Methods for detecting estradiol by mass spectrometry

Abstract

Provided are methods for determining the amount of estradiol in a sample using mass spectrometry. The methods generally involve ionizing estradiol in a sample and detecting and quantifying the amount of the ion to determine the amount of estradiol in the sample.

1 . A method for determining the amount of estradiol in a test sample said method comprising:
 (a) purifying estradiol from said test sample by liquid chromatography;   (b) ionizing the purified estradiol from said test sample to produce one or more ions detectable by tandem mass spectrometry; and   (c) detecting the amount of the estradiol ion(s) by tandem mass spectrometry, wherein the amount of the estradiol ion(s) is related to the amount of estradiol in said test sample;   wherein the limit of quantitation of the method is less than or equal to 80 pg/mL and estradiol is not derivatized prior to mass spectrometry.   
   
   
       2 . The method of  claim 1 , wherein said liquid chromatography is high performance liquid chromatography (HPLC). 
   
   
       3 . The method of  claim 1 , wherein said purifying further comprises high turbulence liquid chromatography (HTLC) followed by high performance liquid chromatography (HPLC). 
   
   
       4 . The method of  claim 1 , wherein said estradiol ions comprise one or more ions selected from the group consisting of ions with a mass/charge ratio of 271.14±0.5, 255.07±0.5, 183.10±0.5, 159.20±0.5, 145.10±0.5, and 133.20±0.5. 
   
   
       5 . The method of  claim 1 , wherein said ionizing comprises generating a precursor ion selected from the group consisting of ions with a mass/charge ratio of 271.14±0.5 and 255.07±0.5, and generating one or more fragment ions selected from the group consisting of ions with a mass/charge ratio of 183.10±0.5, 159.20±0.5, 145.10±0.5, and 133.20±0.5. 
   
   
       6 . The method of  claim 1 , wherein an agent is added to said test sample in an amount sufficient to free estradiol from a protein that may be present in said test sample prior to said ionizing step. 
   
   
       7 . The method of  claim 6 , wherein said agent is added in an amount sufficient to acidify said test sample. 
   
   
       8 . The method of  claim 7 , wherein said agent is formic acid. 
   
   
       9 . The method of  claim 6 , wherein said agent is a salt. 
   
   
       10 . The method of  claim 9 , wherein said salt is ammonium sulfate. 
   
   
       11 . The method of  claim 1 , wherein both glucuronidated and non-glucuronidated estradiol present in said test sample are detected and measured by the method. 
   
   
       12 . The method of  claim 1 , wherein said test sample is body fluid. 
   
   
       13 . A method for determining the amount of estradiol in a body fluid sample by tandem mass spectrometry said method comprising:
 (a) purifying estradiol from said body fluid sample by liquid chromatography;   (b) generating a precursor ion of said estradiol having a mass/charge ratio of 255.07±0.5;   (c) generating one or more fragment ions of said precursor ion, wherein at least one of said one or more fragment ions comprise an ion fragment having a mass/charge ratio of 133.20±0.5: and   (d) detecting the amount of one or more of said ions generated in step (b) or (c) or both and relating the detected ions to the amount of said estradiol in said body fluid sample.   
   
   
       14 . The method of  claim 13 , wherein said method has a limit of quantitation less than or equal to 80 pg/mL. 
   
   
       15 . The method of  claim 13 , wherein said estradiol is not derivatized prior to mass spectrometry. 
   
   
       16 . The method of  claim 13 , wherein said liquid chromatography is high performance liquid chromatography (HPLC). 
   
   
       17 . The method of  claim 13 , wherein said purifying further comprises high turbulence liquid chromatography (HTLC) followed by high performance liquid chromatography (HPLC). 
   
   
       18 . The method of  claim 13 , wherein said precursor ion has a mass/charge ratio of 255.07±0.5, and said one or more fragment ions comprise a fragment ion with a mass/charge ratio of 159.20±0.5. 
   
   
       19 . The method of  claim 13 , wherein an agent is added to said body fluid sample in an amount sufficient to free estradiol from a protein that may be present in said body fluid sample prior to said ionizing step. 
   
   
       20 . The method of  claim 19 , wherein said agent is added in an amount sufficient to acidify said body fluid sample. 
   
   
       21 . The method of  claim 20 , wherein said agent is formic acid. 
   
   
       22 . The method of  claim 13 , wherein both glucuronidated and non-glucuronidated estradiol present in said body fluid are detected and measured by the method. 
   
   
       23 . A method for determining the amount of estradiol in a body fluid sample by tandem mass spectrometry said method comprising:
 (a) purifying estradiol from said body fluid sample by liquid chromatography;   (b) generating a precursor ion of said estradiol having a mass/charge ratio of 271.14±0.5;   (c) generating one or more fragment ions of said precursor ion, wherein at least one of said one or more fragment ions comprise an ion fragment having a mass/charge ratio of 183.10±0.5; and   (d) detecting the amount of one or more of said ions generated in step (b) or (c) or both and relating the detected ions to the amount of said estradiol in said body fluid sample.   
   
