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Application of inductively coupled plasma mass spectrometry to phospholipid

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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
b
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

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 at cellular pH (ethanolamine, choline), leading to negatively

charged or neutral phospholipids due to the negatively charged
Phospholipids (PL) are complex lipids, which contain as their phosphate residue. The amphipathic character of the PL
backbone glycerol esterified in the sn-3 position with a molecules forces them spontaneously into bilayers in aqueous
phosphate residue. The glycerol-3-phosphate is esterified at systems. This tendency to form bilayers is the basis of all
its sn-1 and sn-2 positions with fatty acids (typically C16 to C18 cellular membranes in nature.
carbon atoms), and at its phosphoryl group to an alcohol X, to Phospholipid classes are characterized by the alcohol residue
form various phospholipids as shown in Fig. 1.1 Some of the esterified to the phosphate group. The basic PL structure is
alcohol residues are neutral (e.g. inositol) or positively charged phosphatidic acid, or 1,2 diacylglycerol-3-phosphate. For
example, phosphatidylcholine (PtdCho) describes a phospho-
lipid harboring choline (N,N,N-trimethylaminoethanol-2)
esterified to the phosphate residue. The diversity of phospho-
lipid 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 phosphati-
dylcholines.
Pure synthetic phospholipids are chemically defined and they
can be designated by their systematic name. For example,
abbreviation ‘‘DOPC’’ stands for 1,2-dioleoyl-phosphatidyl-
choline.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-, amino-
DOI: 10.1039/b307545a

or with diol-type stationary phases and all have in common,

that they separate only different chemical classes of phospho-
lipids, 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,
Fig. 1 Chemical structures of most common phospholipids and their hexane, chloroform, tetrahydrofurane, 2-propanol or acetoni-
abbreviations* (R1 and R2 are different fatty acids groups).1 trile. For better separation, gradient elution should be applied,

