Phosphatidylethanols (PEths) are specific, direct alcohol biomarkers with a substantially longer half-life than ethanol, and can be used to distinguish between heavy- and social drinking. More than forty PEth homologues have been detected in blood from heavy drinkers, and PEth 16:0/18:1 is the predominant one.
Trang 1Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/chroma
phospholipids
Marisa Henriques Maria a , Benedicte Marie Jørgenrud b , Thomas Berg b , ∗
a Faculty of Sciences of the University of Lisbon, Campo Grande, Lisboa 1749-016, Portugal
b Department of Forensic Sciences, Division of Laboratory Medicine, Section of Drug Abuse Research, Oslo University Hospital, P.O Box 4950 Nydalen,
N-0424, Lovisenberggt 6 Oslo 0456, Norway
Article history:
Received 26 August 2022
Revised 11 October 2022
Accepted 12 October 2022
Available online 14 October 2022
Keywords:
Phosphatidylethanol
PEth 16:0/18:1
Reversed phase LC-MS/MS
Alcohol
Blood
Phosphatidylethanols(PEths)arespecific,directalcoholbiomarkerswithasubstantiallylongerhalf-life thanethanol,andcanbeusedtodistinguishbetweenheavy-andsocialdrinking.MorethanfortyPEth homologueshavebeendetectedinbloodfromheavy drinkers,and PEth16:0/18:1isthepredominant one.SincePEthsarephospholipidsitcanbedifficulttoisolatethemfromunwantedphospholipidsduring samplepreparation.TominimizepossiblematrixeffectsitisthereforeimportanttoseparatePEthsfrom otherphospholipidsduringLC-MS/MSanalysis.Inthisstudy,wehaveinvestigatedhowtheretentionand chromatographicseparationofeightPEthhomologuesandthephospholipidbackgroundareinfluenced
bychangesinmobilephasecompositionusingtwodifferentLCcolumns,theAcquityBEHC18 column (50× 2.1mmID,1.7μmparticles)andtheKinetexbiphenylcolumn(100× 2.1mmID,1.7μmparticles) Ourfindingsshow thatthe bufferconcentrationoftheaqueous partofthe mobilephasehad ahuge effectontheretentionofPEthhomologuesand separationofPEths fromunwantedphospholipids.By usingabuffer-freemobile phaseconsistingof0.025%ammoniainType1water, pH10.7,assolventA andmethanolassolventB,alleightPEthhomologueswereseparatedfromboththeearlyeluting lyso-phospholipidsandthelaterelutingphospholipidswithtwofattychainsusingtheBEHC18 column.The knowledgeobtainedinthisstudy canbeofgreatimportanceforthoseseekingtodevelopreliableand robustbioanalyticalLC-MS/MSmethodsfordeterminationofPEthhomologues
© 2022TheAuthor(s).PublishedbyElsevierB.V ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/ )
Alcohol is a legal psychoactive substance consumed worldwide
during cultural, religious and social practices, and provides
per-ceived satisfaction to many users However, alcohol use is toxic
for the human body and associated with an increased risk of
var-ious negative health effects, injuries and mortality [1–5] Alcohol
use is also associated with huge economic and social costs
indi-viduals and for the society [6–8] Recently, a growing interest in
phosphatidylethanols (PEths) as biomarkers for alcohol
consump-tion has emerged PEths are a group of direct alcohol
biomark-ers with a substantially longer half-life than ethanol, and they are
formed in various tissues exclusively in the presence of alcohol [9–
∗Corresponding author
E-mail address: rmthbe@ous-hf.no (T Berg)
12] When consuming alcohol, the majority of the dose ( ≈ 92–95%)
is oxidized to acetaldehyde and further to acetate, while about 5%
is excreted unchanged in urine, sweat and breath, and a tiny part is metabolized to PEths and other non-oxidative metabolites [ 10 , 13 ] Still, there is a significant correlation between concentration of PEth in blood and alcohol intake [ 14 , 15 ] PEth concentrations in blood can be used to detect alcohol use up to three-four weeks after abstinence and to distinguish between different drinking pat-terns, such as heavy and social drinking [ 15 , 16 ] The most abundant and frequently analyzed PEth homologue is PEth 16:0/18:1 [ 17 , 18 ] Other PEth homologues frequently found in human blood are PEth 16:0/18:2, PEth 18:0/18:2 and PEth 18:0/18:1 The proportion of PEth homologues appear to differ according to the drinking habits and the time passed after the last alcohol intake Since blood elim-ination half-life of the various PEths is different, it can be impor-tant to include more PEth homologues in cases where one seeks
