The use of hybrid surface technology (HST), applied to the metal surfaces of an ACQUITYTM UPLCTM system and column, designed to mitigate the chelation, poor peak shape and analyte loss seen with acidic phospholipids was investigated.
Trang 1Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/chroma
Application of hybrid surface technology for improving sensitivity and
phosphatidylserine
Giorgis Isaaca, ∗, Ian D Wilsonb, Robert S Plumba
a Scientific Operations, Waters Corporation, Milford, MA, 01757, USA
b Division of Systems Medicine, Dept Metabolism, Digestion and Reproduction, Imperial College, South Kensington, London, SW7 2AZ, UK
Article history:
Received 8 November 2021
Revised 21 February 2022
Accepted 24 February 2022
Available online 25 February 2022
Keywords:
Lipidomics
Metal interaction
Surface treatment
Acidic phospholipids
Phosphatidic acid
Phosphatidylserine
a b s t r a c t
Theuseofhybridsurfacetechnology(HST),appliedtothemetalsurfacesofanACQUITYTM UPLCTM sys-temandcolumn,designedtomitigatethechelation,poorpeakshapeandanalytelossseenwithacidic phospholipids was investigated.Comparedto aconventionalsystem significantimprovementsinboth sensitivity,recoveryandpeakshapewereobtainedfollowingUPLConaCSHC18columnwhentheHST wasusedfortheanalysisoflysophosphatidicacid(LPA),phosphatidicacid(PA),lysophosphatidylserine (LPS), phosphatidylserine (PS), phosphatidylinositol-monophosphates (PIP), ceramide phosphate (CerP) andsphingoidbasephosphate(SPBP).ThebenefitsinchromatographicperformanceprovidedbytheHST wereseenparticularlyatlowconcentrationsoftheseanalytes.TheHSTsystemandcolumnreducedpeak tailingby65–80%andpeakwidthby70–86%forLPAandPA.Moreover,increasedsignalintensitiesofup
to12.7timeswereobservedforLPAwiththeHSTapproachcomparedtotheequivalentuntreatedLC sys-temandcolumn.TheapplicationofthismethodologytotheanalysisofchickeneggPAandbrainporcine
PSextractswereaccompaniedbysimilarimprovementsindataquality
© 2022WatersCorporation.PublishedbyElsevierB.V ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/)
1 Introduction
Phospholipids (PLs) are one of the main components of cell
membranes and perform many important biological functions such
as cell signaling, regulation of apoptosis, lipid synthesis and trans-
port [1–5] The importance of PLs has led to an increased focus
on analytical methods for the characterization of both lipid phe-
notypes (lipidomics) and the individual molecular species How-
ever, analysis of PLs is difficult because they exist in cells and
biological fluids as complex mixtures of lipids formed from dif-
ferent combinations of polar headgroups and hydrophobic fatty
acyl chains The extreme structural diversity of lipids in real bi-
ological samples is challenging for analytical techniques due to
the large difference in physicochemical properties of the individ-
ual lipid species which may be present as acids or bases, and neu-
tral, polar or non-polar forms Currently the two main analytical
strategies used for lipidomics are direct infusion mass spectrome-
try (DIMS) and liquid chromatography coupled to mass spectrome-
try (LC/MS) DIMS can provide high-throughput analysis but suf-
∗ Corresponding author at: Waters Corporation, USA
E-mail address: Giorgis_Isaac@waters.com (G Isaac)
fers from the inability to separate and resolve isobaric/isomeric species and to identify low abundant species due to ion suppres- sion [ 6, 7] For reasons of sample compatibility, specificity and se- lectivity LC/MS has become increasingly important for the iden- tification, and/or quantification of lipids directly from biological extracts The chromatographic separation can be performed us- ing normal-phase (NP), reversed-phase (RP), hydrophilic interac- tion chromatography (HILIC) [6], or via supercritical fluid chro- matography (SFC); (these different analytical approaches are sum- marized in reference [8]) NP methods, employing solvents such
as hexane and chloroform are used for the separation of lipid classes However, NP separations often have long analysis times, can demonstrate limited analyte solubility, use toxic solvents, and lack solvent compatibility with mass spectrometry making the technique increasingly less attractive HILIC and SFC, have similar characteristics to NPLC and can be used for the separation of lipid classes with organic/water-based solvent systems for HILIC and liq- uid carbon dioxide as primary mobile phase with co-solvents for SFC, overcoming many of the challenges of the NP mode [8–10] The successful separation of phosphatidic acids (PA) and phos- phatidylserines (PS) using HILIC has been previously reported [11– 18] and SFC has demonstrated advantages in short analysis time for the separation of lipid classes RPLC on the other hand is
