Methods and Systems for the Quantitative Analysis of Biomarkers

Abstract

Disclosed are methods and systems using liquid chromatography/tandem mass spectrometry (LC-MS/MS and 2D-LC-MS/MS) for the analysis of endogenous biomarkers, including steroid hormones, such as estrone and estradiol, thyroid hormones, such as free thyroxine, and metabolites, such as 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, in biological samples.

1 . A method for determining the presence or amount of free thyroxine in a plurality of samples by tandem mass spectrometry, comprising: (a) dialyzing the plurality of samples to separate the free thyroxine from the protein-bound thyroxine in the samples; (b) generating a precursor ion of the thyroxine in each sample; (b) generating one or more fragment ions of the thyroxine in each sample; and (c) detecting the presence or amount of one or more of the ions generated in step (b) or (c) or both, and relating the detected ions in each sample to the presence or amount of the free thyroxine in the plurality of samples. 
     
     
         2 . The method of  claim 1 , wherein the samples are subjected to a purification step prior to ionization. 
     
     
         3 . The method of  claim 1 , wherein the purification step comprises chromatography. 
     
     
         4 . The method of  claim 3 , wherein the chromatography comprises high performance liquid chromatography (HPLC). 
     
     
         5 . The method of  claim 1 , further comprising at least one of liquid-liquid extraction or dilution of the plurality of samples prior to mass spectrometry. 
     
     
         6 . The method of  claim 1 , further comprising detection of thyroxine over a range of from about 2.0 pg/mL to about 100 pg/mL. 
     
     
         7 . The method of  claim 1 , wherein the dialysing step comprises the use of a buffer, and wherein the buffer comprises and sufficient salts such that the buffer is isotonic. 
     
     
         8 . A method for determining the presence or amount of at least one biomarker of interest in a biological sample, the method comprising:
 providing a biological sample believed to contain at least one biomarker of interest;   chromatographically separating the at least one biomarker of interest from other components in the sample; and   analyzing the chromatographically separated at least one biomarker of interest by mass spectrometry to determine the presence or amount of the at least one biomarker of interest in the sample.   
     
     
         9 . The method of  claim 8 , further comprising partially purifying the biomarker of interest prior to chromatography. 
     
     
         10 . The method of  claim 8 , wherein the at least one biomarker comprises at least one of steroid hormone or a thyroid hormone. 
     
     
         11 . The method of  claim 8 , wherein at least one biomarker comprises free thyroxine. 
     
     
         12 . The method of  claim 11 , wherein the method comprises dialyzing a plurality of samples to separate the free thyroxine from the protein-bound thyroxine in the samples and isolating the portion of the samples comprising the majority of the free thyroxine prior to chromatographically separating the free thyroxine from other components extracted from the plurality of samples. 
     
     
         13 . The method of  claim 12 , further comprising detection of thyroxine over a range of from about 2.0 pg/mL to about 100 pg/mL. 
     
     
         14 . The method of  claim 12 , wherein the dialysing step comprises the use of a buffer, and wherein the buffer comprises and sufficient salts such that the buffer is isotonic. 
     
     
         15 . The method of  claim 8 , wherein the biological sample comprises blood, serum, plasma, urine, or saliva. 
     
     
         16 . The method of  claim 8 , wherein the chromatography comprises high performance liquid chromatography (HPLC). 
     
     
         17 . The method of  claim 8 , wherein the chromatography comprises extraction and analytical liquid chromatography. 
     
     
         18 . The method of  claim 9 , wherein the partial purification comprises liquid-liquid extraction. 
     
     
         19 . The method of  claim 8 , further comprising diluting the sample into a solvent used for liquid chromatography or mass spectrometry. 
     
     
         20 . The method of  claim 8 , wherein the tandem MS/MS spectrometer is operated in an atmospheric pressure chemical ionization (APCI) mode. 
     
     
         21 . A system for determining the presence or amount of at least one biomarker of interest in a test sample, the system comprising:
 a station for providing a test sample suspected of containing one or more hormones or metabolites;   a station for partially purifying the at least one biomarker of interest from other components in the sample;   a station for chromatographically separating the at least one biomarker of interest from other components in the sample; and   a station for analyzing the chromatographically separated one or more hormones or metabolites by mass spectrometry to determine the presence or amount of the one or more hormones or metabolites in the test sample.   
     
     
         22 . The system of  claim 21 , further comprising a station for dialyzing a plurality of samples as a means to separate the hormone or metabolite from protein-bound hormone or metabolite. 
     
     
         23 . The system of  claim 21 , wherein at least one of the stations is controlled by a computer.