Method for detection of coenzyme Q10
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.
1. A method for determining the amount of coenzyme Q10 (CoQ 10 ) or reduced form of coenzyme Q10 (CoQ 10 H 2 ) in a sample, the method comprising analyzing the sample using LC/MS/MS using oxidized deuterated coenzyme Q10 (CoQ 10 -d6) and/or reduced deuterated coenzyme Q10 (CoQ 10 H 2 -d6) as an internal standard, wherein the method comprises the steps of:
a1) adding a known amount of deuterated coenzyme Q10 (CoQ 10 -d6) to the sample,
b1) detecting CoQ 10 and CoQ 10 -d6 by mass spectrometry, and
c1) determining the amount of detected CoQ 10 by comparing it to the known amount of detected CoQ 10 -d6; or
a2) adding a known amount of reduced deuterated coenzyme Q10 (CoQ 10 H 2 -d6) to the sample,
b2) detecting CoQ 10 H 2 and CoQ 10 H 2 -d6 by mass spectrometry, and
c2) determining the amount of detected CoQ 10 H 2 by comparing it to the known amount of detected CoQ 10 H 2 -d6; or
a3) adding a known amount of CoQ 10 -d6 and CoQ 10 H 2 d6 to the sample,
b3) detecting CoQ 10 , CoQ 10 H 2 , CoQ 10 -d6 and CoQ 10 H 2 -d6 by mass spectrometry,
c3) determining the amount of detected CoQ 10 by comparing it to the known amount of detected CoQ 10 H 2 -d6, and
d3) determining the amount of detected CoQ 10 H 2 by comparing it to the known amount of detected CoQ 10 H 2 -d6.
2. The method of claim 1 , wherein CoQ 10 H 2 -d6 is obtained by reacting CoQ 10 -d6 with one or more reducing agents.
3. The method of claim 2 , wherein said reducing agent is sodium borohydride.
4. The method of claim 1 , wherein CoQ 10 -d6 and/or CoQ 10 H 2 -d6 are added to the sample immediately after sample collection.
5. The method of claim 1 , wherein the sample is a biological sample.
6. The method of claim 5 , wherein the biological sample is plasma or serum.
7. The method of claim 5 , wherein the biological sample is a human sample.
8. The method of claim 1 , comprising the steps of:
a1) providing the sample,
b1) adding a known amount of CoQ 10 -d6 to the sample;
c1) extracting the sample,
d1) optionally subjecting the sample to liquid chromatography,
e1) detecting CoQ 10 and CoQ 10 -d6 by mass spectrometry, and
f1) determining the amount of detected CoQ 10 by comparing it to the known amount of detected CoQ 10 -d6; or
a2) providing the sample,
b2) adding a known amount of CoQ 10 H 2 -d6 to the sample,
c2) extracting the sample,
d2) optionally subjecting the sample to liquid chromatography,
e2) detecting CoQ 10 H 2 and CoQ 10 H 2 -d6 by mass spectrometry, and
f2) determining the amount of detected CoQ 10 H 2 by comparing it to the known amount of detected CoQ 10 H 2 -d6; or
a3) providing the sample,
b3) adding known amounts of CoQ 10 -d6 and CoQ 10 H 2 -d6 to the sample,
c3) extracting the sample,
d3) optionally subjecting the sample to liquid chromatography,
e3) detecting CoQ 10 , CoQ 10 -d6, CoQ 10 H 2 and CoQ 10 H 2 -d6 by mass spectrometry,
f3) determining the amount of detected CoQ 10 by comparing it to the known amount of detected CoQ 10 -d6, and
g3) determining the amount of detected CoQ 10 H 2 by comparing it to the known amount of detected CoQ 10 H 2 -d6.
9. The method of claim 8 , wherein step c1, c2 or c3 comprises adding an extraction buffer.
10. The method of claim 9 , wherein the extraction buffer comprises 1-propanol or isopropanol.
11. The method of claim 8 , wherein steps b1-d1, b2-d2 or b3-d3 are carried out using a pre-cooled cryo-block.
12. The method of claim 1 , wherein CoQ 10 -d6 is the compound of the following structural formula:
13. The method of claim 1 , wherein CoQ 10 H 2 -d6 is the compound of the following structural formula: