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isolink AOAC March 2005 - Safford

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isolink AOAC March 2005 - Safford Powered By Docstoc
					     Finnigan™ LC IsoLink

   A new hyphenated technique,
isotope ratio monitoring LC-IRMS
  (irm-LCMS), enables two new
    microanalytical capabilities
        Chuck Douthitt, Andreas Hilkert, Dieter Juchelka
        Thermo Electron
        thermochuck@starband.net, www.thermochuck.org
•   13C-compound specific isotope analysis
    (13C-CSIA)

    and

•    C-analysis of bulk dissolved organic
    13

    carbon (13C-mEA)
• THE GOAL: To measure 13C/12C in intact organic molecules which have
  been chromatographically resolved by HPLC
    – with precision sufficient to resolve the small variations of 13C/12C found in
      nature (natural variation)
    – with a dynamic range sufficient to analyze compounds which have high levels of
      13C-enrichment (isotope tracers or labels)

• WHY HPLC INSTEAD OF GC? Some compounds are either not directly
  amenable or not at all amenable to GC analysis. The requirements for
  derivatization of many compounds can be a serious drawback:
    – Can be a laborious procedure of some complexity
    – added time, added cost
    – introduction of extra carbon of different isotopic composition, which must be
      corrected for
    – isotopic fractionation which must be calibrated and corrected out
• LC IsoLink allows δ13C analysis of individual compounds with:
   – High molecular weight
   – High polarity
   – Thermal instability
   – Low vapor pressure
• WHAT TYPE OF MS? Isotope ratios can be measured on a
  wide variety of analyzers:
• SOURCE*               ANALYZER                DETECTOR Thermo MS
•   various             Quadrupole              EM             MSQ

•   various             Triple Quadrupole       EM             Quantum

•   various             Ion trap                EM             LCQ, LTQ

•   various             TOF                     EM             none

•   various             FTICR                                  LTQ-FTICR

•   various             Double focusing         EM             MAT 95

•   EI                  B, Magnetic sector      Faraday array Delta+, MAT 253

• *EI, CI, FD, APCI, thermospray, electrospray, FAB…
The only class of mass spectrometer that can deliver the high precision that is required to resolve
variations in natural abundance is an “isotope ratio mass spectrometer”. An isotope ratio mass
spectrometer is designed to achieve one purpose: high precision isotope ratios of the light elements
(CNOHS). It is NOT designed for identifying unknown compounds.

•   For measurement of C (13C/12C), the ONLY form in which the sample is admitted into the ion
    volume or ion source is as CO2 (± He):

     –   CO2          + e-                  CO2+ (plus minor fragments, e.g, CO+)

But the CO2+ion beam actually consists of different ion beams (isotopomers and isotopologues)
    which are physically resolved by the magnetic sector:
• 12C16O2+                       13C16O + + 12C16O17O +
                                        2
                                                                              12C16O18O+

• m/z 44                         m/z 45                                       m/z 46
note: The doubly labeled species are not collected (e.g., 13C16O18O+) or are ignored (e.g,
    12C17O17O +)



The special attributes of the IRMS are:
• a)       Sample introduction limited to a simple compound, which for 13C, is CO2. No matter
    what the substrate for which analysis of 13C is desired, it MUST be converted into CO2 prior to
    entering the MS
• b)       Static multicollection of the ion beams in Faraday cups
• c)       Output is principally used to form an isotope ratio
Why NOT a quadrupole or ion trap MS like we are used to?
• With the IRMS, very small changes in stable isotope
  composition can be detected, e.g., change in relative 13C
  content of about 0.001% for as little as 1 nanomole C

• A quadrupole MS is about 1000-5000x less accurate than an
  IRMS and typically only changes of 5% in 13C content can be
  determined.

