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Biochimica et Biophysica Acta 1686 (2004) 108 117

http://www.elsevier.com/locate/bba

High-throughput quantification of phosphatidylcholine and sphingomyelin


by electrospray ionization tandem mass spectrometry coupled with
isotope correction algorithm
Gerhard Liebisch, Bernd Lieser, Jan Rathenberg, Wolfgang Drobnik, Gerd Schmitz*
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, D-93042 Regensburg, Germany
Received 1 June 2004; received in revised form 16 August 2004; accepted 1 September 2004
Available online 18 September 2004

Abstract
The choline head group containing phosphatidylcholine (PC) and sphingomyelin (SPM) are major eukaryotic lipid components playing
an important role in forming membrane microdomains and serve as precursor of signaling molecules. Both lipids can be monitored by
positive ion mode electrospray tandem mass spectrometry using a parent ion scan of m/z 184. Although PC species appear at even m/z and
SPM species at odd m/z, there may be a significant overlap of their isotopes. In order to separate PC and SPM species, an isotope correction
algorithm was established, which utilizes calculated isotope percentages to correct the measured peak intensities for their isotopic overlap.
We could demonstrate that this approach was applicable to correct the isotope overlap resulting from spiked PC and SPM species.
Quantification was achieved by addition of different PC and SPM species prior to lipid extraction. The developed assay showed a precision,
detection limit and robustness sufficient for routine analysis. Furthermore, an analysis time of only 1.3 min combined with automated data
analysis using self-programmed Excel Macros allows high-throughput analysis. In summary, this assay may be a valuable tool for detailed
lipid analysis of PC and SPM species in a variety of sample materials.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Mass spectrometry; Electrospray ionization; Phosphatidylcholine; Sphingomyelin; Phospholipid; High-throughput

1. Introduction
A major component of eukaryotic lipid membranes are
phospholipids, including the glycerol-based phosphatidylcholine (PC) and the sphingosine-based sphingomyelin
(SPM) [1]. These lipids vary in the degree of saturation
and chain length of the fatty acid moiety. In addition, there
exists a minor PC fraction containing an ether bond at sn-1
position instead of the ester bond [2], which may serve as
precursor for the generation of platelet-activating factor
(PAF), a potent lipid mediator [3]. These structural differ-

Abbreviations: CV, coefficient of variation; IS, internal standards; LCAT,


lecithin-cholesterol acyltransferase; LDL, low density lipoprotein; PAF,
platelet-activating factor; PC, phosphatidylcholine; SPM, sphingomyelin
* Corresponding author. Tel.: +49 941 944 6201; fax: +49 941 944
6202.
E-mail address: gerd.schmitz@klinik.uni-regensburg.de (G. Schmitz).
1388-1981/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbalip.2004.09.003

ences may have a great influence on membrane properties,


e.g., by modulating the formation of lipid raft structures [4].
Thus, Triton X-100 detergent-insoluble lipid microdomains
are enriched in saturated PC species compared to the soluble
membrane fraction [5]. PC and SPM also serve as
precursors for a variety of signaling molecules including
lysophosphatidylcholine [6], diacylglycerol [7] and ceramide [8] resulting from agonist induced action of different
lipases [9]. Additionally, SPM exhibits a tight interaction
with cholesterol, which may influence cellular cholesterol
homeostasis [10] as well as lipoprotein function. Thus, the
lecithin-cholesterol acyltransferase (LCAT) reaction in
reconstituted high density lipoproteins is inhibited by
addition of SPM [11]. Moreover, an increased SPM to PC
ratio enhances the susceptibility of low density lipoproteins
(LDL) to secretory sphingomyelinase [12]. Sphingomyelinase mediated ceramide generation leads to the formation of
aggregated LDL, which exhibits a high atherogenic poten-

G. Liebisch et al. / Biochimica et Biophysica Acta 1686 (2004) 108117

tial. In summary, SPM and PC are involved in the regulation


of several cellular processes as well as in the modulation of
lipoprotein function, which may be of special pathophysiological relevance.
A large number of methodologies have been described
for quantitative phospholipid analysis including high
performance liquid chromatography [13] and thin layer
chromatography [1416]. However, these conventional
methods usually do not provide information concerning
the fatty acid chains. Additionally, these methods often are
time-consuming multistep procedures and lack sensitivity
essential for the analysis of cellular subfractions. In the last
years numerous mass spectrometric approaches with different ionization techniques were used to characterize and
quantify phospholipid species [1729]. The most commonly
used technique is electrospray ionization tandem mass
spectrometry (ESI-MS/MS) allowing the quantification of
different phospholipid classes from crude lipid extracts [30].
In positive ion mode, both PC and SPM are monitored by
the collision induced formation of a m/z 184 fragment
specific for the phosphocholine head group, whereas in
negative ion mode only SPM is detected by a fragment of
m/z 168 resulting from an in source demethylation of the
phosphocholine head group [30]. However, SPM analysis in
negative ion mode exhibits a reduced sensitivity compared to
positive ion mode measurement [30]. In positive ion mode
the protonated PC species appear at even m/z and SPM
species at odd m/z values. In principle, the different m/z
allow a discrimination of these lipid classes, but frequently
unseparated lipid extracts reveal a relevant overlap of SPM
and PC isotope peaks. The aim of this study was to develop a
high-throughput ESI-MS/MS method using direct flow
injection of crude lipid extracts, which allows a separate
quantification of PC and SPM by correction of the overlapping isotope peaks.