80 J. Anal. At. Spectrom., 2004, 19, 80–84 This journal is ß The Royal Society of Chemistry 2004
starting from a weak (nonpolar) mobile phase to a strong Experimental
(more polar) mobile phase. However, reverse phase liquid
chromatography can also be used for their separation.5,6 Reagents and sample preparation
Due to the absence of intrinsic molecular properties and the Acetone, hexane and triethylamine all of p.a. purity were
heterogeneity of the substance classes, detection of phospho- purchased from Fluka (Buchs, Switzerland). Methanol, acetic
lipids is a major analytical problem. Different detection acid (96%), chloroform and MgCl2 all of p.a. purity were
systems have been described for phospholipid analysis.7 The purchased from Merck (Darmstadt, Germany). Chemically
refractive index detection suffers from poor detection limits defined phospholipids were purchased from Avanti (Alabaster,
and is only useful for simple mixtures, since gradient elution AL, USA): 1,2-dioleoyl-phosphatidic acid monosodium
cannot be applied due to a baseline drift.8 Reported detection salt (C39H72O8PNa, DOPA), 1,2-dioleoyl-phosphatidylcholine
limits are approximately 20 ng of phosphorus.9 Because many (C44H84NO8P, DOPC), 1,2-dioleoyl-phosphatidylethanol-
solvents used for liquid chromatography are non-transparent amine (C41H78NO8P, DOPE), 1,2-dioleoyl-phosphatidylglycerol
in the UV range (200–210 nm, useful for detection of sodium salt (C42H78O10PNa, DOPG), 1,2-dioleoyl-phosphati-
phospholipids) these detectors have serious constraints with dylserine sodium salt (C42H77NO10PNa, DOPS) and phospha-
respect to the selection of mobile phases.10 To overcome lack of tidylinositol sodium salt isolated from bovine liver
chromophores in the phospholipid molecules, post-column (C47H82O13PNa, PI).
derivatisation was applied by Rastegar et al.6 They derivatised The standard mixture of six phospholipids was prepared by
the sample with Naproxen and derivatives were subjected to diluting each phospholipid standard in a chloroform/methanol
HPLC with UV absorption measurements at 230 nm. The mixture (2/1, v/v). The concentrations of each expressed as
reported detection limit was 0.3 ng (expressed as phosphorus). phosphorus were as follows: 3.3 mg l21 DOPA, 2.9 mg l21
With the introduction of the evaporative light scattering DOPG, 2.7 mg l21 PI, 3.1 mg l21 DOPE, 3.0 mg l21 DOPS and
detector, analysis of all lipid species became easier.8,11,12 2.9 mg l21 DOPC. Approximately 1 g of wild-type yeast cells
However, this detector suffers from a limited linear range, with (Saccharomyces cerevisiae W303, MATa, leu2, ura3, his3, ade2,
400 ng of phosphorus as a lowest sample amount giving linear trp1) were resuspended in 10 ml of deionised water and cells
response.13,14 Lower sample quantities can be detected (10 ng were disrupted in a glass bead homogeniser (B. Braun
of phosphorus), however the calibration response is not Melsungen, Germany). Then 80 ml of chloroform/methanol
linear.15 Another possibility for detection of phospholipids is mixture (2/1, v/v) was added and the suspension was stirred for
hyphenation of HPLC to mass spectrometry, giving better 30 min at room temperature, additionally 20 ml of a MgCl2
selectivity towards the compounds of interest.16 With electro- solution (0.034%) was added for phase separation and it was
spray ionisation mass spectrometry a quantification limit for again stirred for 30 min. After centrifugation for 5 min, the
phosphatidylserine was reported to be 0.05 ng of phosphorus.17 organic phase was transferred to a round bottom flask and
Electrospray ionisation used together with tandem mass evaporated to dryness. Lipids were dissolved in 2 ml of
spectrometry can be used to obtain molecular information chloroform/methanol mixture (2/1, v/v) and the solution was
about the phospholipids without prior chromatographic transferred to a HPLC vial. Solutions were stored at 220 uC.
separation.18–20
It is a common property of all described detectors that their
responses are depending on the molecular structure of species Chromatographic system
being analysed and, therefore, standard compounds are required HPLC separations were carried out using an Agilent 1100
for their quantification. To overcome this problem, an chromatographic system (Waldbronn, Germany) equipped
inductively coupled plasma mass spectrometer (ICP-MS) can with a thermostated autosampler (variable injection loop
be used as a detection technique, since its response is theoretically 0–100 ml), YMC-Pack Diol-120 column (250 6 4.6 mm,
dependant only on the element in question. The hyphenation of 5 mm) (Kyoto, Japan) maintained at 50 uC and a flow rate of
ICP-MS to LC for phospholipid analysis was first described by 0.6 ml min21. The composition of mobile phase A was acetone/
Axelsson et al.21 The authors have reported successful normal hexane/acetic acid/triethlyamine—900/70/14/2 (v/v) and the
phase HPLC separation and ICP-MS detection of three standard composition of mobile phase B was methanol/hexane/acetic
compounds belonging to PC, PE and PG phospholipid classes. acid/triethlyamine—900/70/14/2 (v/v). The following gradient
However, authors did not provide any details on analysis elution program was used: 95% of A at 0 min, 82% of A at
procedure and they did not test this method on real complex 40 min, 55% of A at 42 min, 40% of A at 44 min, 40% of A at
biological samples. Additionally they used a low flow membrane 49 min, 95% of A at 49.5 min and 95% of A at 58 min.
desolvation unit for removal of interfering organic solvents,
which is still not widely accessible in laboratories. It should be
Detection system
noted, that the determination of phosphorus and its compounds
by an ICP-MS is not an easy task because phosphorus has a high The 0.6 ml min21 flow from the HPLC column was split to
ionisation potential and consequently is poorly ionised in the approximately 130 ml min21 before reaching the 7500c ICP-MS
plasma. Additionally, it suffers from polyatomic interferences at (Agilent, Waldbronn, D) equipped with a PFA microcon-
m/z ratio 31 when low mass resolution instruments are used. centric nebulizer, a Scott double pass spray chamber and an
Therefore not only 31P1 ions are measured, but also polyatomic octopole reaction cell (ORC). Since constant intake of sample
ions such as 12C1H316O1.22,23 As a result, poorer detection limits to nebulizer was desired, self-aspiration was minimised by a
are achieved when carbon compounds are in the sample matrix. 69 cm long capillary (0.127 mm id) mounted between splitter
Jiang and Houk24 reported a decrease of the mass spectrometer and nebulizer. According to the desired flow, appropriate
