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, 9 (1), 9822

Monitoring of System Conditioning After Blank Injections in Untargeted UPLC-MS Metabolomic Analysis

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Monitoring of System Conditioning After Blank Injections in Untargeted UPLC-MS Metabolomic Analysis

Teresa Martínez-Sena et al. Sci Rep.

Abstract

Ultra-performance liquid chromatography - mass spectrometry (UPLC-MS) is widely used for untargeted metabolomics in biomedical research. To optimize the quality and precision of UPLC-MS metabolomic analysis, evaluation of blank samples for the elimination of background features is required. Although blanks are usually run either at the beginning or at the end of a sequence of samples, a systematic analysis of their effect of the instrument performance has not been properly documented. Using the analysis of two common bio-fluids (plasma and urine), we describe how the injection of blank samples within a sequence of samples may affect both the chromatographic and MS detection performance depending on several factors, including the sample matrix and the physicochemical properties of the metabolites of interest. The analysis of blanks and post-blank conditioning samples using t-tests, PCA and guided-PCA provides useful information for the elimination of background UPLC-MS features, the identification of column carry over and the selection of the number of samples required to achieve a stable performance.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Scheme of the experimental design employed, in which either three consecutive (A) or one single blank sample (B) are analyzed after every 8 sample replicates for an intense or mild system deconditioning, respectively.
Figure 2
Figure 2
(A) Analysis of the distribution of informative, and uninformative (background contaminant and carryover) features in the plasma data set. (B) Representative intensity profiles in blanks and post-blank samples of UPLC-MS features labelled as informative, column carry over and contaminants; (C) PC1 vs PC2 scores (top) and loadings (bottom) from the PCA of UPLC-MS profiles obtained from the analysis of blanks, using features labelled as carryover or contaminant.
Figure 3
Figure 3
Analysis of the effect of blank injections during the analysis of a batch of plasma samples. (A) PCA scores (PC1 and PC2) as a function of the injection order. Red circles indicate the position of the blanks; (B) Evolution of the δ value and the number of UPLC-MS features showing different mean peak area, RT and peak width using as reference the mean values calculated after the injection of 8 samples for reconditioning. Note: *indicates permutation test p-value < 0.05; (C) Distribution (top) and mean intensity profiles (bottom) of the features included in clusters 1 and 2 (see text for details); (D) Peak area values, RT and extracted ion chromatograms for tryptophan (included in HCA cluster #1) and LysoPC(18:0) (included in HCA cluster #2) in plasma replicates. Red dots: blanks. Colored bold lines in the chromatograms indicate the limits of the XCMS integration window.
Figure 4
Figure 4
(A) Analysis of the distribution of informative, and uninformative (background contaminant and carryover) features in the urine data set. (B) Representative intensity profiles in blanks and post-blank samples of UPLC-MS features labelled as informative or contaminants; (C) PC1 vs PC2 scores (top) and loadings (bottom) from the PCA of UPLC-MS profiles obtained from the analysis of blanks, using features labelled as carryover or contaminant.
Figure 5
Figure 5
Analysis of the effect of blank injections during the analysis of a batch of urine samples. (A) PCA scores (PC1 and PC2) as a function of the injection order. Red circles indicate the position of the blanks; (B) Evolution of the δ value and the number of LC-MS features showing different mean peak area using as reference the mean values calculated after the injection of 8 samples for reconditioning. Note: *indicates permutation test p-value < 0.05; (C) Peak area values, RT and extracted ion chromatograms for tryptophan in urine replicates. Red dots: blanks. Colored bold lines in the chromatograms indicate the limits of the XCMS integration window; (D) Distribution (top) and mean intensity profiles (bottom) of the features included in clusters 1 and 2 (see text for details).

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