Harmonized procedures lead to comparable quantification of total oxylipins across laboratories

Oxylipins are potent lipid mediators involved in a variety of physiological processes. Their profiling has the potential to provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. However, results generated by independent groups are rarely comparable, which increases the need for the implementation of internationally agreed upon protocols. We performed an interlaboratory comparison for the MS-based quantitative analysis of total oxylipins. Five independent labs assessed the technical variability and comparability of 133 oxylipins using a harmonized and standardized protocol, common biological materials (i.e. 7 quality control plasmas), standard calibration series and analytical methods. The quantitative analysis is based on a standard calibration series with isotopically labelled internal standards. Using the standardized protocol the technical variance was within ±15% for 73% of oxylipins, however, most epoxy fatty acids were identified as critical analytes due to high variabilities in concentrations. The comparability of concentrations determined by the labs was examined using consensus value estimates and unsupervised /supervised multivariate analysis (i.e. PCA and PLS-DA). Inter-lab variability was limited and did not interfere with our ability to distinguish the different plasmas. Moreover, all laboratories were able to identify similar differences between plasmas. In summary, we show that using a standardized protocol for sample preparation, low technical variability can be achieved. Harmonization of all oxylipin extraction and analysis steps led to reliable, reproducible and comparable oxylipin concentrations in independent laboratories allowing the generation of biologically meaningful oxylipin patterns.


INTRODUCTION
Eicosanoids and other oxylipins are potent lipid mediators produced via the oxygenation of polyunsaturated fatty acids (PUFA). PUFA can be oxygenated enzymatically by cyclooxygenases to form prostanoids, by lipoxygenases to form hydroperoxy fatty acids which react further to mono-and poly-hydroxylated fatty acids or by cytochrome P450 monooxygenases giving rise to epoxy and hydroxy fatty acids, or nonenzymatically by free radicals during autoxidation (1,2). A major portion of circulating oxylipins (>90%) is found esterified in lipids, e.g. phospholipids, triacylglycerides or cholesterol esters (3)(4)(5).
Oxylipins include hundreds of structurally different molecules which are involved in a variety of physiological processes such as the regulation of blood coagulation (6), endothelial permeability (4), blood pressure and vascular tone as well as the control of kidney function (7) and the immune system (4,8). The function of fat tissue is also regulated by these lipid mediators and it has been shown that oxylipins intervene in the energy homeostasis regulation of insulin and its signaling pathways (6,9). Thus, the oxylipin pattern can provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. Several clinical studies have already demonstrated the utility of oxylipin profiling in the identification of potential disease biomarkers, the characterization of inflammatory and oxidative status or in the monitoring of the effects of diet or drugs (1).
Currently, the analysis of oxylipins is mainly carried out by liquid chromatography coupled with mass spectrometry using reversed phase columns filled with sub-2 μm particles, electrospray ionization and triple quadrupole detectors. This provides an excellent chromatographic separation of the isomeric analytes as well as a fast detection by MS following fragmentation. Furthermore, this allows the quantification of low oxylipin concentrations with highest sensitivity over a large dynamic range (1,(10)(11)(12)(13). Prior to MS analysis, several steps of sample preparation are usually carried out. The samples are often pre-treated with organic solvents to precipitate proteins (12,14,15) or extract lipids (5,16). Moreover, when analyzing total oxylipins, quantified as the sum of free and bound oxylipins, alkaline hydrolysis is performed (5,17,18).
Then, matrix compounds are removed and oxylipins are concentrated via solid phase extraction (SPE) (19-by guest, on November 6, 2020 www.jlr.org Downloaded from 5 21). All steps of the sample preparation procedure have to be optimized to both achieve good oxylipin recoveries and remove the matrix efficiently and thus minimize ion suppression (1,21). The analysis of oxylipins is usually quantitative which requires external calibration with internal standards. However, there are only a small number of companies that sell oxylipin standards and the quality is not always guaranteed (22). Only a few standards are available with verified concentrations (1,22). Other commercially available oxylipin standards show varying purities often resulting in different actual concentration than the stated nominal concentration (22) thus leading to inconsistent results across different studies (23).
There are currently no harmonized protocols for the analysis of oxylipins although it is well established that each analytical choice (i.e. type of biofluid, type of anticoagulant, free or esterified oxylipins, type of sample preparation protocol, type of instrument) can have a major influence on the detection and quantification of oxylipins (1). Therefore after optimization of relevant steps of the oxylipin analysis, standardized and harmonized methods should be established to obtain reliable and comparable results. Vesper et al. mainly recommend for clinical laboratory tests 1) the establishment of reference methods and materials, 2) calibration using the reference system and 3) verification of the uniformity of method results (24).
Using a standardized and harmonized protocol for oxylipin quantification is a mandatory prerequisite to obtain meaningful and reproducible results as oxylipin concentrations obtained from different laboratories are rarely comparable due to varying analytical strategies, the lack of certified analytical calibrators and reference materials (1,10,23). Therefore, the harmonization of oxylipin analysis can enhance the use of oxylipin profiling in clinics. Another crucial step is to assess the technical variability and interlaboratory comparability of each oxylipin quantified. This will allow the identification of potential technically critical oxylipins, to appropriately power clinical studies and to guarantee the relevance of oxylipin profiling involving different laboratories. So far there are only a few studies that have investigated the comparability of targeted metabolomics across laboratories (25)(26)(27)(28), and only one study that included oxylipins (26). In the present study, we used a standardized protocol for the quantitative analysis of total oxylipins (18) due to their higher relevance in a context of biomarker discovery (1). Five independent laboratories were by guest, on November 6, 2020 www.jlr.org Downloaded from 6 involved to assess the technical (intra-laboratory) variability and comparability of 133 oxylipins following a standardized and harmonized protocol for sample preparation and MS analysis and using the same biological material (i.e. 7 quality control (QC) plasmas) and standard calibration series.

