Development and validation of LC-MS/MS method for determination of very long acyl chain (C22:0 and C24:0) ceramides in human plasma

Jiang, Hui; Hsu, Fong‐Fu; Farmer, Marsha; Peterson, Linda R.; Schaffer, Jean E.; Ory, Daniel S.; Jiang, Xuntian

doi:10.1007/s00216-013-7166-9

Cited by 29 publications

(25 citation statements)

References 41 publications

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“…We modified a validated liquid chromatography/tandem mass spectrometry assay to simultaneously quantify C16:0, C22:0, and C24:0 ceramides, which are the most abundant long‐ and very–long‐chain ceramide species in human plasma 9, 13. Linear dynamic ranges for C16:0, C22:0, and C24:0 ceramides in this triplex assay were 0.01 to 2, 0.04 to 8, and 0.1 to 20 μg/mL, respectively, which encompass values reported for each of these species in human plasma 9.…”

Section: Resultsmentioning

confidence: 99%

Ceramide Remodeling and Risk of Cardiovascular Events and Mortality

Peterson

Xanthakis

Duncan

et al. 2018

JAHA

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130

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BackgroundRecent studies suggest that circulating concentrations of specific ceramide species may be associated with coronary risk and mortality. We sought to determine the relations between the most abundant plasma ceramide species of differing acyl chain lengths and the risk of coronary heart disease (CHD) and mortality in community‐based samples.Methods and ResultsWe developed a liquid chromatography/mass spectrometry assay to quantify plasma C24:0, C22:0, and C16:0 ceramides and ratios of these very–long‐chain/long‐chain ceramides in 2642 FHS (Framingham Heart Study) participants and in 3134 SHIP (Study of Health in Pomerania) participants. Over a mean follow‐up of 6 years in FHS, there were 88 CHD and 90 heart failure (HF) events and 239 deaths. Over a median follow‐up time in SHIP of 5.75 years for CHD and HF and 8.24 years for mortality, there were 209 CHD and 146 HF events and 377 deaths. In meta‐analysis of the 2 cohorts and adjusting for standard CHD risk factors, C24:0/C16:0 ceramide ratios were inversely associated with incident CHD (hazard ratio per average SD increment, 0.79; 95% confidence interval, 0.71–0.89; P<0.0001) and inversely associated with incident HF (hazard ratio, 0.78; 95% confidence interval, 0.61–1.00; P=0.046). Moreover, the C24:0/C16:0 and C22:0/C16:0 ceramide ratios were inversely associated with all‐cause mortality (C24:0/C16:0: hazard ratio, 0.60; 95% confidence interval, 0.56–0.65; P<0.0001; C22:0/C16:0: hazard ratio, 0.65; 95% confidence interval, 0.60–0.70; P<0.0001).ConclusionsThe ratio of C24:0/C16:0 ceramides in blood may be a valuable new biomarker of CHD risk, HF risk, and all‐cause mortality in the community.

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Section: Resultsmentioning

confidence: 99%

Ceramide Remodeling and Risk of Cardiovascular Events and Mortality

Peterson

Xanthakis

Duncan

et al. 2018

JAHA

Self Cite

130

106

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“…These hurdles have been resolved in the recent evolvement of mass spectrometry-driven lipid analysis, facilitating rapid and sensitive monitoring of molecular species either by shotgun lipidomics [11][12][13] or LC-based targeted lipidomics [14,15]. Although these methodologies have not in general been designed for clinical routine use, this technology has been utilized to establish clinical relevant applications as for instance the measurement of Cer d18:1/22:0 and Cer d18:1/24:0 [16].…”

Section: Introductionmentioning

confidence: 99%

Development and validation of a high-throughput LC–MS/MS assay for routine measurement of molecular ceramides

et al. 2016

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Monitoring the levels of the ceramides (Cer) d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1 and ratios thereof in human plasma empowers the prediction of fatal outcome of coronary artery disease (CAD). We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for clinical-scaled measurement of the four distinct ceramides. Rapid plasma precipitation was accomplished in 96-well format. Excellent extraction recoveries in the range of 98-109% were achieved for each ceramide. Addition of corresponding D7-labeled ceramide standards facilitated precise quantification of each plasma ceramide species utilizing a novel short 5-min LC-MS/MS method. Neither matrix interference nor carryover was observed. Robust intra- and inter-assay accuracy and precision <15% at five different concentrations were obtained. Linear calibration lines with regressions, R(2) > 0.99, were achieved for all analytes. Short-term bench top, long-term plasma, and extract stability demonstrated that the distinct ceramides were stable in the conditions evaluated. The validity of the methodology was demonstrated by determining the precise ceramide concentrations in a small CAD case-control study. Thus, our LC-MS/MS methodology features simple sample preparation and short analysis time for accurate quantification of Cer d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1, designed for routine analysis.

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“…We decided to use in-house prepared surrogate matrix, containing compounds that were abundantly present in the human serum, in order to satisfy the analytical needs for NCS quantification in both serum and HDL-fraction and provide reliable results that could be used in clinical studies. We opted to test cholesterol, proteins, and cholesterol+proteins solutions (25). The rationale behind this decision was the fact that cholesterol is highly abundant in the NCS lipid extract used for quantification.…”

Section: Discussionmentioning

confidence: 99%

Determination of non-cholesterol sterols in serum and HDL fraction by LC/MS-ms: Significance of matrix-related interferences

Vladimirov

Gojković

Zeljković

et al. 2019

Journal of Medical Biochemistry

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Summary Background Non-cholesterol sterols (NCS) are promising biomarkers for estimation of cholesterol homeostasis properties. In addition, determination of NCS in high-density lipoprotein (HDL) fraction (HDL-NCS) could provide information on cholesterol efflux. However, matrix effects interfere in liquid chromatography–mass spectrometry (LC-MS) analysis of NCS, thereby impairing the method sensitivity. The aims of this study were development, optimization and validation of LC-MS method for quantification of NCS in serum and HDL-NCS. Additionally, matrix effect interferences and methods application in individual serum samples were examined. Methods HDL precipitating reagent was used for HDL isolation. Matrix effect was examined by comparing different surrogates by simple regression analysis. Validation was conducted according to the FDA-ICH guideline. 20 healthy volunteers were recruited for testing of method application. Results The observed matrix effect was 30%, and matrix comparison showed that cholesterol was the dominant contributor to the matrix effect. Cholesterol concentration was adjusted by construction of the calibration curve for serum and HDL fraction (5 mmol/L and 2.5 mmol/L, respectively). The intra- and inter- run variabilities for NCSs were 4.7–10.3% for serum NCS and 3.6–13.6% for HDL-NCS and 4.6–9.5% for serum NCSs and 2.5–9.8% for HDL-NCS, respectively. Recovery studies showed satisfactory results for NCSs: 89.8–113.1% for serum NCS and 85.3–95.8% for HDL-NCS. Conclusions The method was successfully developed and optimized. The matrix interference was solved by customising calibration curves for each method and sample type. The measurement of NCS in HDL fraction was proposed for the first time as potentially useful procedure in biomedical researches.

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Development and validation of LC-MS/MS method for determination of very long acyl chain (C22:0 and C24:0) ceramides in human plasma

Cited by 29 publications

References 41 publications

Ceramide Remodeling and Risk of Cardiovascular Events and Mortality

Ceramide Remodeling and Risk of Cardiovascular Events and Mortality

Development and validation of a high-throughput LC–MS/MS assay for routine measurement of molecular ceramides

Determination of non-cholesterol sterols in serum and HDL fraction by LC/MS-ms: Significance of matrix-related interferences

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