to discriminate between different drinking patterns and between
https://doi.org/10.1016/j.chroma.2022.463566
0021-9673/© 2022 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )
Trang 2M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566
Fig 1 Simplified molecular structure of most common phospholipids; the phospholipids with glycerol backbone (glycerophopholipids) and those with a sphingoid backbone
(sphingomyelin phospholipids) Figure was created based on information from Xia and Jemal and Lordan et al (32, 37) a Lyso-phospholipids have only one tail A hydrogene (H) has then replaced one chain in either 1-sn or 2-sn position, most commonly H has replaced C = O-R in the 2-sn position However, most phospholipids got two tails
b 1-sn position for glycerophospholipids may also be CH 2 -O-CH 2 -CH 2 -R1 (alkyl ether) or CH 2 -O-CH = CH-R1 (vinyl ether) c The oxygen in the red ring can be considered as part of the head group For instance, in phosphatidylethanol the “ethanol” can be considered to include the oxygene attached to the phosphorus, since R3 = ethyl (see also Fig 2 )
recent consumption and older consumption of alcohol [ 19 , 20 ] So
far, nearly 50 different PEth homologues have been found in blood
from heavy drinkers [ 21 ].
For targeted qualitative and quantitative bioanalysis of small
molecules in various biological matrices, LC-MS/MS has been one
of the most valuable analytical techniques used for many years
[ 22–26 ] There are many reversed phase (RP) LC-MS/MS
meth-ods developed for determination of one or more PEth homologues
in blood [ 23 , 27–30 ] However, when analyzing PEths, which is a
group of abnormal glycerolphospholipids, other unwanted
phos-pholipids not removed during sample preparation may generate
different challenges, such as changing column performance,
in-creasing column backpressure, and generate matrix effects [31–35]
Phospholipids are a class of lipids and they are essential
com-ponents in biological membranes, tissue and fluids in both plant
and animal cells [ 33 , 36 ] They are amphiphilic compounds with
both hydrophilic and lipophilic properties Their molecular
struc-ture contains a polar “head” connected to two (sometimes only
one) non-polar chains of various lengths and various degree of
sat-uration ( Fig 1 ).
Hundreds of different phospholipids are described in the
liter-ature In general (see Fig 1 ) for the polar head; pKa ≈ 0–2 for
the phosphate group (acidic), pKa ≈ 9–11 for the amine group
(ba-sic functional head group for cholines, ethanolamines and serines)
and pKa ≈ 3–5 for the carboxyl group (e.g glycerophospholipids
where R1 or R2 = H), with some changes due to hydrogen
bond-ing [37] As seen from Fig 1 there are many sub-classes of
phos-pholipids Two subgroups can be distinguished by their backbones,
the sphingoid base backbone and the glycerol backbone
phospho-lipids Other subgroups can be categorized based on the
num-ber of fatty chains (“di” or “mono”) Lyso-phospholipids are those
with only one non-polar tail, either at the sn-1 position
(1-lyso-phospholipids) or at the sn-2 position (2-lyso-phospholipids)
Sub-groups can also be categorized based on the R3 group attached to
the phosphate-moiety, and the most common phospholipids, ac-counting for 60–70 % of the total plasma phospholipid, is phos-phatidylcholines (PCs) [31]
In bioanalytical LC-MS/MS methods it is easy to remove un-wanted phospholipids during sample preparation, for instance by using liquid-liquid extraction (LLE) or supported liquid extraction (SLE) with an organic solvent(s), such as tert butyl methyl ether (MTBE) or mixtures of heptane/ethylacetate, ([ 31 , 4 , 38 ] However, the PEths will be removed at the same time [ 38 , 39 ] By addition of
an alcohol (e.g.: 2-propanol) to the organic solvent used during LLE
or SLE, PEth recovery can be increased, but other unwanted phos-pholipids will also be extracted and introduced into the LC-MS/MS [ 29 , 38 , 39 ].
