citrate blood sampling tubes improve preanalytical stability?

Journal Pre-proof Implementation of lipidomics in clinical routine: Can fluoride/citrate blood sampling tubes improve preanalytical stability? Lisa Hahnefeld, Robert Gurke, Dominique Thomas, Yannick Schreiber, Stephan M.G. Schäfer, Sandra Trautmann, Isabel Faria Snodgrass, Daniel Kratz, Gerd Geisslinger, Nerea Ferreirós PII:

S0039-9140(19)31226-3

DOI:

https://doi.org/10.1016/j.talanta.2019.120593

Reference:

TAL 120593

To appear in:

Talanta

Received Date: 23 July 2019 Revised Date:

22 November 2019

Accepted Date: 25 November 2019

Please cite this article as: L. Hahnefeld, R. Gurke, D. Thomas, Y. Schreiber, S.M.G. Schäfer, S. Trautmann, I.F. Snodgrass, D. Kratz, G. Geisslinger, N. Ferreirós, Implementation of lipidomics in clinical routine: Can fluoride/citrate blood sampling tubes improve preanalytical stability?, Talanta (2019), doi: https://doi.org/10.1016/j.talanta.2019.120593. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.

150 t1 = +20 min … t5 = +24 h 0.9 ± 0.7 °C

22.0 ± 1.3 °C

rel. change [%]

K3EDTA

Stability after 20 min in ice water

NaF/citrate

100 50 0

n = 10 6♀4♂ BMI < 30 Age 35 ± 10 y

1-AG

2-AG

AEA OEA+VEA

PEA

Plasma K3EDTA

WB K3EDTA

Plasma NaF/citrate

WB NaF/citrate

Implementation of lipidomics in clinical routine: can fluoride/citrate blood sampling tubes improve preanalytical stability? Lisa Hahnefeld1, Robert Gurke1,2, Dominique Thomas1, Yannick Schreiber2, Stephan M. G. Schäfer2, Sandra Trautmann1, Isabel Faria Snodgrass1,2, Daniel Kratz1, Gerd Geisslinger1,2, Nerea Ferreirós1* 1

pharmazentrum frankfurt/ ZAFES, Institute of Clinical Pharmacology, Goethe

University, Frankfurt, Germany 2

Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch for

Translational Medicine and Pharmacology TMP, Frankfurt, Germany

*Corresponding author: Dr. Nerea Ferreirós pharmazentrum frankfurt/ZAFES Institute of Clinical Pharmacology Goethe-University Frankfurt Building 74 / 4th floor / room 4.106b Theodor-Stern-Kai 7 D-60590 Frankfurt am Main / Germany E-mail: [email protected] Tel.: 0049-69-6301-7618 Fax: 0049-69-6301-7331

1

1

Abstract

The impact of preanalytical sample handling on lipid stability has been assessed in human plasma using targeted LC-MS/MS quantification of endocannabinoids, sphingolipids and LPA, complemented by non-targeted lipidomics screening with LCQTOFMS. The study involved incubation of whole blood and plasma from healthy volunteers at room temperature or in ice water for time periods ranging from 20 min to 24 h. The impact of two different anticoagulants, K3EDTA and sodium fluoride/citrate, on lipid stability was evaluated. It was found that the concentrations determined for several endogenous lipids vary when whole blood and plasma samples are processed at room temperature, whereas the concentrations of most lipids were stable for 4 h in ice water. Surprisingly, the detected amounts of endocannabinoids 1- and 2arachidonoyl glycerol and arachidonoyl ethanolamide increased markedly by 60, 95, and 30% in K3EDTA whole blood after storage in ice water for only 20 min. When using sodium fluoride/citrate blood collection tubes, the stability of several lipids, including that of the endocannabinoids, was improved. Accordingly, it is absolutely necessary to keep the blood sampling and plasma processing time below 1 h to avoid ex-vivo formation of endocannabinoids. It is worth mentioning that baseline lipid levels differ when using K3EDTA or sodium fluoride/citrate blood sampling tubes, which emphasizes the importance of traceability of reported plasma concentrations to the used anticoagulant. 1

Abbreviations: 1-AG/2-AG: 1-/2- arachidonoyl glycerol; AEA: arachidonoyl ethanolamide; APCI, atmospheric pressure chemical ionization; Cer, ceramide; CES, collision energy spread; DDA, data dependent acquisition; DG, diglyceride; EDTA: ethylenediaminetetraacetic acid; EMEA: European Medicines Agency; FA, fatty acid; FDA: U.S. Food and Drug Administration; FDR: false discovery rate; GlcCer, glucosylceramide; LacCer: lactosylceramide; LC-QTOFMS, liquid chromatography-quadrupoletime-of-flight mass spectrometry; LPA: lysophosphatidic acid; LPX, lysophospholipids; MTBE, methyl-tertbutyl-ether; O-, ether-species; OEA: oleoyl ethanolamide; PEA: palmitoyl ethanolamide; PBS: phosphate buffered saline; PC, phosphatidylcholine; PCA: principal component analysis; PE, phosphatidylethanolamine; RT: room temperature; SE: sterol ester, SM, sphingomyelin; S1P: sphingosine-1-phosphate; ST, sterol; TG, triglycerides

2

Keywords: preanalytical stability, lipidomics, endocannabinoids, ceramides, mass spectrometry, lysophosphatidic acid

1 Introduction Lipid compounds have been investigated as potential biomarkers for several diseases including cardiovascular diseases and cancer [1-3]. For lipids to be generally utilized as biomarkers, the analytical methods applied have to be thoroughly validated using the strict guidelines given by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) among others. Both institutions recommend stability studies under the pre-analytical conditions, however these are not a requirement [4, 5]. To ensure that the lipid concentrations detected accurately reflect the true endogenous concentrations, the stability of the compounds must be confirmed from the time of sampling until the end of analysis. Small changes in lipid degradation or release will increase the variance and therefore, confidence in the results obtained, especially in clinical trials or animal experiments with a small number of subjects. On the other hand, excessive instability increases the possibility of generating artifactual results [6, 7]. Whereas only a few studies have focussed on the lipidome, several metabolomics studies in which various lipids were included, have been carried out, demonstrating the stability of several lipids in human EDTA whole blood stored in ice water for up to 4 h [6, 8, 9]. Storage at room temperature (RT) on the other hand, leads to alterations in the lipid profile such as an increase in sphingosine-1-phosphate (S1P) concentrations [6, 8, 3

10]. Similar results were observed for the stability of lipids in processed plasma and serum samples, however after separation of the cellular fraction, differences were less evident [10, 11]. Wang et al. investigated the stability of compounds in mouse and rat plasma at RT focusing on the lipidome and found an increase in lysophospholipids and free fatty acids as well as a decrease in diacyl-phospholipids, di- and triglycerides [12]. In addition to such investigations on abundant lipids, stability studies have also been carried out on endogenous lipid mediators. Brunkhorst et al. investigated changes in preanalytical effects on several sphingolipids and found that the concentrations of sphingoid

base-1-phosphates

increased

with

increasing

incubation

time

and

temperature of blood samples, whereas ceramide concentrations were unchanged [13]. On the other hand, quantitation of lysophosphatidic acids (LPA) has proved to be challenging because of their in-vitro formation in human plasma at RT as well as an upward trend after multiple freeze-thaw cycles [14, 15]. In addition, platelet activation in serum leads to increased LPA concentrations [15]. Therefore, storage of samples at 4 °C directly after blood sampling and processing h as been proposed, since enzymatic activity should be suppressed under these conditions, but this assumption has not been confirmed [16]. Another preanalytical problem occurs with endocannabinoids, which are assayed in plasma or serum and are reportedly prone to ex-vivo formation [17, 18]. Therefore, the authors recommend that the blood collection and processing time should be kept to a minimum and the samples should be stored on ice, as incubation of whole blood at 4 °C for 30 min significantly increased en docannabinoid levels [19, 20]. In clinical practice, such short collection and processing times are unlikely to be achievable, so maintenance of the stability of lipid compounds in samples is required.

