WO2015161078A1

WO2015161078A1 - Methods for comprehensive profiling of steroid metabolome

Info

Publication number: WO2015161078A1
Application number: PCT/US2015/026174
Authority: WO
Inventors: Nilesh W. GAIKWAD
Original assignee: The Regents Of The University Of California
Priority date: 2014-04-17
Filing date: 2015-04-16
Publication date: 2015-10-22

Abstract

The present invention provides a method for quantitating at least 100 steroids in a biological sample using ultra-performance liquid chromatography-tandem mass spectrometry. The present invention also provides a method for detecting an imbalance in steroid metabolism or the presence of cancer in an individual.

Description

METHODS FOR COMPREHENSIVE PROFILING OF STEROID

METABOLOME

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/981 ,087, filed April 17, 2014, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Grant No. CA-DNTR- 2104-H, awarded by The United States Department of Agriculture. The Government has certain rights in this invention.

BACKGROUND OF THE IN VENTION

[0003] Steroids, such as androgens, corticosteroids, estrogens and progestogens control many physiological processes in humans including reproduction, secondary sexual characteristics, maturation, gene expression, cardiovascular health and neurological functions (Gniber et al, N. Engl. J. Med., 2002, 346: 340-352). In addition, steroids, especially estrogens, are implicated in the development and/or progression of many diseases, such as breast cancer (demons and Goss, N. Engl, J Med., 2001 , 344:276-285), ovarian cancer (Bulun et al, J Steroid Biochem, Mol Biol, 2007,106: 81-96), prostate cancer (Carruba G., Ann. N. Y. Acad. Set., 2006, 1089, 201 -217), endometrial cancer (Shang Y., Nat. Rev. Cancer, 2010, 6:360-368), osteoporosis (Khosla S., J. Clin. Endocrinol. Metabol, 2010, 95:3569- 3577), neurodegenerative diseases (Chakraborti et al., Rev. Neurosciences, 2010, 18: 395- 416), cardiovascular disease (Bechlioulis et al., Curr. Vascular Pharmacol., 2010, 8: 249- 258) and obesity (Mayes and Watson, Obesity Rev., 2004, 5: 197-2169).

[ΘΘ04] There is a clear association between cumulative exposure of exogenous and endogenous estrogens and the risk of breast and other cancers (Persson I., J. Steroid. Biochem. Mol. Biol, 2000, 74, 357-364). Furthermore, epidemiologic studies have indicated that breast cancer risk is higher in women with early menarche and late menopause, who have longer exposure to estrogens. Estrogen-replacement therapy has also been implicated as a risk factor for breast cancer in postmenopausal women (Feigelson and Henderson, Carcinogenesis, 1996, 17, 2279-2284), Obese postmenopausal women have higher serum concentrations of estrogen and are at risk of breast cancer(Simpson and Brown, Exp. Rev. Endocrinol. Metabol, 2011, 6, 383-395; Cieary and Grossmann, Endocrinology, 2009, 6:2537-2542).

[0005] Evidence suggests that oxidative metabolism of estrogens plays a significant role in the carcinogenicity, which are oxidized to the 2-QH and 4-OH catechol estrogens by the phase I enzymes {see, FIG. 1). 4-hydroxyestrone was found to be carcinogenic in the male Syrian golden hamster kidney tumor model, whereas 2-hydroxylated metabolites were not (Liehr et al, J. Steroid Biochem., 1986, 24:353-356; Li and Li, Fed. Proc, 1987, 46: 1858- 1863). Similarly, Newbold and Liehr have shown that 4-hydroxyestradiol induced uterine tumors in 66% of CD-I mice, whereas mice treated with 2-hydroxy estradiol or E2 had much lower incidence of uterine tumor (Newbold and Liehr, Cancer Res., 2000, 60:235-237). Oxidative enzymes, metal ions, and in some cases molecular oxygen can catalyze the oxidation of catechols to reactive o-quinones, which can cause damage within cells by alkyiation of cellular nucleophiles (proteins, DNA) (Yager and Davidson, N. Engl. J, Med., 2006, 354:270-282). In an earlier study, it was shown that in healthy women the urinary levels of methoxy-estrogens and thiol conjugates of catechol estrogen are high and the level of estrogen-DNA adduces are low. in contrast, women with breast cancer had lower levels of estrogen metabolites and conjugates and higher levels of estrogen-DNA adducts (Gaikwad et al, hit J Cancer, 2008, 122: 1949-1957; Gaikwad et al, Breast Cancer: Basic & Clin. Res., 2009, 3: 1 -8). Taken together, the findings indicate that comprehensive analysis of steroid and steroid metaboiite levels in individuals may be useful for diagnosing disease, such as cancer, neurodegenerative disease {e.g., Alzheimer's disease and Parkinson's disease), cardiovascular disease and obesity.

[0006] The most widely used techniques for measuring steroids include radioimmunoassay (Andrew, Best Pract. Res. Clin. Endocrinol. Metab., 2001, 15: 1-16), gas chromatography- mass spectrometry (GC-MS) (Hoffmann et al., Steroids, 2010, 75: 1067-1074; Sadanalaa et al., Mass Spectrometry Letters, 2012, 3:4-9), and liquid chromatography-mass spectrometr (LC-MS) (Hauser et al, Chromatogr. B, 2008, 862:100-112). Immunoassay-based methods are simple and sensitive, but are subject to problematic issues such as lack of reproducibility, cross-reactivity, limited dynamic range, and matrix effects. Immunoassay also cannot analyze multiple steroids in a single assay (Andrew, Best Pract. Res. Clin. Endocrinol. Metab., 2001 , 15: 1-16; Wudy et al, Pediatr. Res., 1995, 38, 76-80; Tate and Ward, Clin. Biochem. Rev., 2004, 25, 105-120). GC-MS and LC-MS methods have large dynamic range and can measure multiple steroids simultaneously. GC-MS has been used extensively, but steroid measurements from biological samples require elaborate and tedious sample preparation procedures. For instance, prior to GC-MS analysis, steroid derivatization is required to increase the volatility and thermal stability of the molecules and to improve chromatographic separation and detection (Sadanalaa et al., Mass Spectrometry Letters, 2012, 3:4-9; Zhou et al., Environ. Sci. (China), 2007, 19: 879-884). LC-MS has been shown to be useful in determining steroid levels in the biological samples. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are routinely used in qualitative and quantitative analysis of steroids. For instance, LC-ESI-MS methods were used to quantitate 28 steroids in animal tissue (Flores-Valverde and Hill, Anal. Chem., 2008, 80, 8771-8779) and 24 steroid hormones in human urine in a single 50 min LC-ESI-MS ran (Weidong et al., Anal. Chem., 2012, 84: 10245-10251). LC-APCI-MS methods were used to measure the levels of 7 endogenous adrenal steroids in human serum (Carvalho et al., Chromatogr. B, 2008, 872: 154-161), 16 estrogen derivatives in rat hepatocytes, 4 catechol estrogens in rat brains (Mitamura et al, Analyst. , 2000, 125:811-814) and human urine (Xu et al, J Chromatogr B, 2002, 780: 15-330), and 34 anabolic steroids in bovine muscle (Vanhaecke et al, Analytica Chimica Acta, 2011 ,700, 70-77). In addition, LC-atmospheric pressure photo ionization (APPI)- S methods have been used to measure the levels of non-polar steroids in biological samples (Hintikkaa et al, J. Chromatogr. A, 2010, 1217: 8290-8297). As such, there remains a need for a method to comprehensively and globally profile the steroid metabolome (e.g., over 100 steroids and metabolites thereof) in a sample from an individual in a single quantitative assay. The present invention satisfies this need and provides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

[0007] In one aspect, the present invention provides a method for profiling a steroid metabolome in a biological sample. The method comprises (a) extracting at least 100 steroids from the biological sample using liquid-liquid extraction (LLE) to form a steroid extract, wherein the at least 100 steroids in the steroid extract are not derivatized; and (b) detecting the presence and/or level of the at least 100 steroids in the steroid extract using ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS). [0008] In some embodiments, the biological sample is obtained from an individual. In some instances, the individual is a human.

