Concordant inter-laboratory derived concentrations of ceramides in human plasma reference materials via authentic standards

Abstract

In this community effort, we compare measurements between 34 laboratories from 19 countries, utilizing mixtures of labelled authentic synthetic standards, to quantify by mass spectrometry four clinically used ceramide species in the NIST (National Institute of Standards and Technology) human blood plasma Standard Reference Material (SRM) 1950, as well as a set of candidate plasma reference materials (RM 8231). Participants either utilized a provided validated method and/or their method of choice. Mean concentration values, and intra- and inter-laboratory coefficients of variation (CV) were calculated using single-point and multi-point calibrations, respectively. These results are the most precise (intra-laboratory CVs ≤ 4.2%) and concordant (inter-laboratory CVs < 14%) community-derived absolute concentration values reported to date for four clinically used ceramides in the commonly analyzed SRM 1950. We demonstrate that calibration using authentic labelled standards dramatically reduces data variability. Furthermore, we show how the use of shared RM can correct systematic quantitative biases and help in harmonizing lipidomics. Collectively, the results from the present study provide a significant knowledge base for translation of lipidomic technologies to future clinical applications that might require the determination of reference intervals (RIs) in various human populations or might need to estimate reference change values (RCV), when analytical variability is a key factor for recall during multiple testing of individuals.

Fig. 1. Consensus concentrations of ceramides in NIST SRM 1950 across all study participants.

Concentrations of the 4 reported ceramides in NIST SRM 1950. Each circle corresponds to the mean of 1 to 6 replicate measurements from a vial of NIST SRM 1950 shipped to each of the participating laboratories. The error bars depict the ±1 × SD of 3 injection replicates, which in some cases is smaller than the diameter of the plotted circle or absent if only 1 measurement was made. Horizontal bars represent the mean values of the mean measured concentration per NIST vial. The outer green dotted lines indicate used for as cut-off values for outlier removal. The mean ±2 × SD (dashed grey lines) are based on data after outlier removal. Molar concentrations were calculated using the external calibration curve (multi-point calibration). SOP (red) and OTHER (blue) refer to the Standard vs Other methods, respectively. Source data are provided as Source Data File.

Fig. 2. Comparison of single-point and multi-point calibrations.

Molar concentrations derived from single-point vs. multi-point calibration based quantifications. The diagonal line indicates equal results between single-point (horizontal axis) and multi-point (vertical axis) calibration-based quantification. Outlier measurements were removed for each ceramide species in each reference plasma (see Fig. 1 and text). Each circle corresponds to the mean value reported by one laboratory (n = 6). NIST SRM 1950 (SRM, dark blue); hypertriglyceridemic (hTAG, red), diabetic (DB, light orange), and Young African American (YAA, light blue), which are part of NIST RM 8231. Source data are provided as Source Data File.

Fig. 3. Concentration values of ceramides in NIST SRM 1950 and the suite of human plasma RM contained in RM 8231.

Concentrations of the 4 ceramides in different NIST reference plasma samples. Each circle corresponds to the values reported by one laboratory. P values are based on paired two-tailed t-tests of complete datasets. The horizontal bars correspond to the mean ±1 × SD of all shown data per group (number of data points indicated below each group). Outlier measurements were removed for ceramide species in each reference plasma (see Fig. 1 and text). SOP (red) and OTHER (blue) refer to the Standard vs Other methods, respectively. Source data are provided as Source Data File.

Fig. 4. Comparison of concentration values obtained in this and previous studies covering ceramides by MS-platforms in NIST SRM 1950.

Comparison of inter-laboratory and/or cross-platform trials reporting ceramide concentrations in NIST SRM 1950. Each open circle corresponds to the reported value from one laboratory in each of the respective studies referenced by first author and year of publication. (sp) refers to a single, standardized protocol and platform employed by all laboratories while (mp) denotes studies in which multiple protocols and platforms were used. The red boxes and numbers indicate median ±MAD (mean absolute deviation) and the number of participating laboratories, respectively. Depending on the precise nature of the study and/or reporting laboratory, concentrations were either reported with sphingoid-base and fatty acyl chain details (e.g. Cer 18:1;O2/16:0) or as sum compositions (i.e. Cer 34:1;O2). In the latter case, isobaric ceramide species other than the 4 ceramides targeted in this study may therefore have contributed to the reported concentrations. Details of the individual studies are presented in Supplementary Table 3. No outlier removal has been applied for any of the studies. Source data are provided as Source Data File.

Fig. 5. Authentic vs non authentic internal standard normalization and its effects on quantification for SRM 1950 RM.

Authentic vs non authentic internal standard normalization and its effects on bias and variability. The horizontal bars correspond to the mean and ±1 × SD of all shown data per group (number of data points indicated for each group). Data originating from the same laboratory are connected by dashed lines. SOP (red) and OTHER (blue) refer to the Standard vs other methods, respectively. Outliers in the authentic internal standard groups have been removed in all groups for each ceramide species (see Fig. 1 and text). Additionally, dataset 11 was excluded for Cer16:0 as it had 15x higher concentrations than the second highest data point for C24:0 d7 normalization.

Fig. 6. Re-calibration of RM results using SRM 1950 as a shared reference materials.

Concentration of the 4 reported ceramides in NIST RM 8231-1 (DB) plasma before and after re-calibration using the NIST SRM 1950 concentration. Each circle corresponds to one measurement from one NIST DB sample shipped to each of the participating laboratories before (None) and after (SRM 1950) re-calibration with the SRM1950 concentration of the corresponding laboratory (see text). The horizontal bars correspond to the mean ±1 × SD of all shown data per group (number of data points indicated for each group). This plot contains all datasets before and after re-calibration, no outlier removal has been performed.