4

The aim of the present study was to assess the short-term stability of lipids during the preanalytical phase, both in whole blood and plasma, in order to develop feasible standard operating procedures to ensure compound stability in clinical practice. Moreover, K3EDTA, which was proposed as a standard anticoagulant for lipid analysis [9, 10, 17], was compared to sodium fluoride, acidified with citrate, to assess their capacity to improve the stability of lipids and lipid mediators. K3EDTA and citrate prevent coagulation by inhibition of calcium dependent enzymes but the outcomes vary in terms of reaction rate, reaction extent and their respective counter ions [21, 22]. Whereas K3EDTA specifically inhibits enzymes with divalent metal ions, fluoride inhibits a broad range of enzymes including enolase and phosphatases [23-25]. For LPA and endocannabinoid quantitation, preanalytical stability is crucially needed to obtain robust and reliable results. [7, 20]. We further sought to assess the transferability of these preanalytical conditions to the analysis of other lipid groups such as sphingolipids and non-targeted lipidomics screening.

2 Methods 2.1 Materials Water, isopropyl alcohol, methanol, acetonitrile (LC-MS grade), butanol (≥ 99.5 %) hexane (UV/IR grade) and methyl-tert-butyl-ether (MTBE, HPLC-grade) were purchased from Carl Roth (Karlsruhe, Germany). Acetonitrile (ULC-MS grade) was purchased from Biosolve B. V. (Valkenswaard, The Netherlands). Chloroform (analytical reagent grade) was obtained from Fisher Scientific (Schwerte, Germany) and ethyl acetate (Reag. Ph. Eur. ≥ 99.9%) from VWR (Darmstadt, Germany). APCI positive calibration solution was 5

purchased from Sciex (Darmstadt, Germany), ammonium formate (for mass spectrometry, ≥ 99.0 %) and Sulfinpyrazon (100%) from Sigma-Aldrich (Munich, Germany) and formic acid (98-100 %) from AppliChem (Darmstadt, Germany). The endocannabinoids and their internal standards were provided by Cayman Chemical (Ann Arbor, MI, USA). All standards and internal standards for the lysophosphatidic acid, sphingolipid and non-targeted measurement were purchased from Avanti Polar Lipids (Alabaster, AL, USA) (see suppl. material for a detailed list). For the Bradford protein assay Dulbecco's phosphate-buffered saline from Life Technologies Limited (Paisley, UK) and heat shock fractionated Bovine Serum Albumin (≥ 98 %, protease, fatty acid and essentially globulin free, pH 7) as well as Bradford Reagent from SigmaAldrich (Steinheim, Germany) were used.

2.2 Blood sampling Blood samples were taken from healthy volunteers after informed consent was obtained. The study included 6 female and 4 male participants with normal weight and a mean age of 34.6 ± 9.5 years. All study participants were fasted for more than 4 h. Venous blood from the vena brachialis was taken using 9 mL and 2.7 mL K3EDTA and 3.1 mL GlucoExact (containing sodium fluoride and citrate) tubes (all from Sarstedt, Nümbrecht, Germany). General sample processing included storage of the collecting tubes in ice water directly after collection and centrifugation at 2,000 g for 10 min at 4 °C after incubation as stated below. Plasma was collected immediately after centrifugation and stored at -20 °C for approximately 4 h before t ransferring to a -80 °C freezer. Individual patient samples were randomized before storage to improve the validity of the

6

results generated.

2.3 Experimental Setup For the determination of the stability of the different analytes in plasma, two 9 mL K3EDTA and six 3.1 mL NaF/citrate tubes were collected per test subject, directly processed and pooled according to anticoagulant. The plasma was then incubated at room temperature or in ice water for 20 min, 1 h, 2 h, 4 h or 24 h, whereas the t0 samples were directly frozen without incubation. Stability in whole blood was assessed using one 2.7 mL K3EDTA and one 3.1 mL NaF/citrate tube per time point and temperature per test subject. The tubes were incubated at room temperature or in ice water for 20 min, 1 h, 4 h or 24 h and processed as described above. The corresponding reference samples (t0) were stored for approximately one min in ice water until transfer to the centrifuge. The processing time from sampling to freezing for the reference (t0) samples, both for plasma and whole blood, was approximately 25 min. Room temperature was 22.0 ± 1.2 °C and the tem perature in the ice water was 0.9 ± 0.7 °C. The temperatures were assessed as minimum and maximum values per sampling day. The samples stored for 24 h in ice water were incubated in a refrigerator at 0 to 6 °C to avoid overnight thawing of the ice. After incubation, all samples were centrifuged as described above and each sample was split into aliquots. The endocannabinoids were directly measured from one 200 µL aliquot whereas the remaining aliquots were stored at -80 °C. One of these was thawed in the fridge for 1 h and then split into 10, 20 and 100 µL aliquots for the analysis of sphingolipids, non-targeted and LPA. The samples were stored in ice water during the

7

aliquoting and stored at -80 °C until analysis. Before storage, samples were randomized per subject to avoid systematic errors.

2.4 LC-MS methods Quantitation of LPA, sphingolipids and endocannabinoids was achieved as described in previously published papers [20, 26, 27]. A detailed list of analytes and internal standards can be found in supplementary Table S1. Briefly, LPA were extracted from 100 µL plasma using liquid-liquid extraction with 500 µL Na2HPO4/citrate buffer (pH 4) and 800 µL butanol. LC-MS/MS measurement was performed using an Agilent 1200 HPLC System equipped with a Luna C18 (2) chromatographic column (5 µm, 50 x 2 mm ID, Phenomenex, Aschaffenburg, Germany) coupled to a QTrap 5500 hybrid triple quadrupole- ion trap mass spectrometer (Sciex, Darmstadt, Germany). For the sphingolipid quantification, 10 µL sample was extracted using 200 µL 30 mM citric acid and 40 mM Na2HPO4 buffer (pH 4.2) and 600 µL methanol: chloroform: hydrochloric acid (15:83:2, v/v/v). Chromatographic separation was achieved using a Zorbax RRHD Eclipse Plus C18 column (1.8 µm 50 x 2.1 mm ID) on an Agilent 1290 Infinity I UHPLC system (Agilent, Waldbronn, Germany) and a QTrap 5500 for detection (Sciex, Darmstadt, Germany). The endocannabinoids and endocannabinoid-like substances 1arachidonoyl glycerol (1-AG), 2- AG, arachidonoyl ethanolamide (AEA), oleoyl ethanolamide (OEA) and palmitoyl ethanolamide (PEA) were extracted from 200 µL plasma using 400 µL ethyl acetate: hexane (9:1, v/v). Their LC-MS/MS measurement included separation on an Agilent 1290 Infinity I UHPLC system with an Acquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 µm, Waters, Eschborn, Germany) and