Chemicals
Acetonitrile, methanol, iso-propanol (LC-MS grade) and acetic acid (Optima LC-MS grade) were purchased from Fisher Scientific. Ethyl acetate (HPLC grade) was bought from VWR and n-hexane (HPLC grade) was purchased from Carl Roth. The ultra-pure water with a conductivity of >18 MΩ*cm was generated by the Barnstead Genpure Pro system from Thermo Fisher Scientific. The oxylipin standards 10-HODE, 12-HODE, 15-HODE, 9,10,9,12,9,10,9,10,9,12, Several aliquots (100-500 μL) of each QC plasma were sent to the other laboratories to ensure that the assessment of the technical variability is independent of the biological material.

Sample preparation and LC-ESI(-)-MS/MS analysis
All laboratories used the same standardized protocol for sample preparation. Human plasma samples were extracted using solid phase extraction (SPE) following protein precipitation and alkaline hydrolysis as described previously (the detailed standard operation procedure which was provided to all labs can be found in the Supplemental Information, SI Table S1-4) (18). In brief, to 100 μL human plasma 10 μL antioxidant mixture (0.2 mg/mL BHT, 100 μM indomethacin, 100 μM trans-4-(-4-(3-adamantan-1-yl-ureido9cyclohexyloxy)-benzoic acid (t-AUCB) in MeOH) and 10 μL internal standard solution (100 nM of each 9 8(9)-EpETrE in MeOH) were added. Following protein precipitation with iso-propanol and alkaline hydrolysis at 60 °C for 30 min using 0.6 M potassium hydroxide (MeOH/water, 75/25, v/v) samples were extracted using Bond Elut Certify II SPE cartridges (200 mg, 3 mL, Agilent, Waldbronn, Germany) as described (12,13,18). Oxylipins were eluted into glass tubes containing 10 μl of 30% glycerol in MeOH using ethyl acetate/n-hexane/acetic acid (75/25/1, v/v/v). Samples were evaporated and the residue was reconstituted in 50 μL MeOH. The analysis of the samples was performed using a sensitive LC-ESI(-)-MS/MS method with optimized mass spectrometric and chromatographic parameters as described (12,13) which was provided to all participating laboratories. The quantitative analysis was based in all laboratories on the same standard calibration series comprising 133 oxylipins with isotope-labelled internal standards (30) allowing the same analyte/internal standard assignment in the laboratories.

Study design
A comprehensive standard operation procedure (SOP, SI Table S1 Figure S1). Furthermore, all participants were provided a data submission template including information on the analysis and calculation of the concentrations. The oxylipin concentrations in the plasma pools were reported in nM (nanomoles per liter).
For each triplicate determination mean and SD was calculated. If in a triplicate determination the concentration of an analyte in one sample was below LLOQ, the LLOQ threshold was filled in for this by guest, on November 6, 2020 www.jlr.org Downloaded from sample and the mean and SD were calculated. This approach was chosen as the omission of analyte concentrations below LLOQ leads to bias of the results (31). If concentrations in two or all samples were below LLOQ, the concentration of the analyte was set to LLOQ. Moreover, all participants were asked to fill out a short questionnaire with general remarks on the analysis.