response with increasing concentration of organic modifiers in resistance of splitter drain was achieved by a 16 cm long
mobile phase of the LC-ICP-MS system when polyphosphates capillary (0.127 mm id). To prevent deposition of carbon on the
and adenosine phosphates were analysed. interface cones, an optional gas (20% oxygen in argon) was
In this work we present a detailed study of processes applied through a T-piece connecting spray chamber and torch
occurring during the determination of phospholipids by a (narrow injection tube—1.5 mm). Since added oxygen
quadrupole ICP-MS instrument equipped with a conventional promotes corrosion of interface cones, a platinum sampler
double pass spray chamber for desolvation. Additionally, we cone was used. The skimmer cone was made from nickel. The
are demonstrating its use for the characterization and detection detection was carried out by recording m/z ratio 31 at scan rate
of phospholipids from yeast lipid extracts by applying a of 0.3 s per point.
modified chromatographic separation published by Sas et al.3 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 flow rates are applied, condensation is efficient, but transport
tuning procedure, such conditions were chosen that the signal of aerosol particles is compromised and SB ratios are
at m/z 31 was as high as possible and that the mass peak at m/z decreased. This fact was also confirmed by observation of
31 and neighbouring peaks m/z 30 and m/z 32 were clearly the colour of the plasma and the required amount of oxygen to
separated. Further optimisation of detector response was prevent carbon deposition. At higher carrier or make-up gas
performed in flow injection analysis mode with mobile phase A flow rates the plasma was greener and more oxygen was
as a carrier solution and by injection of 2 ml of 90 mg l21 of needed. On the basis of these results it was decided not to use
phosphorus as a DOPE diluted in mobile phase A. To evaluate any make-up gas.
results, signal to background ratios (SB) were calculated as a The optimum sampling point of ions in the plasma is of
quotient between height of phosphorus signal and height of course influenced by the gas flows and sample depth. Therefore
background signal. After having optimal conditions for both parameters were optimised, while no make-up gas flow
detection of phosphorus, helium was added as a collision gas was used. The tested carrier gas flow rates were in the range
to reduce the background signal. from 0.35 to 0.55 l min21 and sampling depths were in the
For the detection of the phospholipids the following opti- range from 5 to 11 mm. The results are presented in Fig. 2 as a
mised conditions were used: Plasma gas 15 l min21, auxiliary two-dimensional plot, where colour intensity is representing SB
gas 1.0 l min21, carrier gas 0.50 l min21, optional gas flow rate ratio. The highest SB ratios were observed when carrier gas
24% (of carrier gas flow rate), rf power 1600 W (reflected power flow rate of 0.50 l min21 was applied and sample depths were
¡10 W), ORC gas (helium) 4.0 ml min21, spray chamber from 5 to 9 mm. These conditions are therefore giving highest
temperature 25 uC and sample depth (torch-interface distance) sensitivity for detection of phospholipids. However, there is an
10 mm. All chromatograms were smoothed before integration. 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
Results and discussion and means shorter life time of the cones. As a compromise
between good sensitivity and system robustness, a carrier gas
Optimisation of detector response
flow rate of 0.50 l min21 and sample depth of 10 mm were
The detection of phospholipids by ICP-MS suffers from two chosen for routine work.
problems. The first one is the stability of the plasma in the Despite optimal conditions for the desolvation process in the
presence of organic solvents. Since phospholipids are not soluble spray chamber, the background signal was still high and was
in aqueous solutions, organic solvents have to be used for sample negatively influencing the limits of detection. Another negative
extraction and chromatographic separation. Despite a modified consequence was noticeable drift of baseline in the chromato-
sample introduction system (cooled spray chamber and torch gram due to change of mobile phase composition during
with narrow injector tube), intake rates should be low, therefore chromatography. This problem was solved with helium as a
splitting of mobile phase before reaching the detector was collision gas. We explored helium flow rates through the ORC
utilised. Additionally, oxygen has to be added to carrier gas to in the range from 0 to 6 ml min21. At each tested helium flow
avoid carbon deposition on interface cones. The second problem rate, the flow injection signal was recorded and SB ratios were
is the formation of carbon-based polyatomic ions in the plasma calculated (Fig. 3). As can be seen, helium improved SB ratio at
with m/z ratio 31 (31CH3O1).22,23 Their formation is unavoid- flow rates between 4 and 6 ml min21. For the practical
able, since organic solvents are in the mobile phase. Therefore, applications a flow rate of 4 ml min21 was applied, giving five-
the optimisation procedure should at the same time maximise the times higher SB ratio than without using any collision gas. At
phosphorus signal and minimise the intensity of the polyatomic higher helium flow rates, suppression of both phosphorus and
ions, to avoid high background in the chromatogram. background signal was too strong and the noise of the signal
The first parameter we optimised was the rf power. We tested was hindering improved SB ratios. At the same time,
powers from 1200 to 1600 W by recording flow injection signal. background was efficiently reduced for about two orders of
The experiment was performed at three different sample magnitude, minimising the problem of baseline drift during the
depths (6, 9 and 12 mm) at constant carrier gas flow rates of chromatography.
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 Chromatographic separation
required to decompose organic matrix and to improve
Successful chromatographic separation of six standard phos-
ionisation of phosphorus. According to this results rf power
pholipids was achieved by utilisation of modified conditions
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 back- Fig. 2 Signal to background ratios (SB) at different carrier gas flow
ground derived from organic vapours in the form of carbon rates (x-axis) and different sample depths (y-axis) (1600 W, no make-up
based polyatomic ions is increased. However, when too low gas).

