DOI chapter:
Grenzgänger, traders and the last hunter-gatherers of the North European Plain
DOI chapter:Demirci, Özge; Lucquin, Alexandre; Klimscha, Florian; Craig, Oliver E.; Raemaekers, Daan C. M.: Lipid residue analysis of ceramics from Hüde I (Lower Saxony, Germany): New data to understand the transition to farming
DOI Page / Citation link:https://doi.org/10.11588/diglit.66745#0072
Ozge Demirci, Alexandre Lucquin, Florian Klimscha, Oliver E. Craig and Daan C. M. Raemaekers
71
sheets were added in order to remove cyclic octaatomic
sulphur that was present in all of the samples. All the
samples were dried under nitrogen at 35 °C. Finally, an
additional internal standard (n-hexatriacontane C36.0,
10 pL) was added to all samples prior to their analysis
by gas chromatography-mass spectrometry (GC-MS)
and gas chromatography-combustion isotope ratio
mass spectrometry (GC-C-IRMS). To control for any
contamination introduced during the sample prepara-
tion, a negative control, containing no ceramic powder,
was prepared and analysed with each sample batch.
Gas Chromatography-Mass Spectrometry
(GC-MS)
GC-MS analysis was undertaken using an Agilent
7890A series Gas Chromatograph coupled to an Agi-
lent 5975C inert XL mass-selective detector equipped
with a quadrupole mass analyser (Agilent technolo-
gies, Cheadle, Cheshire, UK). A split/splitless injector
(used in splitless mode) was maintained at 300 °C. The
column was inserted into the ion source of the mass
spectrometry directly. Helium was used as the carrier
gas, with a constant flow rate at 2 mL/min. The ionisa-
tion energy was 70 eY and spectra were obtained by
scanning between m/z 50 and 800. Samples (n = 37)
were analysed by using an Agilent DB-5 ms (5 °/o phe-
nyl) methylpolysiloxane column (PN 122-5532; 30 m
x 250 pm x 0.25 pm; J&W Scientific technologies,
Folsom, CA, USA). The temperature started at 50 °C
(for 2 min), increasing by 10 °C per minute up to 325
°C. The final temperature was maintained for 15 min.
Compounds were identified by comparing them with
the library of mass spectral data and published data.
In order to identify w-(o-alkylphenyl) alkanoic
acids and isoprenoid fatty acids and to calculate
the ratio of phytanic acid diastereomers, all samples
(n = 37) were also analysed by using a DB23 ms
(50 °/o-Cyanopropyl)-methylpolysiloxane column (60 m
x 0.250 mm x 0.25 pm; J&W Scientific technologies,
Folsom, CA, USA). Briefly, samples were re-dissolved in
hexane and 1 pL was injected with a splitless injector
at 300 °C. The temperature was set to 50 °C for 2 min.
This was followed by a rise of 4 °C per minute up to
140 °C, then 0.5 °C per minute up to 160 °C, and then
20 °C per minute up to 250 °C. The temperature was
then held at 250 °C for 10 min. The SIM (Selective
Ion Monitoring) mode was used in order to target the
specific markers trimethyltridecanoic acid (TMTD), m/z
74, 88, 101, 312 for pristanic acid, m/z 74, 101, 171,
326 for phytanic acid, and m/z 74, 105, 262, 290, 318,
346 for the detection of w-(o-alkylphenyl)alkanoic acids
of carbon lengths C 16 to C 22 (APAA 16-22). In addi-
tion, separation of the two phytanic acid diastereomers
(3S,7R,llR,15-phytanic acid or SRR and 3R,7R,11R,15-
phytanic acid or RRR) was obtained, which enabled the
calculation of the percentage of SRR in total phytanic
acid (SRR °/o) by integrating the m/z 101 ion (48). The
carrier gas used was helium with a flow rate of 1.5 mL/
min. Quantifications for the peak measurements were
calculated by the integration tool on the Agilent Chem-
Station enhanced data analysis software.
