Supporting Information
Practical recommendations for the reduction of memory effects in compoundspecific 15N/14N-ratio analysis of enriched amino acids by gas
chromatography/combustion/isotope ratio mass spectrometry
Klaus J. Petzke1* and Cornelia C. Metges2
1
German Institute of Human Nutrition in Potsdam-Rehbruecke (DIfE), Arthur-Scheunert-Allee
114-116, 14558 Nuthetal, Germany,
2
Research Unit Nutritional Physiology, 'Oskar Kellner', Leibniz Institute for Farm Animal
Biology (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany
*Correspondence to: K. J. Petzke, Deutsches Institut für Ernährungsforschung (DIfE), ArthurScheunert-Allee 114–116, D-14558 Nuthetal, Germany.
E-mail: petzke@dife.de
Additional information about instrumentation, derivatisation method for amino acids used,
and examples of GC-C-IRMS-chromatograms of m/z 28 (nitrogen)
A schematic representation of our gas chromatography/combustion/isotope ratio mass
spectrometry (GC-C-IRMS) system is shown in Fig. S1. For a detailed instrumental description
the reader is referred to the literature.[1–6] All technical features mentioned in the text refer to a
Finnigan-delta S isotope ratio mass spectrometer (Thermo Scientific, Bremen, Germany) coupled
on-line with a 5890A gas chromatograph (Agilent Technologies, Inc., Waldbronn, Germany) via
a combustion interface (Thermo Scientific).
Figure S1. Schematic representation of the GC-C-IRMS system: Finnigan-delta-S isotope ratio
mass spectrometer (Thermo Scientific, Bremen, Germany) coupled on-line with a 6890A gas
chromatograph (Agilent Technologies, Inc., Waldbronn, Germany) via a combustion interface
(Thermo Scientific).
In the gas chromatograph, capillary columns with various stationary phases have been used
depending on the compounds to be separated. Low bleed columns are preferred in order to extend
the life of the oxidation furnace. For 15N/14N analysis, phases containing nitrogen may be used
with caution. For the separation of amino acid derivatives columns with a length of 50 m, an i.d.
of 0.25 or 0.32 mm and a film thickness of 0.25 or 0.52 µm were used (for example: HP Ultra 2,
Agilent J&W GC Columns, Folsom, CA, USA).[5–11] Quantities between 1 and 20 nmol per
amino acid, in 0.5 to 0.75 µL injection volume, were injected in the splitless mode. Usually
>90% of the capillary column effluent is directed into an oxidation oven for further sample
processing. Optional detectors (flame ionization detector, FID; mass selective detector, MSD)
can be used via a splitter to simultaneously monitor or identify compound signals.
Gas isotope ratio mass spectrometry (GIRMS) requires that an analyte is converted into a pure
gas which represents the isotope composition of the original sample. This is performed on-line by
the combustion interface which comprises the oxidation oven alumina tube filled with CuO/Pt or
CuO/NiO/Pt wires, maintained between 800 and 1100°C for the oxidation of eluting compounds
to CO2, H2O, N2, NOx, and a reduction furnace filled with wires of elemental copper and
maintained at about 600°C for the reduction of NOx and scavenging of surplus oxygen. Water is
removed from the carrier using a water-permeable Nafion™ (fluorinated polymer, Permapure,
Toms River, NJ, USA) tube. In the nitrogen mode, CO2 is removed by a liquid N2 cold trap. The
reduction furnace and the liquid N2 trap are not necessary when the carbon isotope composition is
measured. Before the processed sample gas enters the GIRMS system, an open split device
allows pressure adjustment and a second open split is usually used to introduce N2 or CO2
reference gas into the carrier gas stream for calibration purposes and drift corrections at
appropriate time points between the sample peaks. Between the oxidation oven and the reduction
oven, O2 and He can be directed opposite to the carrier gas stream to reoxidize the Cu and Ni in
the oxidation oven. A He inlet backflush valve and a waste valve at the GC allow an outflow of
undesired GC effluents and solvent peaks to prevent early exhaustion of the oxidation oven.[1–3]
The GIRMS system comprises an ion source for sample gas ionization by electron ionization, a
section for ion separation, and a detector unit. Under high vacuum, electrons emitted by a heated
filament in the ion source collide with sample gas molecules resulting in positively charged ions
(CO2+, N2+). A magnetic field sorts ions according to their mass to charge (m/z) ratios. For CO2
and N2, the m/z ratios are 44 (12C16O16O), 45 (13C16O16O or 12C16O17O), 46 (13C16O17O or
12 17
C O17O), 28 (14N14N), 29 (14N15N, 30 (15N15N), respectively. The detector for ion collection
consists of a triple Faraday cup allowing the simultaneous detection of all three isotopomers of
the measured gas. Cups differ in response due to the different natural abundance of the masses
(e.g. 3 x 10-8 , 3 x 10-10, and 3 x 10-11, resistors for m/z 44, 45, 46, respectively). One
discharging ion at the Faraday cups generates one measurable ion current. The ion currents are
continuously monitored and digitized and the peak areas for each isotopomer are integrated. The
results are expressed as ratios (45/44 or 29/28) and may be converted into respective isotopic
ratios (13C/12C or 15N/14N), delta (δ) values, or APE (atom% excess). To increase the yield of
analytical data from the same chromatographic run it is possible to split the outflow of the
combustion unit into two separate GIRMS instruments for the simultaneous measurementof 13C
and 15N.[1–3] Figure S3 shows typical amino acid chromatograms as nitrogen gas peak profile (m/z
28) of plasma free and hydrolyzed protein amino acids.