82 J. Anal. At. Spectrom., 2004, 19, 80–84

Fig. 3 Phosphorus signal (left y-axis, full line), background signal Fig. 4 Separation of six chemically defined phospholipids in standard
(left y-axis, dotted line) and signal to background ratio (SB) (right mixture on YMC Pack Diol-120 column (250 6 4.6 mm, 5 mm) with ICP-
y-axis, full bold line) at different helium flow rates through the octopole MS detection of phosphorus at m/z ratio 31 (5 ml injected, 0.6 ml min21,
reaction cell. each peak corresponds y15 ng of phosphorus).

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 Fig. 5 Separation of phospholipid classes in yeast lipid extract on
compounds should be tested for identification via matching YMC Pack Diol-120 column (250 6 4.6 mm, 5 mm) with ICP-MS
of retention times. Another possible solution would be detection of phosphorus at m/z ratio 31 (2 ml injected, 0.6 ml min21).
application of tandem molecular mass spectrometry for
identification of the unknown signal. Besides the split signal presence of highly polar orthophosphate in the chloroform/
of PC, eight chromatographic signals were obtained in the methanol mixture is very unlikely.
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 Quantification
related to the mobile phase. The signal at y5 min is coming Different volumes (0.5–20 ml) of standard mixtures of the six
from the solvent, since its intensity is correlating with the phospholipids were injected for construction of calibration
amount of injected solvent. This signal might derive from some curves and for the determination of the detection limits. A good
polyatomic ions in the solvent mixture or it is belonging to linearity with correlation coefficients (R) equal or above 0.9997
some impurities in one of the solvents. The origin of the peak at was obtained for all tested compounds and it was generally
y57 min could also be attributed to the mobile phase. Besides covering range from y1.5 to y60 ng of phosphorus (Table 1).
these two signals deriving from the chromatographic system, The exceptions were compounds DOPA and DOPG which had
we clearly found two unknown compounds that did not match linear response only up to y15 ng of phosphorus due to poor
the retention times of any of the phospholipid standards chromatographic separation at higher injected amounts, and
available to us. While X1 (Fig. 5) is a non-identified compounds DOPE and DOPS, with linear range starting at
phosphorous containing compound, we can only speculate 3.0 ng of phosphorus. Limits of detection were estimated as
about the identity of ‘‘X2’’. Its retention time is similar to the peak height equalling three times the baseline noise and are also
retention time of orthophosphate. Therefore, we cannot affirm presented in the Table 1. As expected, limits of detection were
that it belongs to phospholipids, in spite of the fact, that the 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 6.7 A ~ 18000 6 m 2 830 0.9998 1.6–16 0.36 ¡6

DOPG 7.8 A ~ 23400 6 m 2 5650 0.9997 1.4–14 0.21 ¡5
PI 14.2 A ~ 25000 6 m 2 850 0.9999 1.4–55 0.54 ¡7
DOPE 18.3 A ~ 21000 6 m 2 10400 0.9999 3.0–61 1.2 ¡7
DOPS 28.1 A ~ 16500 6 m 2 18600 0.9998 3.0–59 1.2 ¡16
DOPC 35.9 A ~ 19500 6 m 2 230 0.9999 1.5–59 0.50 ¡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)

Semi-quantiative relative
Class Peak area/103 units Mass/ng Concentration/mg l21 Relative amountsa (%) amountsb (%)

PA 65.5 3.7 0.74 1.6 1.2

PI 783 31 6.3 13 15
PE 1440 69 14 29 27
PS 150 10 2.0 4.3 2.8
PC 2380 120 24 51 44
X1 400 — — — 7.6
X2 45.6 — — — 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 Acknowledgements

for compounds having broad peaks (DOPE 1.2 ng and DOPS
1.2 ng of phosphorus). Table 1 additionally contains data on Support by the Ministry of Education, Science and Sports of
reproducibility of peak areas at the lowest point of the the Republic of Slovenia for a travel grant to M.K. and the
calibration curve. Compared to performances of other Austrian Science Fund FWF (SFB Biomembranes F706) to
detectors (especially evaporative light scattering detector) S.D.K. is gratefully acknowledged.
that are routinely used for phospholipids analysis, an ICP-
MS gives superior detection limits. However, it cannot be
compared to molecular mass spectrometry, since it does not
give any structural information. References
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literature data2 one can clearly say that this semi-quantitative 24 S. Jiang and R. S. Houk, Spectrochim. Acta, 1988, 43B, 405–411.
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84 J. Anal. At. Spectrom., 2004, 19, 80–84

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