[0009] In some embodiments, the individual is suspected of having cancer, osteoporosis, a neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease, etc.), a cognitive disorder, a psychiatric disorder, cardiovascular disease, or obesity. In some instances, the cancer is a hormone-related cancer selected from the group consisting of breast cancer, prostate cancer, adrenal cancer, thyroid cancer, pancreatic cancer, gastrointestinal cancer, pituitary cancer, coiorectai cancer, ovarian cancer, endometrial cancer, and osteosarcoma.

[0010] In some embodiments, the biological sample is a whole blood, serum, plasma, saliva, urine, cerebrospinal fluid, amniotic fluid, fine needle aspirate, cancer cells, cultured cells, cell culture media, or tissue biopsy sample.

[001 i] In some embodiments, the at least 100 steroids comprises at least 150 steroids. In other embodiments, the at least 100 steroids comprises from about 150 to about 200 steroids. In yet other embodiments, the at least 100 steroids comprises at least 200 steroids. [0012] In some embodiments, the at least 100 steroids are selected from the group consisting of androgens, corticosteroids, progestogens, estrogens, bile acids, oxysterols, neurosteroids, exogenous steroids, steroid metabolites, steroid derivatives, steroid conjugates, steroid adducts, and combinations thereof. In some instances, the steroid adducts comprise estrogen- DN A adducts. In some instances, the at least 100 steroids comprise 50 or more steroids from Table 1 or 4.

[0013] In some embodiments, the presence and/or level of the at least 100 steroids is detected in about 10 to about 15 minutes.

[0014] In some embodiments, the ultra-performance liquid chromatography (UPLC) comprises a chromatography column at about 45 °C to about 50 °C. [0015] In some embodiments, step (b) further comprises using multiple reaction monitoring (MRM). In some embodiments, step (b) further comprises selecting parent-daughter ion transitions for each of the at least 100 steroids. In some instances, the selected parent- daughter ion transitions comprise 100 or more parent-daughter ion transitions from Table 1 or 4. [0016] In some embodiments, at least 5 MRM functions are selected and at least 5 detection time windows are selected. In some instances, the at least 5 MRM functions and the at least 5 detection time windows are set forth in Table 6. In some aspects, the steroids detected in each of the at least 5 MRM functions are set forth in Table 7. [0017] In some embodiments, at least 30 MRM functions are selected and at least 30 detection time windows are selected. In some instances, the at least 30 MRM functions and the at least 30 detection time windows are set forth in Table 6. In some aspects, the steroids detected in each of the at least 30 MRM functions are set forth in Table 7.

[0018] In another aspect, the present invention provides a method for determining an imbalance in steroid metabolism in an individual. The method comprises (a) profiling a steroid metabolome in a biological sample obtained from the individual in accordance w^rith the method described above to form a steroid metabolome profile for the individual; (b) comparing the steroid metabolome profile for the individual to a steroid metabolome profile for a control sample; and (c) determining an imbalance in steroid metabolism in the individual when a difference between the steroid metabolome profile for the individual and the steroid metabolome profile for the control sample is detected.

[0019] In some embodiments, the control sample is a sample from an individual not having an imbalance in steroid metabolism.

[0020] In some embodiments, the imbalance in steroid metabolism is associated with cancer, osteoporosis, a neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease, etc.), a cognitive disorder, a psychiatric disorder, cardiovascular disease, or obesity. In some instances, the cancer is a hormone-related cancer selected from the group consisting of breast cancer, prostate cancer, adrenal cancer, thyroid cancer, pancreatic cancer, gastrointestinal cancer, pituitary cancer, colorectal cancer, ovarian cancer, endometrial cancer, and osteosarcoma.