• At natural abundance, the total variation in terrestrial material
  is on the order of 0.02%, so this precision is required.
                  Thermo Isotope and Elemental Mass Spectrometers
                                                           RETARDATION                                  Finnigan
                                                           LENS                                         BRAND
                               ION
                                                                   FARADAY ION
SEPARATION   LASER    SAMPLE   SOURCE   kV   HR ANALYZER
                                                                   CUPS   COUNTERS    METHOD   N
                                                                                                        Delta+XP
                                        3-kV
  GC, LC               GAS      EI      10-kV
                                              No      B     Yes    2-12               IRMS     750   Delta+Advantage

                                                                                                      MAT 253
                     SOLID      GD              Yes   BE    Yes     1        1       HR-GDMS 2       ELEMENT GD

                     SOLID      TI              No          Yes
                                                      B             9        9        TIMS     85        Triton
                                        10-kV
                      SOLID                     Yes   EB    Yes                      MC-ICPMS 40        Neptune
                     LIQUID
 GC, LC
 CE, FFF     UV         GAS    ICP              Yes   BE    Yes     1        1       HR-ICPMS 400     ELEMENT2
                                                                                                     ELEMENT XR
                                                No                           1       Q-ICPMS            X series
LC-IRMS: A new strategy was required
                                                      CO2    CO2 CO2
HPLC                                   Gas Dryer
                                                                                   Isotope
                                                                                    Ratio
                                                                                     MS
                                               He
                       Combustion               +                      Open
       Separation of the Reactor      He       H2 O                    Split
        mobile Phase



                                                                   CO2   CO2 CO2
HPLC
                                                Gas Dryer

                                                                                       IRMS
                                                            He
                  Oxidation                                  +                 Open
                   Reactor          Separation He           H2 O               Split
                                      of the
                                    mobile phase
Schematic of the LC IsoLink Interface

                     Two-Head Pumps
        Oxidation                      Acid/Catalyst
        Reagent                                                CO2
                                                          Separation Unit
       Needle
        Port
                                                                         He
                                       T = 99.9°C
                                                                                Waste
                                                                            (mobile phase)
                           T-Piece     Oxidation
                6-Port-                 Reactor                  He + CO2+ H2O
                 Valve
                                                                       He
                                                     Gas
HPLC                                                 Dryer
                                                                    He + H 2O
                          Isotope
                           Ratio MS He + CO2

                                                    Open Split
LC IsoLink: Overview of operation
                HPLC Resolution
                             • Parameters:
  HPLC separation of carbohydrates
                                                                               – HPLC flow
m/z 44 (mV)
                                                                                 300 μl/min
                                                                   d13C (‰)




                                      Mannitol
                                     Glucose
                      Sucrose
     2000




                                  Galactose
                                                                   S: -10.7    – Oxidation reagent:




                                Fructose

                                                 Sorbitol
                                                                   G: -10.2       • 60 μl/min
                                                                   Ga: -24.5        (NH4)2S2O8,
     1500
                                                                   F: -11.5         100g/l
                                                                   M: -11.6    – Column: 700 CH
                                                                   S: - 9.7      Carbohydrate
                                                                                 Column, 90 °C
     1000


                                                                               – Reactor: 99.9 °C

      500                                                                      – CO2 Exchanger:
            0   500   1000                 1500             2000
                      Time (s)                                                    • 1 ml/min He
                                                                                    flow
  HPLC resolution is maintained
                                   Adulteration Determination
                                          δ13C of glucose, sucrose, fructose




                                                                                                                                                                                                           Fructose
                                                                                                                                  m/z 44 (mV)


                                                                    Fructose
m/z 44 (mV)
18500                                                                                                                             20000
                                   Honey 1                                                                                                                           Honey 2
                                                               Glucose

16500
                                                                                                        δ13C (‰)                  18000

                                                                                                         Honey




                                                                                                                                                                                                 Glucose
            Reference gas pulses




                                                                                                                                  16000




                                                                                                                                              Reference gas pulses
14500
                                                                                                        1               2
                                                                                                                                  14000
                                                                                        Sucrose      -25.3            n.d.
12500                                                                                   Glucose      -24.9           -25.1
                                                                                                                                  12000
                                                     Sucrose




                                                                                                                                                                                       Sucrose
                                                                                        Fructose     -24.9           -26.4




                                                                                                                                                                        Injection
                                      Injection




10500                                                                                 Area Fru/Glu    1.42            2.17        10000

                                                                                         Result      pure        adulterated
                                                                                                                                   8000
 8500


                                                                                                                                   6000
 6500                                                                                                                                     0                           500                 1000                        1500
        0                           500                 1000                   1500

                                                  Time (s)                                                   • δ (Fru/Glu) = 1.3 ‰                                                 Time (s)
                                                                                                             • Area (Fru/Glu) too high
                                                                                                             • conclusion: Sugar Added
             Origin of Drugs
Determination of different analgesic compounds.
δ13C of Paracetamol (Acetamidophenol) and Aspirin® (Acetylsalicylic Acid; ASA).

           δ13C (‰)                 HPLC Mode             Bulk Mode
          Tablet Type         Paracetamol      ASA
            A   Country 1           -          -34.2        -33.4
            A Country 2             -          -34.2        -33.5
                 B                  -          -29.1        -27.6
                 C                  -          -27.2        -26.6
                 D1                 -          -26.8        -26.7
                 E                  -          -27.7        -27.6
                 F               -32.3         -32.6        -31.7
                 D2              -28.7         -33.8        -31.3
                 G               -29.2         -32.7        -29.7

                          CONCLUSIONS based on 13C data:
                          • There are 4 sources of ASA
                          • Tablets A1 and A2 are from the same source
                          • Producer D use different ASA sources for D1 and D2
                                                                        Labeled Amino Acids
Mixture of two natural and two labeled amino acids

                                                                                                                                          δ13C     S.D.   SDM
   Amp (mV)
   50000
                                                                                                                                          [‰]       [‰]    [%]   n
                                      [1-13C] Leucine




                                                                                                                                   Ala    -29.03   0.3    0.04   5
   40000
                                                   13
                                                 [1- C] Phenylalanine


   30000
                                                                                                    Internal References
                                                                                                       of Benzoic Acid
               Reference gas pulses




   20000
                                                                                                                          m/z 44
                                                                                                                          m/z 45
                                      Alanine




                                                                                                                                   Leu   4444.86   14     0.25   5
                                                                               Tryptophan
                                                                                                                          m/z 46
   10000



      0
           0                               500              1000           1500             2000   2500
                                                                    Time (s)




                                                                                                                                   Phe   1759.63   13     0.47   5
                                                                                                                                   Try    -21.38   0.2    0.03   5




Good reproducibility; no memory effects by the labeled amino acids
Analysis of leucine, angiotensin III and insulin (data from Nestle, 2004)


Table I: Linearity of model compounds (area versus ng of carbon injected)

 compounds                  range of compound injected                            equation

                     in nmol molecule               in ng carbon         (area versus ng of carbon)

 leucine                 533 to 0.53                  300 to 0.3       Y = 0.194 x + 4.822, r2 = 0.997

 angiotensin III        34.2 to 0.038                 590 to 20        Y = 0.226 x + 0.989, r2 = 0.998

 insulin               0.193 to 0.012                 591 to 35        Y = 0.122 x - 0.421, r2 = 0.999




    Table II: Limits of detection based on shift of δ 13C/12C of model compounds

  compounds         limit of detection (based on shift of δ 13C/12C)   area       δ 13C/12C +/- sd (‰)
                       in pmol molecule         in ng carbon           (V.s)    associated of LOD level

  leucine                    40                        2.9              4          - 22.73 +/- 0.07 ‰

  angiotensin III            38                        20               6           -39.39 +/- 0.25 ‰

  insulin                    12                        35               7          - 19.96 +/- 0.65 ‰
                                             Organic Acids
                       Measurements of mixtures of organic acids

          -16                                        Citric Acid
                                                                                         m/z 44 (mV)




                                                                                                                                             Quinic Acid
                                                                                          10000                                                                          Benzoic Acid
          -18




                                                                                                                             Tartaric Acid
                                                                                           8000
          -20




                                                                                                                                                           Malic Acid
                                                     Malic Acid




                                                                                                                                                                                        Citic Acid
                                                                                           6000




                                                                                                       Reference gas pulse
          -22
d C (‰)




