Quantifying phosphatidylethanol from blood samples
AssigneesLoyola University of Chicago, Loyola University Medical Center
InventorsJames Vincent DeFrancesco, Majid Afshar
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Abstract
Disclosed herein is a method for quantifying phosphatidylethanol (“PEth”), a direct ethanol biomarker, from a blood sample using gas chromatography mass spectrometry. The method disclosed herein is useful for diagnosing acute and chronic alcohol abuse.
Claims30
A method for quantifying phosphatidylethanol (“PEth”) in a blood sample comprising:
- (a) extracting PEth from the blood sample by contacting the blood sample with a solvent comprising one or more of chloroform and an alcohol having 1-4 carbon atoms to form a PEth extract;
- (b) concentrating the PEth extract to form a PEth concentrate;
- (c) derivatizing the PEth in the PEth concentrate with a derivatizing agent to form a single PEth derivative having a structure of formula (I):
- wherein each R1, each R2, and each R3 independently is C1-4alkyl;
- and
- (d) quantifying the amount PEth derivative in the concentrate by gas chromatography mass spectrometry (“GC-MS”).
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The method of claim 19, wherein the silylating agent comprises a mixture of BSTFA and TMCS in a ratio of 99:1 by volume.
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The method of claim 29, wherein the column comprises dimethylpolysiloxane, diphenyl, 1,4-bis(dimethylsiloxy)phenylene dimethylpolysiloxane, or a combination thereof.
The method of claim 30, wherein the column comprises 100% dimethylpolysiloxane, 5%-diphenyl-95%-dimethylpolysiloxane, 50%-diphenyl-50%-dimethylpolysiloxane, 1,4-bis(dimethylsiloxy)phenylene dimethylpolysiloxane, or combinations thereof.
-33. (canceled)
The method of claim 35, wherein the quantifying comprises:
- (i) injecting about 0.133 ng/μL to 5.330 ng/μL of the PEth derivative in chloroform into the GC-MS at an initial temperature of about 70° C. held for about 3 minutes;
- (ii) flowing the PEth derivative through the GC-MS at a rate of about 1.6 mL per minute;
- (ii) ramping the initial temperature to an interim temperature of about 150° C. at a rate of about 5° C. per minute;
- (iv) ramping the interim temperature to a final temperature of about 280° C. at a rate of about 40° C. per minute;
- and
- (v) holding the final temperature for about 5 minutes.
Dependencies are inferred from the claim text.