https://doi.org/10.1016/j.chroma.2022.462921
0021-9673/© 2022 Waters Corporation Published by Elsevier B.V This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )
Trang 2widely used for the separation of individual lipid molecular species
with separations based on their lipophilicity; a characteristic gov-
erned by the carbon chain length and number of double bonds
[19–21]
The PA and PS classes tend to elute as broad peaks under the
RPLC conditions generally used in lipidomics studies [19–22] and
this can, at least in part, be attributed to the metal-analyte inter-
actions of these lipids, particularly the phosphate moieties, with
the metal surfaces of the LC system and the column Thus, the
lipid classes that show these poor chromatographic properties typ-
ically contain functional groups such as phosphate or carboxylate,
groups that can form chelation complexes with iron and other
transition metal ions Phosphorylated and carboxylate lipids en-
compass classes such as the PA, PS, phosphatidylinositols (PI), PI
with mono-, bis and tris-phosphates, phosphatidylglycerols, phos-
phorylated sphingolipids including ceramide-phosphate (CerP) and
sphingoid base-phosphates (SPBP) All these lipids are metal sensi-
tive and readily adsorb to stainless steel surfaces present within
the flow path of the chromatographic system via a Lewis acid-
base interaction, leading to the aforementioned poor peak shape
as well as low recovery and a reduced sensitivity compared to
non-interacting lipids Mitigation of these interactions has, in the
past, involved the addition of metal chelators to the mobile phase
or sample Volatile chelators such as acetylacetone, citric, phos-
phonic or medronic acids have been used for LC/MS analyses
[22–27] However, the use of these chelators can negatively im-
pact both chromatographic selectivity and MS sensitivity Lee et al
used carbamate-embedded C18 as the stationary phase to optimize
peak tailing and separation efficiencies of acidic lipids [28] Al-
ternatively, columns constructed from a metal free material such
as polyether ether ketone (PEEK) have been described for e.g.,
the determination of SPBP and CerP [29] However, the applica-
tion of PEEK is limited by its low-pressure tolerance and whilst
coating stainless steel LC tubing and column walls with PEEK
can provide a partial solution to this problem the issue of the
poor pressure tolerance of frits prepared from PEEK would re-
main a challenge To address these issues, we have been en-
couraged by recent publications [30–32] to investigate the use
of a hybrid surface technology (HST) applied to the metal sub-
strates in UltraPerformance LC (UPLC TM) instruments and columns
This HST is based on an ethylene-bridged siloxane polymer which
has been demonstrated to be well-suited for RP and HILIC For
a detailed description and characteristics of the HST see refer-
ence [31] Based on our previous experience of the analysis of
lipids [19]we have evaluated a UPLC system and ACQUITY TM CSH
C18 column incorporating the HST and compared the results ob-
tained against those from a conventional UPLC system and AC-
QUITY CSH C18 column for the analysis of phosphorylated and
carboxylate lipids (mainly PA, PS, lysophosphatidic acids (LPA),
lysophosphatidylserines (LPS), CerP, SPBP and PI-monophosphate
(PIP))
2 Experimental
2.1 Chemicals and standards
Acetonitrile (ACN), isopropanol (IPA), formic acid (FA), chloro-
form (CHCl 3), methanol (MeOH) and ammonium formate (LC/MS
grade) were purchased from Fisher Scientific (Pittsburgh, PA, USA)
Total recovery vials were supplied by Waters Corporation (Milford,
MA, USA) Ultrapure water was obtained, in-house, from an Elga
PURELAB TM Flex water purification system (High Wycombe, Eng-
land, U.K.) LPA 16:0/0:0, PA 16:0/18:1, LPS 18:1/0:0, PS 16:0/18:1,
CerP 18:1;O2/16:0, SPBP 18:1;O2, PIP (4 ) 18:1/18:1 and differen-
tial ion mobility system suitability Lipidomix TM lipid standards,
egg chicken PA and brain porcine PS extracts were purchased from Avanti Polar Lipids (Birmingham, AL, USA)
2.2 Standard solutions
A stock solution was prepared for PA 16:0/18:1, LPS 18:1/0:0 and PS 16:0/18:1 in chloroform at 1 mg/mL The stock solution for LPA 16:0/0:0 was prepared at a concentration of 0.5 mg/mL in –CHCl 3-MeOH-H 2O (80:20:2, v/v/v) with gentle heating at 40 °C and sonication to facilitate complete dissolution A working mix- ture dilution series of 5, 50 and 250 pg/μL was prepared in IPA- ACN (50:50, v/v) f or the lipid st andards CerP 18:1;O2/16:0 and SPBP 18:1;O2 were prepared in CHCl 3-MeOH (1:1, v/v) and PIP (4 ) 18:1/18:1 in –CHCl 3-MeOH-H 2O (1:1:0.3, v/v/v) Powdered egg chicken PA and brain porcine PS extracts from Avanti Polar Lipids (Birmingham, AL, USA) were dissolved in CHCl 3at a concentration
