Determination of eight phosphatidylethanol homologues in blood by reversed phase liquid chromatography–tandem mass spectrometry – How to avoid co-elution of phosphatidylethanols

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.

Journal of Chromatography A 1684 (2022) 463566 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Determination of eight phosphatidylethanol homologues in blood by reversed phase liquid chromatography–tandem mass spectrometry – How to avoid co-elution of phosphatidylethanols and unwanted 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 Department of Forensic Sciences, Division of Laboratory Medicine, Section of Drug Abuse Research, Oslo University Hospital, P.O Box 4950 Nydalen, N-0424, Lovisenberggt Oslo 0456, Norway b a r t i c l e i n f o 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 a b s t r a c t 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 Since PEths are phospholipids it can be difficult to isolate them from unwanted phospholipids during sample preparation To minimize possible matrix effects it is therefore important to separate PEths from other phospholipids during LC-MS/MS analysis In this study, we have investigated how the retention and chromatographic separation of eight PEth homologues and the phospholipid background are influenced by changes in mobile phase composition using two different LC columns, the Acquity BEH C18 column (50 × 2.1 mm ID, 1.7 μm particles) and the Kinetex biphenyl column (100 × 2.1 mm ID, 1.7 μm particles) Our findings show that the buffer concentration of the aqueous part of the mobile phase had a huge effect on the retention of PEth homologues and separation of PEths from unwanted phospholipids By using a buffer-free mobile phase consisting of 0.025% ammonia in Type water, pH 10.7, as solvent A and methanol as solvent B, all eight PEth homologues were separated from both the early eluting lysophospholipids and the later eluting phospholipids with two fatty chains using the BEH C18 column The knowledge obtained in this study can be of great importance for those seeking to develop reliable and robust bioanalytical LC-MS/MS methods for determination of PEth homologues © 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/) Introduction Alcohol is a legal psychoactive substance consumed worldwide during cultural, religious and social practices, and provides perceived satisfaction to many users However, alcohol use is toxic for the human body and associated with an increased risk of various negative health effects, injuries and mortality [1–5] Alcohol use is also associated with huge economic and social costs individuals and for the society [6–8] Recently, a growing interest in phosphatidylethanols (PEths) as biomarkers for alcohol consumption has emerged PEths are a group of direct alcohol biomarkers 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 patterns, 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 elimination half-life of the various PEths is different, it can be important 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/) M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566 Fig 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 CH2 -O-CH2 -CH2 -R1 (alkyl ether) or CH2 -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 methods 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 phospholipids not removed during sample preparation may generate different challenges, such as changing column performance, increasing column backpressure, and generate matrix effects [31–35] Phospholipids are a class of lipids and they are essential components 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 structure contains a polar “head” connected to two (sometimes only one) non-polar chains of various lengths and various degree of saturation (Fig 1) Hundreds of different phospholipids are described in the literature In general (see Fig 1) for the polar head; pKa ≈ 0–2 for the phosphate group (acidic), pKa ≈ 9–11 for the amine group (basic 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 bonding [37] As seen from Fig there are many sub-classes of phospholipids Two subgroups can be distinguished by their backbones, the sphingoid base backbone and the glycerol backbone phospholipids Other subgroups can be categorized based on the number of fatty chains (“di” or “mono”) Lyso-phospholipids are those with only one non-polar tail, either at the sn-1 position (1-lysophospholipids) or at the sn-2 position (2-lyso-phospholipids) Subgroups can also be categorized based on the R3 group attached to the phosphate-moiety, and the most common phospholipids, accounting for 60–70 % of the total plasma phospholipid, is phosphatidylcholines (PCs) [31] In bioanalytical LC-MS/MS methods it is easy to remove unwanted 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 phospholipids will also be extracted and introduced into the LC-MS/MS [29,38,39] PEths and other phospholipids have similar molecular structures and physico-chemical properties Consequently, they will often 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 analysis, 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 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 Materials and methods 2.1 Chemicals and materials Methanol (MeOH) of LC-MS grade was purchased from Honeywell (Seelze, Germany) Acetonitrile (ACN) of HPLC Far UV grade was purchased from JT Baker (Deventer, The Netherlands) Ethyl M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566 Fig 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 obtained from VWR Chemicals, Prolabo (Leuven, Belgium) Type water (18.2 M ) purified with a Synthesis A 10 milli-Q system from Millipore (Billerica, MA, USA) was used MeOH, vortexed and then placed in the sample organizer for LC– MS/MS analysis Injection volume was 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 solvent manager, sample manager with sample organizer, and a column oven, coupled to a Xevo TQ-S MS/MS, all from Waters (Milford, 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 column (100 × 2.1 mm ID, 1.7 μm particles) from Phenomenex (Torrance, 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 column whereas it was 0.5 mL/min for the tests performed on the Kinetex biphenyl column Injection volume was 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 temperature 500 °C, cone gas flow 300 L/h and desolvation gas flow 10 0 L/hr Acquisition and processing of data were performed using MasslynxTM software (version 4.1, Waters, Milford, MA, USA) Table shows the MRM transitions, cone voltages, collision energies 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 phospholipid 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 and 40, respectively 2.2 Blank blood PEth-free whole blood from employees at the Department of Forensic Sciences at Oslo University Hospital was collected in mL Vacuette® K2E K2EDTA tubes from Greiner bio-one (Kremsmünster, 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 solutions LC-MS/MS analyses of the eight PEth homologues were performed 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 extraction procedure) 2.4 Sample preparation by 96-well SLE that were used for extraction of blood samples Results and discussion 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 eluates collected in 96-collection plates were evaporated to dryness and the residues were reconstituted in 100 μL 2-propanol/ACN or To minimize possible matrix effects, it is important to understand how PEths can be chromatographically resolved from unwanted phospholipids during LC-MS/MS analysis In this case, different mobile phase compositions and gradient profiles were investigated on two different UHPLC columns, and some interesting results were found Each chromatogram shows overlaid chro3 M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566 Table MRM transitions, cone voltages, collision energies and dwell times MRM transitions Analyte PEth 16:0/16:0 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 18:1/18:1 675.5 675.5 701.5 701.5 699.5 699.5 723.5 723.5 715.5 715.5 729.5 729.5 727.5 727.5 727.5 727.5 > > > > > > > > > > > > > > > > 255.2 437.3 255.2 281.2 255.2 279.2 303.2 437.3 269.2 281.2 281.2 283.2 279.2 283.2 281.2 463.3 MS/MS parametersa Cone voltage (V) Collision energy (eV) Dwell time (ms) 45 45 60 60 55 55 50 50 60 60 65 65 50 50 60 60 30 30 40 30 40 30 25 25 35 35 40 40 40 40 40 30 10 10 20 20 20 20 20 20 20 20 20 20 20 20 20 20 Fig 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), mM (b) and mM (c) mM and mM ammonium formate buffers were prepared by dilution of 20 mM buffer using Type 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 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 injecting 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 several injections of the PEth homologues without injecting the dirtier extracted blood samples into the system, the latter may change column performance 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 important 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 M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566 Fig Chromatographic separation between eight PEth homologues and phospholipid background obtained by LC-MS/MS analysis on three different Acquity BEH C18 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, 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 Fig Chromatographic separation between eight PEth homologues and phospholipid background obtained by LC-MS/MS analysis on a BEH C18 column using basic mobile phases with different buffer concentrations; 20 mM (a), mM (b), mM (c) and mM (d) LC-MS/MS analysis were performed using a mobile phase Solvent A solution of ammonium formate buffers, pH 10, in Fig 5a–c, whereas 0.025% ammonia in Type water, pH 10.7, was used in Fig 5d Gradient profile and retention time order were the same as described for Fig M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566 Fig Chromatographic separation between eight PEth homologues and phospholipid background obtained by LC-MS/MS analysis on a BEH C18 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 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 salting out effect (increased retention at higher salt concentrations) Fig shows the retention times of the PEths homologues and phospholipid background obtained by an LC-MS/MS analyses on a BEH C18 column 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 phospholipid background were similar when using both mobile phase pH 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 effect 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 shows good separation between the PEths and the unwanted phospholipids using the mM buffer as solvent A However, further investigations 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 and 4, it is clear, despite 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 investigated using high pH mobile phases by testing a basic mobile phase without any buffer (Fig 5) Fig clearly illustrates reduced retention of the eight PEths when using 0.025 % ammonia in Type water, pH 10.7, compared 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 Solvent A composition This high pH mobile phase consisting of 0.025% ammonia in Type 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 Therefore, this mobile phase was used in a subsequent experiment for investigation of how retention times of PEth homologues varied after analyses of 50, 100, and 150 extracted blood samples (Fig 6) Fig shows a reduction in the retention times over time for all PEth homologues after analyzing several extracted blood samples, 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 samples 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 something worth investigated further in future studies in order to investigate 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 M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566 Fig Chromatographic separation between eight PEth homologues and phospholipid background obtained by LC-MS/MS analysis on a BEH C18 column using basic mobile phases consisting of 0.025% ammonia in Type 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 Retention order for PEth homologues were the same as described in Fig 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 performed (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 composition 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 7b) Gradient 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, phospholipids will normally be retained (“focused”) on the column in RP LC-MS/MS methods as long as the mobile phase contains ≤ 60 % MeOH [23] 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 shows retention of the PEth homologues and the phospholipid background obtained at two mobile phase pH values, both tested with three different buffer concentrations Fig shows similar results as obtained for the BEH C18 columns, the buffer concentration of the aqueous part of the mobile phase had a great effect on the retention of the PEth homologues 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 investigated further comparing ammonium formate buffer to ammonium acetate buffer, but no or only minor changes were observed 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 