PEths and other phospholipids have similar molecular struc-tures and physico-chemical properties Consequently, they will of-ten co-elute during LC-MS/MS analysis It can therefore be of great importance to know and understand how to minimize co-elution between PEths and other phospholipids during LC-MS/MS analy-sis, which to our knowledge is not previously described in other published LC-MS/MS methods In this study, we investigated the chromatographic separation of as much as eight PEth homologues and the phospholipid background using different mobile phase compositions on two different ultra-high performance LC (UHPLC) columns Fig 2 shows the molecular structure of the eight PEth homologues investigated in this study All eight PEth homologues are among the most commonly occurring in human blood.
2.1 Chemicals and materials
Methanol (MeOH) of LC-MS grade was purchased from Honey-well (Seelze, Germany) Acetonitrile (ACN) of HPLC Far UV grade was purchased from JT Baker (Deventer, The Netherlands) Ethyl
Trang 3Fig 2 Molecular structures of the eight PEth homologues that were included in this study
acetate, n-heptane 2-propanol, and nitric acid (p.a,) were obtained
from Merck (Darmstadt, Germany) Formic acid (98%) was acquired
from VWR International AS (Oslo, Norway) Aqueous ammonia ( >
25%), ammonium formate, and ammonium carbonate were
ob-tained from VWR Chemicals, Prolabo (Leuven, Belgium) Type 1
water (18.2 M ) purified with a Synthesis A 10 milli-Q system
from Millipore (Billerica, MA, USA) was used.
2.2 Blank blood
PEth-free whole blood from employees at the Department of
Forensic Sciences at Oslo University Hospital was collected in 4 mL
Vacuette® K2E K2EDTA tubes from Greiner bio-one
(Kremsmün-ster, Austria).
2.3 Preparation of working solution and standard samples with eight
PEth homologues
PEth 16:0/16:0 was purchased from Avanti Polar, while PEth
16:0/18:1, PEth 16:0/18:2, PEth 16:0/20:4, PEth 17:0/18:1, PEth
18:0/18:1, PEth 18:0/18:2, PEth 18:1/18:1 were purchased from
Echelon Biosciences (Salt Lake City, USA) The stock solutions of
the PEths homologues were prepared in MeOH Working solutions
were prepared in MeOH by appropriate dilution of the stock
solu-tions LC-MS/MS analyses of the eight PEth homologues were
per-formed by injection of pure working solutions into the LC-MS/MS
instrument LC-MS/MS analyses of the phospholipid background
were performed by parent ion m/z 184 scan of extracted blank
blood samples prepared by 96-well SLE (see Section 2.4 for
extrac-tion procedure).
2.4 Sample preparation by 96-well SLE that were used for extraction
of blood samples
For investigation of the retention of phospholipid background,
extracted blank whole blood samples analyzed were prepared
by 96-well SLE using [heptane/ethylacetate (1:5, v:v )]/2-propanol
(100:20) as organic solvent, as described in a previous paper [39] ,
except the addition of Triton-X 100 After 96-well SLE the
elu-ates collected in 96-collection plates were evaporated to dryness
and the residues were reconstituted in 100 μL 2-propanol/ACN or
MeOH, vortexed and then placed in the sample organizer for LC– MS/MS analysis Injection volume was 1 μL.
