Search

US-12618857-B2 - Methods for detecting estrone by mass spectrometry

US12618857B2US 12618857 B2US12618857 B2US 12618857B2US-12618857-B2

Abstract

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

Inventors

  • Mildred M. Goldman
  • Richard E. Reitz

Assignees

  • QUEST DIAGNOSTICS INVESTMENTS INCORPORATED

Dates

Publication Date
20260505
Application Date
20220321

Claims (8)

  1. 1 . A method for determining an amount of estrone in a human serum or plasma sample, the method comprising: (a) adding deuterated estrone to a human serum or plasma sample; (b) purifying the estrone and the deuterated estrone from the human serum or plasma sample by high performance liquid chromatography (HPLC); (c) ionizing the purified estrone and deuterated estrone to produce one or more of estrone ions and deuterated estrone ions detectable by mass spectrometry, wherein the deuterated estrone ions comprise an ion having a mass/charge ratio of 159.10±0.5; and (d) detecting the amount of the estrone ions by tandem mass spectrometry, wherein the amount of the estrone ions is correlated to the amount of estrone in the human serum or plasma sample.
  2. 2 . The method of claim 1 , wherein the deuterated estrone is d 4 -estrone.
  3. 3 . The method of claim 1 , wherein the deuterated estrone is 2,4,16,16-d 4 estrone.
  4. 4 . The method of claim 1 , wherein the method further comprises subjecting estrone from the human serum or plasma sample to an additional extraction.
  5. 5 . The method of claim 1 , wherein the method has a limit of quantitation less than or equal to 20 pg/mL.
  6. 6 . The method of claim 1 , wherein the method has a limit of detection less than or equal to 10 pg/mL.
  7. 7 . The method of claim 1 , wherein the ionization is atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI).
  8. 8 . The method of claim 1 , wherein the ionization is in a positive ion mode or a negative ion mode.

Description

CROSS REFERENCE TO RELATED PATENT APPLICATIONS This application is a continuation of U.S. application Ser. No. 16/579,438, filed Sep. 23, 2019, now U.S. Pat. No. 11,280,798, which is a continuation of U.S. application Ser. No. 15/616,657, filed Jun. 7, 2017, now U.S. Pat. No. 10,422,804, which is a continuation of U.S. application Ser. No. 14/533,602, filed Nov. 5, 2014, now U.S. Pat. No. 9,678,087, which is a continuation of U.S. application Ser. No. 12/002,314, filed Dec. 23, 2014, now U.S. Pat. No. 8,916,385, each of which is incorporated by reference in its entirety herein. FIELD OF THE INVENTION The invention relates to the detection of estrone. In a particular aspect, the invention relates to methods for detecting estrone by mass spectrometry. BACKGROUND OF THE INVENTION The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention. Estrone [1,3,5 (10)-estratrien-3-ol-17-one or 3-Hydroxy-1,3,5 (10)-estratrien-17-one] or E1 is a C18 steroid hormone with a molecular weight of 270.37 daltons. Estrone is produced primarily from androstenedione originating from the gonads or the adrenal cortex. Estrone (or E1) is one of the three naturally occurring estrogens, the others being estradiol and estriol, that are natural to the human body. Its molecular formula is C18H22O2. Estrogens are primarily responsible for the growth of female characteristics in puberty and regulating the menstrual cycle. Estrone may be measured in women who have gone through menopause to determine their estrogen levels. It may also be measured in men or women who might have cancer of the ovaries, testicles, or adrenal glands. In premenopausal women estrone levels generally parallel those of estradiol. After menopause estrone levels increase, possibly due to increased conversion of androstenedione to estrone. Methods for detecting specific estrone ions using mass spectrometry have been described. For example Nelson R, et al., Clinical Chem 2004, 50(2):373-84, and Xu X, et al., Nature Protocols 2007, 2(6):1350-1355 disclose methods for detecting various estrone ions using liquid chromatography and mass spectrometry. These methods derivatize estrone prior to detection by mass spectrometry. Methods to detect underivatized estrone by liquid chromatography/mass spectrometry are discussed in Diaz-Cruz S, et al., J Mass Spectrom 2003, 38:917-923, and Nelson R, et al., Clinical Chem 2004, 50(2):373-84. Methods to detect estrone by gas chromatography/mass spectrometry are disclosed in Nachtigall L, et al., Menopause: J of N. Amer. Menopause Society 2000, 7(4):243-250 and Dorgan J, et al., Steroids 2002, 67:151-158. SUMMARY OF THE INVENTION The present invention provides methods for detecting the amount of estrone in a sample by mass spectrometry, including tandem mass spectrometry. In one aspect, methods are provided for determining the amount of estrone in a body fluid sample. The methods may include: (a) purifying estrone in the body fluid sample by liquid chromatography; (b) ionizing estrone in the body fluid sample; and (c) detecting the amount of the estrone ion(s) by mass spectrometry and relating the amount of the detected estrone ion(s) to the amount of estrone in the body fluid sample. In certain preferred embodiments of this aspect, the limit of quantitation of the methods is less than or equal to 500 pg/mL. In other preferred embodiments, estrone is not derivatized prior to mass spectrometry. In certain preferred embodiments, estrone ions are selected from a group of ions with a mass/charge ratio of 269.07±0.5, 145.03±0.5, and 143.02±0.5. In some preferred embodiments, the methods include generating one or more precursor ions of estrone in which at least one of the precursor ions has a mass/charge ratio of 269.07±0.5. In related preferred embodiments, the methods may include generating one or more fragment ions of an estrone precursor ion in which at least one of the fragment ions has a mass/charge ratio of 145.03±0.5, or 143.02±0.5. In some preferred embodiments, the methods may include adding an agent to the body fluid sample in an amount sufficient to free estrone from a protein that may be present in the body fluid sample. In related preferred embodiments, the methods may include acidifying the body fluid sample; preferably acidifying before ionizing; more preferably acidifying before purifying; preferably acidifying with formic acid. In particularly preferred embodiments, the body fluid sample is serum, plasma, or urine. As used herein, unless otherwise stated, the singular forms “a,” “an,” and “the” include plural reference. Thus, for example, a reference to “a protein” includes a plurality of protein molecules. As used herein, the term “purification” or “purifying” does not refer to removing all materials from the sample other than the analyte(s) of interest. Instead, purification refers to a proced