Fast LC-MS/MS analysis of free oxysterols derived from reactive oxygen species in human plasma and carotid plaque.
Clin Chim Acta
; 425: 3-8, 2013 Oct 21.
Article
en En
| MEDLINE
| ID: mdl-23827692
ABSTRACT
BACKGROUND:
A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque.METHOD:
In vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80 µl plasma. For preparation of 10mg atherosclerotic plaques an additional liquid-liquid extraction was included. Free 7-keto-, 7-α/ß-hydroxy-, 5,6-α-epoxy-, 5,6-ß-epoxycholesterol, cholestane-3ß,5α,6ß-triol and cholesterol were separated within 7 min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization.RESULTS:
The detection limit was 0.1 ng/ml and the linearity ranged from 0.5 to 0.75 to 2000 ng/ml for the oxysterols and from 50 to 1000 µg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47 ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n=5).CONCLUSION:
This rapid LC-MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions.Palabras clave
-OHC; -hydroxycholesterol; 5,6-α-EC; 5,6-α-epoxycholesterol; 5,6-ß-EC; 5,6-ß-epoxycholesterol; 7-KC; 7-ketocholesterol; 7-α- and 7-ß-hydroxycholesterol; 7-α/ß-OHC; APCI; Atherosclerotic plaque; BHT; CE; CEA; CID; CV; CXP; DP; EP; Fast chromatography; Free oxysterol; GC-MS; HCl; HPLC; HQC; LC; LCMS/MS; LLE; LLOQ; LOD; LQC; MQC; MRM; NQC; Na(2)SO(4); NaOH; Oxysterol; PQC; QC; ROS; Reactive oxygen species; SPE; Tandem mass spectrometry; Triol; atmospheric pressure chemical ionization; butylated hydroxytoluene; carotid endarterectomy; cholestane-3ß,5α,6ß-triol; coefficient of variation; collision energy; collision exit potential; collision induced dissociation; declustering potential; extensions potential; gas chromatography mass spectrometry; high concentrated quality control; high-performance liquid chromatography; hydrochloric acid; limit of detection; liquid chromatography; liquid chromatography tandem mass spectrometry; liquidliquid extraction; low concentrated quality control; lower level of quantification; medium concentrated quality control; multiple reacting monitoring; native quality control; pathological concentrated quality control; quality controls; reactive oxygen species; s/n; signal-to-noise ratio; sodium hydroxide; sodium sulfate; solid phase extraction
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Arterias Carótidas
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Colesterol
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Especies Reactivas de Oxígeno
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Placa Aterosclerótica
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Hidroxicolesteroles
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Cetocolesteroles
Límite:
Female
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Humans
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Male
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Middle aged
Idioma:
En
Revista:
Clin Chim Acta
Año:
2013
Tipo del documento:
Article