Gas Chromatography-Combustion Isotope
Ratio Mass Spectrometry (GC-C-IRMS)
All samples (n = 37) were analysed by GC-C-IRMS
in duplicates based on the existing protocol (Craig
et al. 2012), in order to measure stable carbon iso-
tope values of two fatty acid methyl esters, methyl
palmitate (C16 0) and methyl stearate (C18,0). Samples
were analysed by using Delta V Advantage isotope
ratio mass spectrometer (Thermo Fisher, Bremen,
Germany), linked to a Trace Ultra gas chromatograph
(Thermo Fisher) with a GC Isolink II interface (Cu/Ni
combustion reactor held at 1000 °C; Thermo Fisher).
All samples were diluted with hexane. Then 1 pL of
each sample was injected into DB5 ms ultra-inert-
fused-silica column (60 m x 0.25 mm x 0.25 pm; J&W
Scientific). The temperature was fixed at 50 °C for
0.5 min and raised by 25 °C/min to 175 °C, then raised
by 8 °C/min to 325 °C. The temperature was then held
at 325 °C for 20 min. Ultra-high-purity-grade helium
was used as the carrier gas with a constant flow rate
at 2 mL/min. Eluted products were ionized in the
mass spectrometer by electron ionization, and the
ion intensities of m/z 44,45 and 46 were recorded for
automatic computation of 13C/12C ratio of each peak
in the extracts (Heron et al. 2015). Isodat software
(version 3.0; Thermo Fisher) was used for the compu-
tation, based on the comparison with a standard refer-
ence gas (CO2) with known isotopic composition that
was repeatedly measured. The results of the analyses
were recorded in per mil (°/oo) relative to an interna-
tional standard, Vienna Pee Dee belemnite (VPDB).
N-alkanoic acid ester standards of known isotopic
composition (Indiana standard F8-3) were used to
determine the instrument accuracy. The mean ± stan-
dard deviation (SD) values of these n-alkanoic acid
ester standards were -29.60 ± 0.21 °/oo, and -23.02 ±
0.29 °/oo for the methyl ester of C16 0 (reported mean
value vs. VPDB -29.90 ± 0.03 °/oo) and C18 0 (reported
mean value vs. VPDB -23.24 ± 0.01 °/oo), respectively.
Precision was determined on a laboratory standard
mixture injected regularly between samples (28 mea-
surements). The mean ± SD values of n-alkanoic acid
71
sheets were added in order to remove cyclic octaatomic
sulphur that was present in all of the samples. All the
samples were dried under nitrogen at 35 °C. Finally, an
additional internal standard (n-hexatriacontane C36.0,
10 pL) was added to all samples prior to their analysis
by gas chromatography-mass spectrometry (GC-MS)
and gas chromatography-combustion isotope ratio
mass spectrometry (GC-C-IRMS). To control for any
contamination introduced during the sample prepara-
tion, a negative control, containing no ceramic powder,
was prepared and analysed with each sample batch.
Gas Chromatography-Mass Spectrometry
(GC-MS)
GC-MS analysis was undertaken using an Agilent
7890A series Gas Chromatograph coupled to an Agi-
lent 5975C inert XL mass-selective detector equipped
with a quadrupole mass analyser (Agilent technolo-
gies, Cheadle, Cheshire, UK). A split/splitless injector
(used in splitless mode) was maintained at 300 °C. The
column was inserted into the ion source of the mass
spectrometry directly. Helium was used as the carrier
gas, with a constant flow rate at 2 mL/min. The ionisa-
tion energy was 70 eY and spectra were obtained by
scanning between m/z 50 and 800. Samples (n = 37)
were analysed by using an Agilent DB-5 ms (5 °/o phe-
nyl) methylpolysiloxane column (PN 122-5532; 30 m
x 250 pm x 0.25 pm; J&W Scientific technologies,
Folsom, CA, USA). The temperature started at 50 °C
(for 2 min), increasing by 10 °C per minute up to 325
°C. The final temperature was maintained for 15 min.
Compounds were identified by comparing them with
the library of mass spectral data and published data.