   
       24 . The method of  claim 23 , wherein said method has a limit of quantitation less than or equal to 80 pg/mL. 
   
   
       25 . The method of  claim 23 , wherein said estradiol is not derivatized prior to mass spectrometry. 
   
   
       26 . The method of  claim 23 , wherein said liquid chromatography is high performance liquid chromatography (HPLC). 
   
   
       27 . The method of  claim 23 , wherein said purifying further comprises high turbulence liquid chromatography (HTLC) followed by high performance liquid chromatography (HPLC). 
   
   
       28 . The method of  claim 23 , wherein said precursor ion has a mass/charge ratio of 271.14±0.5, and said one or more fragment ions comprise a fragment ion with a mass/charge ratio of 145.10±0.5. 
   
   
       29 . The method of  claim 23 , wherein an agent is added to said body fluid sample in an amount sufficient to free estradiol from a protein that may be present in said test sample prior to said ionizing step. 
   
   
       30 . The method of  claim 29 , wherein said agent is a salt. 
   
   
       31 . The method of  claim 30 , wherein said salt is ammonium sulfate. 
   
   
       32 . The method of  claim 23 , wherein both glucuronidated and non-glucuronidated estradiol present in said body fluid sample are detected and measured by the method. 
   
   
       33 . A method for determining the amount of estradiol in a test sample said method comprising:
 (a) acidifying said test sample with an agent in an amount sufficient to free estradiol from a protein that may be present in said test sample;   (b) purifying estradiol from said test sample by liquid chromatography;   (c) ionizing the purified estradiol from said test sample to produce one or more ions detectable by tandem mass spectrometry; and   (d) detecting the amount of the estradiol ion(s) by tandem mass spectrometry in positive ion mode, wherein the amount of the estradiol ion(s) is related to the amount of estradiol in said test sample.   
   
   
       34 . The method of  claim 33 , wherein said method has a limit of quantitation less than or equal to 80 pg/mL. 
   
   
       35 . The method of  claim 33 , wherein said estradiol is not derivatized prior to mass spectrometry. 
   
   
       36 . The method of  claim 33 , wherein said liquid chromatography is high performance liquid chromatography (HPLC). 
   
   
       37 . The method of  claim 33 , wherein said purifying further comprises high turbulence liquid chromatography (HTLC) followed by high performance liquid chromatography (HPLC). 
   
   
       38 . The method of  claim 33 , wherein said acidifying agent is formic acid. 
   
   
       39 . The method of  claim 33 , wherein said estradiol ions comprise one or more ions selected from the group consisting of ions with a mass/charge ratio of 255.07±0.5, 159.20±0.5, and 133.20±0.5. 
   
   
       40 . The method of  claim 33 , wherein said ionizing comprises generating a precursor ion with a mass/charge ratio of 255.07±0.5, and generating one or more fragment ions selected from the group consisting of ions with a mass/charge ratio of 159.20±0.5, and 133.20±0.5. 
   
   
       41 . The method of  claim 33 , wherein both glucuronidated and non-glucuronidated estradiol present in said test sample are detected and measured by the method. 
   
   
       42 . The method of  claim 33 , wherein said test sample is body fluid. 
   
   
       43 . A method for determining the amount of estradiol in a test sample said method comprising:
 (a) adding an agent to said test sample in an amount sufficient to free estradiol from a protein that may be present in said test sample;   (b) purifying estradiol from said test sample by liquid chromatography;   (c) ionizing the purified estradiol from said test sample to produce one or more ions detectable by tandem mass spectrometry; and   (d) detecting the amount of the estradiol ion(s) by tandem mass spectrometry in negative ion mode, wherein the amount of the estradiol ion(s) is related to the amount of estradiol in said test sample.   
   
   
       44 . The method of  claim 43 , wherein said method has a limit of quantitation less than or equal to 80 pg/mL. 
   
   
       45 . The method of  claim 43 , wherein said estradiol is not derivatized prior to mass spectrometry. 
   
   
       46 . The method of  claim 43 , wherein said liquid chromatography is high performance liquid chromatography (HPLC). 
   
   
       47 . The method of  claim 43 , wherein said purifying further comprises high turbulence liquid chromatography (HTLC) followed by high performance liquid chromatography (HPLC). 
   
   
       48 . The method of  claim 43 , wherein said agent is a salt. 
   
   
       49 . The method of  claim 48 , wherein said salt is ammonium sulfate. 
   
   
       50 . The method of  claim 43 , wherein said estradiol ions comprise one or more ions selected from the group consisting of ions with a mass/charge ratio of 271.14±0.5, 183.10±0.5, and 145.10±0.5. 
   
   
       51 . The method of  claim 43 , wherein said ionizing comprises generating a precursor ion with a mass/charge ratio of 271.14±0.5, and generating one or more fragment ions selected from the group consisting of ions with a mass/charge ratio of 183.10±0.5, and 145.10±0.5. 
   
   
       52 . The method of  claim 43 , wherein both glucuronidated and non-glucuronidated estradiol present in said test sample are detected and measured by the method. 
   
   
       53 . The method of  claim 43 , wherein said test sample is body fluid.