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Application of inductively coupled plasma mass spectrometry to phospholipid
analysis
Article in Journal of Analytical Atomic Spectrometry · January 2004
DOI: 10.1039/B307545A
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Application of inductively coupled plasma mass spectrometry to
phospholipid analysis
Miroslav Kovačevič,a Regina Leber,b Sepp D. Kohlweinb and Walter Goessler*c
a
National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia
Institute of Molecular Biology, Biochemistry and Microbiology, Karl Franzens
University Graz, Schubertstrasse 1, A-8010 Graz, Austria
c
Institute of Chemistry-Analytical Chemistry, Karl Franzens University Graz,
Universitaetsplatz 1, A-8010 Graz, Austria. E-mail: walter.goessler@uni-graz.at
b
Received 2nd July 2003, Accepted 9th October 2003
First published as an Advance Article on the web 25th November 2003
Phospholipids are the main constituents of membranes in all types of prokaryotic and eukariotic cells. Due to
their complexity and heterogeneity in biological samples, qualitative and quantitative analyses of membrane
phospholipids in cellular extracts represent major analytical challenges, mainly due to suitable and sensitive
detection methods. The inductively coupled plasma mass spectrometer (ICP-MS) is a suitable detector for
selective determination of phospholipids as they all contain phosphorus. Phospholipids are extractable with
organic solvents, therefore liquid chromatography with an organic mobile phase was used for separation of
different lipid species. Solvent load to the plasma was reduced by splitting the mobile phase prior to reaching
the nebulizer, by chilling the spray chamber to 25 uC and by optimisation of carrier gas flow for maximum
condensation of organic vapours. Despite desolvation, oxygen was added to prevent carbon deposition on
interface cones. To reduce polyatomic interferences at m/z ratio 31 (e.g. 31CH3O1) and to improve detection
limits, helium was used as a collision gas. The achieved absolute detection limits were between 0.21 and 1.2 ng
of phosphorus and were superior to those obtained by an evaporative light scattering detector, which provides
an alternative detection system for lipid analysis. The usefulness of the developed method was demonstrated by
analysis of lipid extracts from the yeast Saccharomyces cerevisiae.
Introduction
DOI: 10.1039/b307545a
Phospholipids (PL) are complex lipids, which contain as their
backbone glycerol esterified in the sn-3 position with a
phosphate residue. The glycerol-3-phosphate is esterified at
its sn-1 and sn-2 positions with fatty acids (typically C16 to C18
carbon atoms), and at its phosphoryl group to an alcohol X, to
form various phospholipids as shown in Fig. 1.1 Some of the
alcohol residues are neutral (e.g. inositol) or positively charged
80
Fig. 1 Chemical structures of most common phospholipids and their
abbreviations* (R1 and R2 are different fatty acids groups).1
J. Anal. At. Spectrom., 2004, 19, 80–84
at cellular pH (ethanolamine, choline), leading to negatively
charged or neutral phospholipids due to the negatively charged
phosphate residue. The amphipathic character of the PL
molecules forces them spontaneously into bilayers in aqueous
systems. This tendency to form bilayers is the basis of all
cellular membranes in nature.
Phospholipid classes are characterized by the alcohol residue
esterified to the phosphate group. The basic PL structure is
phosphatidic acid, or 1,2 diacylglycerol-3-phosphate. For
example, phosphatidylcholine (PtdCho) describes a phospholipid harboring choline (N,N,N-trimethylaminoethanol-2)
esterified to the phosphate residue. The diversity of phospholipid molecular species is brought about by the diversity of fatty
acids esterified to the glycerol backbone. Yeast, for instance,
produces some 20 different molecular species of phosphatidylcholines.