8

measurement on a QTrap 6500+ (Sciex, Darmstadt, Germany). All quantitations were performed using an ESI Turbo-V-source with negative ion source voltage for LPA and positive ion source voltage for sphingolipid and endocannabinoid measurement. For the current study, the chromatographic gradient was shortened and the sample volume was increased for endocannabinoid quantification compared to the previously published method [20] to increase sensitivity (detailed information can be found in the supplementary material). Due to the shortened gradient, OEA and its isomer vaccenic acid ethanolamide (VEA) were not separated and were therefore, quantified as a sum parameter.

LC-QTOFMS measurement For the non-targeted screening, lipids were extracted using a modified MTBE extraction protocol by Matyash et al. [28]. Protein precipitation was induced by adding 150 µL of internal standards (see suppl. material) in methanol to 20 µL of human plasma, followed by 500 µL MTBE. Phase separation was induced by addition of 125 µL 50 mM ammonium formate, the mixture was vortexed for 1 min and centrifuged for 5 min at 20,000 g at room temperature. After transfer of the upper organic phase, the aqueous phase was re-extracted with 200 µL of a mixture of MTBE: methanol: water (10:3:2.5, v/v/v, upper phase). The combined organic phases were then split into two 290 µL aliquots for measurement in both polarity modes and dried under a nitrogen stream at 45 °C. The aliquots were stored at -80 °C pending a nalysis and reconstituted in 120 µL methanol before measurement. Chromatographic separation was performed using a Zorbax RRHD Eclipse Plus C8

9

column (1.8 µm 50 x 2.1 mm ID, Agilent, Waldbronn, Germany) with a SecurityGuard Ultra C8 pre-column (Phenomenex, Aschaffenburg, Germany) on a Nexera X2 system (Shimadzu Corporation, Kyoto, Japan) equipped with two LC-30AD pumps, a SIL-30 AC autosampler, a DGU-20A5R degassing unit and a CTO-30A column oven. The column was maintained at 40 °C. A 17 min linear gradient w ith a flow rate of 0.3 mL/min is described in the supplementary material. The injection volumes were 2 µL for positive and 5 µL for negative ionization mode. For measurements in positive ionization mode, a peek-tee was implemented directly before the ESI probe to reduce the flow from 300 µL/min to approximately 75 µL/min. Samples were analyzed on a TripleTOF 6600 equipped with a DuoSpray ion source (both Sciex, Darmstadt, Germany). Electrospray ionization was used for sample ionization in positive and negative ionization mode, whereas the atmospheric pressure chemical ionization (APCI) probe was used to inject the calibration solution, a 1:1 mixture (v/v) of APCI positive calibration solution and 1 µg/mL sulfinpyrazone in acetonitrile, every ten samples to ensure a mass error below 5 ppm. A detailed description of the source applied and acquisition parameters can be found in the supplementary material. For verification of the system stability, samples from the first four subjects were pooled for quality control samples, which were injected at the start and at the end of a run and after every 10th sample.

2.5 Data acquisition and processing All quantitative data were acquired using triple quadrupole MS with Analyst software

10

v1.6.3 and peak integration was performed by MultiQuant software v 3.0.2. Acceptance criteria and quality assurance measures were applied as described previously [20]. The lower and upper limits of quantification for all analytes can be found in the supplementary material. Non-targeted QTOF data were acquired using Analyst TF v1.7.1 and peak alignment and filtering was done using MarkerView software v1.2.1 with a mass and retention time tolerance of 10 ppm and 0.15 min, respectively. Lipid compounds were identified with MasterView v1.1 and LibraryView v1.0.1 software using the exact mass ± 5 ppm, isotopic distribution and comparison of MS/MS fragmentation patterns with a custom made compound library cross-checked with LIPID MAPS (http://www.lipidmaps.org), METLIN (http://metlin.scripps.edu) and the Human Metabolome Database (HMDB, version 4.0). All data acquisition and processing software was purchased from Sciex (Darmstadt, Germany). For the non-targeted analysis of QTOF data, samples were normalized to the signal of all internal standards and for the analysis of previously identified lipids peak areas were corrected using one internal standard per lipid class. Features which were identified as isotopes by MarkerView software were excluded from further data analysis. In addition, features for which > 20 % of the samples had a peak area smaller than the extracted blanks as well as features with a relative standard deviation above 20 % for the quality control samples were all excluded, resulting in 3198 features for both positive and negative ionization mode together. The semitargeted analysis was performed for previously identified lipids using MultiQuant software v 3.0.2. The semi-targeted results were normalized to one internal standard per lipid class.

11

2.6 Determination of protein content For further characterization of the plasma samples, the protein content was analyzed using a Bradford microplate assay [29]. A standard curve of bovine serum albumin (BSA) in phosphate buffered saline (PBS) was employed for quantification (range: 0.05 – 0.8 mg/mL, R2 > 0.99). The directly processed NaF/citrate and K3EDTA plasma samples of the 10 healthy volunteers were thawed, diluted by a factor of 200 with PBS and immediately used for quantification. The blank signal of the standard curve was calculated using pure PBS. To exclude anticoagulant interference with the assay, the blank signal of the respective tubes was evaluated by filling three unused blood sampling tubes each with PBS via the vacuum technique and utilizing those samples as additional blanks for the plasma samples. 10 µL of sample was mixed with 250 µL Bradford reagent and mixed for 5 min at RT. The absorption was measured at 595 nm using an Infinite F200 PRO plate reader (Tecan, Männedorf, Switzerland) and the value of the corresponding PBS blanks (n = 3) was set as zero. The pH of the K3EDTA and NaF/citrate plasma was assessed using a HI 221 pH meter (Hanna Instruments, Vöhringen, Germany).

2.7 Statistical analysis Statistical analysis was performed with R v3.4.2 in the R Studio environment v1.1.383 [30, 31] and GraphPad Prism v5.0 (GraphPad Software Inc., San Diego, CA, USA). The results of the NaF/citrate tubes were multiplied by 1.16 to compensate for the dilution due to the liquid anticoagulant, as indicated by the manufacturer. The differences 12

between two groups were evaluated using paired t-test or paired Wilcoxon test and normal distribution was checked by Kolmogorov-Smirnov test. More than two groups in a time series were compared by linear mixed effect models followed by Tukey’s test and false discovery rate (FDR) adjustment using the “nlme” and “multcomp” package in R [32, 33]. For further identification of aligned features in the non-targeted screening, the paired t-test was performed after the Kolmogorov-Smirnov test. Features with an FDR adjusted p-value below 0.1 for comparison of the 24 h of incubation time to the directly processed samples, were identified as described above.

3 Results and discussion 3.1 Stability of sphingolipids Previous studies indicated the suitability of plasma using K3EDTA as anticoagulant as a matrix for the reliable determination of sphingolipids [13]. The quantitative LC-MS/MS analysis of sphingolipids demonstrated the stability of all investigated ceramides (n=8) as well as glucosyl- (n=3) and lactosylceramides (n=5) in K3EDTA whole blood and plasma stored in ice water and at room temperature (detailed results can be found in the supplementary material, the investigated compounds are listed in Table S1). These findings were confirmed by the non-targeted analysis. However, amounts of sphingosine-1-phosphate increased significantly after incubation of K3EDTA and NaF/citrate whole blood at RT, but not in ice water or plasma stored at RT (see Figure 1), as already described previously. It may be assumed that S1P is released from red blood cells and platelets in whole blood at ambient temperature [8, 13]. Therefore, the findings of Brunkhorst et al. could be confirmed in the present study [13]. The

13

sphingolipids, including S1P can be quantified reliably in K3EDTA plasma, but the samples must be kept in ice water until separation of the blood cells. Afterwards, the processed plasma can be extracted at RT.

3.2 Stability of lysophosphatidic acids using K3EDTA as anticoagulant The data presented here indicate the importance of these investigations for LPA 16:0, 18:1, 18:2 and 20:4 as an increase of about 80% after 1 h of incubation time at RT was determined both in whole blood and in plasma using K3EDTA as anticoagulant. The course of the LPA increase was comparable for the ten healthy subjects as well as between the analyzed compounds. Arachidonic acid containing LPA 20:4, as an example, is shown in Figure 2. More detailed information for the LPA can be found in the supplementary material. Keeping the samples on ice stabilized the LPA concentrations for up to 4 h in plasma and in whole blood, but a significant increase was detectable after 24 h. Therefore, sample processing in ice water is recommended, but longer processing times should be avoided. The results of the present investigation support previous findings of elevated LPA levels in plasma and whole blood at RT as well as improved stability in plasma stored on wet ice [7, 14, 15]. Furthermore, our study demonstrated that direct storage of whole blood samples in ice water improves the accuracy of LPA quantification by reducing ex-vivo formation. As the LPA increase at RT was similar in plasma and whole blood, this was presumably related to enzymatic degradation of phosphatidic acids, which were not analyzed in this study.

14

3.3 Stability of endocannabinoids using K3EDTA as anticoagulant The preanalytical stability of endocannabinoids has been investigated several times, always showing that they are prone to ex-vivo generation [17, 19, 20]. The current study shows that the endocannabinoids 2-AG, AEA, PEA and OEA/VEA are stable in K3EDTA plasma at RT and in ice water for up to 4 h. However, the concentration of 1AG increased significantly after only 20 min at RT and consequently plasma samples should be kept on ice during lipid extraction. In whole blood samples stored in ice water, an increase of 60 % in 1-AG, 95 % in 2-AG and 30 % in AEA after 20 min and further a 30 % increase in PEA levels after 1 h of incubation time could be observed when compared to directly processed samples (approx.. 25 min processing time until freezing of the samples, Figure 3 B). The different behavior in plasma compared to that in whole blood, as well as the decrease in 1-AG and 2-AG between 1 h and 4 h on ice and the decrease in 1-AG and 2-AG at RT, suggest multiple underlying mechanisms, presumably involving cellular activity as well as the isomerization from 2-AG to 1-AG [34]. The instructions to proceed with blood sampling as fast as possible thus seem to be insufficient, especially when sampling takes place in different locations and is done by different personnel. The resulting deviation in endocannabinoid concentrations can skew results in smaller clinical trials and greatly impair study quality when unequal blood sampling times between groups occur. Thus, pooling K3EDTA blood sampling tubes to generate samples for endocannabinoid analysis cannot be recommended and other anticoagulants should be used instead (see section 3.5).

15

3.4 Lipidomics analysis using K3EDTA as anticoagulant The lipidomic analysis with LC-QTOFMS encompassed the non-targeted analysis of all features (distinctive m/z to retention time values) present in the samples, as well as a semi-targeted analysis of 223 from 431 previously identified lipids covering more than 15 different lipid classes (details are listed in the supplementary table S6). The semi-targeted analysis of K3EDTA whole blood and plasma stored at RT showed a time-dependent

increase

in

free

fatty

acids

(FA),

diglycerides

(DG),

lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). For some of these species, relative concentrations increased by more than 20 % after 4 h storage at RT compared to directly processed samples, as can be seen in the representative examples displayed in Figure 4. The increase in FA 20:4 and FA 20:5 exceeded that in the other fatty acids in plasma, whereas a decrease could be observed in whole blood (see supplementary table S10). Moreover, a decrease in phosphatidylcholines (PC), phosphatidylethanolamines (PE) and triglycerides (TG) was observed after 24 h in whole blood and plasma. The plasma concentrations of these molecules are overall much larger than those of LPC, LPE and DG. Therefore, a small hydrolysis ratio of TG would not significantly influence the TG concentration, it would however, substantially alter DG concentrations, thereby explaining the rapid increase in the hydrolysis products.

The

relative

concentrations

of

lysophosphatidylinositols

and

lysophosphatidylglycerols were also increased after 24 h storage at RT, but they did not alter more than 20 % during the first 4 h of incubation. The observed lipid degradation could be prevented by keeping the whole blood and plasma samples in ice water (supplementary table S11). No clear difference was found after 24 h of storage time.

16

Additionally, the stability of lipid compounds could be improved with NaF/citrate as an additive, which is further explained in section 3.5. The stability of lipids in K3EDTA whole blood and plasma for the non-targeted analysis was also evaluated in order to identify potential differences overlooked during the semitargeted analysis. Of 3198 aligned features, both from positive and negative ionization mode, 360 features differed between 24 h and control samples in whole blood at RT with an FDR value < 0.1 (paired t-test). Among these features, 73 lipids could be unequivocally identified and showed that with this added information, acylcarnitines also increased in a time- and temperature dependent manner, as for semi-targeted analysis (Figure 5). The same evaluation of whole blood samples kept in ice water, revealed no significant changes except for an increase in lineolyl carnitine (FDR < 0.05, paired ttest). The analysis of the plasma samples showed that the acylcarnitines were stable both at RT and in ice water, confirming the results from the semi-targeted approach. Similar results were observed by Kamlage et al., showing a significant increase in LPC in plasma after 16 h at RT [9]. After 5 h at RT, no significant increase in LPC was shown, though it may be assumed that relative concentrations were already elevated. Wang et al. observed no alterations in human plasma after storage at room temperature for 4 h, but these results were limited to the plasma of one subject [12]. The data presented in this study suggest that short handling times and storage of plasma samples at RT for lipid extraction do not impair lipidomic analysis. However, if processing times exceeding 2 h are expected, for example during aliquoting for different measurements, samples should be kept in ice water in order to reduce variability. The results obtained further confirm the results of investigations by Yin et al. and their

17

recommendations that whole blood samples should be kept in ice water and processed in less than 4 h [8].