[0021] In yet another aspect, the present invention provides a method for diagnosing cancer in an individual. The method comprises (a) profiling a steroid metabolome in a biological sample obtained from the individual in accordance with the method described above to form a steroid metabolome profile for the individual; (b) comparing the steroid metabolome profile for the individual to at least one steroid metabolome profile for at least one type of cancer; and (c) determining the type of cancer in the individual when a similarity between the steroid metabolome profile for the individual and one of the at least one steroid metabolome profiles for the at least one type of cancer is detected.

[0022] In some embodiments, the at least one type of cancer is selected from the group consisting of breast cancer, lung cancer, prostate cancer, adrenal cancer, thyroid cancer, pancreatic cancer, gastrointestinal cancer, pituitary cancer, colorectal cancer, ovarian cancer, liver cancer, kidney cancer, endometrial cancer, neuroendocrine cancer, bone cancer, hematological cancer, and a combination thereof.

[0023] In some embodiments, the at least one steroid metabolome profile for the at least one type of cancer comprises a plurality of steroid metabolome profiles for different types of cancer. In some instances, the different types of cancer comprise at least two cancer types selected from the group consisting of breast cancer, lung cancer, prostate cancer, adrenal cancer, thyroid cancer, pancreatic cancer, gastrointestinal cancer, pituitary cancer, colorectal cancer, ovarian cancer, liver cancer, kidney cancer, endometrial cancer, neuroendocrine cancer, bone cancer, hematological cancer, and a combination thereof. [0024] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the follo wing detailed description and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 shows exemplary steroid metabolic pathways. Carcinogenic pathways are shown in the bottom center panel, whereas protective pathways are shown in the panels to the immediate left and right.

[0026] FIG. 2 shows the liquid-liquid extraction (LLE) recovery of standard 101 steroids and estrogen-related compounds. The 100 μΐ aliquots from 100 mg human breast tissue homogenates were spiked with the total 12.5 pg/μΐ of 101 steroids and estrogen-related compounds before and after (control ) LLE with 100 μΐ CH₃OH, final solvent ratio CI I :{)! ! :! f_.-O (1 : 1). The recovery of each compound was determined by comparing the experimental values to the controls. The data represents the average of three measurements.

[0027J FIGS. 3A-S show chromatograms of representative steroids and estrogen metabolites, which were detected in a single injection of breast tissue extract. The metabolites shown are androstenolone (FIG. 3A), progesterone (FIG. 3B), Cortisol (FIG. 3C), cortexone (FIG. 3D), androstenedione (FIG. 3E), estrone (Ej) (FIG. 3F), estradiol (E₂) (FIG. 3G), l l ot-Hydroxy E₂ (FIG . 3H), 1 1-ketoestrone (FIG. 31), 3 -methoxy estrone (FIG. 3 J), 4- hydroxyestrone (FIG. 3K), 4-hydroxyestradiol (FIG. 3L), 4-hydroxy-E 1-1 -N-3-adenine (FIG. 3M), 4-hydroxy-E2-l-N-3-adenine (FIG. 3N), 2-hydroxyestriol (FIG. 30), 16a- Hydroxyestrone (FIG. 3P), 6-ketoestriol (FIG. 3Q), E 9N3Ade (FIG. 3R), and E₂-9-N3Ade (FIG. 3S).