2. Material and methods


2.1. Apparatus
Samples were quantified by direct flow injection
analysis using a HTS PAL autosampler (Zwingen, Switzerland) and an Agilent 1100 binary pump (Waldbronn,
Germany) with a solvent mixture containing 10 mM
ammonium acetate in methanol/chloroform=3:1 (v/v). A
flow gradient was performed starting with a flow of 55 Al/
min for 0.1 min followed by 30 Al/min for 1.0 min and an
increase to 250 Al/min for 0.2 min. The triple quadrupole
mass spectrometer (Quattro Ultima, Micromass, Manchester, UK) was equipped with a electrospray ion source
operated in positive ion mode using the following settings:
capillary voltage 3.5 kV, cone voltage 110 V, collision
energy 30 V with a collision gas pressure of 0.13 Pa
argon. Quantification was achieved by a parent ion scan of
m/z 184 in positive ion mode specific for phosphocholine

109

containing lipids [30]. Mass resolution was above unit


resolution.
2.2. Reagents
Methanol and chloroform were all HPLC grade from
Merck (Darmstadt, Germany), ammonium acetate was of
the highest analytical grade available purchased from Fluka
(Buchs, Switzerland). Phosphatidylcholine standards were
1-acyl-2-acyl-sn-glycero-3-phosphocholines except 1-Ohexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine
(PC O 36:4) from Avanti Polar Lipids (Alabaster, AL, USA)
and sphingomyelin standards were from Sigma (Taufkirchen, Germany) all with purities higher than 99%.
2.3. Sample preparation
EDTA plasma samples were used for analysis. Prior to
lipid extraction, 100 Al of a chloroform solution containing
25 ng/Al of each 1,2-dimyristoyl-sn-glycero-3-phosphocholine (PC 28:0) and 1,2-dibehenoyl-sn-glycero-3-phosphocholine (PC 44:0) was placed into a glass centrifuge tube
and evaporated. For calibration samples, defined quantities
of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(PC 34:1), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC 34:0), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (PC 36:4), 1,2-distearoyl-sn-glycero-3-phosphocholine
(PC 36:0), 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PC 38:4) as well as N-palmitoyl-sphingosylphoshorylcholine (SPM 34:1) and N-stearoyl-sphingosyl
phoshorylcholine (SPM 36:1) were added and vacuumdried. Twenty microliters of EDTA plasma was extracted
according to the procedure described by Bligh and Dyer
[31]. In brief, plasma was diluted with water to 800 Al and
3 ml of methanol/chloroform=2:1 (v/v) was added. For
phase separation 1 ml of each water and chloroform was
added. After centrifugation the chloroform phase was
separated and dried. The residue was dissolved in 10 mM
ammonium acetate in methanol/chloroform=3:1 (v/v)
resulting in a 75-fold dilution corresponding to the initial
plasma volume. Twenty microliters of this solution was
injected and data were acquired for 1.3 min.
2.4. Data analysis
Data analysis was performed with MassLynx software
including the NeoLynx tool (Micromass). All scans at half
peak height of the total ion count were averaged using
NeoLynx, which generates centroided peak data from
continuum spectra and allows to pick intensities of certain
m/z. Neolynx includes background subtraction and
smoothing according to Savitzky Golay of the combined
spectra. All scans averaged were in the range of constant
30 Al/min flow. NeoLynx results were exported to Excel
spreadsheets and further processed by self-programmed
Excel Macros.

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G. Liebisch et al. / Biochimica et Biophysica Acta 1686 (2004) 108117

Due to the isotope overlap of PC and SPM species, a


correction of the peak intensities using calculated isotope
distributions was performed. For each species the relative
abundance of isotopes was calculated using an isotope
model implemented in MassLynx (Micromass), which
utilizes the following relative abundances: 2H0.015%,
13
C1.100%, 15N0.3660%, 17 O0.0380%, 18 O
0.2000%. The isotope distribution was calculated for each
species without respecting the phosphocholine head group,
because only molecules generating a monoisotopic m/z 184
phosphocholine head group fragment were monitored.
These isotope distribution percentages were implemented
into Excel Macros correcting the peak intensities in a
sequential algorithm starting from low mass species (five
isotope peaks including the monoisotopic were used). This
algorithm is shown in detail in the manuscript appendix.

3. Results and discussion


Both phosphatidylcholine (PC) and sphingomyelin
(SPM) are monitored by a parent ion scan of m/z 184 in
positive ion mode [30]. Our aim was to establish a high-

throughput methodology for a separate quantification of PC


and SPM species using a triple quadrupole mass spectrometer equipped with an electrospray ion source and direct
flow injection. Human plasma was selected as an easily
accessible sample material for method evaluation. A parent
ion scan of m/z 184 of crude plasma lipid extract revealed
~100 lipid species with PC species appearing at even m/z
and SPM species at odd m/z (Fig. 1). Although the PC peaks
could not be attributed to a certain pair of fatty acids, the
mass information allows the attribution to a defined number
of carbon atoms and double bounds in the O-linked fatty
acids. Thus, peaks with even m/z close to m/z 758, 782 and
810 represent PC species with 34, 36 and 38 carbon atoms
in the fatty acid moiety, respectively. The minor peaks close
to m/z 690, 718, 746, 770, 796 could be attributed to PC
species with a odd number of carbon atoms in the fatty acid
moiety, but may also represent 1-O-alkyl-2-acyl-PC species
[32]. SPM peaks may be assigned to a certain species
assuming that the sphingolipid backbone in mammal
consists mainly of a dihydroxy sphingosine base with 18
carbon atoms containing one double bond (d18:1) [33].
However, it has been shown, that human plasma contains
also significant amounts of other sphingoid bases including

Fig. 1. Parent ion scan of m/z 184 of crude plasma lipid extract. The spectrum shows a typical parent ion scan of m/z 184 from crude plasma lipid extract
averaged from a 1.3-min measurement. The internal standards PC 28:0 and PC 44:0 were added prior to lipid extraction. The PC species appeared at even and
the SPM species at odd m/z values. The inset shows a zoom of the area m/z 805818. The combined spectra were smoothed according to Savitzky Golay.

G. Liebisch et al. / Biochimica et Biophysica Acta 1686 (2004) 108117

d16:1 and d18:2 [34]. Therefore, SPM species were referred


to as the sum of sphingoid base and N-linked fatty acid (in
Table 4 also an assignment based on d18:1 is shown).
3.1. Isotope correction algorithm
Although PC and SPM appear at different m/z, there
exists a significant overlap of their isotope peaks. In
particular, the peak group at m/z 810 showed a substantial
overlap of PC species containing 38 carbon atoms in the
fatty acid moiety and SPM species containing 42 carbon
atoms in the sphingoid base and the N-linked fatty acid (Fig.
1, insert). Therefore, an automated data analysis algorithm
was programmed to correct the measured peak intensities
for the isotopic overlap. In order to allow high sample
throughput, a simple liquidliquid extraction procedure and
direct flow injection with a run time of 1.3 min per sample
were selected as analysis settings. A prerequisite for an
isotope correction approach was a sufficient accuracy and
precision of the isotope profile under the selected analysis
conditions. Especially for signals with a low intensity, a
smoothing algorithm is required prior generation of centroided peak data, because otherwise peaks may be generate
from noise spikes. Therefore, algorithms for noise reduction
and spectrum smoothing implemented in the mass spectrometry software were applied for generation of centroided
peak intensities. Different standard samples covering the
mass range of the naturally occurring species (PC 34:0, PC
36:4, PC 38:4, SPM 36:1 each 300 AM) were measured in
triplicate using a parent ion scan of m/z 184 with the
conditions described above. The ratio isotope peak to
monoisotopic peak was calculated for each species. This
peak ratio showed a coefficient of variation (CV) b2.5% for
the M+1 isotope, b6.5% for M+2, b11% for M+3 and b35%
for M+4 for all measured species. Additionally, the
measured profile was compared with the calculated isotope
profile. The calculation of the isotope profile was performed