3.5 Lipid stability using sodium fluoride/citrate as anticoagulant The results shown so far indicate that time and temperature dependent variations in lipid profile can hinder the applicability of lipids as biomarkers when using K3EDTA sampling tubes, as illustrated by endocannabinoids. However, the stability of endocannabinoids and related compounds was improved by NaF/citrate additives, as can be seen in Figure 3. The plasma samples were stable at RT for up to 20 min and up to 4 h in ice water. Afterwards, an increase in the 1-AG concentrations of 40% and more could be observed (see supplementary tables S7 and S8). In addition, the endocannabinoids were stable in the whole blood samples stored in ice water for up to 1 h. Stabilization of ethanolamine concentration by NaF was explained by blockade by fluoride of the enzymes responsible for ethanolamine generation from the PE that was used [35]. Surprisingly, an opposite trend was observed for 1-AG and 2-AG in whole blood at RT and in ice water when comparing the two different blood sampling additives (see Figure 3 A+B and supplementary material), indicating multiple underlying conversion processes. A possible explanation for the unusual increase in 1- and 2- AG in NaF/citrate whole blood at RT might be the inhibition of AG degradation with unhindered release or formation, whereas in K3EDTA blood the degradation of AGs continues to occur. Another explanation could be the differences in coagulation mechanisms between citrate and K3EDTA, a potential subject for further investigations. The addition of citrate lowered the pH of the plasma to approximately 6.2 compared to

18

7.6 in K3EDTA plasma, which slows down the base-catalyzed isomerization of 2-AG to 1-AG [36]. Moreover, it can be concluded that acidified NaF inhibits the diglyceride lipases relevant for 1-AG and 2-AG generation in plasma, although not as effectively as their inhibition of phospholipases. Using NaF/citrate as anticoagulant is also favorable for the determination of LPAs, as no increase in the LPA levels was observed in NaF/citrate plasma and whole blood stored at RT (Figure 2). Additionally, principal component analysis of the non-targeted measurement of the NaF/citrate whole blood samples stored at room temperature showed a clear subject-wise clustering with no apparent differences due incubation times for up to 24 h (see suppl. fig. S3). Of 3084 aligned features from both ionization modes, 208 features were altered between 24 h at RT and control samples with an FDR value < 0.1 (paired t-test). Thirty-eight of these 208 features could be unequivocally identified as lipids, including several DG, LPC and LPE, which were increased, and steryl esters and TG, which were mildly decreased. Still, the fold changes were smaller compared to the results in K3EDTA whole blood (see Figure 5), suggesting improved lipid stability with the NaF/citrate additives. Exceptionally, the fatty acids 20:4 and 20:5 decreased in K3EDTA whole blood and increased in K3EDTA plasma, whereas they decreased in NaF/citrate whole blood and were unchanged in the respective plasma samples after 24 h. Furthermore, a time dependent increase in acylcarnitine 16:0, 18:1 and 18:2 was observed both in K3EDTA and in NaF/citrate whole blood stored at RT. These different behaviors indicate multiple concurrent reactions, which are affected differently by the anticoagulants and pH. As mentioned before, samples with K3EDTA as anticoagulant intended for the analysis of sphingolipids, LPA and a non-targeted

19

screening can be stabilized adequately by placing them in ice water. Therefore, use of NaF/citrate tubes is not required, unless continuous storage in ice water cannot be ensured in clinical practice.

3.6 Baseline levels in plasma using K3EDTA or NaF/citrate as additives Another important outcome of the present study is the difference in the lipid profiles in K3EDTA and NaF/citrate plasma. The concentrations of 206 of 248 analytes were significantly different, when the blood was drawn into either K3EDTA or NaF/citrate tubes (paired t-test with FDR adjustment, n=10). Table 1 provides an overview of the relative results from blood sampling tubes for different lipid classes, showing lower levels of most lipids in the sodium fluoride tubes. The lipid classes were clearly affected differently, so a simple dilution effect due to the liquid anticoagulant or incomplete filling can be excluded. Both tubes were treated equally, during processing of the whole blood samples into plasma, so differences as a result of the plasma processing are possible but unlikely. In contrast to a previously reported metabolomics study, no differences could be observed due to cluster formation of the counter ions [21]. Comparison of phosphate buffered saline solution (PBS) in K3EDTA and sodium fluoride/citrate blood collection tubes showed no apparent differences in the base peak chromatogram measured with a QTOFMS, while the same comparison with human plasma samples showed quantitative but no qualitative differences (see suppl. fig. S1). Investigation of the internal standards failed to reveal either potassium adducts or differences in the relative amount of sodium adducts and protonated species (suppl. Fig. S2). Furthermore, the peak areas of the isotope-labeled internal standards were unchanged and could not compensate for the concentration differences, hence the alterations must 20

have been present prior to lipid extraction. Whereas endocannabinoids and related compounds like AEA could have been increased in K3EDTA plasma due to fast ex-vivo formation, this does not explain the decreased levels of steryl esters (SE) and triglycerides (TG), which are quite stable. On the other hand, many lipids like SE and TG, which are bound to lipoproteins due to their low solubility in water, were decreased in NaF/citrate plasma, whereas the levels of more water soluble lipids like free fatty acids and lysophospholipids were similar in both tubes. A study of the total protein concentrations in both plasma types via Bradford assay showed that protein levels are significantly decreased by 11 % in the NaF/citrate tubes compared to K3EDTA (suppl. Fig. S4). Therefore, the (lipo-) protein solubility could have been affected directly by the NaF, which can be used as an antichaotropic or salting-out agent [37, 38]. Previous investigations comparing EDTA and NaF plasma (most probably with EDTA as anticoagulant) did not mention differences in lipid levels, hence the acidification due to citrate will further decrease their protein solubility [8]. Additionally, NaF concentration could play a role, but this is proprietary in the case of the NaF/citrate tubes. The coprecipitation of albumin and plasma lipids under acidic conditions was assumed by Roehrig et al., who added the chaotropic salt potassium thiocyanate as a counter measure [39].

3.7 Comparison of endocannabinoid blood sampling protocols Using NaF/citrate sampling tubes in contrast to K3EDTA sampling tubes for the endocannabinoid quantification resulted in prolonged processing times for the whole blood samples with storage times of up to 1 h in ice water, simplifying implementation into clinical practice [19, 34] (see Table 2). Additionally, both the arachidonoyl glycerols 21

and the ethanolamides are stable under these conditions. Pastor et al. used orlistat, a diglyceride lipase inhibitor, to prevent ex-vivo formation of monoacylglycerols in K3EDTA plasma, but this method is not likely to stabilize the ethanolamides [17]. Use of phenylmethylsulfonyl fluoride (PMSF) to improve preanalytical stability for lipidomics analysis has been proposed by Wang et al., but addition of this toxic compound after blood sampling is impractical and unrealistic in clinical practice and endocannabinoids were not analyzed in this study [12]. Likewise, Roehrig et al. showed the stability of endocannabinoids and related compounds in whole blood and plasma using acidification with acetic acid and addition of 2-(N-morpholino) ethanesulfonic acid and potassium thiocyanate after blood sampling [39]. Although they are less toxic than PMSF, addition of these additives will still be inconvenient in clinical practice. The effects of adding the compounds to the anticoagulant tube prior to sampling need to be investigated first and the patients’ safety needs to be prioritized. In contrast, NaF/citrate tubes are already commercially available and ready for clinical use.