affinity towards compounds with double bonds due to dipole-dipole interactions 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 Acquity BEH C18 columns, which are pH stable within pH values M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566 Fig 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), mM (b) and 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 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 Conclusions in biological samples The effects of these parameters on different LC-MS/MS systems should be further investigated Since PEths are phospholipids and difficult to isolate from unwanted 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 separation 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 retention of the PEth homologues were basically unaltered using mobile phase pH 5–10 This finding was as expected since PEths with their acidic pKa value at approximately 1.5–2.0 are completely ionized above pH However, the buffer concentration of the aqueous part of the mobile phase had a huge effect on the retention of PEth homologues, while the unwanted phospholipids seemed almost unaffected In conclusion it was found that LC-MS/MS analysis on the Acquity BEH C18 column (50 × 2.1 mm ID, 1.7 μm particles) using a buffer free mobile phase consisting of 0.025% ammonia in Type 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 phospholipids All PEth homologue peaks were narrow and symmetrical Optimization of the gradient profile was also important in order 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 Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper CRediT authorship contribution statement Marisa Henriques Maria: Data curation, Investigation, Writing – review & editing Benedicte Marie Jørgenrud: Writing – review & editing Thomas Berg: Conceptualization, Data curation, Investigation, 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 LCMS/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 M.H Maria, B.M Jørgenrud and T Berg Journal of Chromatography A 1684 (2022) 463566 References [24] L Novakova, Challenges in the development of bioanalytical liquid chromatography-mass spectrometry method with emphasis on fast analysis, J Chromatogr A 1292 (2013) 25–37 S0021-9673(12)01338-6 [pii], doi:10.1016/j.chroma.2012.08.087 [25] F.T Peters, Recent advances of liquid chromatography-(tandem) mass spectrometry in clinical and forensic toxicology, Clin Biochem 44 (1) (2011) 54–65, doi:10.1016/j.clinbiochem.2010.08.008 [26] D Remane, D.K Wissenbach, F.T Peters, Recent advances of liquid chromatography-(tandem) mass spectrometry in clinical and forensic toxicology - an update, Clin Biochem 49 (13) (2016) 1051–1071 -14, doi:10.1016/j clinbiochem.2016.07.010 [27] T.N Andreassen, H Havnen, O Spigset, B.M Hasle Falch, R.B Skråstad, High throughput UPLC(R)-MSMS method for the analysis of phosphatidylethanol (PEth) 16:0/18:1, a specific biomarker for alcohol consumption, in whole blood, J Anal Toxicol 42 (1) (2018) 33–41, doi:10.1093/jat/bkx075 [28] L.D Muller, S Foreid, A comparison of an optimized automated sample preparation of PEth in blood pretreated by freezing versus manual preparation in whole blood, analyzing by UHPLC - MS/MS, J Pharm Biomed Anal 212 (2022) 114635, doi:10.1016/j.jpba.2022.114635 [29] D Oppolzer, M Barroso, E Gallardo, Bioanalytical procedures and developments in the determination of alcohol biomarkers in biological specimens, Bioanalysis (3) (2016) 229–251, doi:10.4155/bio.15.240 [30] A Isaksson, L Walther, T Hansson, A Andersson, J Stenton, A Blomgren, High-throughput LC-MS/MS method for determination of the alcohol use biomarker phosphatidylethanol in clinical samples by use of a simple automated extraction procedure-preanalytical and analytical conditions, J Appl Lab Med (6) (2018) 880–892, doi:10.1373/jalm.2017.024828 [31] M Jemal, Z Ouyang, Y.Q Xia, Systematic LC-MS/MS bioanalytical method development that incorporates plasma phospholipids risk avoidance, usage of incurred sample and well thought-out chromatography, Biomed Chromatogr 24 (1) (2010) 2–19, doi:10.1002/bmc.1373 [32] Y.Q Xia, M Jemal, Phospholipids in liquid chromatography/mass spectrometry bioanalysis: comparison of three tandem mass spectrometric techniques for monitoring plasma phospholipids, the effect of mobile phase composition on phospholipids elution and the association of phospholipids with matrix effects, Rapid Commun Mass Spectrom 