Large-scaled human serum sphingolipid profiling by using reversed-phase liquid chromatography coupled with dynamic multiple reaction monitoring of mass spectrometry: Method development and application in hepatocellular carcinoma
•Using tert-butyl methyl ether with mild alkaline hydrolysis for sphingolipid extraction.•UHPLC-dynamic multiple reaction monitoring method for serum sphingolipid profiling.•84 serum endogenous sphingolipids can be detected in a single LC run within 10min. Sphingolipids are a family of bioactive mol...
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| Published in | Journal of Chromatography A Vol. 1320; pp. 103 - 110 |
|---|---|
| Main Authors | , , , , , , |
| Format | Journal Article |
| Language | English |
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Amsterdam
Elsevier B.V
13.12.2013
Elsevier |
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| Abstract | •Using tert-butyl methyl ether with mild alkaline hydrolysis for sphingolipid extraction.•UHPLC-dynamic multiple reaction monitoring method for serum sphingolipid profiling.•84 serum endogenous sphingolipids can be detected in a single LC run within 10min.
Sphingolipids are a family of bioactive molecules with high structural diversity and complexity. They not only serve as integral components of cellular membrane, but also play pivotal roles in signaling and other cellular events. It is desirable for the development of sensitive, robust and structural-specific analytical approaches enabling rapid determination of as many sphingolipid species as possible. Herein we present an analytical method for large-scaled profiling of sphigolipids in human serum, which consisted of an improved extraction protocol using tert-butyl methyl ether combined with mild alkaline hydrolysis, and an ultra high performance reversed-phase liquid chromatography-dynamic multiple reaction monitoring-mass spectrometric (RPLC-dynamic MRM-MS) method. In total 84 endogenous sphingolipid species covering six subcategories (i.e. free sphingoid base, dihydroceramide, ceramide, hexosylceramide, lactosylceramide, and sphingomyelin), were separated and quantified in a single run within 10min. A broad linear range over 2.5–4 orders of magnitude (r2>0.99), a limit of detection of 0.01–0.17pmol/mL, and a limit of quantitation of 0.02–0.42pmol/mL were obtained for each subcategory. Average recovery of each subcategory was within 85.6–95.6%. Median values of coefficient of variation (CV) of all detected 84 sphingolipids were 3.9% and 6.8% for intraday and interday precision, respectively. This method was exemplarily applied in a study regarding dysregulated sphingolipid homeostasis in hepatocellular carcinoma. The establishment of this method provides a useful tool for serum-based high throughput screening of sphingolipid biomarkers and mechanism investigation of sphingolipid metabolic regulation in human disease. |
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| AbstractList | •Using tert-butyl methyl ether with mild alkaline hydrolysis for sphingolipid extraction.•UHPLC-dynamic multiple reaction monitoring method for serum sphingolipid profiling.•84 serum endogenous sphingolipids can be detected in a single LC run within 10min.
Sphingolipids are a family of bioactive molecules with high structural diversity and complexity. They not only serve as integral components of cellular membrane, but also play pivotal roles in signaling and other cellular events. It is desirable for the development of sensitive, robust and structural-specific analytical approaches enabling rapid determination of as many sphingolipid species as possible. Herein we present an analytical method for large-scaled profiling of sphigolipids in human serum, which consisted of an improved extraction protocol using tert-butyl methyl ether combined with mild alkaline hydrolysis, and an ultra high performance reversed-phase liquid chromatography-dynamic multiple reaction monitoring-mass spectrometric (RPLC-dynamic MRM-MS) method. In total 84 endogenous sphingolipid species covering six subcategories (i.e. free sphingoid base, dihydroceramide, ceramide, hexosylceramide, lactosylceramide, and sphingomyelin), were separated and quantified in a single run within 10min. A broad linear range over 2.5–4 orders of magnitude (r2>0.99), a limit of detection of 0.01–0.17pmol/mL, and a limit of quantitation of 0.02–0.42pmol/mL were obtained for each subcategory. Average recovery of each subcategory was within 85.6–95.6%. Median values of coefficient of variation (CV) of all detected 84 sphingolipids were 3.9% and 6.8% for intraday and interday precision, respectively. This method was exemplarily applied in a study regarding dysregulated sphingolipid homeostasis in hepatocellular carcinoma. The establishment of this method provides a useful tool for serum-based high throughput screening of sphingolipid biomarkers and mechanism investigation of sphingolipid metabolic regulation in human disease. Sphingolipids are a family of bioactive molecules with high structural diversity and complexity. They not only serve as integral components of cellular membrane, but also play pivotal roles in signaling and other cellular events. It is desirable for the development of sensitive, robust and structural-specific analytical approaches enabling rapid determination of as many sphingolipid species as possible. Herein we present an analytical method for large-scaled profiling of sphigolipids in human serum, which consisted of an improved extraction protocol using tert-butyl methyl ether combined with mild alkaline hydrolysis, and an ultra high performance reversed-phase liquid chromatography-dynamic multiple reaction monitoring-mass spectrometric (RPLC-dynamic MRM-MS) method. In total 84 endogenous sphingolipid species covering six subcategories (i.e. free sphingoid base, dihydroceramide, ceramide, hexosylceramide, lactosylceramide, and sphingomyelin), were separated and quantified in a single run within 10min. A broad linear range over 2.5–4 orders of magnitude (r²>0.99), a limit of detection of 0.01–0.17pmol/mL, and a limit of quantitation of 0.02–0.42pmol/mL were obtained for each subcategory. Average recovery of each subcategory was within 85.6–95.6%. Median values of coefficient of variation (CV) of all detected 84 sphingolipids were 3.9% and 6.8% for intraday and interday precision, respectively. This method was exemplarily applied in a study regarding dysregulated sphingolipid homeostasis in hepatocellular carcinoma. The establishment of this method provides a useful tool for serum-based high throughput screening of sphingolipid biomarkers and mechanism investigation of sphingolipid metabolic regulation in human disease. Sphingolipids are a family of bioactive molecules with high structural diversity and complexity. They not only serve as integral components of cellular membrane, but also play pivotal roles in signaling and other cellular events. It is desirable for the development of sensitive, robust and structural-specific analytical approaches enabling rapid determination of as many sphingolipid species as possible. Herein we present an analytical method for large-scaled profiling of sphigolipids in human serum, which consisted of an improved extraction protocol using tert-butyl methyl ether combined with mild alkaline hydrolysis, and an ultra high performance reversed-phase liquid chromatography-dynamic multiple reaction monitoring-mass spectrometric (RPLC-dynamic MRM-MS) method. In total 84 endogenous sphingolipid species covering six subcategories (i.e. free sphingoid base, dihydroceramide, ceramide, hexosylceramide, lactosylceramide, and sphingomyelin), were separated and quantified in a single run within 10min. A broad linear range over 2.5-4 orders of magnitude (r(2)>0.99), a limit of detection of 0.01-0.17pmol/mL, and a limit of quantitation of 0.02-0.42pmol/mL were obtained for each subcategory. Average recovery of each subcategory was within 85.6-95.6%. Median values of coefficient of variation (CV) of all detected 84 sphingolipids were 3.9% and 6.8% for intraday and interday precision, respectively. This method was exemplarily applied in a study regarding dysregulated sphingolipid homeostasis in hepatocellular carcinoma. The establishment of this method provides a useful tool for serum-based high throughput screening of sphingolipid biomarkers and mechanism investigation of sphingolipid metabolic regulation in human disease. Sphingolipids are a family of bioactive molecules with high structural diversity and complexity. They not only serve as integral components of cellular membrane, but also play pivotal roles in signaling and other cellular events. It is desirable for the development of sensitive, robust and structural-specific analytical approaches enabling rapid determination of as many sphingolipid species as possible. Herein we present an analytical method for large-scaled profiling of sphigolipids in human serum, which consisted of an improved extraction protocol using tert-butyl methyl ether combined with mild alkaline hydrolysis, and an ultra high performance reversed-phase liquid chromatography-dynamic multiple reaction monitoring-mass spectrometric (RPLC-dynamic MRM-MS) method. In total 84 endogenous sphingolipid species covering six subcategories (i.e. free sphingoid base, dihydroceramide, ceramide, hexosylceramide, lactosylceramide, and sphingomyelin), were separated and quantified in a single run within 10 min. A broad linear range over 2.5-4 orders of magnitude (r2 > 0.99), a limit of detection of 0.01-0.17 pmol/mL, and a limit of quantitation of 0.02-0.42 pmol/mL were obtained for each subcategory. Average recovery of each subcategory was within 85.6-95.6%. Median values of coefficient of variation (CV) of all detected 84 sphingolipids were 3.9% and 6.8% for intraday and interday precision, respectively. This method was exemplarily applied in a study regarding dysregulated sphingolipid homeostasis in hepatocellular carcinoma. The establishment of this method provides a useful tool for serum-based high throughput screening of sphingolipid biomarkers and mechanism investigation of sphingolipid metabolic regulation in human disease. |
| Author | Dai, Weidong Li, Jia Zhao, Xinjie Hu, Chunxiu Lu, Xin Chen, Shili Xu, Guowang |
| Author_xml | – sequence: 1 givenname: Jia surname: Li fullname: Li, Jia – sequence: 2 givenname: Chunxiu surname: Hu fullname: Hu, Chunxiu – sequence: 3 givenname: Xinjie surname: Zhao fullname: Zhao, Xinjie – sequence: 4 givenname: Weidong surname: Dai fullname: Dai, Weidong – sequence: 5 givenname: Shili surname: Chen fullname: Chen, Shili – sequence: 6 givenname: Xin surname: Lu fullname: Lu, Xin – sequence: 7 givenname: Guowang surname: Xu fullname: Xu, Guowang email: xugw@dicp.ac.cn |
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| Keywords | Tert-butyl methyl ether Sphingolipid profiling Multiple reaction monitoring MRM Lipidomics Sph APCI dHCer MTBE Cer RPLC HexCer LacCer Serum SM ESI Liquid chromatography–mass spectrometry NPLC HILIC Ultra performance liquid chromatography Biological fluid Tandem mass spectrometry Chemical analysis Biological marker Lipids Hepatic disease Hepatocellular carcinoma Solvent extraction Electrospray Blood Sample preparation Quantitative analysis Human Validation Healthy subject Liquid liquid extraction Coupled method Multiple reaction monitoring mode Liquid chromatography-mass spectrometry Sphingolipid Patient Malignant tumor Reversed phase chromatography Digestive diseases Blood serum Chemometrics Cancer Principal component analysis electrospray ionization ceramide hydrophilic interaction liquid chromatography reversed phase liquid chromatography normal phase liquid chromatography hexosylceramide dihydroceramide lactosylceramide free sphingoid base atmospheric-pressure chemical ionization sphingomyelin |
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| Snippet | •Using tert-butyl methyl ether with mild alkaline hydrolysis for sphingolipid extraction.•UHPLC-dynamic multiple reaction monitoring method for serum... Sphingolipids are a family of bioactive molecules with high structural diversity and complexity. They not only serve as integral components of cellular... |
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| SubjectTerms | alkaline hydrolysis bioactive properties Biological and medical sciences biomarkers blood serum Carcinoma, Hepatocellular - blood cell membranes ceramides Chromatography, Reverse-Phase - methods detection limit hepatoma homeostasis Host-tumor relations. Immunology. Biological markers human diseases Humans Lipidomics Liquid chromatography–mass spectrometry Liver Neoplasms - blood mass spectrometry Medical sciences monitoring Multiple reaction monitoring Reproducibility of Results reversed-phase liquid chromatography screening Serum Sphingolipid profiling Sphingolipids - blood sphingomyelins Tandem Mass Spectrometry - methods Tert-butyl methyl ether Tumors |
| Title | Large-scaled human serum sphingolipid profiling by using reversed-phase liquid chromatography coupled with dynamic multiple reaction monitoring of mass spectrometry: Method development and application in hepatocellular carcinoma |
| URI | https://dx.doi.org/10.1016/j.chroma.2013.10.064 https://www.ncbi.nlm.nih.gov/pubmed/24210299 https://www.proquest.com/docview/1635025462 https://www.proquest.com/docview/1694493479 |
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