2.5 Instrumental analysis
LC-MS/MS analyses were performed on an Acquity UPLC I-class system with flow through needle (FTN), comprised of a binary sol-vent manager, sample manager with sample organizer, and a col-umn oven, coupled to a Xevo TQ-S MS/MS, all from Waters (Mil-ford, MA, USA) Chromatographic separations were performed on a Acquity BEH C18 column (50 × 2.1 mm ID, 1.7 μm particles) from Waters (Milford, MA, USA) and a Kinetex biphenyl core shell col-umn (100 × 2.1 mm ID, 1.7 μ m particles) from Phenomenex (Tor-rance, CA, USA) at a column temperature of 60 °C Mobile phase flow rate was 0.6 mL/min for all tests on the Acquity BEH C18 col-umn whereas it was 0.5 mL/min for the tests performed on the Kinetex biphenyl column Injection volume was 1 μL in all tests Electrospray ionization (ESI)-MS/MS detection was performed in negative ESI (ESI−) with multiple reaction monitoring (MRM) using argon as collision gas MS/MS settings were as follows; capillary voltage 2.6 kV, source temperature 150 °C, desolvation gas tem-perature 500 °C, cone gas flow 300 L/h and desolvation gas flow
10 0 0 L/hr Acquisition and processing of data were performed us-ing MasslynxTM software (version 4.1, Waters, Milford, MA, USA) Table 1 shows the MRM transitions, cone voltages, collision ener-gies and dwell times used for LC-MS/MS analysis of the eight PEth homologues For determination of PEth homologue retention times, LC-MS/MS analyses were performed in MRM mode by injection of pure working solutions In contrast, determination of general phos-pholipid background was performed by parent ion scan of m/z 184
of extracted blood samples prepared by 96-SLE (see Section 2.4 ), using positive ESI, cone voltage 50 V, capillary voltage 1.25 kV, MS and MS/MS mode collision energy of 2 and 40, respectively.
To minimize possible matrix effects, it is important to under-stand how PEths can be chromatographically resolved from un-wanted phospholipids during LC-MS/MS analysis In this case, dif-ferent mobile phase compositions and gradient profiles were in-vestigated on two different UHPLC columns, and some interest-ing results were found Each chromatogram shows overlaid
Trang 4chro-M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566
Table 1
MRM transitions, cone voltages, collision energies and dwell times
MRM transitions MS/MS parameters a
Fig 3 Chromatographic separation of eight PEths and phospholipid background by LC-MS/MS analysis using an acidic mobile phase (pH 5, left hand side) and a basic mobile
phase (pH 10, right hand side) Concentration of ammonium formate in the aqueous part of the mobile phase were 20 mM (a), 5 mM (b) and 2 mM (c) 2 mM and 5 mM ammonium formate buffers were prepared by dilution of 20 mM buffer using Type 1 water Gradient profile: 60% B in 0.0–0.2 min, 60–88% B in 0.2–0.3 min, 88–98% B in 0.3–3.8 min, 98–100% B in 3.8–3.9 min, 100% B in 3.9–6.4 min, 100–60%B in 6.4–6.5 min, 60% B in 6.5–7.0 min Retention time order for PEth homologues were; 1: PEth 16:0/20:4, 2: PEth 16:0/18:2, 3: PEth 16:0/16:0, 4: PEth 16:0/18:1, 5: PEth 18:1/18:1, 6: PEth 18:0/18:2, 7: PEth 17:0/18:1, 8: PEth 18:0/18:1
matograms from two subsequently LC-MS/MS analyses; one by
in-jecting working solutions with the eight PEth homologues (MRM
mode) and injection of extracted blood sample for determination
of the phospholipid background (parent ion m/z 184 scan, red
broad peaks) By doing this it was possible to do several injections
of the PEth homologues without injecting the dirtier extracted
blood samples into the system, the latter may change column
per-formance and give retention times variation over time.