[0028] The present invention is based, in part, on the discovery that the steroid metabolome (steroi dome) can be detected and quantitated in a biological sample using liquid-liquid extraction followed by ultra performance liquid chromatography-tandem mass spectrometry. The present invention is particularly advantageous as it enables global steroid metabolic profiling for the simultaneous measurement of over 100 endogenous and exogenous steroids (e.g., about 200 or more steroids) including androgens, corticosteroids, progestogens, estrogens, bile acids, oxysterols, neurosteroids, exogenous steroids, steroid metabolites, steroid derivatives, steroid conjugates, steroid adduets, as well as exogenous steroid derivatives. Currently available methods are limited to detecting less than 30 steroids in a single assay. The present invention provides methods for determining a steroid metabolic profile and/or detecting an alteration {e.g., an imbalance) in steroid metabolism in a biological sample taken from an individual. Further, the methods can be used to diagnose cancer or determine whether an individual has a specific type of cancer. II. Definitions

[0029 J The terms "a," "an," or "the" as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such ceils and reference to "the agent" includes reference to one or more agents known to those skilled in the art, and so forth.

[0030] The terms "subject", "patient" or "individual" are used herein interchangeably to refer to a human or animal. For example, the animal subject may be a mammal, a primate, a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance. [0031] The term "steroid" refers to a compound having a basic molecular structure of 17-C atoms arranged in four rings (e.g., perhydrocyclopentaphenanthrene scaffold) and may have side chains attached to it. The term "steroid" includes any compound produced from the metabolism of a steroid or used to generate a steroid (e.g., steroid precursor). [0032] The term "neurosteroid" refers to a steroid compound (e.g., an estrogen, androgen and progestagen) that is synthesized in the central nervous system (CNS), either de novo from cholesterol or f om steroid hormone precursors. Neurosteroids can affect brain function by modulating neurotransmission. They can also affect neuronal survival, neuronal and glial differentiation, and myelination in the CNS. [0033] The term "oxysterol" refers to an oxidized derivative of cholesterol.

[0034] The term "steroid metabolome" or "steroid metabolic profile" refers to the concentrations (e.g., amounts) of steroids, steroid precursors, and steroid metabolites (e.g., compounds of steroid anabolic pathways and compounds of steroid catabolic pathways) that are in a biological system (e.g., a cell, tissue, biological fluid, or whole subject). An individual's steroid metabolome may be specific for a particular condition (e.g., a healthy condition, diseased condition, treatment condition, etc.).

[0035] The term "biological sample" refers to any sample comprising biological material from any biological source that may contain an analyte (e.g., steroid, derivative thereof, metabolite thereof) of interest. For example, "biological sample" may include whole blood, serum, plasma, saliva, urine, feces, bile, tears, perspiration, sperm, vaginal fluid, or a tissue sample. In some embodiments, the biological sample is derived, e.g., by biopsy, from cells, tissues or organs.

[0036] The term "derivatization" as used herein, refers to a process of chemically modifying a compound (e.g., steroid) by reacting it with a chemical agent to render the compound into a more detectable compound. Derivatization can be performed during the sample preparation process. In some instances, derivatization can enhance the chromatographic behavior and the extractability or a compound, and/or stabilize the compound.

[0037] The term "liquid chromatography" or "LC" means a process of selective retention of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid {i.e., mobile phase), as this fluid moves relative to the stationary phase(s).

[0038] The term "high performance liquid chromatography" or "HPLC" (also sometimes known as "high pressure liquid chromatography") refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column,

[0039] As used herein, the term "ultra high performance liquid chromatography," "ultra high pressure liquid chromatography," "ultra performance liquid chromatography ," "UPLC" or "UHPLC" refers to HPLC which occurs at much higher pressures than traditional HPLC techniques. For UPLC, column particles of < 2.0 um size, e.g., 1.9 um, 1.8 μηι, 1 .7 um, 1 ,6 um, or 1 ,5 μτη, can be used at a working pressure of, preferably, from approximately 400 to 1,000 bar, or more preferably from 580 to 750 bar.

[0040] The term "mass spectrometry" or "MS" refers to an analytical technique to identify compounds by their mass. MS refers to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m/z". MS technology generally includes ( 1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. The compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge ("z").