In order to identify w-(o-alkylphenyl) alkanoic
acids and isoprenoid fatty acids and to calculate
the ratio of phytanic acid diastereomers, all samples
(n = 37) were also analysed by using a DB23 ms
(50 °/o-Cyanopropyl)-methylpolysiloxane column (60 m
x 0.250 mm x 0.25 pm; J&W Scientific technologies,
Folsom, CA, USA). Briefly, samples were re-dissolved in
hexane and 1 pL was injected with a splitless injector
at 300 °C. The temperature was set to 50 °C for 2 min.
This was followed by a rise of 4 °C per minute up to
140 °C, then 0.5 °C per minute up to 160 °C, and then
20 °C per minute up to 250 °C. The temperature was
then held at 250 °C for 10 min. The SIM (Selective
Ion Monitoring) mode was used in order to target the
specific markers trimethyltridecanoic acid (TMTD), m/z
74, 88, 101, 312 for pristanic acid, m/z 74, 101, 171,
326 for phytanic acid, and m/z 74, 105, 262, 290, 318,
346 for the detection of w-(o-alkylphenyl)alkanoic acids
of carbon lengths C 16 to C 22 (APAA 16-22). In addi-
tion, separation of the two phytanic acid diastereomers
(3S,7R,llR,15-phytanic acid or SRR and 3R,7R,11R,15-
phytanic acid or RRR) was obtained, which enabled the
calculation of the percentage of SRR in total phytanic
acid (SRR °/o) by integrating the m/z 101 ion (48). The
carrier gas used was helium with a flow rate of 1.5 mL/
min. Quantifications for the peak measurements were
calculated by the integration tool on the Agilent Chem-
Station enhanced data analysis software.
Gas Chromatography-Combustion Isotope
Ratio Mass Spectrometry (GC-C-IRMS)
All samples (n = 37) were analysed by GC-C-IRMS
in duplicates based on the existing protocol (Craig
et al. 2012), in order to measure stable carbon iso-
tope values of two fatty acid methyl esters, methyl
palmitate (C16 0) and methyl stearate (C18,0). Samples
were analysed by using Delta V Advantage isotope
ratio mass spectrometer (Thermo Fisher, Bremen,
Germany), linked to a Trace Ultra gas chromatograph
(Thermo Fisher) with a GC Isolink II interface (Cu/Ni
combustion reactor held at 1000 °C; Thermo Fisher).
All samples were diluted with hexane. Then 1 pL of
each sample was injected into DB5 ms ultra-inert-
fused-silica column (60 m x 0.25 mm x 0.25 pm; J&W
Scientific). The temperature was fixed at 50 °C for
0.5 min and raised by 25 °C/min to 175 °C, then raised
by 8 °C/min to 325 °C. The temperature was then held
at 325 °C for 20 min. Ultra-high-purity-grade helium
was used as the carrier gas with a constant flow rate
at 2 mL/min. Eluted products were ionized in the
mass spectrometer by electron ionization, and the
ion intensities of m/z 44,45 and 46 were recorded for
automatic computation of 13C/12C ratio of each peak
in the extracts (Heron et al. 2015). Isodat software
(version 3.0; Thermo Fisher) was used for the compu-
tation, based on the comparison with a standard refer-
ence gas (CO2) with known isotopic composition that
was repeatedly measured. The results of the analyses
were recorded in per mil (°/oo) relative to an interna-
tional standard, Vienna Pee Dee belemnite (VPDB).
N-alkanoic acid ester standards of known isotopic
composition (Indiana standard F8-3) were used to
determine the instrument accuracy. The mean ± stan-
dard deviation (SD) values of these n-alkanoic acid
ester standards were -29.60 ± 0.21 °/oo, and -23.02 ±
0.29 °/oo for the methyl ester of C16 0 (reported mean
value vs. VPDB -29.90 ± 0.03 °/oo) and C18 0 (reported
mean value vs. VPDB -23.24 ± 0.01 °/oo), respectively.
Precision was determined on a laboratory standard
mixture injected regularly between samples (28 mea-
surements). The mean ± SD values of n-alkanoic acid