Pure synthetic phospholipids are chemically defined and they
can be designated by their systematic name. For example,
abbreviation ‘‘DOPC’’ stands for 1,2-dioleoyl-phosphatidylcholine.2
A major goal of research on phospholipids is to understand
the significance of these compounds in the functioning of
cellular membranes. The majority of studies are based on
HPLC analysis of lipid extracts. Since phospholipids are
soluble only in organic solvents, normal phase liquid
chromatography is the most frequently used separation
method.3 The columns are packed either with silica-, aminoor with diol-type stationary phases and all have in common,
that they separate only different chemical classes of phospholipids, rather than molecular species differing in the fatty acid
composition.4 Mobile phases commonly employed are usually
mixtures of different solvents, such as methanol, acetone,
hexane, chloroform, tetrahydrofurane, 2-propanol or acetonitrile. For better separation, gradient elution should be applied,
This journal is ß The Royal Society of Chemistry 2004
starting from a weak (nonpolar) mobile phase to a strong
(more polar) mobile phase. However, reverse phase liquid
chromatography can also be used for their separation.5,6
Due to the absence of intrinsic molecular properties and the
heterogeneity of the substance classes, detection of phospholipids is a major analytical problem. Different detection
systems have been described for phospholipid analysis.7 The
refractive index detection suffers from poor detection limits
and is only useful for simple mixtures, since gradient elution
cannot be applied due to a baseline drift.8 Reported detection
limits are approximately 20 ng of phosphorus.9 Because many
solvents used for liquid chromatography are non-transparent
in the UV range (200–210 nm, useful for detection of
phospholipids) these detectors have serious constraints with
respect to the selection of mobile phases.10 To overcome lack of
chromophores in the phospholipid molecules, post-column
derivatisation was applied by Rastegar et al.6 They derivatised
the sample with Naproxen and derivatives were subjected to
HPLC with UV absorption measurements at 230 nm. The
reported detection limit was 0.3 ng (expressed as phosphorus).
With the introduction of the evaporative light scattering
detector, analysis of all lipid species became easier.8,11,12
However, this detector suffers from a limited linear range, with
400 ng of phosphorus as a lowest sample amount giving linear
response.13,14 Lower sample quantities can be detected (10 ng
of phosphorus), however the calibration response is not
linear.15 Another possibility for detection of phospholipids is
hyphenation of HPLC to mass spectrometry, giving better
selectivity towards the compounds of interest.16 With electrospray ionisation mass spectrometry a quantification limit for
phosphatidylserine was reported to be 0.05 ng of phosphorus.17
Electrospray ionisation used together with tandem mass
spectrometry can be used to obtain molecular information
about the phospholipids without prior chromatographic
separation.18–20
It is a common property of all described detectors that their
responses are depending on the molecular structure of species
being analysed and, therefore, standard compounds are required
for their quantification. To overcome this problem, an
inductively coupled plasma mass spectrometer (ICP-MS) can
be used as a detection technique, since its response is theoretically
dependant only on the element in question. The hyphenation of
ICP-MS to LC for phospholipid analysis was first described by
Axelsson et al.21 The authors have reported successful normal
phase HPLC separation and ICP-MS detection of three standard
compounds belonging to PC, PE and PG phospholipid classes.
However, authors did not provide any details on analysis
procedure and they did not test this method on real complex
biological samples. Additionally they used a low flow membrane
desolvation unit for removal of interfering organic solvents,
which is still not widely accessible in laboratories. It should be
noted, that the determination of phosphorus and its compounds
by an ICP-MS is not an easy task because phosphorus has a high
ionisation potential and consequently is poorly ionised in the
plasma. Additionally, it suffers from polyatomic interferences at
m/z ratio 31 when low mass resolution instruments are used.
Therefore not only 31P1 ions are measured, but also polyatomic
ions such as 12C1H316O1.22,23 As a result, poorer detection limits
are achieved when carbon compounds are in the sample matrix.
Jiang and Houk24 reported a decrease of the mass spectrometer
response with increasing concentration of organic modifiers in
mobile phase of the LC-ICP-MS system when polyphosphates
and adenosine phosphates were analysed.
In this work we present a detailed study of processes
occurring during the determination of phospholipids by a
quadrupole ICP-MS instrument equipped with a conventional
double pass spray chamber for desolvation. Additionally, we
are demonstrating its use for the characterization and detection
of phospholipids from yeast lipid extracts by applying a
modified chromatographic separation published by Sas et al.3
Experimental
Reagents and sample preparation
Acetone, hexane and triethylamine all of p.a. purity were
purchased from Fluka (Buchs, Switzerland). Methanol, acetic
acid (96%), chloroform and MgCl2 all of p.a. purity were
purchased from Merck (Darmstadt, Germany). Chemically
defined phospholipids were purchased from Avanti (Alabaster,
AL, USA): 1,2-dioleoyl-phosphatidic acid monosodium
salt (C39H72O8PNa, DOPA), 1,2-dioleoyl-phosphatidylcholine
(C44H84NO8P, DOPC), 1,2-dioleoyl-phosphatidylethanolamine (C41H78NO8P, DOPE), 1,2-dioleoyl-phosphatidylglycerol
sodium salt (C42H78O10PNa, DOPG), 1,2-dioleoyl-phosphatidylserine sodium salt (C42H77NO10PNa, DOPS) and phosphatidylinositol sodium salt isolated from bovine liver
(C47H82O13PNa, PI).