Statistical analysis
Several statistical methodologies were used to assess the intra-and interlaboratory analytical variabilities.
Coefficients of variations (CV) were calculated to determine the intra-and inter-day variability for each laboratory and each QC plasma. Multivariate methods were applied to assess the interlaboratory variability. To assess the ability of laboratories to identify similar differences in the oxylipin profiles between two different QC plasma and therefore to provide similar biological interpretation, ratios between QC plasma 2,

Analytical variance of oxylipin analysis
All participating laboratories were able to simultaneously quantify 133 oxylipins in the standard calibration series using the provided LC-MS/MS method. Deviations from the SOP and analytical instruments used can be found in the SI (SI Table S5). Oxylipins were analyzed in seven different QC plasmas in triplicate (i.e. 3 different samples were prepared) on two consecutive days to determine the analytical variance of the method. In all laboratories, an average of 84 oxylipins (63%) were above the limit of quantification (LLOQ, Table 1).
Regarding the oxylipin pattern of QC plasma 1 to 6, the determined oxylipin concentrations were in a similar range (0.16 nM to 547 nM) while in QC plasma 7, mainly for hydroxy-PUFA and multi-hydroxylated oxylipins, as well as for linoleic acid derived epoxy-PUFA and oxo-PUFA, 10 to 25-fold higher concentrations were found.
The results of the intra-day and inter-day variability of the oxylipin analysis in the reference laboratory (laboratory 1) is shown in Figure 1. For this laboratory, the variabilities were assessed for 81 quantified oxylipins. Overall, low intra-day variability (CV < 15%) was observed for 85% of oxylipins. The inter-day variability was also low (CV < 15% for 73% of oxylipins). However, for epoxy-PUFA as well as oxo-and trihydroxy-PUFA a variance up to > 25% was observed. This higher variability did not correlate with concentration. There was no difference between the different types of plasma.
Increased intra-day and inter-day variability for epoxy-PUFA could also be observed in the other participating laboratories (SI Figure S2-5). For laboratories 2, 3, 4 and 5, the variabilities were assessed for 69, 73, 94 and 82 quantified oxylipins, respectively. These laboratories presented overall higher variabilities, except for the laboratory 2, with low intra-day variability (CV < 15%) for 81% of oxylipins for the day 2 and low inter-day variability (CV < 15%) for 72% of oxylipins (SI Figure S2) which is consistent to laboratory 1. Of note, for laboratories 2 to 5, the intra-day variability for day 1 was higher than for day 2 which impacted the inter-day variability (SI Figure S2-5). For example, laboratory 3 (SI Figure S3) showed by guest, on November 6, 2020 www.jlr.org

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13 the highest inter-day variability (only 39% of oxylipins with a CV < 15%), which is due to the very high intra-day variability for day 1 (CV < 15% for 36% of oxylipins) compared to day 2 (CV < 15% for 61% of oxylipins). For laboratories 4 and 5, the inter-day variability was also high with a CV < 15% only for 52% and 44% of the oxylipins, respectively.

Laboratory comparison
The quantified oxylipin concentrations in all QC plasmas by all laboratories can be found in the SI (SI Table S6).
Unsupervised multivariate analysis (i.e. PCA) was first performed to assess the variability of the overall oxylipin profiles obtained in each laboratory and its major determinant (i.e. type of QC plasma, laboratory).
The initial PCA shows that the main variability (40.5% on the 1 st component) is related to the type of QC plasma with QC plasma 7 -B2 being different from the others. The variability on the 2 nd component is much lower (i.e.18.6%), and mainly driven by laboratory 4 (SI Figure S6A). The loading plot shows that the discrimination of laboratory 4 is based mainly on epoxy-PUFA (SI Figure S6B). Epoxy-PUFA have already been noted for their high technical variability. Moreover, in laboratory 4 the samples could either not be measured directly, or they had to be injected several times due to technical issues (as described in SI Table S5) the latter of which results in increased concentrations of epoxy-PUFA (SI Figure S7). Therefore, The consensus values were evaluated using the median of means (MEDM) approach to assess the interlaboratory comparability. The consensus values were deemed acceptable when the coefficient of dispersion (COD = 100*u/MEDM) was less than 40% as described in Bowden et al (26). Means ± SD for each laboratory and MEDM consensus values ± u were plotted for oxylipins with an acceptable consensus value. The MEDM ± u and COD for oxylipins quantified in all QC plasmas can be found in the SI (SI Table   S7-8, SI Figure S9-14).
In total 78 oxylipins were reported for QC plasma 1 in all laboratories, with an acceptable consensus value for 17 oxylipins (22%) (Figure 4). Of note, the same analysis was performed without laboratory 4 (due to the issues encountered during sample analysis) and shows that an acceptable consensus value is obtained by guest, on November 6, 2020 www.jlr.org

