Mass spectrometry assay for estrogenic compounds

Abstract

Methods are provided for detecting the amount of one or more HRT panel analytes (i.e., estrone (E1), estrone sulfate (E1s), 17α-estradiol (E2a), 17β-estradiol (E2b), estradiol sulfate (E2s), estriol (E3), equilin (EQ), 17α-dihydroequilin (EQa), 17β-dihydroequilin (EQb), Equilenin (EN), 17α-dihydroequilenin (ENa), 17β-dihydroequilenin (ENb), and Δ8,9-dehydroestrone (dE1)) in a sample by mass spectrometry. The methods generally involve ionizing one or more HRT panel analytes in a sample and quantifying the generated ions to determine the amount of one or more HRT panel analytes in the sample. In methods where amounts of multiple HRT panel analytes are detected, the amounts of multiple analytes are detected in the same sample injection.

1 . A method for determining the level of circulating estrogenic compounds in an individual undergoing hormone replacement therapy (HRT), wherein said circulating estrogenic compounds comprise two or more estrogenic compounds selected from the group consisting of estrone (E1), estrone sulfate (E1s), 17α-estradiol (E2a), 17β-estradiol (E2b), estradiol sulfate (E2s), estriol (E3), equilin (EQ), 17α-dihydroequilin (EQa), 17β-dihydroequilin (EQb), equilenin (EN), 17α-dihydroequilenin (ENa), 17β-dihydroequilenin (ENb), and Δ8,9-dehydroestrone (dE1),
 the method comprising:
 a. ionizing a sample comprising the body fluid of an individual undergoing HRT as a single sample injection under conditions suitable to produce one or more ions detectable by mass spectrometry from each of two or more estrogenic compounds; 
 b. determining the amounts of one or more ions from each of the two or more estrogenic compounds by tandem mass spectrometry; and 
 c. using the amounts of one or more ions from each of the two or more estrogenic compounds to determine the amounts of each of the two or more estrogenic compounds in the body fluid sample of the individual. 
   
     
     
         2 . The method of  claim 1 , further comprising calculating a ratio of the combined levels of one or more sulfated estrogenic compounds compared to the combined levels of one or more non-sulfated estrogenic compounds,
 wherein the one or more sulfated estrogenic compounds comprise one or more estrogenic compounds selected from the group consisting of estrone sulfate (E1s) and estradiol sulfate (E2s), and if the non-sulfated estrogenic compound(s) are predominant, a decrease in HRT dosage is indicated.   
     
     
         3 . The method of  claim 2 , wherein the non-sulfated estrogenic compound(s) comprise the non-sulfated versions of the sulfated estrogenic compound(s). 
     
     
         4 . The method of  claim 1 , further comprising calculating a ratio of the combined levels of one or more sulfated estrogenic compounds compared to the combined levels of one or more non-sulfated estrogenic compounds,
 wherein the one or more sulfated estrogenic compounds comprise one or more estrogenic compounds selected from the group consisting of estrone sulfate (E1s) and estradiol sulfate (E2s), and if the sulfated estrogenic compound(s) are predominant, an increase in HRT dosage is indicated.   
     
     
         5 . The method of  claim 4 , wherein the non-sulfated estrogenic compound(s) comprise the non-sulfated versions of the sulfated estrogenic compound(s). 
     
     
         6 . The method of  claim 1 , wherein if the two or more estrogenic compounds are only two estrogenic compounds, the two estrogenic compounds exclude the combination of E1 and E3 or the combination of E1S and E2S, and if the two or more estrogenic compounds are only three estrogenic compounds, the three estrogenic compounds exclude the combination of E1, E2b and E3. 
     
     
         7 . The method of  claim 1 , wherein the amounts of two or more estrogenic compounds are determined. 
     
     
         8 . The method of  claim 1 , wherein the amounts of four or more estrogenic compounds are determined. 
     
     
         9 . The method of  claim 1 , wherein the amounts of six or more estrogenic compounds are determined. 
     
     
         10 . The method of  claim 1 , wherein the amounts of eight or more estrogenic compounds are determined. 
     
     
         11 . The method of  claim 1 , wherein the amounts of ten or more estrogenic compounds are determined. 
     
     
         12 . The method of  claim 1 , wherein the amounts of twelve or more estrogenic compounds are determined. 
     
     
         13 . The method of  claim 1 , wherein the sample has been purified by liquid chromatography prior to ionization. 
     
     
         14 . The method of  claim 13 , wherein liquid chromatography is selected from the group consisting of high performance liquid chromatography, ultra high performance liquid chromatography, and turbulent flow liquid chromatography. 
     