3.1 Influence of mobile phase pH and mobile phase buffer concentration on an Acquity BEH C18 column
When optimizing RP LC separation, mobile phase pH, gradient profile, choice of organic modifier and choice of column, are impor-tant factors For ionizable compounds (acids, bases) a mobile phase
pH that increases ionization will reduce retention, and vice versa These effects are especially observed at pH values near the pKa
Trang 5Fig 4 Chromatographic separation between eight PEth homologues and phospholipid background obtained by LC-MS/MS analysis on three different Acquity BEH C 18 columns (50 × 2.1 mm ID, 1.7 μm particles) using an acidic mobile phase consisting of ammonium formate buffer (pH 5) as solvent A and MeOH as solvent B On all three columns, mobile phase buffer concentration of 2, 5 and 20 mM was tested, as depicted in figure None of the three columns were complete new before the tests Gradient profile and retention time order for PEths were the same as described for Fig 3
Fig 5 Chromatographic separation between eight PEth homologues and phospholipid background obtained by LC-MS/MS analysis on a BEH C 18 column using basic mobile phases with different buffer concentrations; 20 mM (a), 5 mM (b), 2 mM (c) and 0 mM (d) LC-MS/MS analysis were performed using a mobile phase Solvent A solution of ammonium formate buffers, pH 10, in Fig 5 a–c, whereas 0.025% ammonia in Type 1 water, pH 10.7, was used in Fig 5 d Gradient profile and retention time order were the same as described for Fig 3
Trang 6M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566
Fig 6 Chromatographic separation between eight PEth homologues and phospholipid background obtained by LC-MS/MS analysis on a BEH C 18 column using basic mobile phases consisting of 0.025% ammonia (solvent A, pH 10.7) and MeOH (solvent B) Retention times for PEth homologues and phospholipid background shown for LC-MS/MS analysis before analysis of extracted samples (a), after injection of 50 extracted blood samples (b), after injection of 100 extracted blood samples (c), and after injection of
150 extracted blood samples (d) Gradient profile and retention time order were the same as described inin Fig 3 caption
value of the compound [40–44] Since the PEth homologues in this
study have an acidic functional group with pKa value ≈ 1-2, the
retention times of the PEths were not expected to be influenced
much by changes in the mobile phase pH at pH values above 3-4.
Concerning the mobile phase buffer concentration, changing ionic
strength can be a significant parameter for controlling retention of
ionized compounds and for neutral compounds by generating
salt-ing out effect (increased retention at higher salt concentrations).
Fig 3 shows the retention times of the PEths homologues and
phospholipid background obtained by an LC-MS/MS analyses on a
BEH C18column using an acidic (pH 5) and a basic (pH 10) mobile
phase, both tested with three different buffer concentrations.
The retention times of the PEths homologues and
phospho-lipid background were similar when using both mobile phase pH
5 and pH 10 However, reducing the buffer concentration clearly
reduced the retention of all eight PEth homologues and improved
separation between the PEth homologues and the phospholipid
background (broad red peaks), probably due to salting out
ef-fect at higher buffer concentrations Interestingly, retention of the
unwanted phospholipids seemed almost unaffected by both the
change in both mobile phase pH and by the change in the mobile
phase buffer concentration The results presented in Fig 3 shows
good separation between the PEths and the unwanted
phospho-lipids using the 2 mM buffer as solvent A However, further
inves-tigations revealed that retention times of the PEth homologues and
also the separation between PEth and the unwanted phospholipids
were not stable over time, even though column type (Acquity BEH
C18(50 × 2.1 mm ID, 1.7 μm particles)), gradient profile, column
temperature, mobile phase composition and flow were the same
( Fig 4 ).
Based on the results observed in Figs 3 and 4 , it is clear, de-spite the variation of the retention times, that reducing the buffer concentration in the aqueous part of the mobile phase resulted in reduced retention times for the PEths This issue was further in-vestigated using high pH mobile phases by testing a basic mobile phase without any buffer ( Fig 5 ).