of 1 mg/mL Working solutions of the egg chicken PA and brain porcine PS extracts were prepared in IPA-ACN (50:50, v/v) at a con- centration of 10 ng/μL The differential ion mobility system suit- ability Lipidomix lipid standards contained 1 mg/mL of each lipid and (0.25 mg/mL PI) in CHCl 3-MeOH (1:1, v/v) It contained a certi- fied mixture of 14 lipids comprising LPC 18:1/0:0, PC 14:1/14:1, PE 14:1/14:1, PS 14:1/14:1, PG 14:1/14:1, PI 14:1/14:1, PA 14:1/14:1, SM d18:1/18:1, Cer d18:1/18:1, cholesterol, CE 19:0, DG 14:1/14:1, TG (18:1/18:1/18:1) and CL 14:1/14:1/14:1/14:1 A stock solution was prepared in IPA-ACN (50:50, v/v) at 0.05 mg/mL for each lipid and 0.0125 mg/mL for PI A working Lipidomix lipid standard 100 pg/μL solution (25 pg/μL PI) was prepared in IPA-ACN (50:50, v/v) All operations involving preparing CHCl 3-based solutions were carried out in a fume hood
2.3 Chromatographic separation and mass spectrometry conditions
Chromatographic separations were performed using two sepa- rate, but similar, chromatography systems Either a conventional ACQUITY UPLC I-Class chromatography system coupled with AC- QUITY CSH C18 column (2.1 × 100 mm, 1.7 μm, p/n 186005297)
or an ACQUITY Premier UPLC System with ACQUITY Premier CSH C18 Column (2.1 × 100 mm, 1.7 μm, p/n 186009461) constructed with HST (both columns packed with the same batch of stationary phase) were employed Both systems were equipped with a binary solvent delivery system and flow through needle (FTN) autosam- pler (Waters Corporation, Milford, MA) The mobile phase consisted
of (A) –ACN-H 2O-1 M aqueous ammonium formate (600:390:10, v/v/v) in 0.1% FA and (B) IPA-ACN-1 M aqueous ammonium formate (900:90:10, v/v/v) in 0.1% FA The chromatography columns were operated at a flow rate of 0.4 mL/min and eluted using a multi- phase gradient ( Table S1) where the initial conditions were 1:1 A:B the mobile phase composition B was then gradual increased over
11 min using the following profile; 50–53% B from 0 to 0.5 min, 53–55% B 0.5–4 min, 55–65% B 4–7 min, held at 80% B 7–7.5 min, 80–99% B 7.5–10 min, held at 99% B 10–11 min, followed by re- turning to the starting conditions for re-equilibration giving a total run time of 12 min Various injection volumes were used, with a sample and column temperature of 10 °C and 55 °C respectively The column effluent was monitored using a SYNAPT TM XS mass spectrometer (Waters Corporation, Wilmslow, UK) operated in “V” mode and negative electrospray ionization (ESI) using the follow- ing instrument settings: capillary 2.5 kV, desolvation temperature
500 °C, desolvation gas 800 L/h, cone gas 50 L/h, cone voltage 30 V, and source temperature 120 °C The MS data were acquired using data independent acquisition in resolution and continuum mode with a mass range 10 0 −1200 m/z The lock mass was acquired and applied using a solution of 200 pg/ μL leucine enkephalin The low collision energy was set to 6 eV, and for high energy, a transfer lin- ear ramp of 25 −45 eV was used LC/MS data acquisition and pro-
Trang 3Fig. 1 Negative ion ESI mode base peak extracted ion chromatograms at a concentration of 50 pg/μL (A) LPA 16:0/0:0 m/z 409.2355 using the conventional system/CSH C18
column (B) LPA 16:0/0:0 m/z 409.2355 using the HST system/CSH C18 column (C) PA 16:0/18:1 m/z 673.4808 using the conventional system/CSH C18 column and (D) PA 16:0/18:1 m/z 673.4808 using the HST system/CSH C18 column Inset figures show the chemical structure of LPA and PA
cessing were performed using MassLynx TM V4.2 software (Waters
Corporation, Wilmslow, UK)
3 Results and discussion
As noted in the introduction, in order to investigate the poten-
tial benefits of the HST for the phosphorylated and carboxylated
lipid classes we took a previously optimized method developed us-
ing the ACQUITY CSH C18 phase [19] as our starting point The
initial experiments were conducted to compare the conventional
and HST systems, using the differential ion mobility system suit-
ability Lipidomix (described in the experimental section) as a stan-
dard mixture This enabled the lipid separation to be evaluated and
provided a benchmark for instrument performance when changing
between conventional and HST systems For the purposes of this
comparison all the samples were prepared in one vial and divided
into separate vials for the conventional and HST systems/columns
In addition, the same batch of mobile phase and stationary phase
was employed for all of these analyses with the same chromato-
graphic conditions employed for all instrumental configurations
The three configurations evaluated were: (i) conventional LC sys-
tem with conventional CSH C18 column, (ii) conventional LC sys-
tem with HST CSH C18 column and (iii) HST LC system with HST
CSH C18 column
3.1 Peak shape and tailing factor comparison
Under the RP LC conditions generally used in lipidomic stud-
ies the acid phospholipids LPA, PA, LPS and PS tend to elute as
quite broad peaks However, with suitable modification of the mo-
bile phase, using mobile phase additives such as phosphoric acid
[22], this peak tailing can be attenuated or eliminated However,
these mobile phase additives such as phosphoric acid can compete
with analyte ionization, especially in negative ion mode thus the
approach of employing a metal surface barrier was an attractive
alternative To evaluate whether or not the chromatographic per-
formance was improved using the HST system and CSH C18 col-
umn we measured the lipid standards LPA 16:0/0:0, PA 16:0/18:1,
LPS 18:1/0:0 and PS 16:0/18:1 and compared the results with those
obtained using a conventional system and CSH C18 column Rep-
resentative negative ion ESI mode base peak extracted ion chro-
matograms of LPA 16:0/0:0, PA 16:0/18:1, LPS 18:1/0:0 and PS
16:0/18:1, at a concentration of 50 pg/μL, are displayed in Figs 1 and 2 As these figures show the HST system/column showed sig- nificant improvement in peak tailing, sensitivity and recovery of these lipids compared to the conventional system/column The av- erage ( n = 3) peak tailing factor (PTF) and full width at half max- imum (FWHM) of LPA 16:0/0:0, PA 16:0/18:1, LPS 18:0/0:0 and PS 16:0/18:1 were calculated for the standard solution prepared at a concentration of 250 pg/μL These data are provided for both HST and conventional systems in Table 1 showing that the HST sys- tem/column reduced peak tailing by 65–80% for LPA 16:0/0:0, PA 16:0/18:1, LPS 18:0/0:0 and PS 16:0/18:1 compared to the conven- tional configuration The peak width at FWHM was reduced by 70– 86% for LPA and PA; and by 25–50% for LPS and PS using the HST system/column combination compared to the conventional config- uration These improvements in chromatographic performance for these acidic phospholipids are most probably due to the elimina- tion of the interactions of phosphate and carboxylate groups with the metal surfaces provided by the metal surface barrier [31] Inter- estingly both LPA and LPS showed a significantly greater improve- ment in PTF and peak width compared to their corresponding PA and PS respectively with the HST system/column These improve- ments in chromatographic performance can be attributed to the fact that the phosphate groups in LPA and the carboxylate group
in LPS are, due to their chemical structure, more exposed to metal surfaces compared to PA and PS respectively LPA and LPS eluted earlier than PA and PS and, due to the shallow gradient in the region where the LPA and LPS eluted, they would be expected to have reduced PTF’s and peak widths at FWHM
3.2 Peak signal intensity and sensitivity comparison
The increase in sensitivity obtained with the HST sys- tem/column can be clearly seen for the analysis of LPA 16:0/0:0 shown in Figs.1A and B at a concentration of 50 pg/μL As these figures clearly show there was a noticeable improvement in peak intensity for these analytes when the HST system/column was used compared to the conventional system Similarly, Figs 1C and D
(PA), 2 A and B (LPS) and 2 D and C (PS) also showed improved sensitivity, peak shape and recovery obtained with the HST sys- tem/column compared to the conventional system/column The chromatographic performance of the two systems was compared
at 5, 50, and 250 pg/μL for LPA 16:0/0:0, PA 16:0/18:1, LPS 18:1/0:0
Trang 4Fig. 2 Negative ion ESI mode base peak extracted ion chromatograms at a concentration of 50 pg/μL (A) LPS 18:1/0:0 m/z 522.2832 using the conventional system/CSH
C18 column (B) LPS 18:1/0:0 m/z 522.2832 using the HST system/CSH C18 column (C) PS 16:0/18:1 m/z 760.5129 using the conventional system/CSH C18 column (D) PS 16:0/18:1 m/z 760.5129 using the HST system/CSH C18 column Inset figures show the chemical structure of LPS and PS
Table 1
Calculated average ( n = 3) peak tailing factor (PTF) and full width at half maximum (FWHM) for LPA 16:0/0:0, PA 16:0/18:1, LPS 18:0/0:0 and PS 16:0/18:1 at a concentration
of 250 pg/μL using the conventional system and CSH C18 column compared to the hybrid surface technology (HST) system and CSH C18 column
System
Conventional
system and
CSH C18
column
HST system
and CSH C18