111

for molecules containing a monoisotopic phosphocholine


head group, since only these molecules are monitored in a
parent ion scan of m/z 184. All analyzed species revealed
differences between the measured mean of the isotope
profile and the calculated isotope percentages below 1.0%,
0.4%, 0.1% and 0.1% for the M+1, M+2, M+3 and M+4
isotope, respectively. In summary, the selected analysis
conditions should provide a sufficient precision and
accuracy to allow an intensity correction of the measured
species peaks.
Based on these findings, PC and SPM species isotope
profiles were calculated for all species occurring in the
analyzed plasma lipid extracts. Within overlapping peak
groups the presence of certain species could not be excluded
without calculation; therefore, a series of species with a
varying number of double bonds were included. These
percentages were enclosed in a self-programmed Excel
Macro, which corrects the measured peak intensities for the
isotopic overlap of adjacent lipid species in a sequential
algorithm starting from low mass species (s. appendix).
Subsequently, this approach was tested with human
plasma as sample material. For quantification an addition
of internal standards (IS) is crucial, because the ESI process
and consequently the signal intensity are highly affected by
matrix components. In analogy to Brqgger et al. [30], not
naturally occurring PC 28:0 and PC 44:0 were added as IS
prior lipid extraction and quantification was achieved by
calculation of the ratio counts(analyte) to counts(IS). For
species like SPM 40:3 adjacent to intense PC species (PC
36:4) the ratio to the IS was dramatically reduced from 0.47
without correction (Table 1A) to 0.03 after running the
isotope correction algorithm (Table 1B). Additionally,
increasing concentrations of PC 36:4, PC 38:4 and
SPM 36:1 were added to human plasma (Table 1), which
also significantly increase the concentration of adjacent
species without isotope correction (Table 1A). For example,
the addition of 300 AM PC 36:4 increased the peak ratio

Table 1
Isotope correction of overlapping isotope peaks
m/z

731

732

782

783

784

810

811

812

813

814

815

C (mM)

SPM 36:1

PC 32:1

PC 36:4

SPM 40:3

PC 36:3

PC 38:4

SPM 42:3

PC 38:3

SPM 42:2

PC 38:2

SPM 42:1

0.306
0.366
0.452
0.577
0.731

0.999
1.205
1.539
1.931
2.460

0.470
0.568
0.721
0.900
1.128

0.865
0.882
0.923
0.964
0.998

0.569
0.793
1.126
1.513
2.037

0.428
0.533
0.693
0.879
1.123

0.390
0.419
0.461
0.527
0.584

0.542
0.546
0.565
0.574
0.588

0.260
0.269
0.275
0.284
0.279

0.157
0.159
0.166
0.166
0.164

0.982
1.186
1.517
1.907
2.434

0.030
0.037
0.042
0.046
0.040

0.748
0.741
0.744
0.743
0.724

0.528
0.752
1.086
1.472
2.000

0.173
0.173
0.177
0.182
0.180

0.245
0.247
0.249
0.269
0.266

0.397
0.395
0.406
0.397
0.403

0.036
0.044
0.044
0.053
0.046

0.088
0.087
0.093
0.089
0.090

A. Uncorrected ratios
0
0.149
0.045
0.310
0.113
0.549
0.188
0.821
0.300
1.229

B. Ratios after overlap correction


0
0.136
0.247
0.045
0.279
0.245
0.113
0.493
0.238
0.188
0.735
0.258
0.300
1.098
0.255

The displayed values are ratios counts(analyte) to counts(PC 28:0 as internal standard). SPM 36:1, PC 36:4 and PC 38:4 were added at the indicated
concentrations to plasma samples prior to lipid extraction. Table A shows the uncorrected ratio and table B the ratios after correction by calculated isotope
percentages using self-programmed Excel Macros as described in Materials and methods.

112

G. Liebisch et al. / Biochimica et Biophysica Acta 1686 (2004) 108117

Table 2
Cholesterol and concentration dependence of the calibration lines
Dilution

PC 34:0
Slope

PC 36:0
Intercept

PC 34:1

PC 36:4

PC 38:4

SPM 34:1

SPM 36:1

Slope

Intercept

Slope

Intercept

Slope

Intercept

Slope

Intercept

Slope

Intercept

Slope

Intercept

Plasma cholesterol-100 mg/dL


50
5.43
0.043
100
5.44
0.044
200
5.34
0.031
400
5.32
0.045
Mean
5.38
0.041
CV%
1.1
16.7

5.32
5.25
5.38
5.30
5.31
1.0

0.028
0.027
0.022
0.023
0.025
11.9

4.82
4.84
4.82
4.71
4.80
1.2

1.076
1.075
1.080
1.094
1.081
0.8

5.23
5.25
5.12
5.07
5.17
1.7

0.730
0.725
0.719
0.740
0.729
1.2

6.12
6.14
6.10
5.93
6.07
1.6

0.262
0.277
0.268
0.282
0.272
3.3

3.12
3.05
2.87
2.69
2.93
6.6

0.369
0.369
0.363
0.343
0.361
3.4

2.81
2.76
2.63
2.41
2.65
6.7

0.110
0.106
0.105
0.106
0.107
1.8

Plasma cholesterol-314 mg/dL


50
5.40
0.042
100
5.47
0.046
200
5.44
0.046
400
5.32
0.057
Mean
5.41
0.048
CV%
1.2
13.9

5.40
5.20
5.20
5.10
5.23
2.4

0.027
0.014
0.016
0.008
0.016
46.8

4.90
4.90
4.90
4.80
4.88
1.0

1.158
1.158
1.169
1.152
1.159
0.6

5.30
5.40
5.20
5.10
5.25
2.5

1.437
1.434
1.422
1.432
1.431
0.4

6.20
6.30
6.30
6.10
6.23
1.5

0.620
0.625
0.626
0.622
0.623
0.4

3.10
3.11
3.05
2.78
3.01
5.2

0.570
0.548
0.527
0.527
0.543
3.8

2.80
2.81
2.64
2.58
2.71
4.3

0.151
0.148
0.150
0.141
0.148
3.2

Calibration lines were established by standard addition of naturally occurring PC and SPM species to plasma prior to lipid extraction. The ratio counts(analyte)
to counts(PC 28:0 as internal standard) was plotted against the spiked concentration in AM plasma and the regression line was calculated by linear least square
fit. Regression line coefficients were all above 0.99 and the displayed values show the slope and intercept of the regression line. The calibration lines consisted
of five different concentrations ranging from 0 to 300 AM. Sample dilution corresponds to the extracted plasma volume.

SPM 40:3 to PC 28:0 from 0.47 to 1.13 without isotopic


correction. In contrast, after correction of the isotope
overlap, no significant increase was observed for species
adjacent to spiked species (Table 1B).
In order to further validate this approach, both a
saponified plasma sample and its non-saponified control
were quantified as described below using the isotope
correction algorithm (Table 4B). Total PC concentration
decreased from 2.7 to 0.03 mM after saponification (data
not shown). SPM species concentrations showed a good
correlation with or without saponification, except low
concentrated species (under 5 AM), which reside at high
m/z in overlapping peak clusters such as SPM 38:0, SPM
39:1, SPM 40:0. The higher difference may be in part due to
the used algorithm, because an imprecision in high intensity
peaks will affect adjacent small peaks to a greater extent.
Moreover, peak intensities at high m/z in overlapping peak
clusters are subjected to more correction steps than peak

intensities at low m/z. Therefore, intensity imprecision has a


greater impact on peak intensities at high m/z in overlapping
peak clusters.
Taken together, these data strongly support the validity of
the performed isotope correction and consequently provide
the basis for a separate quantification of PC and SPM
species using a parent ion scan of m/z 184.
3.2. Quantification of PC and SPM species
In order to achieve real quantitative data, it has to be
considered that the response of phospholipid species
decreases with increasing acyl chain length [30,35].
Moreover, Koivusalo et al. [35] described that the degree
of acyl chain unsaturation influences the response of
phospholipid species. At high concentrations polyunsaturated species show a higher response compared to
saturated species, but this effect disappears with sample

Table 3
Precision intra day and inter day of PC and SPM species
Phosphatidylcholine

Sphingomyelin
Saturated

IS
PC 28:0
PC 44:0
PC 28:0
PC 44:0

Mean
CV%
Mean
CV%
mean
CV%
mean
CV%

0.05
3.7
0.05
6.3
0.06
11.7
0.05
10.1

Monounsaturated
0.47
3.0
0.44
5.8
0.56
9.2
0.49
10.9

Polyunsaturated
2.01
3.3
1.93
6.3
2.17
8.8
1.95
11.5

O-alkyl
0.17
2.7
0.16
5.8
0.17
9.7
0.15
10.8

Total
2.53
3.2
2.42
6.2
2.78
8.8
2.50
11.2

Total
IS
PC 28:0
PC 44:0
Inter day
PC 28:0
PC 44:0

mean
CV%
mean
CV%

0.56
3.6
0.56
6.1

mean
CV%
mean
CV%

0.59
8.7
0.56
12.1

The intra day values are mean in mM and the coefficient of variation (CV) from 10 sample aliquots measured in series using PC 28:0 and PC 44:0 as internal
standard, respectively. The inter day values are mean in mM and CV of a plasma sample calculated from seven different runs.

G. Liebisch et al. / Biochimica et Biophysica Acta 1686 (2004) 108117

dilution. Thus, we established calibration lines (Table 2) by


addition of saturated (PC 34:0, PC 36:0) and unsaturated
(PC 34:1, PC 36:4, PC 38:4) PC species as well as SPM
species (SPM 34:1, SPM 36:1). Since cholesterol concentration may be used as surrogate marker for the total lipid
content in the plasma, calibration lines were established for
plasma either with a low (100 mg/dl) or a high cholesterol
concentration (314 mg/dl), in order to analyze the
influence of the lipid content on the species response.
Additionally, these calibration lines were measured at
different dilutions ranging from 50- to 400-fold related to
the extracted plasma volume (Table 2). The generated
calibration lines were linear in the analyzed concentration
range. Except for a slight decrease at 400-fold dilution, the
calibration line slopes revealed no significant influence of
the plasma cholesterol level as well as the sample dilution.
Compared to the analyzed PC species, the SPM species
showed an almost 50% lower response. Although a slight
decrease of the calibration line slope was observed for
long chain saturated species from PC 36:0 to PC 34:0 and
from SPM 36:1 to SPM 34:1, no clear correlations were
observed between the species response and the acyl chain
length or the degree of unsaturation for the measured lipid
species. In addition, the calibration line slope of the alkyl
bond containing PCO 36:4 was not significantly different
from the slope of the diacyl PC species (data not shown).
These results were not in accordance with the study by
Koivusalo et al. [35]. However, this may be due to different
analysis conditions including the solvent mixtures and the
sample matrix used. Thus, Koivusalo et al. measured lipid
standards in a methanol/chloroform=2:1 (v/v) mixture,
whereas the present study analyzes a complex mixture of
plasma lipids in 10 mM ammonium acetate in methanol/
chloroform=3:1 (v/v). For the following experiments, a 75fold dilution of the plasma extract was selected, which
provides sufficient ion counts also for minor species.
In order to achieve exact concentrations for all observed
species, each single species would require calibration.
Since such an approach is not feasible, several PC and
SPM species were selected for calibration from the mass
range of the naturally occurring species (Table 2). The
established calibration lines were also applied to adjacent
species. Despite a tendency towards higher PC and SPM
concentrations using PC 28:0 as IS for the calculation
(Table 3), the single species concentrations resembled each
other closely (data not shown). In addition, the assay
shows a good reproducibility with a significantly higher
precision for concentrations calculated using PC 28:0 as IS
(Table 3). Thus, concentrations of major PC/SPM species
measured in one run and calculated with PC 28:0 revealed
a coefficient of variations (CV) below 4%. Minor species
with concentrations ranging from 1 to 10 AM exhibited a
CV between 5% and 10% within one run (data not
shown). The CV observed from day to day was ~10% for
major species and ~15% for minor species for both PC and
SPM calculated using PC 28:0 as IS. The higher precision

113

obtained for concentrations calculated using PC 28:0 as IS


may be related to a higher signal intensity for PC 28:0,
which was about 50% higher than the PC 44:0 signal
under the used experimental set up. Moreover, no
significant correlation was observed between the precision
and the molecular weight of the species (data not shown).
Therefore, the signal intensity seems to be more important
for the measurement precision than the chain length of the
IS used for calculation. Consequently, the IS showing the
higher signal intensity could be used for quantification and
the second IS added in a constant ratio could serve as an
internal quality control monitoring a proper ionization
response. Thus, the ratio PC 44:0 to PC 28:0 showed a
variation below 5% within different plasma samples in one
run. In addition, the calibration line slopes, linearity and
intercepts were not influenced by 250 injections of
extracted plasma samples showing the stability of the
established methodology.
The effect of the matrix has to be considered for the
determination of the limit of detection, because matrix
suppression effects have to be taken into account when
complex mixtures like crude lipid extracts are investigated. Since a PC- and SPM-free plasma matrix was not
available, the limit of detection was determined from the
baseline noise at analyte-free regions m/z 710, m/z 747,
and m/z 886, selected from the mass range of the
naturally occurring PC and SPM species. The resulting
detection limit was between 0.3 and 0.6 AM for both IS
calculated from the mean+3 S.D. at the particular m/z
values in a measurement of 10 aliquots.
Finally, PC and SPM species were analyzed in the
plasma of 20 healthy blood donors (Table 4). For both PC
and SPM the majority was comprised by a view species:
PC 34:2, PC 34:1, PC 36:4, PC 36:3, PC 36:2 account for
about 70% of PC and SPM 34:1, SPM 36:1, SPM 38:1,
SPM 40:2, SPM 40:1, SPM 42:3, SPM 42:2, SPM 42:1
comprised 73% of SPM. Assuming that the main sphingoid
backbone consists of a dihydroxy sphingosine base with 18
carbon atoms containing one double bond (d18:1) [33], the
majority of SPM contains a saturated amide linked fatty
acid. In total we found 103 PC and SPM species peaks with
a mean above a detection limit of 0.6 AM (Table 4).
Considering that these peaks may consist of different
combinations of fatty acids as well as two bond types for
PC or different combinations fatty acid and sphingoid bases
for SPM, human plasma may contain several hundred
distinct PC and SPM species.

4. Conclusion
In order to achieve a separate quantification of PC and
SPM using a parent ion scan of m/z 184, an automated
sequential isotope correction algorithm was developed. A
detailed evaluation of this approach revealed a good
precision as well as a low limit of detection. Combined