[0041] The term "tandem mass spectrometry" or "MS/MS" refers to a technique wherein a precursor ion (e.g., a parent ion) generated from an analyte of interest can be filtered in an MS instrument, and the precursor ion subsequently fragmented to yield one or more fragment ions (e.g., daughter ions or product ions) that are then analyzed in a second MS procedure. By careful selection of precursor ions, only ions produced by certain analytes are passed to the fragmentation chamber, where collisions with atoms of an inert gas produce the fragment ions. Both the parent ions and the daughter ions are produced in a reproducible fashion under a given set of iomzation/fragmentation conditions. [0042] The term "eiectrosprav ionization" or "ESF refers to methods in which a solution is passed along a short, length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber, which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

[0043] The term "multiple reaction monitoring" or "MRM" is a detection mode for a mass spectrometric instrument in which transitions, a precursor ion and one or more fragment ions, are selectively detected. The mass-to-charge ratio of the ions is determined using a quadrupole analyzer. For example, in a "quadrupole" or "quadrupole ion trap" instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and the mass/charge (m/z) ratio. The voltage and amplitude may be selected so that only ions having a particular mass/charge ratio travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments may act as both a "mass filter" and as a "mass detector" for the ions injected into the instrument.

[0044] The term "multiple reaction monitoring function" or "MRM function" refers to a scheduled MRM analysis wherein the transition(s) of an analyte(s) are acquired only during a defined elution time window. An MRM function is performed according to a set of optimal MRM coordinates for one or more analytes (e.g., a preselected target anaiyte, its parent and daughter fragments, their corresponding mass-to-charge ratios, the fragment intensity ratios, the associated collision energy, and detection times). Analytes detected and quantified in a particular MRM function can be selected based on their retention times and other MRM coordinates.

III. Detailed Description of the Embodiments A. Steroids

[0045] Steroids are a wide group of natural and synthetic organic compounds that have a basic 17 carbon atom structure arranged in a four ring system. Non-limiting examples of steroids include sterols (e.g., cholesterol), bile acids, corticosteroids (e.g., glucocorticoids and mineralcorticoids), and sex steroids (e.g., progestogens, androgens, and estrogens). They are mostly produced in the ovaries, testes, and the adrenal glands. Neurosteroids are synthesized and metabolized in neurons and glial cells of the nervous system.

[0046] Steroids can control many physiological processes ranging from metabolism and digestion to the development and function of the sexual organs. Disturbances in the metabolism of one or more steroids can cause the development and/or progression of various diseases, such as cancer, multiple sclerosis, neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, etc.), cardiovascular diseases, adrenal diseases, etc. For example, the formation of carcinogenic estrogen metabolites such as estrogen-DNA adducts have been linked to breast cancer (Cavaiieri and Rogan, J. Steroid Biochem Mol Biol, 125: 169-180, 2011 ; Gaikwad et al, Int J Cancer, 122: 1949-57, 2008). Neurosteroids appear to play a role in learning and memory in cognitive aging (Vallee et al, Brain Research Reviews, 2001, 37:301-312) and changes in neurosteroid. levels have been detected in patients with Alzheimer's disease, Parkinson's disease, and multiple sclerosis (Luchetti et a!., Neuroscience, 201 1 , 191 :6-21 ), as well as in patients with psychiatric disorders (Mac enzie et al , Cell Mol Neurobiol, 2007, 27(5):541 -74).

[0047] Liquid chromatography-mass spectrometry (LC-MS) techniques can be used to measure steroid hormones; however, currently available methods can evaluate only a limited number of steroids {e.g., <30 steroids) in a single analysis. Provided herein is a method for measuring at least 100 steroids in a biological sample in a single UPLC-tandem MS run. Also, the steroids do not require derivatization prior to liquid chromatography. Furthermore, the steroid measurement can be obtained from a UPLC-tandem MS run of about 10 minutes to about 1 minutes.