The precision reached by a GIRMS instrument is 10-5 to 10-4 APE which is based on the low
molecular weights of the measured gases (<70 Da) and on the feature that the difference between
the isotopic ratios of a sample and a reference gas is measured, rather than the absolute isotopic
ratios. However, information on stable isotope enrichments at specific intramolecular positions is
lost at the expense of the high precision because GIRMS requires, contrary to GC-MS, that the
whole molecule of interest is converted into pure gases.
2.0000
1.8000
1.6000
1.4000
histidine
lysine
tyrosine
phenylalanine
internal standard
glutamate
aspartate
threonine
methionine
0.4000
proline
isoleucine
0.6000
serine
leucine
0.8000
valine
alanine
1.0000
glycine
28 (v)
1.2000
0.2000
0.0000
1500
2000
3000
2500
3500
4000
Time (s)
Figure S2. GC-C-IRMS chromatogram of m/z 28 (nitrogen) representing protein-bound amino
acids of liver protein measured as N-pivaloyl-i-propyl esters (Column: HP Ultra 2, Injection
volume: 0.5 µL; He-flow rate: 1 mL min–1; I.S.: Internal standard, α-aminoadipic acid, ~1.5
nmol/injection).
Figure S3. Gas chromatography/combustion/isotope ratio mass spectrometry chromatogram of
m/z 28 (nitrogen) of N-pivaloyl-i-propyl amino acid esters of human hair protein hydrolysate
(Column: HP Ultra 2; Injection volume: 0.5 µL; He-flow rate: 1.5 mL min–1; I.S.: Internal
standard, α-aminoadipic acid, ~1.5 nmol/injection).
Figure S4. Chemical structure of N-pivaloyl-i-propyl ester (NPP) of leucine. For further
information about typical amino acid derivatives used for GC analysis in stable isotope research,
see Metges and Petzke.[3]
A brief description of the sample pre-treatments and derivatisation of plasma free and protein
hydrolysate amino acids to N-pivaloyl-i-propyl esters as routinely used is given here. Plasma is
centrifuged at 3000 g. The supernatant (500 µL) is acidified with 1 mL 0.1 N HCl, and αaminoadipic acid (internal standard) is added (200–500 nmol). Sample proteins obtained from
acid precipitation of 50 µL of plasma, isolated by affinity column chromatography or by acid
precipitation of finely ground tissue samples or of freeze-dried digest or food samples (about 1
mg of protein) are hydrolysed (6 N HCl, 110°C, 24 h, PTFE capped Pyrex vials, addition of
internal standard), evaporated to dryness with a stream of nitrogen at 80°C and are dissolved in
0.1 N HCl. After applying free or hydrolysate amino acids to individual columns filled with
Dowex AG 50W-X8 resin (Na+ form, 200 mesh), amino acids are eluted by 2 mL 4 N NH4OH
plus 1 mL double-distilled H2O.
An aliquot of amino acids consisting of a laboratory standard mixture or of sample amino acids
and corresponding to a total of about 3–8 µmol is dissolved in 1 mL esterification reagent (1 M
thionylchloride solution in 2-propanol, freshly prepared) and heated for 60 min at 100°C in a 16
× 100 mm tube. The product is dried under a stream of nitrogen at 60°C and dissolved in 100 µL
pyridine (water-free). After adding 100 µL of pivaloyl chloride, the solution is acylated for 30
min at 60°C and 2 mL dichloromethane is added after cooling. The mixture is passed dropwise
through a 4 cm silica gel (60, 200–400 mesh) column (4 mm i.d.) to remove excess acylation
reagent and impurities which may affect the GC performance. The filtrate is evaporated with a
gentle stream of nitrogen at room temperature and the residue is dissolved in ethyl acetate for
sample injection.[5,12]
For the separation of NPP amino acid derivatives an HP Ultra 2 capillary column (50 m, 0.32 mm
i.d.) with He as carrier gas (1 mL min–1) is preferred. A volume of 0.5 µL is injected splitless.
The injector temperature is 280°C and the following oven temperature gradient program is used:
70°C, held 1 min; 70-220°C, ramp 3°C min–1; 220–300°C, ramp 10°C min–1; held 8 min.
Reference N2 or CO2 gas pulses with known isotopic composition are introduced at specific time
points during the gas chromatographic run for calibration of the sample amino acid nitrogen or
carbon (see Figs. S2 and S3). Data processing is performed by ISODAT vendor-provided
software (Thermo Scientific). The slope sensitivity for peak start and stop definition is usually set
at 0.2 and 0.4 mV s–1, respectively. The integration time is 0.25 s.
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[3]
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