Identification of differences between plasma pools
To assess the ability of the labs to identify similar differences (in magnitude and direction) between two plasmas, we compared the ratios calculated between different QC plasmas by each lab. For this purpose, the concentration of each oxylipin for a given QC plasma was divided by the oxylipin concentration obtained for another QC plasma.
First, we compared the differences observed between two very contrasted QC plasmas, i.e. QC plasma 7 (commercial plasma rich in hydroxy-PUFA and linoleic acid metabolites) vs QC plasma 1 (plasma prepared from fresh EDTA blood of healthy donors immediately stored at -80 °C). For 98% of the oxylipins (matrix consisting of 60 oxylipins) the ratio between QC plasma 7 vs QC plasma 1 was similar for the five laboratories. The only noticeable difference occurred between laboratory 2 vs laboratory 3 for 13-oxo-ODE (ratio 5-15 and < 0.5, respectively) whereas the laboratories 1, 4 and 5 obtained very similar ratios (ratio 1.33-5; Figure 5A).
A second laboratory comparison was made with the ratios calculated between the QC plasmas obtained in obese individuals with or without hypertriglyceridemia (plasma 5 vs plasma 6). For 92% of the oxylipins (matrix consisting of 61 oxylipins) the ratios were very similar between the 5 laboratories. However, ratios in opposite directions were obtained for 9,10,11-TriHOME, 11,12-DiHETE, 9-HODE, 13-HODE and 15-oxoETE ( Figure 5B).

Interlaboratory variability in the LC-MS/MS specific results
Knowing that the different laboratories use different mass spectrometers, the contribution of this factor on the overall variability of the oxylipin profiles was assessed (i.e. 42 oxylipins which were detected at a concentration above LLOQ in all laboratories). The oxylipin profiles were generated from samples prepared  Table S9, Figure S18). Of note, the intra-MS variabilities (i.e. variability within each independent triplicate) were generally higher for the samples prepared in Lab 5 which impairs the detection of valid differences between the two sets of triplicate analyses.

