Most cited article - PubMed ID 32078000
Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography-mass spectrometry and hydrophilic interaction liquid chromatography-mass spectrometry
Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 μm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.
- Keywords
- High-throughput lipidomics, Mass spectrometry, Plasma, Quantitation, Reversed-phase, Serum, Ultrahigh-performance liquid chromatography,
- MeSH
- Chromatography, Reverse-Phase * methods MeSH
- Mass Spectrometry methods MeSH
- Liquid Chromatography-Mass Spectrometry MeSH
- Humans MeSH
- Lipidomics * methods MeSH
- Lipids * analysis MeSH
- High-Throughput Screening Assays methods MeSH
- Software MeSH
- Tandem Mass Spectrometry methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Lipids * MeSH
Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100 × 3 mm; 1.7 μm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4 min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8 min including column equilibration, which enables the analysis of 160 samples per day.
- Keywords
- High-throughput lipidomics, Mass spectrometry, Plasma, Quantitation, Serum, Ultrahigh-performance supercritical fluid chromatography, Validation,
- MeSH
- Mass Spectrometry methods MeSH
- Humans MeSH
- Lipidomics * methods MeSH
- Lipids * analysis blood MeSH
- Chromatography, Supercritical Fluid * methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Lipids * MeSH
A Gram-stain-negative, aerobic and rod-shaped bacteria, designated ZY210820T, was isolated from the nasal cavity of a goat with respiratory disease in Yunnan Province, PR China. The strain grew at pH 6.0-9.0 (optimum pH 7.0), at 24-39 °C (optimum 37 °C) and with 0.5-2.0% (w/v) NaCl (optimum 1.0% NaCl). Phylogenetic analysis of 16S rRNA gene sequences showed that the strain formed a separated branch within the family Moraxellaceae with the highest similarity of 93.1% to Acinetobacter kanungonis JCM 34131T. Phylogenomic analysis of 327 single-copy protein sequences revealed that the strain belonged to the family Moraxellaceae and constituted a separated branch. The genomic G + C content was 35.5%. The highest orthologous average nucleotide identity (OrthoANI), digital DNA-DNA hybridization (dDDH), and average amino acid identity (AAI) values between the strain and the type strains in the family Moraxellaceae were 72.5% (Acinetobacter calcoaceticus DSM 30006T), 37.0% (Fluviicoccus keumensis JCM 19370T), and 64.0% (Acinetobacter kanungonis JCM 34131T), respectively. The predominant polar lipids were phosphatidylglycerol, phosphatidylethanolamine, cardiolipin, monolysocardiolipin, and diacylglycerol. The strain contained C12:0, C16:0, summed feature 3 (C16:1ω7c and/ or C16:1ω6c), C17:0, and C15:1ω6c as the major fatty acid (> 5%) and CoQ-9 as the major respiratory quinone. The results of the polyphasic analysis revealed that strain ZY210820T represents a novel species of a new genus in the family Moraxellaceae, and the name Nasibacterium caprae gen. nov., sp. nov. is proposed. The type strain is ZY210820T (= CCTCC AB 2021475T = NBRC 115474T).
- Keywords
- Nasibacterium caprae, Goat, Respiratory disease, Taxonomic position, Virulence gene,
- Publication type
- Journal Article MeSH
Chemical derivatization involves the reaction of an analyte with a derivatization agent to modify its structure, improving the peak shape, chromatographic performance, structural analysis, ionization efficiency, and sensitivity. A novel derivatization method using 3-(chlorosulfonyl)benzoic acid is developed for the determination of monoacylglycerols, diacylglycerols, free sterols, and tocopherols using the reversed-phase ultra-high-performance liquid chromatography-tandem mass spectrometry (RP-UHPLC/MS/MS) method in the negative ion mode. The chromatographic and mass spectrometric properties of derivatized lipids are investigated by using 29 lipid standards spanning four lipid classes. The derivatization process is optimized using pooled plasma spiked by 9 internal standards, achieving an optimal yield with a reaction time of 40 min at 60 °C. The stability of the derivatives is confirmed, with short-term stability maintained for 10 h at 4 °C and long-term stability preserved for 5 days at -80 °C. The repeatability and reproducibility are verified by one/two operator(s), which underscores the simplicity and robustness of the method, and calibration curves with high linear regression coefficients illustrate the accuracy of the method. The derivatization approach, which combines RP-UHPLC/MS/MS and the use of specific fragmentation patterns, significantly reduces limits of detection, reaching 15-25 pmol/mL for free sterols in plasma. The optimized method is applied to the analysis of human plasma, leading to the identification of 92 lipid species in the targeted lipid classes. This represents a substantial improvement in sensitivity and detection capabilities compared to those of previously reported methods.
Multidimensional chromatography offers enhanced chromatographic resolution and peak capacity, which are crucial for analyzing complex samples. This study presents a novel comprehensive online multidimensional chromatography method for the lipidomic analysis of biological samples, combining lipid class and lipid species separation approaches. The method combines optimized reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) in the first dimension, utilizing a 150 mm long C18 column, with ultrahigh-performance supercritical fluid chromatography (UHPSFC) in the second dimension, using a 10 mm long silica column, both with sub-2 μm particles. A key advantage of employing UHPSFC in the second dimension is its ability to perform ultrafast analysis using gradient elution with a sampling time of 0.55 min. This approach offers a significant increase in the peak capacity. Compared to our routinely used 1D methods, the peak capacity of the 4D system is 10 times higher than RP-UHPLC and 18 times higher than UHPSFC. The entire chromatographic system is coupled with a high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) in both full-scan and tandem mass spectrometry (MS/MS) and with positive- and negative-ion polarities, enabling the detailed characterization of the lipidome. The confident identification of lipid species is achieved through characteristic ions in both polarity modes, information from MS elevated energy (MSE) and fast data-dependent analysis scans, and mass accuracy below 5 ppm. This analytical method has been used to characterize the lipidomic profile of the total lipid extract from human plasma, which has led to the identification of 298 lipid species from 16 lipid subclasses.
- MeSH
- Humans MeSH
- Lipidomics * methods MeSH
- Lipids * analysis MeSH
- Chromatography, Supercritical Fluid methods MeSH
- Tandem Mass Spectrometry * methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Lipids * MeSH
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.
- MeSH
- Ceramides * blood MeSH
- Mass Spectrometry methods MeSH
- Calibration MeSH
- Laboratories * standards MeSH
- Humans MeSH
- Lipidomics methods MeSH
- Reference Standards * MeSH
- Reproducibility of Results MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Ceramides * MeSH
Pancreatic cancer has the worst prognosis among all cancers. Cancer screening of body fluids may improve the survival time prognosis of patients, who are often diagnosed too late at an incurable stage. Several studies report the dysregulation of lipid metabolism in tumor cells, suggesting that changes in the blood lipidome may accompany tumor growth. Here we show that the comprehensive mass spectrometric determination of a wide range of serum lipids reveals statistically significant differences between pancreatic cancer patients and healthy controls, as visualized by multivariate data analysis. Three phases of biomarker discovery research (discovery, qualification, and verification) are applied for 830 samples in total, which shows the dysregulation of some very long chain sphingomyelins, ceramides, and (lyso)phosphatidylcholines. The sensitivity and specificity to diagnose pancreatic cancer are over 90%, which outperforms CA 19-9, especially at an early stage, and is comparable to established diagnostic imaging methods. Furthermore, selected lipid species indicate a potential as prognostic biomarkers.
- MeSH
- CA-19-9 Antigen blood MeSH
- Ceramides blood MeSH
- Humans MeSH
- Lipidomics methods MeSH
- Lysophosphatidylcholines blood MeSH
- Lipid Metabolism genetics MeSH
- Multivariate Analysis MeSH
- Biomarkers, Tumor blood genetics MeSH
- Pancreatic Neoplasms blood diagnosis mortality pathology MeSH
- Proportional Hazards Models MeSH
- Sensitivity and Specificity MeSH
- Sphingomyelins blood MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Case-Control Studies MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- CA-19-9 Antigen MeSH
- Ceramides MeSH
- Lysophosphatidylcholines MeSH
- Biomarkers, Tumor MeSH
- Sphingomyelins MeSH
Early detection of cancer is one of the unmet needs in clinical medicine. Peripheral blood analysis is a preferred method for efficient population screening, because blood collection is well embedded in clinical practice and minimally invasive for patients. Lipids are important biomolecules, and variations in lipid concentrations can reflect pathological disorders. Lipidomic profiling of human plasma by the coupling of ultrahigh-performance supercritical fluid chromatography and mass spectrometry is investigated with the aim to distinguish patients with breast, kidney, and prostate cancers from healthy controls. The mean sensitivity, specificity, and accuracy of the lipid profiling approach were 85%, 95%, and 92% for kidney cancer; 91%, 97%, and 94% for breast cancer; and 87%, 95%, and 92% for prostate cancer. No association of statistical models with tumor stage is observed. The statistically most significant lipid species for the differentiation of cancer types studied are CE 16:0, Cer 42:1, LPC 18:2, PC 36:2, PC 36:3, SM 32:1, and SM 41:1 These seven lipids represent a potential biomarker panel for kidney, breast, and prostate cancer screening, but a further verification step in a prospective study has to be performed to verify clinical utility.
- MeSH
- Early Detection of Cancer MeSH
- Adult MeSH
- Heparin chemistry MeSH
- Mass Spectrometry MeSH
- Kidney metabolism MeSH
- Middle Aged MeSH
- Humans MeSH
- Lipidomics * MeSH
- Lipids chemistry MeSH
- Young Adult MeSH
- Biomarkers, Tumor metabolism MeSH
- Prostatic Neoplasms metabolism MeSH
- Breast Neoplasms metabolism MeSH
- Area Under Curve MeSH
- Prospective Studies MeSH
- Prostate metabolism MeSH
- Breast metabolism MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Reproducibility of Results MeSH
- Retrospective Studies MeSH
- ROC Curve MeSH
- Aged MeSH
- Models, Statistical MeSH
- Case-Control Studies MeSH
- Chromatography, Supercritical Fluid MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Heparin MeSH
- Lipids MeSH
- Biomarkers, Tumor MeSH
Glycosphingolipids (GSL) represent a highly heterogeneous class of lipids with many cellular functions, implicated in a wide spectrum of human diseases. Their isolation, detection, and comprehensive structural analysis is a challenging task due to the structural diversity of GSL molecules. In this work, GSL subclasses are isolated from human plasma using an optimized monophasic ethanol-water solvent system capable to recover a broad range of GSL species. Obtained deproteinized plasma is subsequently purified and concentrated by C18-based solid-phase extraction (SPE). The hydrophilic interaction liquid chromatography coupled to electrospray ionization linear ion trap tandem mass spectrometry (HILIC-ESI-LIT-MS/MS) is used for GSL analysis in the human plasma extract. Our results provide an in-depth profiling and structural characterization of glycosphingolipid and some phospholipid subclasses identified in the human plasma based on their retention times and the interpretation of tandem mass spectra. The structural composition of particular lipid species is readily characterized based on the detailed interpretation of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) spectra and further confirmed by specific fragmentation behavior following predictable patterns, which yields to the unambiguous identification of 154 GSL species within 7 lipid subclasses and 77 phospholipids representing the highest number of GSL species ever reported in the human plasma. The developed HILIC-ESI-MS/MS method can be used for further clinical and biological research of GSL in the human blood or other biological samples.