     
         15 . The method of  claim 1 , wherein the sample has been purified by turbulent flow liquid chromatography and either high performance liquid chromatography or ultra high performance liquid chromatography prior to ionization. 
     
     
         16 . The method of  claim 1 , wherein the sample comprises serum. 
     
     
         17 . The method of  claim 1 , further comprising determining one or more internal standards by tandem mass spectrometry, wherein each internal standard comprises an isotopically labeled estrogenic compound. 
     
     
         18 . The method of  claim 1 , further comprising determining one or more internal standards by tandem mass spectrometry, wherein each internal standard is selected from the group consisting of d 4 -estrone (E1-d4), d 2 -17α-estradiol (E2a-d2), d 5 -17β-estradiol (E2b-d5), d 3 -estriol (E3-d3), d 4 -equilin (EQ-d4), d 5 -17β-dihydroequilin (EQb-d5), d 3 -equilenin (EN-d3), d 3 -17β-dihydroequilenin (ENb-d3), d 4 -estrone sulfate (E1s-d4), d 4 -17β-estradiol sulfate (E2bs-d4), and d 2 -Δ8,9-dehydroestrone (dE1-d2). 
     
     
         19 . A method for determining in a sample the amount of one or more analytes selected from the group consisting of estrone (E1), estrone sulfate (E1s), 17α-estradiol (E2a), 17β-estradiol (E2b), estradiol sulfate (E2s), estriol (E3), equilin (EQ), 17α-dihydroequilin (EQa), 17β-dihydroequilin (EQb), equilenin (EN), 17α-dihydroequilenin (ENa), 17β-dihydroequilenin (ENb), and Δ8,9-dehydroestrone (dE1),
 wherein when said one or more is only one, said one excludes E1, the method comprising:
 a. subjecting the sample to turbulent flow liquid chromatography to obtain a sample enriched in the one or more analytes subject to determination; 
   b. ionizing sample obtained from step a. as a single sample injection under conditions suitable to produce one or more ions detectable by mass spectrometry from each of the one or more analytes;
 c. determining the amounts of one or more ions from each of the one or more analytes by tandem mass spectrometry; and 
 d. using the amounts of one or more ions from each of the one or more analytes to determine the amounts of each of the one or more analytes in the sample. 
   
     
     
         20 . The method of  claim 19 , wherein said sample has been subjected to high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC) prior to ionization. 
     
     
         21 . The method of  claim 19 , wherein the sample comprises a biological fluid. 
     
     
         22 . The method of  claim 19 , wherein the sample comprises serum. 
     
     
         23 . The method of  claim 19 , wherein if the one or more analytes are only two analytes, the two analytes exclude the combination of E1 and E3 or the combination of E1S and E2S, and if the one or more analytes are only three analytes, the three analytes exclude the combination of E1, E2b and E3. 
     
     
         24 . A method for determining in a sample the amount of two or more analytes selected from the group consisting of estrone (E1), estrone sulfate (E1s), 17α-estradiol (E2a), 17β-estradiol (E2b), estradiol sulfate (E2s), estriol (E3), equilin (EQ), 17α-dihydroequilin (EQa), 17β-dihydroequilin (EQb), equilenin (EN), 17α-dihydroequilenin (ENa), 17β-dihydroequilenin (ENb), and Δ8,9-dehydroestrone (dE1),
 wherein if the two or more analytes are only two analytes, the two analytes exclude the combination of E1 and E3 or the combination of E1S and E2S, and if the two or more analytes are only three analytes, the three analytes exclude the combination of E1, E2b and E3;   the method comprising:
 a. ionizing a single sample injection under conditions suitable to produce one or more ions detectable by mass spectrometry from each of the two or more analytes; 
 b. determining the amounts of one or more ions from each of the two or more analytes by tandem mass spectrometry; and 
 c. using the amounts of one or more ions from each of the two or more analytes to determine the amounts of each of the two or more analytes in the sample. 
   
     
     
         25 . The method of  claim 24 , wherein the sample has been subjected to liquid chromatography prior to ionization. 
     
     
         26 . The method of  claim 25 , wherein liquid chromatography is selected from the group consisting of high performance liquid chromatography, ultra high performance liquid chromatography, and turbulent flow liquid chromatography. 
     
     
         27 . The method of  claim 24 , wherein the sample has been subjected to turbulent flow liquid chromatography and either high performance liquid chromatography or ultra high performance liquid chromatography prior to ionization. 
     
     
         28 . The method of  claim 24 , wherein the sample comprises a biological fluid. 
     
     
         29 . The method of  claim 24 , wherein the sample comprises a serum.