3.8 Outlook There is a substantial number of diseases and other factors like age, which could affect the stability of lipids, due to alterations in metabolic pathways. In this study, we focused on healthy subjects, but alterations in lipid stability in many diseases will undoubtedly be investigated in the future. Additionally, lipid analysis depends on many cofactors, like nutrition and fasting state and therefore, poses many (pre-) analytical challenges that need to be addressed [40]. Future biomarker investigations need to account for these factors to facilitate reliable assays of biomarker candidates. The blood sampling protocols described here can be used for larger clinical trials, but before potential 22

biomarkers can be transferred to clinical practice, sample stability will need to be demonstrably robust.

4 Conclusion Based on the present study, we recommend the following blood sampling protocol for the measurement of lysophosphatidic acids, sphingolipids and non-targeted screening. The K3EDTA blood tubes must be placed in ice water directly after blood sampling and further processed within 2 h. The plasma fraction can be separated by centrifugation at 2,000 g for 10 min at 4 °C and the collected plasma can be stored for 2 h on ice. In case of the endocannabinoids, NaF/citrate should be used as anticoagulant to improve the stability of the analytes. Whole blood can be stored for up to 1 h before centrifugation at 2,000 g for 10 min at 4 °C. The separated plasma c an be stored for 2 h in ice water. The data presented here show the importance for reliable results in lipid analysis of careful study planning and implementation of standard operation procedures for blood sampling and processing. Investigations of lipids as potential biomarkers can only produce valid outcomes if lipid-specific characteristics are considered. Nevertheless, requirements for the validation of a biomarker for clinical practice are high and especially in multicenter trials, adherence to the schedule for sample processing could be difficult. These obstacles should be considered before proposing a lipid (mediator) as a potential biomarker.

Competing interests The authors declare that they have no conflict of interests. 23

Acknowledgments The authors would like to thank Carlo Angioni and Dominik Schmidt for their assistance in conducting the experiments as well as Prof. Klaus Scholich for discussion. We also thank Prof. Michael J. Parnham for his help. The work was supported by the Deutsche Forschungsgemeinschaft

Sonderforschungsbereich

SFB

1039/Z01

“Krankheitsrelevante Signaltransduktion durch Fettsäurederivate und Sphingolipide” and by the LOEWE Center “Translationale Medizin und Pharmakologie”.

References [1] Á. López-López, Á. López-Gonzálvez, T.C. Barker-Tejeda, C. Barbas, A review of validated biomarkers obtained through metabolomics, Expert Review of Molecular Diagnostics 18(6) (2018) 557-575. [2] K. Furukawa, Y. Ohmi, Y. Ohkawa, R.H. Bhuiyan, P. Zhang, O. Tajima, N. Hashimoto, K. Hamamura, K. Furukawa, New era of research on cancer-associated glycosphingolipids, Cancer Sci 110(5) (2019) 1544-1551. [3] V.M. Martin Gimenez, S.E. Noriega, D.E. Kassuha, L.B. Fuentes, W. Manucha, Anandamide and endocannabinoid system: an attractive therapeutic approach for cardiovascular disease, Ther Adv Cardiovasc Dis 12(7) (2018) 177-190. [4] EMA/CHMP/EWP/192217, Guideline on bioanalytical method validation, 2009 Rev. 1 Corr. 2 (2009). [5] F. US FDA UDoHaHS, Center for Drug Evaluation and Research, Rockville, MD, USA, Bioanalytical Method Validation - Guidance for Industry, (2018). [6] V.V. Hernandes, C. Barbas, D. Dudzik, A review of blood sample handling and preprocessing for metabolomics studies, Electrophoresis 38(18) (2017) 2232-2241. [7] T. Yagi, M. Shoaib, C. Kuschner, M. Nishikimi, L.B. Becker, A.T. Lee, J. Kim, Challenges and Inconsistencies in Using Lysophosphatidic Acid as a Biomarker for Ovarian Cancer, Cancers (Basel) 11(4) (2019). [8] P. Yin, A. Peter, H. Franken, X. Zhao, S.S. Neukamm, L. Rosenbaum, M. Lucio, A. Zell, H.U. Haring, G. Xu, R. Lehmann, Preanalytical aspects and sample quality assessment in metabolomics studies of human blood, Clin Chem 59(5) (2013) 833-45. [9] B. Kamlage, S.G. Maldonado, B. Bethan, E. Peter, O. Schmitz, V. Liebenberg, P. Schatz, Quality markers addressing preanalytical variations of blood and plasma processing identified by broad and targeted metabolite profiling, Clin Chem 60(2) (2014) 399-412. [10] P. Yin, R. Lehmann, G. Xu, Effects of pre-analytical processes on blood samples used in metabolomics studies, Anal Bioanal Chem 407(17) (2015) 4879-92. [11] B. Kamlage, S. Neuber, B. Bethan, S. Gonzalez Maldonado, A. Wagner-Golbs, E. 24