Technical variability of oxylipins
In each participating laboratory, the precision and reproducibility of the analytical method was determined using seven QC plasmas. In the reference laboratory (laboratory 1), the lowest inter-day variance (inter-day) was observed (< 15% for 73% of oxylipins, Figure 1). The SOP and LC-MS method have been developed here and thus this laboratory is best trained in the procedures. The achieved analytical variances meet the criteria of international guidelines, i.e. analytes above LLOQ should have a precision of < 15% (32, 33).
Low inter-day variances for 72% of oxylipins (< 15%, SI Figure S2) were also observed in laboratory 2, whose personnel was trained in the reference laboratory. In laboratories where the analysis was carried out only based on the SOP slightly higher variances (> 20%, SI Figure S3 between-run (reinjection of same sample on ten consecutive days) variability of oxylipins, whereby the variability was in a range of 1-24% in each case (37). comparing clinical studies, it becomes apparent that the quality of the analytical standards used for quantification is a critical parameter, which may lead to non-comparable results (23). In order to solve this issue Hartung et al. recently described a strategy to verify the concentration of commercially available standards (22). Because this strategy was not used in previous studies, interlaboratory comparisons can today only be evaluated under consideration of relative results. A common evaluation strategy in lipidomic studies is the normalization to a standard material, such as the NIST SRM plasma. This normalization significantly improves variance and reproducibility (25,27). In our study we show that by normalization (the QC plasmas were normalized to QC plasma 1) and subsequent evaluating of relative results no difference between laboratories are identified ( Figure 5, SI Figure S15-17).
The major field of application of targeted oxylipin metabolomics are biological studies aiming to provide a relative comparison of results such as case and control groups (40)(41)(42). When comparing the two QC plasmas of obese subjects with and without hypertriglyceridemia in this study, for most oxylipins comparable ratios were identified, while for only 8% of the oxylipins a trend in the opposite direction was observed ( Figure 5B). Thus, our data clearly demonstrate that LC-MS oxylipin quantification is suitable to characterize differences in biological samples e.g. in clinical studies.
The comparability of interlaboratory results after standardization to a reference material leads to the fact that a harmonization of methods to a standard material is increasingly desired. Many describe the importance of standard reference materials such as the NIST SRM plasma to ensure interlaboratory comparability and to harmonize data sets (10,24,25,27,43). However, the introduction of a common standard material does not solve the problem that absolute concentrations are not comparable. This is most clearly shown by the Using standardized and harmonized protocols for oxylipin quantification can greatly reduce variance and promote the generation of meaningful and reproducible results when data is to be compared across multiple laboratories. In the absence of such harmonization efforts, robust performance based quality assurance protocols must be implemented to allow the harmonization of data sets processed using discrete protocols and will be critical to enhance the use of oxylipin profiling in clinics. Our study is the first where Sample preparation was carried out according to the same optimized protocol in all laboratories, also taking into account the use of materials from the same manufacturers, as different sample preparation procedures can influence the amount of quantified oxylipins (1,23). Moreover, the used protocol for sample preparation was optimized to yield high reproducibility following the evaluation of various saponification techniques (1,18). Furthermore, the integration of peaks was manually validated to ensure optimum precision as automatic peak integration may lead to incorrect peak integration. Especially the integration of peaks close to LLOQ or with unusual peak shapes using automatic integration may lead to deviating results.
With our study we show that the harmonization of parameters that can cause technical variability leads to comparable absolute oxylipin concentrations obtained in different laboratories. If the quantified oxylipin concentrations for all plasma pools in all laboratories are presented in a PCA model, for 3 of 5 laboratories no differences between the results can be observed ( Figure 2). The analysis instead reveals differences between the plasma pools indicating that biological differences could be detected in batch samples analyzed in different laboratories. Notably laboratory 4, showing the highest variability and most extreme deviation from the other results, experienced a MS/MS turbo pump failure mid analysis leading to a prolonged delay between sample processing and data acquisition, representing a serious deviation from the described protocols. To better reveal a difference between laboratories, the analysis tool can be forced to display such differences as shown by the PLS-DA model (Figure 3). If laboratory 4 results are excluded the consensus values estimates were acceptable for 73% of the oxylipins in QC plasma 1. In general, excluding laboratory 4 increased the number of acceptable consensus values (SI Table S7 -8). It is particularly enlightening that the long post extraction acquisition delays and repeated injections led to higher concentrations of epoxy-PUFA in these samples (SI Figure S7-8). Furthermore, excluding the epoxy-PUFA from the analysis, higher levels and variance in hydroxy-PUFA were also observed in laboratory 4 data ( Figure 3). Therefore, extreme caution is generally suggested in the timing of post preparation delays in data acquisition.
Oxylipins have the potential to provide a wealth of information regarding human health and disease and are a promising technology for translation into clinical applications. The changes in the oxylipin profile are of particular importance, as physiological effects are not attributed to a single oxylipin but to an interplay of many oxylipins or a general shift of the oxylipin profile (1,45). In addition, the oxylipins in biological samples come in a concentration range of several orders of magnitude (> 4) with differences in polarity and stability. Therefore, the targeted metabolomic analysis of these potent lipid mediators requires sensitive and precise methods to detect as many different oxylipins as possible and to detect even the smallest concentration differences (1,(46)(47)(48).
The presented study is unique in evaluating comparability and reproducibility of oxylipin analysis. We are able to show for the first time that the standardization and harmonization of the processing protocol as well as the analysis not only allows an interlaboratory comparison in terms of relative results, but also the absolute concentrations obtained are comparable. In the lipidomics community there is an increasing demand for standardized methods (1, 10). Our study could be used as first step for the development of an internationally agreed upon oxylipin quantification procedures and benchmarks. Moreover, the standardization of routine performances will allow direct comparisons of data sets generated at various laboratories.
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CONCLUSION
Our study is the first to investigate the technical variability and interlaboratory comparability, of the targeted metabolomics analysis of total oxylipins. Epoxy-and oxo-PUFA appear particularly sensitive to analytical sample handling, and delayed post-processing analyses are to be avoided. In addition, when analyzing total oxylipins special care should be taken during the drying steps when using non-end capped silica materials.
However, our findings show that reproducible results with low variability can be obtained using standardized protocols for sample preparation and analysis, and that specific training of personnel in these complex protocols reduces variability. This will be crucial to appropriately power experimental designs and to enhance the identification of reliable and relevant biomarkers of disease.
Overall, we could show that with appropriate standardization a direct comparison of absolute concentrations obtained in different laboratories is possible which opens a new door for the quantitative analysis of oxylipins and into clinical applications.

Limitations of this study
The small number of participating laboratories in this exercise is a significant limitation. However, the five participating laboratories represent three countries and laboratories in academic, governmental, and industrial environments arguing that the findings have broad implications. The lack of a commercially available certified reference material for total oxylipins also limits future direct comparisons to the data The range of ratio goes from <0.5 to more than 15. B) Heatmap of ratio between QC plasma 5 -Ob-H and QC plasma 6 -Ob+H. The range of ratio goes from < 0.5 to more than 2.
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