column
∗ The tailing factor was calculated as the ratio of the full width at 5% of peak height and two times of the front half width [33]
# The full width at half maximum is the peak width, measured in seconds, at 50% of the maximum peak height
and PS 16:0/18:1 molecular species ( Figures S1-S4) and at 250
pg/μL for CerP 18:1;O2/16:0, SPBP 18:1;O2 and PIP (4 ) 18:1/18:1
( Figures S5) The LPA and PA were undetectable at 5 pg/μL with
the conventional system/column due to analyte loss caused by in-
teraction with metal surfaces This result contrasts with the re-
sults for the HST system/column where these low concentration
injections gave peaks that were clearly visible Even though the in-
creased sensitivity provided may not be required for quantification
at high concentrations of these analytes the improved peak shape
is still helpful in reducing peak overlap
The peak intensities obtained for LPA 16:0/0:0 at a concentra-
tion of 250 pg/μL using either a (i) the conventional LC system/CSH
C18 column or (ii) the conventional LC system with HST CSH C18
column or, (iii) the HST LC system with HST CSH C18 column were
compared in negative ion ESI mode ( m/z 409.235) The extracted
ion chromatograms obtained using these three separate configura-
tions are displayed in Fig.3 The increase in intensity obtained us-
ing the HST for LPA 16:0/0:0 is also given in Figure S6 as bar graph
with the average ( n= 3) of the total ion intensity for each system
configuration used Using the conventional LC system, a marked
increase in signal intensities was observed for the HST column
rather than the untreated column However, the greatest increase
in response was obtained for the HST system/column configura-
tion which showed a 12.7 fold increase over the conventional sys-
tem/column combination Use of the HST CSH C18 column and the untreated conventional LC system also provided increased intensi- ties for the phosphorylated and carboxylate lipid standards eval- uated However, these increases were modest when compared to the HST system/column configuration ( Figure S6)
3.3 Application to PA and PS lipid extracts
This new HST technology was also applied to the analysis of egg (chicken) PA and brain (porcine) PS extracts and the results compared to those obtained with the conventional system/column ( Figs.4A-D and 5A-D) The results of this comparison clearly show that the PA lipid species at m/z 671.47 (16:1_18:1 and 16:0_18:2) and 673.48 (16:0_18:1) eluted with very broad tailing peaks using the conventional system and column ( Fig.4A) These results were compared to the data acquired with HST system/column ( Fig.4B), where the peaks showed significant improvements in shape and intensity The identities of the fatty acid constituents were con- firmed via fragment ion information using the HST negative ion ESI mode LC/MS data independent acquisition where lipid identifica- tion was performed based on low energy exact precursor ion mass and corresponding high energy characteristic fragment ions The molecular species level information was provided only when the presence of the fatty acyl chain was confirmed using fragment ion
Trang 5Fig 3 Overlayed negative ESI base peak extracted ion chromatogram of LPA
16:0/0:0 at a concentration of 250 pg/μL using the conventional system/CSH C18
column, the conventional system/HST CSH C18 column or the HST system/CSH C18
column
information ( Figure S7). The HST system/column provided four ad-
ditional baseline separated peaks at retention times of 3.1, 3.3, 4.7
and 5.8 min respectively which were not observed with the con-
ventional system Based on exact mass and negative ion ESI mode
high energy fragment ion information these peaks were identified
as PA 38:6 m/z 719.46, PA 36:4 m/z 695.46, PA 36.2 m/z 699.49
and PA 18:0_18:1 m/z 701.51 lipids In contrast, it was difficult
to make confident molecular species identification with the data
obtained using the conventional system/column ( Fig.4A) as there
was a large amount overlap of the fragment ions because of peak
tailing as well as very low MS signal intensity The data displayed
in Figs.4C and D show extracted ion chromatograms for the lipid
PA 16:0_18:1, from which it can be seen that an average peak
FWHM of 34.0 ( n= 3) was obtained with the conventional sys-
tem/column ( Fig.4C) whereas that for the HST system/column pro-
duced an average peak width of only 10.8 ( n = 3) ( Fig.4D) Sim- ilarly, the data displayed in Fig.5 shows the results obtained for the analysis of the porcine brain PS extract, analyzed using both conventional and HST systems In this application the HST sys- tem/column not only provided reduced peak tailing compared to the conventional system/column but also allowed the visualization