The standard mixture of six phospholipids was prepared by
diluting each phospholipid standard in a chloroform/methanol
mixture (2/1, v/v). The concentrations of each expressed as
phosphorus were as follows: 3.3 mg l21 DOPA, 2.9 mg l21
DOPG, 2.7 mg l21 PI, 3.1 mg l21 DOPE, 3.0 mg l21 DOPS and
2.9 mg l21 DOPC. Approximately 1 g of wild-type yeast cells
(Saccharomyces cerevisiae W303, MATa, leu2, ura3, his3, ade2,
trp1) were resuspended in 10 ml of deionised water and cells
were disrupted in a glass bead homogeniser (B. Braun
Melsungen, Germany). Then 80 ml of chloroform/methanol
mixture (2/1, v/v) was added and the suspension was stirred for
30 min at room temperature, additionally 20 ml of a MgCl2
solution (0.034%) was added for phase separation and it was
again stirred for 30 min. After centrifugation for 5 min, the
organic phase was transferred to a round bottom flask and
evaporated to dryness. Lipids were dissolved in 2 ml of
chloroform/methanol mixture (2/1, v/v) and the solution was
transferred to a HPLC vial. Solutions were stored at 220 uC.
Chromatographic system
HPLC separations were carried out using an Agilent 1100
chromatographic system (Waldbronn, Germany) equipped
with a thermostated autosampler (variable injection loop
0–100 ml), YMC-Pack Diol-120 column (250 6 4.6 mm,
5 mm) (Kyoto, Japan) maintained at 50 uC and a flow rate of
0.6 ml min21. The composition of mobile phase A was acetone/
hexane/acetic acid/triethlyamine—900/70/14/2 (v/v) and the
composition of mobile phase B was methanol/hexane/acetic
acid/triethlyamine—900/70/14/2 (v/v). The following gradient
elution program was used: 95% of A at 0 min, 82% of A at
40 min, 55% of A at 42 min, 40% of A at 44 min, 40% of A at
49 min, 95% of A at 49.5 min and 95% of A at 58 min.
Detection system
The 0.6 ml min21 flow from the HPLC column was split to
approximately 130 ml min21 before reaching the 7500c ICP-MS
(Agilent, Waldbronn, D) equipped with a PFA microconcentric nebulizer, a Scott double pass spray chamber and an
octopole reaction cell (ORC). Since constant intake of sample
to nebulizer was desired, self-aspiration was minimised by a
69 cm long capillary (0.127 mm id) mounted between splitter
and nebulizer. According to the desired flow, appropriate
resistance of splitter drain was achieved by a 16 cm long
capillary (0.127 mm id). To prevent deposition of carbon on the
interface cones, an optional gas (20% oxygen in argon) was
applied through a T-piece connecting spray chamber and torch
(narrow injection tube—1.5 mm). Since added oxygen
promotes corrosion of interface cones, a platinum sampler
cone was used. The skimmer cone was made from nickel. The
detection was carried out by recording m/z ratio 31 at scan rate
of 0.3 s per point.
The system was optimised by pumping mobile phase A
J. Anal. At. Spectrom., 2004, 19, 80–84
81
containing 2 mg l21 of phosphorus as a DOPE. During the
tuning procedure, such conditions were chosen that the signal
at m/z 31 was as high as possible and that the mass peak at m/z
31 and neighbouring peaks m/z 30 and m/z 32 were clearly
separated. Further optimisation of detector response was
performed in flow injection analysis mode with mobile phase A
as a carrier solution and by injection of 2 ml of 90 mg l21 of
phosphorus as a DOPE diluted in mobile phase A. To evaluate
results, signal to background ratios (SB) were calculated as a
quotient between height of phosphorus signal and height of
background signal. After having optimal conditions for
detection of phosphorus, helium was added as a collision gas
to reduce the background signal.