23 (14) (2009) 2125–2138, doi:10.1002/rcm 4121 [33] O.A Ismaiel, T Zhang, R.G Jenkins, H.T Karnes, Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry, J Chromatogr B Anal Technol Biomed Life Sci 878 (31) (2010) 3303–3316, doi:10.1016/j.jchromb.2010.10.012 [34] H Jiang, H Cao, Y Zhang, D.M Fast, Systematic evaluation of supported liquid extraction in reducing matrix effect and improving extraction efficiency in LC-MS/MS based bioanalysis for 10 model pharmaceutical compounds, J Chromatogr B Anal Technol Biomed Life Sci 891-892 (2012) 71–80, doi:10.1016/j jchromb.2012.02.031 [35] L Novakova, H Vlckova, A review of current trends and advances in modern bio-analytical methods: chromatography and sample preparation, Anal Chim Acta 656 (1) (2009) 8–35 -2S0003-2670(09)01355-5 [pii], doi:10.1016/j.aca 20 09.10.0 04 [36] J.L Little, M.F Wempe, C.M Buchanan, Liquid chromatography-mass spectrometry/mass spectrometry method development for drug metabolism studies: examining lipid matrix ionization effects in plasma, J Chromatogr B Anal Technol Biomed Life Sci 833 (2) (2006) 219–230, doi:10.1016/j.jchromb.2006.02 011 [37] R Lordan, A Tsoupras, I Zabetakis, Phospholipids of animal and marine origin: structure, function, and anti-inflammatory properties, Molecules (11) (2017) 22, doi:10.3390/molecules22111964 [38] T Berg, E Eliassen, B Jørgenrud, S Kabashi, A Petukhov, S.T Bogstrand, Determination of phosphatidylethanol 16:0/18:1 in whole blood by 96-well supported liquid extraction and UHPLC-MS/MS, J Clin Lab Anal 33 (1) (2019) e22631, doi:10.1002/jcla.22631 [39] B Jorgenrud, E Skadeberg, J de Carvalho Ponce, H Furuhaugen, T Berg, Determination of the alcohol biomarker phosphatidylethanol 16:0/18:1 and 33 compounds from eight different drug classes in whole blood by LC-MS/MS, J Pharmacol Toxicol Methods 107 (2021) 106939, doi:10.1016/j.vascn.2020.106939 [40] T Berg, E Lundanes, A.S Christophersen, D.H Strand, Determination of opiates and cocaine in urine by high pH mobile phase reversed phase UPLC-MS/MS, J Chromatogr B Anal Technol Biomed Life Sci 877 (4) (2009) 421–432, doi:10 1016/j.jchromb.2008.12.052 [41] H.I.S Kafeenah, R Osman, N.K.A Bakar, Effect of mobile phase pH on the electrospray ionization efficiency and qualitative analysis of pharmaceuticals in ESI + LC-MS/MS, J Chromatogr Sci 57 (9) (2019) 847–854, doi:10.1093/ chromsci/bmz061 [42] J.N Mess, M Lahaie, M Furtado, F Garofolo, Effect of high pH mobile phase on the sensitivity of multiple drugs by LC positive electrospray ionization MS/MS, Bioanalysis (8) (2009) 1419–1430, doi:10.4155/bio.09.133 [43] A Tan, J.C Fanaras, Use of high-pH (basic/alkaline) mobile phases for LC-MS or LC-MS/MS bioanalysis, Biomed Chromatogr 33 (1) (2019) e4409, doi:10.1002/ bmc.4409 [44] R LoBrutto, et al., Effect of the eluent pH and acidic modifiers in highperformance liquid chromatography retention of basic analytes, J Chromatogr A 913 (1) (2001) 173–187 -2, doi:10.1016/s0 021-9673(0 0)01012-8 [1] S.T Bogstrand, H Gjerde, P.T Normann, I Rossow, Ø Ekeberg, Alcohol, psychoactive substances and non-fatal road traffic accidents–a case-control study, BMC Public Health 12 (2012) 734, doi:10.1186/1471- 2458- 12- 734 [2] S Kabashi, V Vindenes, E.A Bryun, E.A Koshkina, A.V Nadezhdin, E.J Tetenova, A.J Kolgashkin, A.E Petukhov, S.N Perekhodov, E.N Davydova, D Gamboa, T Hilberg, A Lerdal, G Nordby, C Zhang, S.T Bogstrand, Harmful alcohol use among acutely ill hospitalized medical patients in Oslo and Moscow: a cross-sectional study, Drug Alcohol Depend 204 (2019) 107588, doi:10.1016/j drugalcdep.2019.107588 [3] J Rehm, The risks associated with alcohol use and alcoholism, Alcohol Res Health 34 (2) (2011) 135–143 PMCID: PMC3307043 [4] J Rehm, C Mathers, S Popova, M Thavorncharoensap, Y Teerawattananon, J Patra, Global burden of disease and injury and economic cost attributable to alcohol use and alcohol-use disorders, Lancet 373 (9682) (2009) 2223–2233, doi:10.1016/S0140- 6736(09)60746- [5] World Health OrganizationGlobal Status Report on Alcohol and Health, World Health Organization, 2014 [6] J Manthey, S.A Hassan, S Carr, C Kilian, S Kuitunen-Paul, J Rehm, What are the economic costs to society attributable to alcohol use? A systematic review and modelling study, Pharmacoeconomics 39 (7) (2021) 809–822, doi:10.1007/ s40273- 021- 01031- [7] H.J Navarro, C.M Doran, A.P Shakeshaft, Measuring costs of alcohol harm to others: a review of the literature, Drug