114

PC Species
Assignment
Diacyl
Assignment
Diacyl;
Alkyl/Acyl

PC
29:1
PC O
30:1

PC
29:0
PC O
30:0

0.7
0.3
0.03

0.8
0.5
0.03

0.9
0.3
0.04

Assignment
Diacyl
Assignment
Diacyl;
Alkyl/Acyl

PC
35:2
PC O
36:2

PC
35:4
PC O
36:1

PC
35:0
PC O
36:0

PC
36:5
PC
36:5

PC
36:4
PC
36:4

PC
36:3
PC
36:3

PC
36:2
PC
36:2

PC
36:1
PC
36:1

Mean
S.D.
% of total

13.4
3.1
0.54

8.5
2.2
0.35

1.2
0.3
0.05

20.0
7.6
0.81

235.8
60.0
9.57

175.2
51.3
7.11

314.8
74.6
12.77

54.2
16.7
2.20

Assignment
Diacyl
Assignment
Diacyl;
Alkyl/Acyl

PC
39:6
PC O
40:6

PC
39:5
PC O
40:5

PC
39:4
PC O
40:4

PC
40:7
PC
40:7

PC
40:6
PC
40:6

PC
40:5
PC
40:5

PC
40:4
PC
40:4

PC
40:3
PC
40:3

4.1
1.1
0.17

3.3
0.9
0.14

2.6
0.6
0.11

Mean
S.D.
% of total

Mean
S.D.
% of total

PC
26:0
PC
26:0

SPM Species
Sum of
sphingoid base
and FA

SPM
30:1

SPM
32:1

PC
30:1
PC
30:1
4.5
2.9
0.18

5.7
1.7
0.23

SPM
33:1

PC
30:0
PC
30:0
6.6
3.5
0.27

24.8
6.3
1.00

SPM
34:2

PC
31:1
PC O
32:1
3.4
1.1
0.14

11.1
3.3
0.45

SPM
34:1

PC
31:0
PC O
32:0
4.1
1.2
0.17

4.7
1.4
0.19

SPM
34:0

PC
32:2
PC
32:2
6.2
2.7
0.25

3.2
1.1
0.13

PC
32:1
PC
32:1

PC
32:0
PC
32:0

PC
33:3
PC O
34:3

PC
33:2
PC O
34:2

PC
33:1
PC O
34:1

PC
33:0
PC O
34:0

PC
34:4
PC
34:4

PC
34:3
PC
34:3

PC
34:2
PC
34:2

PC
34:1
PC
34:1

29.4
17.1
1.19

20.0
5.7
0.81

9.2
2.3
0.37

13.1
3.5
0.53

11.2
2.5
0.45

1.9
0.6
0.08

2.2
1.1
0.09

19.5
7.1
0.79

645.8
140.1
26.20

334.9
93.4
13.59

PC
36:0
PC
36:0

PC
37:5
PC O
38:5

PC
37:4
PC O
38:4

PC
37:3
PC O
38:3

PC
37:2
PC O
38:2

PC
37:1
PC O
38:1

PC
38:7
PC
38:7

PC
38:6
PC
38:6

PC
38:5
PC
38:5

PC
38:4
PC
38:4

16.9
3.9
0.69

13.1
2.3
0.53

4.1
1.2
0.17

2.6
0.8
0.10

1.3
0.9
0.05

2.3
0.8
0.09

87.9
23.9
3.57

PC
40:1
PC
40:1

PC
40:0
PC
40:0

PC
41:6
PC O
42:6

PC
41:5
PC O
42:5

PC
41:4
PC O
42:4

PC
42:5
PC
42:5

PC
42:4
PC
42:4

1.2
0.3
0.05

2.0
0.5
0.08

1.3
0.4
0.05

1.2
0.3
0.05

2.5
1.5
0.10
PC
40:2
PC
40:2
2.5
0.9
0.10

SPM
35:2

3.7
1.6
0.15

SPM
35:1

1.3
0.5
0.05

SPM
35:0

SPM
36:3

SPM
36:2

SPM
36:1

SPM
36:0

60.1
14.8
2.44
PC
42:0
PC
42:0

0.7
0.3
0.03

PC
35:5
PC O
36:5

PC
35:4
PC O
36:4

PC
35:3
PC O
36:3

11.4
4.2
0.46

18.1
6.0
0.74

8.2
2.1
0.33

PC
38:3
PC
38:3

PC
38:2
PC
38:2

PC
38:1
PC
38:1

PC
38:0
PC
38:0

129.0
32.5
5.23

54.8
17.2
2.22

10.5
3.7
0.43

11.0
3.6
0.45

7.1
1.9
0.29

PC
43:6
PC O
44:6

PC
43:5
PC O
44:5

PC
43:4
PC O
44:4

PC
43:3
PC O
44:3

2.5
0.7
0.10

0.7
0.2
0.03

3.0
1.8
0.12

0.8
0.3
0.03

SPM
37:1

1.5
0.4
0.06

SPM
38:3

PC
34:0
PC
34:0
4.6
3.1
0.19

SPM
38:2

SPM
38:1

SPM
38:0

SPM
39:2

G. Liebisch et al. / Biochimica et Biophysica Acta 1686 (2004) 108117

Table 4
PC and SPM (panel A) species in human plasma; SPM species (panel B) concentration after saponification of PC

FA based on
sphingoid
base d18: 1

SPM
12:0

SPM
14:0

SPM
15:0

SPM
16:1

SPM
16:0

SPM dih
16:0

Conc. Mean
Conc. S.D.
% of total
Conc.
Saponified
% of
unsaponified

0.6
0.2
0.1
0.6

13.1
3.6
2.3
13.1

7.2
1.8
1.2
8.7

20.5
4.6
3.5
24.2

140.6
28.9
24.3
200.3

7.0
1.9
1.2
14.7

Conc. Mean
Conc. S.D.
% of total
Conc.
Saponified
% of
unsaponified

83

99

107

95

SPM
39:1

SPM
40:4

SPM
40:3

SPM
40:2

SPM
21:0

SPM
22:3

SPM
22:2

3.9
1.4
0.7
3.9

1.0
0.7
0.2

10.4
5.4
1.8
3.9

85

26

SPM
17:0

SPM
dih
17:0

SPM
18:2

SPM
18:1

SPM
18:0

SPM
dih
18:0

SPM
19:0

SPM
20:2

SPM
20:1

SPM
20:0

SPM
dih
20:0

SPM
21:1

0.6
0.2
0.1
0.9

4.2
1.3
0.7
6.9

1.1
0.4
0.2
2.5

0.9
0.4
0.1
2.2

13.0
3.3
2.3
17.4

27.1
6.6
4.7
41.9

2.6
1.4
0.5
6.7

3.1
1.4
0.5
4.5

0.6
0.4
0.1
0.7

7.9
2.7
1.4
10.1

31.8
13.8
5.5
25.0

10.1
6.9
1.8
2.9

0.7
0.3
0.1

99

132

140

118

103

99

95

SPM
40:1

SPM
40:0

SPM
41:3

SPM
41:2

SPM
41:1

SPM
41:0

SPM
42:4

SPM
22:1

SPM
22:0

SPM dih
22:0

SPM
23:2

SPM
23:1

SPM
23:0

SPM
dih
23:0

34.1
9.2
5.9
34.4

43.3
12.3
7.5
43.5

1.3
0.5
0.2

13.7
3.3
2.4
16.1

16.5
4.0
2.9
16.0

0.9
0.5
0.1
0.5

102

94

4.1
2.6
0.7
2.0
64

111

100

72

92

108

95

112

114

56

SPM
42:3

SPM
42:2

SPM
42:1

SPM
42:0

SPM
43:2

SPM
43:1

SPM
44:3

SPM
44:2

SPM
24:3

SPM
24:2

SPM
24:1

SPM
24:0

SPM
dih
24:0

SPM
25:1

SPM
25:0

SPM
26:2

SPM
26:1

4.4
2.3
0.8
5.3

36.5
8.9
6.3
39.2

80.5
19.4
13.9
108.4

27.4
6.6
4.7
33.1

0.9
0.8
0.2

0.6
0.4
0.1

4.4
2.1
0.8
3.2

0.7
0.4
0.1

0.8
0.5
0.1
2.2

96

98

97

109

80

131

PC and SPM (panel A) species were quantified in 20 healthy blood donors (IS PC 28:0). PC species were assigned according to the sum of both fatty acids containing either two acyl bonds or one alkyl and one
acyl bond (for species with an odd number of C-atoms). SPM species were assigned either as sum of sphingoid base and N-linked fatty acid or based on a dihydroxy sphingosine base with 18 carbon atoms
containing one double bond (d18:1) indicating the C-atoms and double bonds of the N-linked fatty acid. Species assignment based on a dihydroxy sphingosine base with 18 carbon atoms without double bond
(d18:0) was termed Dihydrosphingomyelin (SPM dih). The displayed values are mean and S.D. in AM and the proportion of total in percent. Only species above a detection limit of 0.6 AM were shown.
In panel B, SPM species concentrations of a saponified plasma sample are shown, which were compared to a non-saponified control. For saponification the plasma sample was treated with 1 M sodium hydroxide
in 90% ethanol for 1 h at 60 8C and IS were added after saponification.