For the detection of the phospholipids the following optimised conditions were used: Plasma gas 15 l min21, auxiliary
gas 1.0 l min21, carrier gas 0.50 l min21, optional gas flow rate
24% (of carrier gas flow rate), rf power 1600 W (reflected power
¡10 W), ORC gas (helium) 4.0 ml min21, spray chamber
temperature 25 uC and sample depth (torch-interface distance)
10 mm. All chromatograms were smoothed before integration.
Results and discussion
Optimisation of detector response
The detection of phospholipids by ICP-MS suffers from two
problems. The first one is the stability of the plasma in the
presence of organic solvents. Since phospholipids are not soluble
in aqueous solutions, organic solvents have to be used for sample
extraction and chromatographic separation. Despite a modified
sample introduction system (cooled spray chamber and torch
with narrow injector tube), intake rates should be low, therefore
splitting of mobile phase before reaching the detector was
utilised. Additionally, oxygen has to be added to carrier gas to
avoid carbon deposition on interface cones. The second problem
is the formation of carbon-based polyatomic ions in the plasma
with m/z ratio 31 (31CH3O1).22,23 Their formation is unavoidable, since organic solvents are in the mobile phase. Therefore,
the optimisation procedure should at the same time maximise the
phosphorus signal and minimise the intensity of the polyatomic
ions, to avoid high background in the chromatogram.
The first parameter we optimised was the rf power. We tested
powers from 1200 to 1600 W by recording flow injection signal.
The experiment was performed at three different sample
depths (6, 9 and 12 mm) at constant carrier gas flow rates of
0.68 l min21 and without any make-up gas flow. As expected,
when highest rf power was used, SB ratio was highest at
all three tested sample depths, since high temperature is
required to decompose organic matrix and to improve
ionisation of phosphorus. According to this results rf power
of 1600 W was chosen for further optimisation and routine
measurements.
In order to find optimum carrier and make-up gas flows,
several combinations of these gas flows were tested by flow
injection technique at 1600 W of rf power. The whole
experiment was conducted at three different sample depths
(6, 9 and 12 mm). Carrier gas flows were tested in the range
from 0.2 to 0.70 l min21 at four different make-up gas flows
(0, 0.10, 0.15 and 0.20 l min21). The signal to background
ratios are not improved by the addition of a make-up gas.
The usage of a make-up gas only shifts the optimal carrier
gas flow rate to lower values. That means the signal to
background ratio is only depending on total flow of gases
through the spray chamber and the most important process in
spray chamber is condensation of organic vapours. When too
high total gas flows are applied, the residence time of the
aerosol in the spray chamber is shorter, the amount of
condensed organic matter is lower and therefore the background derived from organic vapours in the form of carbon
based polyatomic ions is increased. However, when too low
82
J. Anal. At. Spectrom., 2004, 19, 80–84
flow rates are applied, condensation is efficient, but transport
of aerosol particles is compromised and SB ratios are
decreased. This fact was also confirmed by observation of
the colour of the plasma and the required amount of oxygen to
prevent carbon deposition. At higher carrier or make-up gas
flow rates the plasma was greener and more oxygen was
needed. On the basis of these results it was decided not to use
any make-up gas.
The optimum sampling point of ions in the plasma is of
course influenced by the gas flows and sample depth. Therefore
both parameters were optimised, while no make-up gas flow
was used. The tested carrier gas flow rates were in the range
from 0.35 to 0.55 l min21 and sampling depths were in the
range from 5 to 11 mm. The results are presented in Fig. 2 as a
two-dimensional plot, where colour intensity is representing SB
ratio. The highest SB ratios were observed when carrier gas
flow rate of 0.50 l min21 was applied and sample depths were
from 5 to 9 mm. These conditions are therefore giving highest
sensitivity for detection of phospholipids. However, there is an
empirical rule that the intensive green coloured zone on the
front end of the plasma should end before reaching the tip of
the sampler cone.25 This requires higher amounts of oxygen
and means shorter life time of the cones. As a compromise
between good sensitivity and system robustness, a carrier gas
flow rate of 0.50 l min21 and sample depth of 10 mm were
chosen for routine work.