of three additional peaks eluting with retention times of 5.3, 5.6 and 7.9 min These additional peaks were identified based on ex- act mass, fragment ions and theoretical isotopic distribution as PS 38:2 m/z 814.55, PS 40:3 m/z 840.577 and PS 44:2 m/z 898.654 re- spectively In contrast, these minor PS species were not detected using the conventional system/column The peak width at half height from the two systems was calculated for PS 18:0_18:1 m/z
788.545 ( Figs 5C and D) with the conventional system/column having produced a peak FWHM of 10.8 ( n = 3) ( Fig.5C) com- pared to value of 7.4 ( n= 3) for HST system/column ( Fig.5D)
As can be seen from these examples, and in contrast to previ- ous methods using conventional RP columns [19–21], the HST sys- tem/column enabled the simultaneous analysis of phosphate and carboxylate-containing lipid species in biological extracts without evidence of significant binding or peak tailing and without com- promising the determination of other lipid classes The HST sys- tem/column increased the sensitivity, recovery, and coverage of these biologically important lipids Previous RP LC approaches have not sufficiently addressed the simultaneous analyses of these acidic lipids (LPA, PA, LPA, PS, CerP, SPBP and PIP) with other phospho- lipid classes The application and performance of the HST sys- tem/column to other lipid classes was evaluated using the differ- ential ion mobility system suitability Lipidomix that contains PA and PS lipid species together with other lipid classes The data displayed in Fig 6 shows a representative negative ion ESI-base peak extracted ion chromatogram of LPC 18:1/0:0, PE 14:1/14:1, SM d18:1/18:1 and Cer d18:1/18:1 analyzed using conventional sys- tem/column versus the HST system/column These results showed that, for non-phosphorylated lipid classes, the HST system/column provided comparable retention behavior in terms of peak tailing, shape, and signal intensity to the conventional system/column con- figuration ( Table S2)
As we have noted, the complexity of the lipid profile in biolog- ical matrices makes the RPLC/MS analysis of phosphorylated and carboxylated lipids challenging due to metal-analyte interactions However, the accurate measurement of these lipids may well be
Fig 4 Avanti polar lipids egg chicken PA extract (10 ng/μL) measured using (A) the conventional system/CSH C18 column or (B) the HST system/CSH C18 column Corre-
sponding extracted ion chromatograms for PA 16:0_18:1 at m/z 673.481 using (C) the conventional system/CSH C18 column with average peak FWHM 34.0 s ( n = 3) or (D) the HST system/CSH C18 column with average peak FWHM 10.8 s ( n = 3)
Trang 6Fig 5 Avanti polar lipids porcine brain PS extract (10 ng/μL) measured using (A) the conventional system/CSH C18 column or (B) the HST system/CSH C18 column Corre-
sponding extracted ion chromatograms for PS 18:0_18:1 at m/z 788.545 using (C) the conventional system/CSH C18 column with average peak FWHM 10.8 s ( n = 3) or (D) the HST system/CSH C18 column with average peak FWHM 7.4 s ( n = 3)
Fig 6 The Avanti Differential Ion Mobility System Suitability standard Lipidomix Kit was used to evaluate the effect of the HST system/column on other lipid classes Negative
ESI base peak extracted ion chromatograms of LPC 18:1/0:0, PE 14:1/14:1, SM d18:1/18:1 and Cer d18:1/18:1 at a concentration of 100 pg/μL using (A) the conventional system/CSH C18 column (B) the HST system/CSH C18 column
critical for the evaluation of their dysregulation in disease states
Elimination of these unwanted interactions is especially important
for low abundant signaling lipid classes such as LPA, PA, LPS, PS,
CerP, SPBP and PIP where the systemic concentrations are in the
low ng/mL range and metal chelation can result in significant at-
tenuation of the signal, or even its complete loss, as illustrated in
the examples shown here As discussed, metal free materials have
been used to replace the metal components in chromatography
systems Thus, PEEK components have been successfully employed
for the analysis of phosphate-containing sphingolipids [29] and
mobile phase additives are also an option [22–27] However, the
limitations of these approaches are clear, and there are obvious
advantages to simply eliminating the problem from the system, re-
moving the need for the analyst to investigate and mitigate these
unwanted analyte-surface interactions
4 Conclusions
The HST system/column combination clearly provided a sig-
nificant improvement in sensitivity, reduced peak tailing and in-