                                                                                           4000
          -24
 13




                                                    Quinic Acid                            2000

          -26
                                                                                              0
                                                                                                  0    200                   400                           600          800     1000                 1200   1400
          -28                                                                                                                                                    Time (s)

                                                    Tartaric Acid
          -30
                       relates to 64 ng of carbon (tartaric acid)
          -32
                0200    500     1000     1500     2000      2500    3000   3500   4000
                                                Amount (ng)

                       Good linearity; good reproducibility
                                         Nucleotides                                          m/z 44 (mV)
                                                                                              10000


                    δ13C analysis of nucleotide mixtures                                      9000

                                                                                              8000                          3'-CMP
                                                                                                                                      2',3'-GMP
                                                                                                                                                    3'-AMP


                                                                                              7000



          • Cytidine-3„-monophosphate (3„-CMP)
                                                                                              6000

                                                                                              5000




                                                                                                            Reference gas
                                                                                              4000

          • Guanosine-2„,3„-cyclic monophosphate (2„,3„-GMP)                                  3000

                                                                                              2000

          • Adenosine-3„-monophosphate (3„-AMP)                                               1000

                                                                                                 0
                                                                                                      0               100       200      300      400    500   600     700
                                                                                                                                      Time (s)
          -18                                                                350


          -19                                                                300
                                                                                       y = 0.03x - 0.7008
          -20
                                                                                          R2 = 0.9949
                                                                             250




                                                                 Area (Vs)
d C (‰)




          -21                                                                200
 13




          -22                                                                150
                                                3'-AMP

          -23                                   2',3'-GMP                    100

                                                3'-CMP
          -24                                                                50


          -25                                                                 0
                0       5000           10000             15000                     0   2000     4000                        6000       8000         10000      12000
                       Amount Nucleotide (ng)                                                 Amount 2',3'-GMP (ng)
    Compounds and Compound Classes Analyzed to date
             with the Finnigan LC IsoLink
         1. Volatile fatty acids (VFA)
•            Acetate. Formate. Lactate. Proprionate
•        2. Carbohydrates and Polyols (sugar alcohols)
•            Sucrose, Glucose. Galactose. Fructose. Mannitol, Sorbitol
•        3. Organic Acids
•            Citric acid, Lactic Acid, Malic acid, Quinic acid, Tartaric acid
•        4. Amino Acids (underivatized)
•            Alanine, Aspartic acid, Leucine (leucine and 13C-leucine), Phenylalanine, Tryptophan
•        5. Peptides and Proteins
•            Di-peptide, Tetra-peptide
•            Angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Ile)
•        Insulin (C254H377N65O75S6)
•        6. Purine, Pyrimidines, Nucleosides, Nucleotides, Nucleic Acids
     –           Nucleotides
             •   Polynucleotides        3’-CMP, 2’,3’- GMP,   3’-AMP
             •   Mononucleotides
     –     Nucleic Acids                RNA
•      7. Pharmaceutical Compounds
     –     Paracetamol (acetamidophenol)
     –     Aspirin (acetylsalicylic acid)
•      8. Aromatic acids
     –     Benzoic acid
   Compound classes amenable to analysis but not yet analyzed
• 9. Xanthines
    – Caffeine, Theophylline
•   10    Drugs (e.g. Amphetamines)
•   11    Alcohols
•   12.   Amino Sugars
•   13.   Polar lipids (e.g. Phospholipids)
•   14.   Flavor components
•   15.   Vitamins
•   16.   Pigments
• Types of measurements  modes of measurement
• 1. HPLC       Isotope ratios: CSIA by irm-LCMS
  – B.       Quantitation (extensive measurement, ppm)
  – C.       Ratios of compounds (intensive measurement, e.g.,
    fructose/glucose)


• 2. Flow Injection  Isotope ratios: BSIA by m-EA
  –  can be used for referencing
  –  DOC and DIC