Alcohol Depend 114 (2) (2011) 87–99 -3, doi:10.1016/j.drugalcdep.2010.11.009 [8] E.E Bouchery, H Harwood, J Sacks, C.J Simon, R.D Brewer, Economic costs of excessive alcohol consumption in the US, 2006, Am J Prev Med 41 (5) (2011) 516–524, doi:10.1016/j.amepre.2011.06.045 [9] L Gustavsson, ESBRA 1994 award lecture phosphatidylethanol formation: specific effects of ethanol mediated via phospholipase D, Alcohol Alcohol 30 (4) (1995) 391–406 PMID: 8540905 [10] M.A Javors, N Hill-Kapturczak, J.D Roache, T.E Karns-Wright, D.M Dougherty, Characterization of the pharmacokinetics of phosphatidylethanol 16:0/18:1 and 16:0/18:2 in human whole blood after alcohol consumption in a clinical laboratory study, Alcohol Clin Exp Res 40 (6) (2016) 1228–1234, doi:10.1111/acer 13062 [11] C Alling, L Gustavsson, E Anggard, An abnormal phospholipid in rat organs after ethanol treatment, FEBS Lett 152 (1) (1983) 24–28, doi:10.1016/ 0014- 5793(83)80474- [12] S Aradottir, C Lundquist, C Alling, Phosphatidylethanol formation in rat organs after ethanol treatment, Biochim Biophys Acta 793 (1) (1984) 119–122, doi:10.1111/j.1530-0277.2002.tb02569.x13 [13] T.M Maenhout, M.L De Buyzere, J.R Delanghe, Non-oxidative ethanol metabolites as a measure of alcohol intake, Clin Chim Acta 415 (2013) 322–329, doi:10.1016/j.cca.2012.11.014 [14] H Andresen-Streichert, A Müller, A Glahn, G Skopp, M Sterneck, Alcohol biomarkers in clinical and forensic contexts, Dtsch Arztebl Int 115 (18) (2018) 309–315, doi:10.3238/arztebl.2018.0309 [15] S Aradottir, G Asanovska, S Gjerss, P Hansson, C Alling, PHosphatidylethanol (PEth) concentrations in blood are correlated to reported alcohol intake in alcohol-dependent patients, Alcohol Alcohol 41 (4) (2006) 431–437, doi:10 1093/alcalc/agl027 [16] F.M Wurst, N Thon, S Aradottir, S Hartmann, G.A Wiesbeck, O Lesch, K Skala, M Wolfersdorf, W Weinmann, C Alling, Phosphatidylethanol: normalization during detoxification, gender aspects and correlation with other biomarkers and self-reports, Addict Biol 15 (1) (2010) 88–95, doi:10.1111/j 1369-160 0.20 09.0 0185.x [17] A Nalesso, G Viel, G Cecchetto, D Mioni, G Pessa, D Favretto, S.D Ferrara, Quantitative profiling of phosphatidylethanol molecular species in human blood by liquid chromatography high resolution mass spectrometry, J Chromatogr A 1218 (46) (2011) 8423–8431, doi:10.1016/j.chroma.2011.09.068 [18] Y Zheng, O Beck, A Helander, Method development for routine liquid chromatography-mass spectrometry measurement of the alcohol biomarker phosphatidylethanol (PEth) in blood, Clin Chim Acta 412 (15) (2011) 1428– 1435 -16, doi:10.1016/j.cca.2011.04.022 [19] M Lopez-Cruzan, J.D Roache, N Hill-Kapturczak, T.E Karns-Wright, D.M Dougherty, J.J Sanchez, W Koek, M.A Javors, Pharmacokinetics of phosphatidylethanol 16:0/20:4 in human blood after alcohol intake, Alcohol Clin Exp Res 42 (11) (2018) 2094–2099, doi:10.1111/acer.13865 [20] A Helander, U Hermansson, O Beck, Dose-response characteristics of the alcohol biomarker phosphatidylethanol (PEth)-a study of outpatients in treatment for reduced drinking, Alcohol Alcohol 54 (6) (2019) 567–573, doi:10 1093/alcalc/agz064 [21] H Gnann, C Engelmann, G Skopp, M Winkler, V Auwärter, S Dresen, N Ferreirós, F.M Wurst, W Weinmann, Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS, Anal Bioanal Chem 396 (7) (2010) 2415–2423, doi:10.10 07/s0 0216- 010- 3458- [22] R.N Xu, L Fan, M.J Rieser, T.A El-Shourbagy, Recent advances in highthroughput quantitative bioanalysis by LC-MS/MS, J Pharm Biomed Anal 44 (2) (2007) 342–355, doi:10.1016/j.jpba.2007.02.006 [23] M Meng, L Wang, T Voelker, S Reuschel, K.C Van Horne, P Bennett, A systematic approach for developing a robust LC-MS/MS method for bioanalysis, Bioanalysis (1) (2013) 91–115, doi:10.4155/bio.12.295 ... B in 0. 0–0 .2 min, 6 0–8 8% B in 0. 2–0 .3 min, 8 8–9 8% B in 0. 3–3 .8 min, 9 8–1 00% B in 3. 8–3 .9 min, 100% B in 3. 9–6 .4 min, 10 0–6 0%B in 6. 4–6 .5 min, 60% B in 6. 5–7 .0 Retention time order for PEth homologues. .. in 0. 2–0 .3 min, 8 4–9 6% B in 0. 3–4 .0 min, 9 6–1 00% B in 4. 0–4 .1 min, 100% B in 4. 1–7 .5 min, 10 0–1 0%B in 7. 5–7 .6 min, 10% B in 7. 6–8 .2 Mobile phase flow rate was 0.5 mL/min PEth homologues retention... illustration of the both gradient profiles used are included in figure (c) Gradient profiles: 60% B in 0. 0–0 .2 min, 6 0–8 4 (or 88)% B in 0. 2– 0.3 min, 84 (or 88) – 98% B in 0. 3–3 .8 min, 9 8–1 00% B in 3. 8–3 .9