creased the recovery of phosphorylated and carboxylate lipids compared to a conventional system/CSH C18 column The HST sys- tem/column combination reduced peak tailing by 65–80% and peak width by 70–86% for LPA and PA Moreover, increased signal inten- sities of up to 12.7 times were observed for LPA with the HST ap- proach compared to the untreated system This methodology can
be applied to other phosphorylated and carboxylated lipid classes, such as PIP with mono, di and tri phosphates, CerP and SPBP In contrast to most of the previous methodologies using conventional
RP columns the HST allowed for the simultaneous analysis of these acidic phospholipid classes at the same time as other lipid classes without adverse effects on the coverage of these biologically im- portant lipids
Author declaration of interest statment
We wish to confirm that there are no known conflicts of inter- est associated with this publication and there has been no signifi- cant financial support for this work that could have influenced its outcome
Trang 7We confirm that the manuscript has been read and approved by
all named authors and that there are no other persons who satis-
fied the criteria for authorship but are not listed We further con-
firm that the order of authors listed in the manuscript has been
approved by all of us
We confirm that we have given due consideration to the pro-
tection of intellectual property associated with this work and that
there are no impediments to publication, including the timing of
publication, with respect to intellectual property In so doing we
confirm that we have followed the regulations of our institutions
concerning intellectual property
We further confirm that any aspect of the work covered in this
manuscript that has involved either experimental animals or hu-
man patients has been conducted with the ethical approval of all
relevant bodies and that such approvals are acknowledged within
the manuscript
Declaration of Competing Interest
None
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi: 10.1016/j.chroma.2022.462921
CRediT authorship contribution statement
Giorgis Isaac: Conceptualization, Methodology, Data curation,
Writing – original draft Ian D Wilson: Writing – review & edit-
ing Robert S Plumb: Supervision, Writing – review & editing
References
[1] C.L Stace , N.T Ktistakis , Phosphatidic acid- and phosphatidylserine-binding
proteins, Biochim Biophys Acta Mol Cell Biol Lipids 1761 (2006) 913–926
[2] R Welti , W Li , M Li , Y Sang , H Biesiada , H.-.E Zhou , C.B Rajashekar ,
T.D Williams , X Wang , Profiling Membrane Lipids in Plant Stress Responses
Role of Phospholipase D Alpha in Freezing-Induced Lipid Changes in Arabidop-
sis, J Biol Chem 277 (2002) 31994
[3] E Cífková, R Hájek , M Lísa , M Holcapek , Hydrophilic interaction liquid chro-
matography-mass spectrometry of lysophosphatidic acids, lysophosphatidylser-
ines and other lipid classes, J Chromatogr A 1439 (2016) 65–73
[4] A Shevchenko , K Simons ,Lipidomics: coming to grips with lipid diversity, Nat
Rev Mol Cell Biol 11 (8) (2010) 593–598
[5] X Han , Lipidomics for studying metabolism, Nat Rev Endocrinol 12 (11)
(2016) 668–679
[6] T Xu , C Hu , Q Xuan , G Xu , Recent advances in analytical strategies for mass
spectrometry-based lipidomics, Anal Chim Acta 1137 (2020) 156–169
[7] B Brugger , G Erben , R Sandhoff, F.T Wieland , W.D Lehmann , Quantitative
analysis of biological membrane lipids at the low picomole level by nano-elec-
trospray ionization tandem mass spectrometry, Proc Natl Acad Sci U.S.A 94
(1997) 2339–2344
[8] M Holcapek , G Liebisch , K Ekroos , Lipidomic Analysis, Anal Chem 90 (2018)
4249–4257
[9] E Cífková, M Hol ˇcapek , M Lísa , M Ov ˇca ˇcíková, A Ly ˇcka , F Lynen , P Sandra ,
Nontargeted quantitation of lipid classes using hydrophilic interaction liquid
chromatography-electrospray ionization mass spectrometry with single inter-
nal standard and response factor approach, Anal Chem 84 (2012) 10064–10070
2012
[10] E Cífková, M Hol ˇcapek , M Lísa , D Vrána , B Melichar , V Študent , Lipidomic
differentiation between human kidney tumors and surrounding normal tissues
using HILIC-HPLC/ESI-MS and multivariate data analysis, J Chromatogr B Ana-
lyt Technol Biomed Life Sci 10 0 0 (2015) 144-21
[11] A Triebl , M Trötzmüller , A Eberl , P Hanel , J Hartler , H.C Köfeler , Quantita-
tion of phosphatidic acid and lysophosphatidic acid molecular species using
hydrophilic interaction liquid chromatography coupled to electrospray ioniza-
tion high resolution mass spectrometry, J Chromatogr A 1347 (2014) 104–110