• 3. combination of HPLC + FI  for mass balance
• Application areas of the LC IsoLink
• INDUSTRIAL & REGULATORY (Authenticity, Adulteration, Point of
  Origin, Quality Control)
• 1. Pharmaceuticals       aspirin
• 2. Foodstuffs
    – a.        Fruit juices       sugars
    – b.        Honey              sugars
    – c.        Coffee, tea, chocolate      xanthines, caffeine
•   FORENSICS
•   3. Forensics Drugs (amphetamines), amino acids in blood
•   4. Doping control Steroids, hormones
•   BIOGEOCHEMISTRY
•   5. Microbial ecology       Acetate
•   6. Chemical oceanography Pigments
•   7. Plant biology           Organic acids, carbohydrates
•   8. Soil science            Amino acids, carbohydrates
•   BIOCHEMISTRY
•   9. Molecular Biology       RNA
•   10. Metabolic Studies
    Conclusions from Nestle study1
•   1) .. the assessment of the [13C] isotopic enrichment of angiotensin III and insulin can be
    performed without the tedious steps of hydrolysis (into amino acids) and derivatization.

•   2) The limits of detection were 12 pmol for insulin and 38 pmol for angiotensin III with good
    precision (sd< 0.7 ‰).

•    3) The dynamic ranges for
•   Leucine:          53 nmol/mL - 533 μmol/mL
•   Angiotensin III : 4 nmol/mL - 3 μmo/mL
•   Insulin:          1.2 nmol/mL - 19 nmol/mL.
•
•   4) These promising results will allow us to investigate further developments to assess the
    isotopic enrichment of bio-molecules using HPLC-IRMS.

121stMontreux LC/MS Symposium, Montreux, Switzerland, 11/8-10/2004
Godin J-P., Fay L-B. and G. Hopfgartner, Direct analysis of underivatised amino acids and
   peptides by HPLC-isotope ratio mass spectrometry
   Summary and Conclusions
             HPLC      IRMS




                                          LC IsoLink
Thermo has developed a new concept for irm-LCMS
• Can measure 13C from HPLC as long as the mobile phase is H2O
• The technique has been shown to be quantitative, linear, precise and
  accurate
• Avoids derivatization required for GC analysis
    • Amino acids
    • Carbohydrates
• Can analyze biomolecules up to 6000 amu without requirement for
  hydrolysis and derivatization
                               LC IsoLink


                         Thermo         Spectrasystem
 HPLC Pump                                P1000         Knauer
                         Surveyor

HPLC Autosampler   Thermo      CTC HTC PAL
                   Surveyor      (LCPAL)

6 port valve       included      included

Column Oven        included         Mistral

 Alternative         PDA
 detectors          (DSQ)
 Data system       Excalibur
•       required for mEA mode (bulk analysis)
•                                                   Thermo         SpectraSystem
•       I   LC pump                         Surveyor MS Pump         P1000       Knauer
•                                                                     
•       required for HPLC mode (compound specific isotope analysis
•       2    Autosampler                    Surveyor           LC PAL
•       3    Mixing valve                   included           included
•       4    Column oven                    included           Mistral
•       5    PDA                            available          no
•       6    Excalibur control              yes                no
•       7. LCQ integration possible         no
•
•       LC Pump
    –           Requirements for the LC IsoLink
            •          FLOW RATES:                      Typical flow rates are between 200 and 600 μl/min. The maximum flow through the interface, which can be applied, is 700
                       μl/min. You have to take into account the flow of the reagent pumps, which could be set between 30 and 100 μl/min.
            •          REQUIREMENTS: In order to get reliable and precise results the pump must have a low pulsation for a low flow and low background. Any residue of organics
                       results in a dramatic high background (all C is quantitatively converted to CO2 and then measured).
            •          ISOCRATIC vs. GRADIENTS: At the moment, all LC IsoLink applications are done isocratically. Nevertheless, almost all customers, who would perform CSIA
                       or run the HPLC as a standalone system, want the possibility to run gradients.
    –           Pumps available
            •          The Surveyor MS Pump is a quaternary, low-pressure mixing pump with built-in solvent degassing and pulse dampening systems. The pumping system provides
                       optimum performance in the lower flow rate ranges needed for MS. It can run precise gradients from 25 to 2000 µL/min. The pump includes four high precision
                       proportioning valves, which contain sapphire and ceramic sealing surfaces that produce high precision solvent compositions at low flow rates.
            •          The SpectraSystem P1000 is a rugged, reliable and easy-to-use, single-solvent pump, i.e. an isocratic pump, with flow rates up to 10 ml/min. DOWNSIDE: The
                       P1000 is an isocratic pump and there is no Excalibur data system control possible.
•       Autosamplers
    –           The Surveyor autosampler includes a 6-port-valve and a column oven, for columns up to 25 cm long.
    –           The LCPAL comes with a 6-port valve but without a column oven
•       Column Ovens
    –           The Surveyor column oven, which is internal to the Surveyor MS pump takes columns up to 25 cm long
    –           The Mistral, an external column oven, takes columns up to 30 cm long
                                                             Reproducibility
                                        Bulk Injection of benzoic acid