Peter, O. Schmitz, P. Schatz, Impact of Prolonged Blood Incubation and Extended Serum Storage at Room Temperature on the Human Serum Metabolome, Metabolites 8(1) (2018). [12] X. Wang, X. Gu, H. Song, Q. Song, X. Gao, Y. Lu, H. Chen, Phenylmethanesulfonyl fluoride pretreatment stabilizes plasma lipidome in lipidomic and metabolomic analysis, Anal Chim Acta 893 (2015) 77-83. [13] R. Brunkhorst, W. Pfeilschifter, S. Patyna, S. Buttner, T. Eckes, S. Trautmann, D. Thomas, J. Pfeilschifter, A. Koch, Preanalytical Biases in the Measurement of Human Blood Sphingolipids, Int J Mol Sci 19(5) (2018). [14] J.M. Onorato, P. Shipkova, A. Minnich, A.F. Aubry, J. Easter, A. Tymiak, Challenges in accurate quantitation of lysophosphatidic acids in human biofluids, J Lipid Res 55(8) (2014) 1784-96. [15] K. Nakamura, T. Kishimoto, R. Ohkawa, S. Okubo, M. Tozuka, H. Yokota, H. Ikeda, N. Ohshima, K. Mizuno, Y. Yatomi, Suppression of lysophosphatidic acid and lysophosphatidylcholine formation in the plasma in vitro: proposal of a plasma sample preparation method for laboratory testing of these lipids, Anal Biochem 367(1) (2007) 20-7. [16] Y. Yatomi, M. Kurano, H. Ikeda, K. Igarashi, K. Kano, J. Aoki, Lysophospholipids in laboratory medicine, Proc Jpn Acad Ser B Phys Biol Sci 94(10) (2018) 373-389. [17] A. Pastor, M. Farre, M. Fito, F. Fernandez-Aranda, R. de la Torre, Analysis of ECs and related compounds in plasma: artifactual isomerization and ex vivo enzymatic generation of 2-MGs, J Lipid Res 55(5) (2014) 966-77. [18] D. Luque-Córdoba, M. Calderón-Santiago, M.D. Luque de Castro, F. PriegoCapote, Study of sample preparation for determination of endocannabinoids and analogous compounds in human serum by LC–MS/MS in MRM mode, Talanta 185 (2018) 602-610. [19] F. Fanelli, V.D. Di Lallo, I. Belluomo, R. De Iasio, M. Baccini, E. Casadio, D.I. Gasparini, M. Colavita, A. Gambineri, G. Grossi, V. Vicennati, R. Pasquali, U. Pagotto, Estimation of reference intervals of five endocannabinoids and endocannabinoid related compounds in human plasma by two dimensional-LC/MS/MS, J Lipid Res 53(3) (2012) 481-93. [20] R. Gurke, D. Thomas, Y. Schreiber, S.M.G. Schäfer, S.C. Fleck, G. Geisslinger, N. Ferreirós, Determination of endocannabinoids and endocannabinoid-like substances in human K3EDTA plasma – LC-MS/MS method validation and pre-analytical characteristics, Talanta (2019). [21] T. Barri, L.O. Dragsted, UPLC-ESI-QTOF/MS and multivariate data analysis for blood plasma and serum metabolomics: effect of experimental artefacts and anticoagulant, Anal Chim Acta 768 (2013) 118-28. [22] R. Gambino, J. Piscitelli, T.A. Ackattupathil, J.L. Theriault, R.D. Andrin, M.L. Sanfilippo, M. Etienne, Acidification of Blood Is Superior to Sodium Fluoride Alone as an Inhibitor of Glycolysis, Clinical Chemistry 55(5) (2009) 1019-1021. [23] J. Qin, G. Chai, J.M. Brewer, L.L. Lovelace, L. Lebioda, Fluoride inhibition of enolase: crystal structure and thermodynamics, Biochemistry 45(3) (2006) 793-800. [24] D.T. Waugh, Fluoride Exposure Induces Inhibition of Sodium-and PotassiumActivated Adenosine Triphosphatase (Na(+), K(+)-ATPase) Enzyme Activity: Molecular Mechanisms and Implications for Public Health, Int J Environ Res Public Health 16(8) 25

(2019). [25] R.W. Dharmaratne, Exploring the role of excess fluoride in chronic kidney disease: A review, Hum Exp Toxicol (2018) 960327118814161. [26] N. Brunkhorst-Kanaan, K. Klatt-Schreiner, J. Hackel, K. Schroter, S. Trautmann, L. Hahnefeld, S. Wicker, A. Reif, D. Thomas, G. Geisslinger, S. Kittel-Schneider, I. Tegeder, Targeted lipidomics reveal derangement of ceramides in major depression and bipolar disorder, Metabolism 95 (2019) 65-76. [27] M.-S. Wegner, L. Gruber, N. Schömel, S. Trautmann, S. Brachtendorf, D. Fuhrmann, Y. Schreiber, C. Olesch, B. Brüne, G. Geisslinger, S. Grösch, GPER1 influences cellular homeostasis and cytostatic drug resistance via influencing long chain ceramide synthesis in breast cancer cells, The International Journal of Biochemistry & Cell Biology 112 (2019) 95-106. [28] V. Matyash, G. Liebisch, T.V. Kurzchalia, A. Shevchenko, D. Schwudke, Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics, J Lipid Res 49(5) (2008) 1137-46. [29] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analytical Biochemistry 72(1-2) (1976) 248-254. [30] R Development Core Team, R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna, Austria, 2008. [31] RStudio Team, RStudio: Integrated Development for R., RStudio, Inc., Boston, 2016. [32] F.H. Bretz, Torsten; Westfall, Peter, Multiple Comparisons Using R, Boca Raton: CRC Press2010. [33] B.D. Pinheiro J, DebRoy S, Sarkar D, R Core Team, nlme: Linear and Nonlinear Mixed Effects Models, R package version 3.1-140, 2019. [34] A.A. Zoerner, S. Batkai, M.T. Suchy, F.M. Gutzki, S. Engeli, J. Jordan, D. Tsikas, Simultaneous UPLC-MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction, J Chromatogr B Analyt Technol Biomed Life Sci 883-884 (2012) 16171. [35] A.T. Maccarone, J. Duldig, T.W. Mitchell, S.J. Blanksby, E. Duchoslav, J.L. Campbell, Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry, J Lipid Res 55(8) (2014) 1668-77. [36] C.A. Rouzer, K. Ghebreselasie, L.J. Marnett, Chemical stability of 2arachidonylglycerol under biological conditions, Chemistry and Physics of Lipids 119(1) (2002) 69-82. [37] K.D. Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34(3) (2004) 300-311. [38] L.A. Ferreira, V.N. Uversky, B.Y. Zaslavsky, Effects of the Hofmeister series of sodium salts on the solvent properties of water, Physical Chemistry Chemical Physics 19(7) (2017) 5254-5261. [39] W. Roehrig, S. Achenbach, B. Deutsch, M. Pischetsrieder, Quantification of 24 circulating endocannabinoids, endocannabinoid-related compounds and their phospholipid precursors in human plasma by UHPLC-MS/MS, Journal of Lipid 26

Research (2019). [40] M.K. Townsend, Y. Bao, E.M. Poole, K.A. Bertrand, P. Kraft, B.M. Wolpin, C.B. Clish, S.S. Tworoger, Impact of Pre-analytic Blood Sample Collection Factors on Metabolomics, Cancer Epidemiol Biomarkers Prev 25(5) (2016) 823-829.

27

Figure captions Graphical abstract: Experimental setup and results for the stability study of lipids in human plasma at room temperature and in ice water as well as the stability in whole blood at room temperature and in ice water. The experiment was conducted for each test subject (n = 10) with two different blood sampling tubes with either K3EDTA or sodium fluoride/citrate as anticoagulant. Time-dependent changes could be observed for endocannabinoids stored in ice water as well as for lysophosphatidic acids stored at room temperature.

Figure 1: Plasma concentrations of sphingosine-1-phosphate after incubation of human whole blood (A, B) and processed plasma (C, D) at room temperature (A, C) and in ice water (B, D). Storage times were 20 min, 1 h, 2 h, 4 h and 24 h. The results are shown as mean ± SEM (n = 10).

Figure 2: Plasma concentrations of lysophosphatidic acid 20:4 after incubation of human whole blood (A, B) and processed plasma (C, D) at room temperature (A, C) and in ice water (B, D). Storage times were 20 min, 1 h, 2 h, 4 h and 24 h. The results are shown as mean ± SEM (n = 10).