Despite optimal conditions for the desolvation process in the
spray chamber, the background signal was still high and was
negatively influencing the limits of detection. Another negative
consequence was noticeable drift of baseline in the chromatogram due to change of mobile phase composition during
chromatography. This problem was solved with helium as a
collision gas. We explored helium flow rates through the ORC
in the range from 0 to 6 ml min21. At each tested helium flow
rate, the flow injection signal was recorded and SB ratios were
calculated (Fig. 3). As can be seen, helium improved SB ratio at
flow rates between 4 and 6 ml min21. For the practical
applications a flow rate of 4 ml min21 was applied, giving fivetimes higher SB ratio than without using any collision gas. At
higher helium flow rates, suppression of both phosphorus and
background signal was too strong and the noise of the signal
was hindering improved SB ratios. At the same time,
background was efficiently reduced for about two orders of
magnitude, minimising the problem of baseline drift during the
chromatography.
Chromatographic separation
Successful chromatographic separation of six standard phospholipids was achieved by utilisation of modified conditions
Fig. 2 Signal to background ratios (SB) at different carrier gas flow
rates (x-axis) and different sample depths (y-axis) (1600 W, no make-up
gas).
Fig. 3 Phosphorus signal (left y-axis, full line), background signal
(left y-axis, dotted line) and signal to background ratio (SB) (right
y-axis, full bold line) at different helium flow rates through the octopole
reaction cell.
described by Sas et al.3 A typical chromatogram is presented in
Fig. 4. Almost all six phospholipid standards were base-line
separated. However, when a yeast lipid extract was analysed
(Fig. 5), a splitting of the largest signal at y36–39 min
belonging to PC class was observed. All attempts (changing the
gradient) to improve the chromatographic resolution of these
two signals were unsuccessful. Only the higher signal at the low
retention time side matched the retention time of DOPC. There
are two possible explanations. The first could be that we
separated various species of PC containing fatty acids with
different chain length. The second possibility is that the
compound eluting at y38 min (marked with a ?, Fig. 5)
corresponds to another class of yeast phospholipids that have
until now not been identified. In that case a better separation is
essential for accurate quantification and more standard
compounds should be tested for identification via matching
of retention times. Another possible solution would be
application of tandem molecular mass spectrometry for
identification of the unknown signal. Besides the split signal
of PC, eight chromatographic signals were obtained in the
chromatogram of the yeast lipid extract. Four of them were
identified as PA, PI, PE, PS, and PC, but four peaks were
unclassified. The signals at retention time y5 and y57 min are
related to the mobile phase. The signal at y5 min is coming
from the solvent, since its intensity is correlating with the
amount of injected solvent. This signal might derive from some
polyatomic ions in the solvent mixture or it is belonging to
some impurities in one of the solvents. The origin of the peak at
y57 min could also be attributed to the mobile phase. Besides
these two signals deriving from the chromatographic system,
we clearly found two unknown compounds that did not match
the retention times of any of the phospholipid standards
available to us. While X1 (Fig. 5) is a non-identified
phosphorous containing compound, we can only speculate
about the identity of ‘‘X2’’. Its retention time is similar to the
retention time of orthophosphate. Therefore, we cannot affirm
that it belongs to phospholipids, in spite of the fact, that the
Fig. 4 Separation of six chemically defined phospholipids in standard
mixture on YMC Pack Diol-120 column (250 6 4.6 mm, 5 mm) with ICPMS detection of phosphorus at m/z ratio 31 (5 ml injected, 0.6 ml min21,
each peak corresponds y15 ng of phosphorus).
Fig. 5 Separation of phospholipid classes in yeast lipid extract on
YMC Pack Diol-120 column (250 6 4.6 mm, 5 mm) with ICP-MS
detection of phosphorus at m/z ratio 31 (2 ml injected, 0.6 ml min21).
presence of highly polar orthophosphate in the chloroform/
methanol mixture is very unlikely.
Quantification
Different volumes (0.5–20 ml) of standard mixtures of the six
phospholipids were injected for construction of calibration
curves and for the determination of the detection limits. A good
linearity with correlation coefficients (R) equal or above 0.9997
was obtained for all tested compounds and it was generally
covering range from y1.5 to y60 ng of phosphorus (Table 1).