Fig 5 clearly illustrates reduced retention of the eight PEths when using 0.025 % ammonia in Type 1 water, pH 10.7, com-pared to using mobile phases with ammonium formate buffer,
pH 10, at various concentrations The retention of the unwanted phospholipids seemed almost unaffected by the changes in Sol-vent A composition This high pH mobile phase consisting of 0.025% ammonia in Type 1 water as solvent A and MeOH as solvent B seemed to be the best choice for separation of all eight PEth homologues from the late eluting phospholipids There-fore, this mobile phase was used in a subsequent experiment for investigation of how retention times of PEth homologues var-ied after analyses of 50, 100, and 150 extracted blood samples ( Fig 6 ).
Fig 6 shows a reduction in the retention times over time for all PEth homologues after analyzing several extracted blood sam-ples, while retention of the unwanted phospholipids remained the same A reason for the changes in the PEths retention times might
be due to background components from the extracted blood sam-ples bonding to and changing the column stationary phase surface The challenge with drifting retention times was only tested using the basic buffer free mobile phase However, this issue is some-thing worth investigated further in future studies in order to inves-tigate how retention times can be kept as stable as possible over time Almost all LC-MS/MS analyses of the eight PEths in this study
Trang 7Fig 7 Chromatographic separation between eight PEth homologues and phospholipid background obtained by LC-MS/MS analysis on a BEH C 18 column using basic mobile phases consisting of 0.025% ammonia in Type 1 water (pH 10.7) and MeOH LC-MS/MS analyses were performed using two similar gradients, “Gradient 84–98” (a) and
“Gradient 88–98 (b) Graphic illustration of the both gradient profiles used are included in figure (c) Gradient profiles: 60% B in 0.0–0.2 min, 60–84 (or 88)% B in 0.2– 0.3 min, 84 (or 88) – 98% B in 0.3–3.8 min, 98–100% B in 3.8–3.9 min, 100% B in 3.9–6.4 min, 100–60%B in 6.4–6.5 min, 60% B in 6.5–7.0 min Retention order for PEth homologues were the same as described in Fig 3 caption
were based on injection of pure working solutions only However,
a few LC-MS/MS analyses of extracted blood sample mixed (1:1,
v:v ) with working solution containing the eight PEths, were
per-formed (data not shown) Generally, improved signal/noise values
and higher peak responses were observed using the buffer free
mobile phase However, the influence of mobile phase
composi-tion on signal/noise and peak responses for PEth homologues in
extracted blood samples should be investigating more thoroughly
in future studies.
In Fig 7 , chromatograms for the eight PEth homologues, the
lyso-phospholipids and the other later eluting phospholipids using
two different mobile phase gradients, is depicted.
The best separation of PEth homologues and the phospholipids
was obtained by using the “84-98 gradient profile” ( Fig 7 b)
Gra-dient profiles used in these tests started at 60% MeOH which for
many compounds would lead to early elution and poor separation.
However, as mentioned by Meng et al., for RP LC analysis,
phos-pholipids will normally be retained (“focused”) on the column in
RP LC-MS/MS methods as long as the mobile phase contains ≤ 60
% MeOH [23]
3.2 Influence of mobile phase pH and mobile phase buffer
concentration on a Kinetex biphenyl column
All previous tests shown in Figs 3–7 were performed on
Ac-quity BEH C18 columns, which are pH stable within pH values
2–12 However, a few tests were also performed on a Kinetex biphenyl column, which is stable and recommended for use with mobile phases with a pH between 1.5 and 8.5 Fig 8 shows re-tention of the PEth homologues and the phospholipid background obtained at two mobile phase pH values, both tested with three different buffer concentrations.
Fig 8 shows similar results as obtained for the BEH C18
columns, the buffer concentration of the aqueous part of the mo-bile phase had a great effect on the retention of the PEth homo-logues and the separation between the PEth homologues and the phospholipid background Meanwhile, the change in mobile phase buffer concentration had minimal effects on the retention of the phospholipid background Retention time changes were also in-vestigated further comparing ammonium formate buffer to am-monium acetate buffer, but no or only minor changes were ob-served As can also be seen in Fig 8 , the mobile phase with
pH 3.1 lead to slightly increased retention times of the PEths This is most probably due to the increase in lipophilicity as
a consequence of reduced ionization at lower pH values (pKa value for the PEth homologues ≈ 1.5–2) When comparing the retention order obtained on the BEH C18 columns versus the Kinetec biphenyl column, PEth homologues with double bonds generally had increased retention compared to the other PEth homologues on the Kinetex biphenyl column This was also as expected, since the biphenyl stationary phase has more affin-ity towards compounds with double bonds due to dipole-dipole interactions.
