M

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HPLC

IRMS

Gas Dryer Open Split

He He+H20

Oxidation Reactor

Separationof the mobile phase

Oxidation Reagents

C12H22O11 C6H12O6 CO2 CO2 CO2

ChemicalOxidation in Water,

… A Completely New Concept

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Amino acids and peptides are the basic elements for building proteins.Their structure and metabolism are of major interest in medicine andbiology. Either naturally occurring isotope differences or labelingexperiments can be used to give insight into their pathways.

Because pharmaceuticals are produced atvarious sites with different prices anddifferent formulations, it is essential for boththe producers and consumers to be able to

distinguish the points of origin for originalsand generics. Aspirin® and Paracetamol arerepresentatives of mass produced drugswhich have a wide range of origin and price.

Sugars are one of the biggest and mostcommon families of molecules in life. Theyserve as energy resource, backbone of DNAand RNA, carrier of biologically activecompounds and as structure building

material in plants. Sugars can carryinformation of their origin and processing.If isotopically labeled, they tell us abouttheir pathways and use.

The Finnigan LC IsoLink has its ownreferencing unit for direct isotope ratiocomparison of sample and reference gas forhighest precision and accuracy. This is theonly way to achieve such high precision ofe.g. < ± 1.5 ppm of 13C (< 0.15 ‰ in theδ-notation). For the purpose of sample-standard referencing, a cylinder ofcalibrated CO2 gas is used for extendedperiods of time. An inert fused silicacapillary supplies the reference gas in theµL/min range into the miniaturized opensplit, the Reference Gas Injection Port.

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Drugs on a C18 reversed phaseendcapped column

Reference capillary actuator

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Waste line

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Principle of reference gas injection

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Finnigan LC IsoLinkThe Link from

HPLC to Isotope Ratio M

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Isodat 2.0

The complete analytical setup is fullyautomated and controlled by Isodat 2.0. Theoptional LC system, including an optionalautosampler, is either fully integrated (e.g.Finnigan Surveyor™ LC) or synchronized bycontact closure. The Finnigan LC IsoLink andthe mass spectrometer are under fullcomputer control, resulting in analyses withno user interaction. When coupled to anoptional autosampler, the system can beoperated fully unattended for high samplethroughput and high productivity.

The interface does not require anymanual intervention during operation exceptfor refilling of the reagents. Data evaluationand report generation are fully automatedby Isodat 2.0. Since the interface has onlyfew variables, e.g. flow of oxidizing agentand He-flow, method development andoperation are also exceedingly simple.

• Easy and fast method and sequencesetup for Isotope Ratio MS and FinniganLC IsoLink

• Complete control and automation of allinterface functions before and duringdata acquisition

• Automated peak and backgrounddetection

• Dedicated peak detection and backgroundsubtraction algorithms

• Fully automated correction of the GCelution shift of isotopomers

• Fully automated ion correction forisobaric ion contribution such as 12C17O16Oon 13C16O16O

• Full access to all raw and processed data• Complete raw data integrity and secure

sample identity

• Fully customizable and multiple exports ofevaluated data to Excel, otherspreadsheet programs and databases(LIMS)

• All printouts are fully customizable dueto object oriented print-out templatesusing the unique Isodat 2.0 ResultWorkshop package

• Full network compatibility with direct andfast access to Windows® tools

• Complete Isodat 2.0 system backupand retrieval within minutes using theIsodat 2.0 Version Handler

• Based on the unique “Plug and Measure”concept of the new Isotope Ratio MSgeneration of (Finnigan DELTAplus XP,Finnigan DELTAplusAdvantage and FinniganMAT 253), the Finnigan LC IsoLinkinterface is immediately recognized andoperational

Accuracy, precision andsensitivityAccuracy and precision has been testedusing standards with certified isotoperatios. The δ13C value of 369 ng of benzoicacid (254 ng of carbon) analyzed byrepetitive loop injection has been shown tobe correct within 0.08 ‰ with areproducibility of 0.11 ‰. These valuescompare favorably with the accuracy andprecision achievable with bulk analysis byElemental Analyzer Isotope Ratio MS – butrequire 100 times less sample. Linearity hasbeen evaluated by the injection of variousamounts of sucrose, ranging from 3.25 µg to325 ng and the isotope ratio is shown to beindependent of the sample size. Thesensitivity of the integrated irm-LC/MSsystem is sufficient to allow the isotoperatio determination of 13C/12C on sampleamounts as low as 50 ng in direct injectionmode. The minimum amount in HPLC modedepends on the background created by theHPLC column bleed.

Acceptance specificationsAfter installation, the system performanceof the Finnigan LC IsoLink will bedemonstrated by direct loop injection, toshow an external precision of < 0.3 ‰ forδ 13C (n=5). This requires an organic-freeHPLC pump.

Analytical Performance

Mass spectrometer:Finnigan™ DELTA series or Finnigan™ MAT 253

Finnigan 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 Finnigan 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 ‰

Glucose/Fructose (HPLC separation)600 ng each in 20 µL of water

e.g. 3.3 nmol Glucose

20 nmol (240 ng) C as CO2 < 0.3 ‰

BR30039_E04/05C

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DIN EN ISO 9001:2000

Thermo Electron (Bremen) GmbH is certified DIN EN ISO 2001:9000

Power230 V single phase, 8 A

Gases• He, better 99.999%• CO2, better 99.995%

Supplies• Water (HPLC grade) • Reagent• Catalyst

Other supplies recommended• Columns from Thermo Electron

Corporation• Finnigan Surveyor

HPLC system with MS pump

Literature1) R.J. Caimi and J.T. Brenna, 1993,

Anal. Chem., 65, 3497-35002) W.A. Brand and P. Dobberstein, 1996,

Isotopes Environ. Health Stud. 1996,32, 275-283

3) B.L. Osborne and F.P. Abramson,43rd ASMS Conference, Atlanta, USA,1991, 21.-26.05.1994, 875

Analytical data is available in theFinnigan LC IsoLink Application Notes.

Installation Requirements

930

870

Deltaplus

Family

Finnigan Surveyor

HPLC System

LC IsoLink

360 360

Top view with dimensions in mm