G. Liebisch et al. / Biochimica et Biophysica Acta 1686 (2004) 108117

Sum of
sphingoid
base and FA
FA based on
sphingoid
base d18: 1

84

SPM
17:1

115

116

G. Liebisch et al. / Biochimica et Biophysica Acta 1686 (2004) 108117

with a simple sample preparation, a short analysis time and a


highly automated data processing software, this method is
well suited for high-throughput PC and SPM analysis.
Together with other assays recently established for highthroughput lipid analysis [3638], this method may be a
useful tool for the investigation of the complex glycerophospholipid and sphingolipid metabolism in clinical as
well as basic biochemical studies. An application of this
assay has already been shown in different studies utilizing a
variety of sample material such as analysis of lipid microdomains [5], clinical studies using plasma samples [39] and
analysis of different tissue samples from fatty acid transport
protein 4- (Fatp 4) deficient mice [40].
Acknowledgements
We thank Dr. Thomas Langmann for critical reading of
the manuscript and Doreen Mqller for expert technical
assistance. We are grateful to Volker Krey for performing
initial MS studies. This work was supported by Deutsche
Forschungsgemeinschaft (SFB 585-A4).
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Biochimica et Biophysica Acta 1734 (2005) 86 89


http://www.elsevier.com/locate/bba

Erratum

Erratum to bHigh-throughput quantification of phosphatidylcholine


and sphingomyelin by electrospray ionization tandem mass
spectrometry coupled with isotope corrections algorithmQ
[Biochimica et Biophysics Acta, 1686 (2004) 108117]
Gerhard Liebisch, Bernd Lieser, Jan Rahtenberg, Wolfgang Drobnik, Gerd SchmitzT
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, D-93042 Regensburg, Germany
Received 1 June 2004; received in revised form 16 August 2004; accepted 1 September 2004
Available online 27 January 2005

Due to our mistake, Table 3 and the last sentence of


the first paragraph of section 3.2 contained errors and the
Appendix A was not published. Therefore we publish the
corrected Table 3, the last sentence of the first paragraph of
section 3.2 and the Appendix A on the coming pages.

The Publisher apologizes for the inconvenience.


In addition, the calibration line slope of the alkyl
bond containing PC O 36:4 was not significantly
different from the slope of the diacyl PC species (data
not shown).

Table 3
Precision intra day and inter day of PC and SPM species
Phosphatidylcholine
IS
Intraday
PC 28:0
PC 44:0
Interday
PC 28:0
PC 44:0

Sphingomyelin
Saturated

Monounsaturated

Polyunsaturated

O-alkyl

Total

Total

Mean
CV%
Mean
CV%

0.05
3.7
0.05
6.3

0.47
3.0
0.44
5.8

2.01
3.3
1.93
6.3

0.17
2.7
0.16
5.8

2.53
3.2
2.42
6.2

0.56
3.6
0.56
6.1

mean
CV%
mean
CV%

0.06
11.7
0.05
10.1

0.56
9.2
0.49
10.9

2.17
8.8
1.95
11.5

0.17
9.7
0.15
10.8

2.78
8.8
2.50
11.2

0.59
8.7
0.56
12.1

The intraday values are mean in mM and the coefficient of variation (CV) from 10 sample aliquots measured in series using PC 28:0 and PC 44:0 as internal
standard, respectively. The inter day values are mean in mM and CV of a plasma sample calculated from seven different runs.

DOI of original article 10.1016/j.bbalip.2004.09.003.


T Corresponding author. Tel.: +49 941 944 6201; fax: +49 941 944
6202.
E-mail address: gerd.schmitz@klinik.uni-regensburg.de (G. Schmitz).
1388-1981/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbalip.2005.01.002

Erratum

87

Appendix A. Excel data sheet structure


The following data sheet structure is used for the isotope correction algorithm described below.
Calculated isotope percentages (ISO_Sheet)
Species

[M+H]+

Monoisotopic

1 Isotope

2 Isotope

3 Isotope

4 Isotope

PC 34:0
PC 34:1
PC 34:2
PC 34:3
PC 34:4
SPM 38:0
SPM 38:1
SPM 38:2
SPM 38:3

762.6
760.6
758.6
756.6
754.5
761.6
759.6
757.6
755.6

0.6507255
0.6509208
0.6511161
0.6513115
0.6515069
0.6440499
0.6442431
0.6444365
0.6446298

0.2758635
0.275751
0.2756384
0.2755257
0.2754129
0.2822972
0.2821886
0.2820799
0.2819712

0.062164
0.062102
0.0620399
0.0619778
0.0619156
0.0628892
0.0628255
0.0627618
0.062698

0.0098257
0.009811
0.0097963
0.0097816
0.0097668
0.009494
0.0094789
0.0094639
0.0094489

0.0011971
0.0011947
0.0011924
0.00119
0.0011876
0.0010815
0.0010793
0.001077
0.0010748

Uncorrected intensities sheet (Uncorr_Sheet)


File Name

PC 34:4

SPM 38:3

PC 34:3

SPM 38:2

PC 34:2

SPM 38:1

PC 34:1

SPM 38:0

Target Mass
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Sample 11
Sample 12
Sample 13
Sample 14
Sample 15
Sample 16
Sample 17
Sample 18
Sample 19
Sample 20
Sample 21
Sample 22
Sample 23
Sample 24
Sample 25

754.5
25580
21120
50380
79400
43870
50340
82970
79690
54650
58330
65980
75910
49970
67880
60880
67490
76610
56350
66360
94010
83370
75580
77730
88880
75830

755.6
17020
23490
29320
38710
32560
37490
52150
66740
51350
49570
35880
46050
51970
50000
65190
36650
44750
40780
38950
84020
61030
65470
51300
62610
48260

756.6
264500
231100
352600
436000
356500
608200
445300
774100
450200
782300
422900
484400
529900
497200
450600
738900
659800
597200
542000
642200
771900
553300
706400
787200
500000

757.6
263800
284100
321700
409800
442100
539600
533200
571100
535700
579500
505800
467600
608800
574100
492200
548700
502000
503600
491800
671900
718600
627100
576400
812700
533900

758.6
5475000
9264000
12230000
13580000
11650000
15640000
13690000
20240000
17820000
20870000
16130000
16140000
17720000
15420000
14380000
17930000
19410000
17030000
16920000
17250000
21320000
17270000
24800000
19150000
16840000

759.6
2676000
4490000
5726000
6600000
5612000
7664000
7137000
9482000
8304000
9821000
7894000
7652000
8566000
7915000
7322000
8421000
9164000
8291000
8093000
8714000
10170000
8783000
11470000
9179000
8010000

760.6
4910000
4639000
5370000
7880000
8725000
12140000
9569000
12360000
10960000
13850000
11220000
9751000
8869000
11340000
10810000
14170000
15660000
14470000
12090000
12430000
15840000
11330000
11120000
13370000
12330000

761.6
2090000
1907000
1986000
3123000
3402000
5046000
3878000
4889000
4187000
5669000
4433000
3812000
3336000
4382000
4473000
5678000
6374000
5699000
4847000
4915000
6288000
4446000
4165000
5288000
5027000

Species are sorted according to increasing m/z.