Figure 3: Plasma concentrations of 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (AEA) after incubation of human whole blood (A, B) and processed plasma (C, D) at room temperature (A, C) and in ice water (B, D). Storage times were 20 min, 1 h, 2 h, 4 h and 24 h (24 h data not shown). The results are shown as mean ± SEM (n = 10).

Figure 4: Relative lipid concentrations in human plasma after storage at room temperature. Storage times were 20 min, 1 h, 2 h, 4 h and 24 h. Peak area to internal standard ratios were compared to the respective reference values (t0) of the same subject (n = 10) and are reported as mean ± SEM (%). Both axis were truncated to enhance clarity of short-term stability. Results obtained from K3EDTA plasma (solid lines) were compared to the results from sodium fluoride/citrate plasma (dashed lines). DG: diglyceride, FA: fatty acid, LPC: lysophosphatidylcholine, LPE: lysophosphatidylethanolamine, suffix “O-“: plasmanyl species.

Figure 5: Heatmap of human K3EDTA and NaF/citrate whole blood incubated at room temperature. Shown are the fold changes compared to directly processed samples. The lipids were identified via non-targeted LC-QTOFMS screening. Shown are the 73 unequivocally identified lipids out of the 360 changed features in K3EDTA whole blood 28

with a FDR < 0.1 (paired t-test) between 24 h of incubation time and control samples (a total of 3198 aligned features was processed, n = 10). DG: diglyceride, FA: fatty acid, LPC: lysophosphatidylcholine, LPE: lysophosphatidylethanolamine, suffix “O-“: plasmanyl species, LPI: lysophosphatidylinositol, PC: phosphatidylcholine, PE: phosphatidylethanolamine, SM: sphingomyeline, TG: triglyceride.

29

Table 1. Differences in lipid levels in sodium fluoride/citrate and K3EDTA plasma from 10 healthy subjects. Results are reported as mean and standard deviation in percent of the quotients of NaF/citrate and K3EDTA obtained concentration values per subject and the summarized lipids. The relative quantification regards the peak area to internal standard ratio with one IS per lipid group (*). Sodium fluoride results were multiplied with 1.16 to correct for the dilution due to the liquid anticoagulant. Lipid/Lipid class

Abbreviation

Mean [%]

SD [%]

# analytes

1-Arachidonoyl glycerol

1-AG

44.6

12.22

1

2-Arachidonoyl glycerol

2-AG

94.9

45.17

1

Arachidonoyl ethanolamide

AEA

65

10.06

1

Oleoylethanolamide

OEA+VEA

80.3

6.88

1

Palmitoylethanolamide

PEA

83.6

7.37

1

Lysophosphatidic acid 16:0

LPA 16:0

86.9

11.84

1

Lysophosphatidic acid 18:1

LPA 18:1

99.2

19.57

1

Lysophosphatidic acid 18:2

LPA 18:2

91.5

15.58

1

Lysophosphatidic acid 20:4

LPA 20:4

96.2

20.09

1

Ceramides

Cer

59.4

20.97

9

Glucosylceramides

GlcCer

61.3

20.5

4

Lactosylceramides

LacCer

71.5

33.09

5

Sphingosine-1-phosphate Sterol 27:1_OH (cholesterol)*

S1P

90.4

16.63

1

ST 27:1_OH

70.3

10.51

1

Diglycerides*

DG

64.1

17.35

11

Fatty acids*

FA

97.8

12.73

14

Lysophosphatidylcholines*

LPC

84.1

7.65

20

Alkyl-lysophosphatidylcholines*

LPC O

84.2

5.79

4

Lysophosphatidylethanolamines*

LPE

81.8

11.11

7

Alkyl-lysophosphatidylethanolamines*

LPE O

76.4

9.63

2

Lysophosphatidylglycerols*

LPG

93.2

11.83

3

Lysophosphatidylinositols*

LPI

91

27.88

5

Phosphatidylcholines*

PC

72.7

9.58

26

Alkyl-phosphatidylcholines*

PC O

92.7

15

10

Phosphatidylethanolamines*

PE

65

10.48

14

Alkyl-phosphatidylethanolamines*

PE O

67.4

10.48

6

Phosphatidylinositols*

PI

81.7

8.92

10

Steryl ester*

SE

71.9

14.13

11

Sphingomyelins*

SM

65.7

12.93

27

Triglycerides*

TG

44.7

18.03

40

Table 2. Comparison of preanalytical protocols for the endocannabinoid quantification. The time between the blood sampling and the stabilization of the samples is crucial for valid results. The listed storage conditions showed the best stability for the respective protocol.

Parameter

Standard protocol [19, 34]

Pastor et al. [17]

Roehrig et al. [39]

Current protocol

Anticoagulant

K2EDTA/K3EDTA

K2EDTA

K2EDTA

NaF/citrate

Whole blood volume

1.6 – 10 mL

10 mL

n. s.

3.1 mL

-

Additional step

-

-

Transfer of 1mL blood + 7 µL 50% acetic acid and 0.5 M MES

Storage time & condition (whole blood)

↑ after 20 min in ice water

0 min

4 h on ice*

1 h in ice water

Plasma separation (at 4°C)

10 min at 2,000 or 4,655 g

15 min at 2,800 g

10 min at 2,000 g

10 min at 2,000 g

Additional step

-

600 µL plasma + 3.35 µM Orlistat

0.5M KSCN at pH4.7

-

Storage time & condition (plasma)

2 h in ice water

n. s.

60 min at RT

4 h in ice water

* Spiked 2-AG-d8 decreased after > 20 min; n. s. = not specified; MES =2-(N-morpholino) ethanesulfonic acid; KSCN = potassium thiocyanate.

150 140 130 120 110

relative concentration (%)

100 90 80 0

1

2

3

4

5

DG 32:1 EDTA

DG 34:1 EDTA

DG 34:2 EDTA

DG 32:1 NaF

DG 34:1 NaF

DG 34:2 NaF

0

1 FA 18:1 EDTA

2

FA 18:1 NaF

3 FA 20:4 EDTA

4 5 FA 20:5 EDTA

FA 20:4 NaF

FA 20:5 NaF

150 140 130 120 110 100 90 80 0

1

2

3

4

5

0

1

2

3

4

LPC 18:0 EDTA

LPC 20:4 EDTA

LPE 18:2 EDTA

LPE 20:4 EDTA

LPC O-16:1 EDTA

LPC 18:0 NaF

LPE O-18:1 EDTA

LPE 18:2 NaF

LPC 20:4 NaF

LPC O-16:1 NaF

LPE 20:4 NaF

LPE O-18:1 NaF

storage time (h)

5

K3EDTA

NaF/citrate

Highlights - Stability study in human whole blood and plasma using different anticoagulants - LC-MS analysis of endocannabinoids, LPA, sphingolipids, and non-targeted screening - LPA increased in K3EDTA blood and plasma after 20 min at RT, but not on wet ice - Endocannabinoids increased in K3EDTA whole blood stored on ice after 20 min - Combination of fluoride and citrate improves lipid stability

Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: