Validated comprehensive analytical method for quantification of coenzyme A activated compounds in biological tissues by online solid-phase extraction LC/MS/MS.
We report a robust, reliable, and comprehensive analytical method for the identification and quantification of the entire class of coenzyme A (CoA) activated substances, particularly short-, medium-, and long-chain acyl-CoAs derived from various biological tissues. This online SPE-LC/MS/MS-based method is characterized by a simple three-step sample preparation: (1) addition of buffer, organic solvents, and internal standards; (2) homogenization; and (3) centrifugation. The supernatant is injected directly into the SPE-LC/MS/MS system. Identification of CoA activated compounds is performed by accurate mass determination within the HPLC run. Method validation for short-, medium-, and long-chain acyl-CoA fatty acids revealed excellent quality. Accuracy was found to be between 87 and 107% and precision was between 0.1 and 12.8% in mouse skeletal muscle. The lower limit of quantification for all investigated compounds was well below 3.1% of estimated physiological levels in 200 mg of mouse tissue. Comparable results were obtained for mouse liver, mouse brown white adipose tissue and rat liver. For all investigated tissues, no matrix effect was observed.
Magnes
Christoph
C
Institute of Medical Technologies and Health Management, Joanneum Research, Auenbruggerplatz 20,8036 Graz, Austria.
Suppan
Maria
M
Pieber
Thomas R
TR
Moustafa
Tarek
T
Trauner
Michael
M
Haemmerle
Guenter
G
Sinner
Frank M
FM
D002623:Chemistry Techniques, Analytical / Q000379:methods* / Q000592:standards; D002853:Chromatography, Liquid; D003065:Coenzyme A / Q000378:metabolism*; D043844:Crown Ethers; D004563:Electrochemistry / Q000379:methods*; D009842:Oligopeptides / Q000032:analysis*; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
D043844:Crown Ethers; D009842:Oligopeptides; C002564:dibenzo-18-crown-6; D003065:Coenzyme A