m/z 44 (mV)

                                                                                   Amount
6000                                                                       Benzoic Acid Carbon   δ13C     S.D.
                                                      COOH                     [ng]       [ng]   [‰]      [‰]     n
                                                                               188        130    -28.68   0.11   10
           Reference gas pulses




4000                                                                           300        207    -29.34   0.05   10
                                                                               469        323     -29.3   0.07   10
                                                                               938        646    -29.54   0.05   10
2000                                                                          1500       1033     -29.6   0.04   10
                                                                              1875       1291    -29.38   0.05   10
                                                                              2344       1614    -29.24   0.08   10
   0
                                                                              2907       2002    -29.29   0.12   10
       0                          500      1000      1500    2000   2500
                                                  Time (s)                    Mean               -29.3    0.07   80
            e.g., 10 x bulk injections of 188 ng benzoic acid




                                        Reliable reproducibility of the δ13C values
Comparison between irm-GCMS and
 • irm-GCMS
            irm-LCMS
              Elution
           of separated                                CO2   CO2 CO2
  GC        compounds
                                          Gas Dryer
                                                                               Isotope
                                                                                Ratio
                                                He
                                                                                 MS
                          Combustion             +                     Open
                            Reactor      He     H2 O                   Split



  •    Helium is used as a carrier gas
        – Separation of compounds             Dry combustion (oxidation)
        – Transfer to the IRMS                in the He phase
  •    Helium has
        – No impact on combustion
        – No effects in the IRMS
 Comparison between irm-GCMS and
            irm-LCMS
• irm-LCMS-first approaches: remove the mobile phase
                                                     CO2   CO2 CO2
  HPLC
                                     Gas Dryer
                                                                              Isotope
                                                                               Ratio
                                                                                MS
                                              He
                        Combustion             +                     Open
             Separation   Reactor    He       H2 O                   Split
               of the
             mobile phase


 • Solvents as carrier for           First approaches (‟91, ‟93)
     – Separation of compounds
                                          –“Moving Wire” – Drying system
 • Solvents are                            (difficult to use)
     – Oxidized                           –“Particle Beam” Separation
     – Hazardous to IRMS                   (low sensitivity, fractionation)
 • No solvents to reactor or
   IRMS
                                           Linearity
                δ13C of different amounts of benzoic acid

          -26


                                Benzoic Acid (MW): C7H6O2
                                Molecular Weight:    122
                                                                           COOH
          -27
                                Carbon Content:      69 %
                                 300 ng MW ≡ 2.5 nmol MW
                                              ≡ 207 ng carbon
                                              ≡ 17 nmol carbon
          -28
d C (‰)




          -29
 13




          -30
                                                                                   S.D. = 0.13 ‰

          -31


                                                  Linearity Range
          -32
                0   250   500    750     1000     1250    1500      1750    2000   2250   2500   2750   3000
                                                   Benzoic Acid (ng)
                          Quantitation
Correlation between signal area and injected amount of benzoic acid

              500




              400

                              Area = 0.1877x + 5.6754
                                     R2 = 0.999
              300
  Area (Vs)




              200




              100




               0
                    0   500             1000            1500    2000   2500

                                     Amount Benzoic Acid (ng)


          Quantitative Carbon Analysis

				
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