Trang 8M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566
Fig 8 Chromatographic separation of eight PEth homologues and phospholipid background on a Kinetex Biphenyl column (100 × 2.1 mm ID, 1.7 μm particles), using acidic
mobile phases with a buffer concentration of 20 mM (a), 5 mM (b) and 2 mM (c) Mobile phase composition and pH of solvent A as described in the figure Phospholipid background was obtained by parent ion m/z 184 scan Gradient profile: 10% B in 0.0–0.2 min, 10–84% B in 0.2–0.3 min, 84–96% B in 0.3–4.0 min, 96–100% B in 4.0–4.1 min, 100% B in 4.1–7.5 min, 100–10%B in 7.5–7.6 min, 10% B in 7.6–8.2 min Mobile phase flow rate was 0.5 mL/min PEth homologues retention order (shortest retention time first): 1: PEth 16:0/16:0, 2: PEth 16:0/18:2, 3: PEth 16:0/20:4, 4: PEth 16:0/18:1, 5: PEth 17:0/18:1, 6: PEth 18:0/18:2, 7: PEth 18:1/18:1, 8: PEth 18:0/18:1
Since PEths are phospholipids and difficult to isolate from
un-wanted phospholipids during sample preparation, it is important
to know how to separate them chromatographically to minimize
the possibility of matrix effects In this study, retention and
sepa-ration of eight PEth homologues and the phospholipid background
were investigated by LC-MS/MS analysis using two different UHPLC
columns and mobile phases with different pH values and different
mobile phase buffer concentrations Our findings show that the
re-tention of the PEth homologues were basically unaltered using
mo-bile phase pH 5–10 This finding was as expected since PEths with
their acidic pKa value at approximately 1.5–2.0 are completely
ion-ized above pH 5 However, the buffer concentration of the
aque-ous part of the mobile phase had a huge effect on the retention of
PEth homologues, while the unwanted phospholipids seemed
al-most unaffected In conclusion it was found that LC-MS/MS
analy-sis on the Acquity BEH C18column (50 × 2.1 mm ID, 1.7 μm
parti-cles) using a buffer free mobile phase consisting of 0.025%
ammo-nia in Type 1 water (pH 10.7) as solvent A and MeOH as solvent B,
separated all eight PEth homologues from the phospholipids, both
the early eluting lyso-phospholipids and the later eluting
phos-pholipids All PEth homologue peaks were narrow and
symmetri-cal Optimization of the gradient profile was also important in
or-der to separate the eight PEths from the phospholipids This study
demonstrates the effect various mobile phase buffer concentrations
and gradient profile have on the retention and separation of PEth
homologues and phospholipid background, which can be of great
importance for those working with RP LC-MS/MS analysis of PEths
in biological samples The effects of these parameters on different LC-MS/MS systems should be further investigated.
The authors declare that they have no known competing finan-cial interests or personal relationships that could have appeared to influence the work reported in this paper.
– review & editing Benedicte Marie Jørgenrud: Writing – review
& editing Thomas Berg: Conceptualization, Data curation, Investi-gation, Writing – original draft, Writing – review & editing.
Data availability
Data will be made available on request.
Acknowledgments
The authors like to thank Galina Nilsson for assistance and valuable help in the laboratory and Lena Kristoffersen, Dag Helge Strand and Kristin Gaare for fruitful discussion regarding LC-MS/MS bioanalysis of PEth homologues in blood The authors also like to thank Tao Angell-Petersen McQuade for valuable comments and critical reading of the manuscript.
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