[12] E Cífková, R Hájek , M Lísa , M Holcapek , Hydrophilic interaction liq- uid chromatography-mass spectrometry of (lyso)phosphatidic acids, (lyso)phosphatidylserines and other lipid classes, J Chromatogr A 1439 (2016) 65–73
[13] S.L Abidi , T.L Mounts , High-performance liquid-chromatography of phospha- tidic-acid, J Chromatogr B-Biomed Appl 671 (1995) 281–297
[14] C Bure , S Ayciriex , E Testet , J.M Schmitter , A single run LC–MS/MS method for phospholipidomics, Anal Bioanal Chem 405 (2013) 203–213
[15] A A Karlsson , P Michelsen , A Larsen , G Odham , Normal-phase liquid chro- matography class separation and species determination of phospholipids uti- lizing electrospray mass spectrometry tandem mass spectrometry, Rapid Com- mun Mass Spectrom 10 (1996) 775–780
[16] M Narvaez-Rivas , E Gallardo , J.J Rios , M Leon-Camacho , A new high-perfor- mance liquid chromatographic method with evaporative light scattering detec- tor for the analysis of phospholipids Application to Iberian pig subcutaneous fat, J Chromatogr A 1218 (2011) 3453–3458
[17] L.Q Pang , Q.L Liang , Y.M Wang , L Ping , G.A Luo , Simultaneous determination and quantification of seven major phospholipid classes inhuman blood using normal-phase liquid chromatography coupled with electrospray mass spec- trometry and the application in diabetes nephropathy, J Chromatogr B 869 (2008) 118–125
[18] S Uran , A Larsen , P.B Jacobsen , T Skotland , Analysis of phospholipid species
in human blood using normal-phase liquid chromatography coupled with elec- trospray ionization ion-trap tandem mass spectrometry, J Chromatogr B758 (2001) 265–275
[19] C.W Damen , G Isaac , J Langridge , T Hankemeier , R.J Vreeken , Enhanced lipid isomer separation in human plasma using reversed-phase UPLC with high-res- olution ion-mobility MS detection, J Lipid Res 55 (2014) 1772–1783 [20] P.A Vorkas , G Isaac , M.A Anwar , A.H Davies , E.J Want , J.K Nichol- son , E Holmes , Untargeted UPLC-MS profiling pipeline to expand tissue metabolome coverage: application to cardiovascular disease, Anal Chem 87 (2015) 4184–4193
[21] T Cajka , O Fiehn , Increasing lipidomic coverage by selecting optimal mo- bile-phase modifiers in LC–MS of blood plasma, Metabolomics 12 (2016) 1–11 [22] H Ogiso , T Suzuki , R Taguchi , Development of a reverse-phase liq- uid chromatography electrospray ionization mass spectrometry method for lipidomics, improving detection of phosphatidic acid and phosphatidylserine, Anal Biochem 375 (2008) 124–131 2008
[23] J.A Anspach , S Rao , B Rivera , Bioinert Versus Biocompatible: the Benefits of Different Column Materials in Liquid Chromatography Separations, LCGC 36 (2018) 24–29
[24] J.C Heaton , D.V McCalley , Some factors that can lead to poor peak shape in hydrophilic interaction chromatography, and possibilities for their remediation,
J Chromatogr A 1427 (2016) 37–44 (put as 24) [25] K.T Myint , T Uehara , K Aoshima , Y Oda , Polar Anionic Metabolome Anal- ysis by Nano-LC/MS with a Metal Chelating Agent, Anal Chem 81 (2009) 7766–7772
[26] D Winter , J Seidler , Y Ziv , Y Shiloh , W.D Lehmann , Citrate Boosts the Per- formance of Phosphopeptide Analysis by UPLC-ESI-MS/MS, J Proteome Res 8 (2009) 418–424
[27] O.L Knittelfelder , B.P Weberhofer , T.O Eichmann , S.D Kohlwein , G.N Rech- berger , A versatile ultra-high performance LC-MS method for lipid profiling, J Chromatogr B Analyt Technol Biomed Life Sci 951–952 (2014) 119–128 [28] J Lee , Y Kim , M Moon , Enhancement of acidic lipid analysis by nanoflow ultrahigh performance liquid chromatography-mass spectrometry, Anal Chim Acta 1166 (2021) 338573
[29] S.G B Gowda , K Ikeda , M Arita , Facile determination of sphingolipids under alkali condition using metal-free column by LC-MS/MS, Anal Bioanal Chem
410 (2018) 4793–4803 [30] R.S Plumb , L.A Gethings , A King , L.G Mullin , G Maker , R Trengove , I.D Wil- son , Hybrid organic/inorganic hybrid surface technology for increasing the per- formance of LC/MS(MS)-based drug metabolite identification studies: applica- tion to gefitinib and metabolites in mouse plasma and urine, J Pharm Biomed Anal 200 (2021) 114076
[31] M DeLano , T.H Walter , M.A Lauber , M Gilar , M.C Jung , J.M Nguyen , C Bois- sel , A.V Patel , A Bates-Harrison , K.D Wyndham , Using hybrid organic-inor- ganic surface technology to mitigate analyte interaction with metal surface in UHPLC, Anal Chem 93 (2021) 5773–5781
[32] N Tanna , L.G Mullin , P.D Rainville , I.D Wilson , R.S Plumb , Improving LC/MS/MS-based bioanalytical method performance and sensitivity via a hy- brid surface barrier to mitigate analyte - Metal surface interactions, J Chro- matogr B 1179 (2021)
[33] U.D Neue , Theory of peak capacity in gradient elution, J Chromatogr A 1079 (2005) 153–161