Methods and kits for detection of coenzyme Q10
Inventors
Luan, Shen • Narain, Niven Rajin • Sarangarajan, Rangaprasad • Tanna, Nikunj Narendra
Assignees
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Publication Number
US-11209422-B2
Publication Date
2021-12-28
Expiration Date
Abstract
The invention provides methods for rapid and quantitative extraction and detection of coenzyme Q10 in a sample readily adaptable to high throughput screening methods. The invention further provides reagents and kits for practicing the methods of the invention.
Core Innovation
The invention relates to methods and kits for rapid and quantitative extraction and detection of coenzyme Q10 (CoQ10) from biological samples. The workflow uses a two-buffer phase-separation approach followed by spectroscopic analysis, including UV spectroscopy at about 275 nm, to enable quantitative measurement. The described workflow is suitable for high-throughput screening and automation.
The extraction workflow is characterized by selecting a first extraction buffer and a second extraction buffer for phase separation. The buffers include polar protic and aqueous solvent classes and also solvent classes with surfactant/detergent, and the non-polar solvent class is selected from non-polar organic solvents such as hexane, toluene, acetonitrile, or related solvents. The approach supports separation and recovery of CoQ10 from sample matrices using a two-buffer phase-separation workflow.
For quantification, the invention uses isotopically labeled deuterated internal standards including CoQ10-d6 and CoQ10H2-d6 to quantify oxidized and reduced forms and assess oxidation extent, including the reduced form (CoQ10H2, ubiquinol) and oxidized form (ubiquinone). Detection can include spectroscopic analysis or LC/MS/MS, and the document reports supporting data comparing spectrophotometric versus LC/MS/MS quantitation and reporting assay performance/standard curves for cells and multiple sample types such as plasma/serum, nasal wash, and tissues.
Claims Coverage
The partial claim set provides two independent claim families. Each independent claim is directed to an isotopically labeled analog corresponding to either the oxidized form (CoQ10-d6) or the reduced form (CoQ10H2-d6), with dependent claims adding an LC/MS/MS mixture context and specifying biological sample types, including bodily fluid subtypes and human.
Isotopically labeled analog of the oxidized form of coenzyme Q10 (CoQ10-d6)
An isotopically labeled analog of the oxidized form of coenzyme Q10 CoQ10-d6 of the following formula.
Isotopically labeled analog of the reduced form of coenzyme Q10 (CoQ10H2-d6)
An isotopically labeled analog of the reduced form of coenzyme Q10 CoQ10H2-d6 of the following formula.
Across the independent claims, the inventive subject matter is the provision of isotopically labeled deuterated analogs for the oxidized form (CoQ10-d6) and the reduced form (CoQ10H2-d6). Dependent claims further define an LC/MS/MS analysis mixture including a biological sample and the labeled analog, and they narrow the biological sample to bodily fluid types such as blood, serum, plasma, and to human samples.
Stated Advantages
Increased extraction efficiency compared with methanol-only extraction, including reported multi-fold improvements.
Rapid and quantitative extraction and detection of CoQ10 from biological samples.
Spectroscopic detection using UV spectroscopy at about 275 nm, suitable for high-throughput screening and automation.
Documented Applications
High-throughput screening and automation for extracting and detecting CoQ10 from biological samples.
Quantitative CoQ10 analysis in sample types including cells, plasma/serum, nasal wash, and tissues.
CoQ10 quantification using LC/MS/MS or spectroscopic analysis, including use of internal standards to quantify oxidized and reduced forms and assess oxidation extent.
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