Corrected intensities sheet (Corr_Sheet)


File Name

PC 34:4

SPM 38:3

PC 34:3

SPM 38:2

PC 34:2

SPM 38:1

PC 34:1

SPM 38:0

Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample

21927
19750
48420
72354
41060
44771
78131
76314
50659
55322
62920
70673

6939
14837
8416
6558
14578
17326
18046
33729
29048
25515
8602
15010

259259
222687
344252
426020
346127
596180
429819
751981
432546
765781
413055
470943

153108
188152
174519
227831
293633
285019
348428
248550
349119
252228
329273
265837

5383170
9160199
12120640
13439508
11488247
15458176
13496180
20059015
17625507
20686251
15946326
15978476

378315
590500
572762
882027
714836
1083316
1383214
954818
802011
1027737
1105122
854809

4228649
3504376
3961046
6208986
7312318
10187323
7671280
10025480
8923389
11423696
9210945
7849348

180468
226716
69467
204077
61221
391628
289673
246561
62788
417677
182709
162557

1
2
3
4
5
6
7
8
9
10
11
12

88

Erratum

Appendix A (continued)
File Name

PC 34:4

SPM 38:3

PC 34:3

SPM 38:2

PC 34:2

SPM 38:1

PC 34:1

SPM 38:0

Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample

46207
63541
58064
67490
76610
55025
64668
86666
73552

31601
22175
40019
8120
12364
17225
11237
45751
27755

511560
481316
427483
728934
647111
584392
530883
613706
752441

388613
367362
306588
238536
225900
253878
265151
406506
396454

17500671
15212958
14204431
17755983
19249221
16862911
16753139
17012838
21074324

1111820
1431823
1272462
870129
983446
1118857
967043
1463035
1198603

6707858
9257038
8893926
12092202
13390624
12368389
10065277
10161052
13299784

121959
91293
366935
202956
315407
96110
236220
211134
219164

13
14
15
16
17
18
19
20
21

Appendix B. Isotope correction algorithm


Variable Description:
Samplenumber=Number of samples analyzed
Speciesnumber=Number of PC and SPM species
Uncorr_LabelLine=Line of the species label in Uncorr_Sheet
Uncorr_MassLine=Line of the target mass in Uncorr_Sheet
Uncorr_DataCol=First column of the intensity data in Uncorr_Sheet
Corr_DataLine=First line of the intensity data in Corr_Sheet
Corr_DataCol=First column of the intensity data in Corr_Sheet
Algorithm in Visual Basic
Intensities are first transferred without correction from Uncorr_Sheet to the Corr_Sheet. The intensities then are
manipulated using the algorithm below according to the isotope percentages defined in the ISO_Sheet.
Sub Isotopecorrection()
Dim Isotopepercentage(0 To 4) As Single, Moleculemass1 As Single, Moleculemass2 As Single
Dim Speciesname As String, Isotope As Integer, Column_Corr as Integer
For Species=0 To Speciesnumber-1 (REM: Loop for the species)
Speciesname=Worksheets(bUncorr_SheetQ).Cells(Uncorr_LabelLine, Uncorr_DataCol+Species)
(REM: Selects the name of the species, whose isotope were corrected)
IsotopeLine=Function_ IsotopeLine (Speciesname)
(REM: Function, which delivers the line of the selected species in the ISO_Sheet)
Moleculemass1=Worksheets(bUncorr_SheetQ).Cells(Uncorr_MassLine, Uncorr_DataCol+Species)
(REM: m/z of the selected species is picked)
Isotopepercentage(0)=Worksheets(bISO_SheetQ).Cells(IsotopeLine, 3)
(REM: Picks the monoisotopic percentage from the ISO_Sheet for the selected species)
Column_Cor=1
(REM: Counts the colums in the Corr_Sheet)
If IsotopeLineN0 Then
Isotopepercentage(0)=Worksheets(bIsotopeQ).Cells(IsotopeLine, 3)
(REM: Picks the monoisotopic percentage from the ISO_Sheet for the selected species)
For IsotopeNumber=1 To 4 (REM: Loop for the isotope peaks)
Isotopepercentage(IsotopeNumber)=Worksheets(bIsotopeQ).Cells(IsotopeLine, 3+IsotopeNumber)
Moleculemass2=Worksheets(bUncorr_SheetQ).Cells(Uncorr_MassLine, Uncorr_Int_Col+Species+Column_
Cor)
If Abs(Moleculemass2-Moleculemass1-IsotopeNumber)b0.2 Then
(REM: Only if the absolute value of the difference between isotope target mass and selected species mass
and the isotope number is below 0.2 a correction is performed)
For Sample=0 To Samplenumber-1 (REM: Loop for the samples)
If Worksheets(bCorr_SheetQ).Cells(Corr_DataLine+Sample, Corr_DataCol+Species+Column_Cor)N0 Then
Worksheets(bCorr_SheetQ).Cells(Corr_DataLine+Sample, Corr_DataCol+Species+Column_Cor).Value=_
Worksheets(bCorr_SheetQ).Cells(Corr_DataLine+Sample, Corr_DataCol+Species+Column_Cor)-_

Erratum

Worksheets(bCorr_SheetQ).Cells(Corr_DataLine+Sample, Corr_DataCol+Species)
_/Isotopepercentage(0)* Isotopepercentage(IsotopeNumber)
(REM: Correction formula)
End If
Next Sample
Column_Cor=Column_Cor+1
End If
Next IsotopeNumber
End If
Next Species
End Sub

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89