The exceptions were compounds DOPA and DOPG which had
linear response only up to y15 ng of phosphorus due to poor
chromatographic separation at higher injected amounts, and
compounds DOPE and DOPS, with linear range starting at
3.0 ng of phosphorus. Limits of detection were estimated as
peak height equalling three times the baseline noise and are also
presented in the Table 1. As expected, limits of detection were
lowest for those compounds having well defined peak shapes
Table 1 Calibration parameters (expressed as a mass of phosphorus)
Compound
Retention time/min
Calibration curve
R
Linear range/ng
LOD/ng
Reproducibilitya (%)
DOPA
DOPG
PI
DOPE
DOPS
DOPC
6.7
7.8
14.2
18.3
28.1
35.9
A ~ 18000 6 m 2 830
A ~ 23400 6 m 2 5650
A ~ 25000 6 m 2 850
A ~ 21000 6 m 2 10400
A ~ 16500 6 m 2 18600
A ~ 19500 6 m 2 230
0.9998
0.9997
0.9999
0.9999
0.9998
0.9999
1.6–16
1.4–14
1.4–55
3.0–61
3.0–59
1.5–59
0.36
0.21
0.54
1.2
1.2
0.50
¡6
¡5
¡7
¡7
¡16
¡14
a
At lowest point of calibration curve.
J. Anal. At. Spectrom., 2004, 19, 80–84
83
Table 2 Peak areas, calculated masses and concentrations, relative amounts of identified compounds and semi-quantitatively determined relative
amounts of all phospholipids in yeast lipid extract (all values are expressed as phosphorus)
Class
Peak area/103 units
Mass/ng
Concentration/mg l21
Relative amountsa (%)
Semi-quantiative relative
amountsb (%)
PA
PI
PE
PS
PC
X1
X2
65.5
783
1440
150
2380
400
45.6
3.7
31
69
10
120
—
—
0.74
6.3
14
2.0
24
—
—
1.6
13
29
4.3
51
—
—
1.2
15
27
2.8
44
7.6
0.9
a
Relative amounts of identified compounds. b Relative amounts determined by semi-quantitative procedure.
(DOPA 0.36 ng and DOPG 0.21 ng of phosphorus) and highest
for compounds having broad peaks (DOPE 1.2 ng and DOPS
1.2 ng of phosphorus). Table 1 additionally contains data on
reproducibility of peak areas at the lowest point of the
calibration curve. Compared to performances of other
detectors (especially evaporative light scattering detector)
that are routinely used for phospholipids analysis, an ICPMS gives superior detection limits. However, it cannot be
compared to molecular mass spectrometry, since it does not
give any structural information.
The usefulness of the developed method for analysis of
phospholipids was demonstrated on a complex lipid extract
from yeast as presented in Fig. 5. Each identified compound
was quantified by using calibration curves given in Table 1. The
results are presented in Table 2 as peak areas and as calculated
masses and concentrations of each identified compound in the
sample extract. It should be noted, that all masses and
concentrations are expressed as phosphorus and that peak
coeluting with chemical class of PC was integrated and
considered as belonging to PC class. According to the
extraction method, calculating the concentration of each
class of phospholipids in yeast is impossible, since many
steps in the extraction procedure are not quantitative. If such
data were required, then the extraction procedure should be
modified or an internal standard should be added before the
extraction. Since properties of yeast sample are defined by its
phospholipid composition, relative amounts of identified
compounds were calculated and are given as percentages in
Table 2.
Theoretically, the response of an ICP-MS is element depending, meaning it is the same for all compounds regardless to their
structure. The determined response factors, which are part of
calibration curves in Table 1, have values from 16500 to 25000
peak area units per ng of phosphorus. Deviations from theory are
expected, since we used a gradient elution program, giving a
different matrix composition for each compound resulting in
different nebulisation efficiencies. Moreover, the changing matrix
composition might also change the ionisation efficiency of
phosphorus in the plasma. Therefore simplification of quantification procedure by using a calibration curve based only on one
compound should be used with critical evaluation of obtained
results and it already belongs in the field of semi-quantitative
analysis. In cases, when we are interested only in obtaining
approximate ratios between classes of phospholipids in the
sample, only peak areas without any calibration can be used with
an awareness of possible errors, of course. To show the usefulness
of such a quick semi-quantitative analysis, a yeast lipid extract
was also treated in that way. Peak areas of all peaks found in the
chromatogram were summed, their relative amounts were
calculated and are also presented in Table 2. Compared to
literature data2 one can clearly say that this semi-quantitative
approach gives good agreement.
84
J. Anal. At. Spectrom., 2004, 19, 80–84
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Acknowledgements
Support by the Ministry of Education, Science and Sports of
the Republic of Slovenia for a travel grant to M.K. and the
Austrian Science Fund FWF (SFB Biomembranes F706) to
S.D.K. is gratefully acknowledged.
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