[0048] The method provided herein can be used to detect steroids from all classes, including those based on a structural classification or a functional classification. Structural classification divides steroids into the six classes: gonane (having 17 carbons), estrane (having 18 carbons), androstane (having 19 carbons), pregnane (having 21 carbons), cholane (having 24 carbons), and cholestane (having 27 carbons). Functional classification divides steroids into five classes such as estrogens (having 18 carbons), androgens (having 19 carbons), corticosteroids (having 21 carbons), progesterone (having 21 carbons) and sterols (having 27 carbons). Of note, the method provided herein can measure sterols, such as cholesterol which is a precursor of steroid hormones, such as progestogens (e.g., progesterone), glucocorticoids (e.g., Cortisol), mineralocorticoids (e.g., aldosterone), androgens, estrogens, and bile acids,

[0049] In some embodiments, the method described herein is used determine the presence and/or level of steroids such as androgens, corticosteroids, progestogens, estrogens, bile acids, oxysterols, neurosteroids, exogenous steroids, steroid metabolites, steroid derivatives, steroid conjugates, steroid adducts, e.g., estrogen-DNA adducts, and combinations thereof. As a non-limiting example, the steroids set forth in Table 1 or Table 4 can be detected and/or measured,

[0050] In some embodiments, the steroids measured in a biological sample include one or more of the following: pregnenolone; 17-hydroxypregnenolone; androstenolone (DHE A); 5- androstenediol; progesterone; 17-hydroxyprogesterone; cortexolone; Cortisol ; cortexone; corticosterone; aldosterone; androstenedione; testosterone; allodihydrotestosterone; estrone ( El ); estradiol (E2); 1 la-hydroxy_El ; 1 l p-hydroxy_El ; 1 la- hydroxy _E2; 1 1 β- hydroxy _E2; 9,1 1-dehydro EI ; 9, 1 1-dehydro E2; 11-ketoestrone; 3-methoxyestrone; estradiol-3- glucuronate; estradiol- 17-glucuronate; 4-hydroxyestrone; 4-hydroxyestradiol; 4- methoxyestrone; 4-methoxyestradiol; 4-methoxyestriol; 4-hydroxy-El-2-glutatione; 4- hydroxy~E2-2-glutatione; 4-hydroxy-El -2-cysteine; 4-hydroxy-E2-2-cysteine; 4-hydroxy-El- 2-N-acetyl cysteine; 4-hydroxy-E2-2-N-acetylcysteine; 4-hydroxy-E 1-1 -N-3-adenine; 4- hydroxy-E2- 1 -N-3-adenirte; 4-Hydroxy-E 1 - 1 -N-7-guanine; 4-hydroxy-E2- 1 -N-7-guanine; 2- hydroxyestrone; 2-hydroxyestradiol; 2-hydroxy-El-l +4-giutatiotie; 2-hydroxy-E2- 1+4- glutatione; 2-hydroxy-E 1-1 +4-c ysteine; 2-hy droxy-E2- 1 +4-cysteine; 2-hydroxy-E 1-1 +4-N- acetylcysteine; 2-hydroxy-E2-1 +4-N-acerylcysteme; 2-hydroxy-E 1 -6-N-3-adenine; 2- hydroxy-E2-6-N-3-adenine; 2-hydroxyestrioi; 3-methoxy-2-OH-estrone; 2-methoxy-3-OH- estrone; 2-methoxy-3-OH-estradiol; 2,3-dimethoxyestrone; 2,3-dimethoxyestradiol; 6a- hydroxyestradiol; 6P-hydroxyestradiol; 6-ketoestrone; 6-ketoestradiol; 6-dehydroestradiol; 6- dehydroestrone; 16a-hydroxyestrone; 17-epiestriol; estriol; 16,17-epiestriol; 16-epiestriol; estriol-3 -sulfate; estriol-3 -glucuronate; 3-methoxyestrio ; 16-keto- 17P-estradiol; 6- ketoestriol; 7dsehydroestradiol; equilin; dihydroequilin-3-S04; equilin-3~S04; estrone-9- ₃- Ade; estradiol-9-N₃-Ade; estradiol-3 -acetate; estrone-3-acetate; estradioi-3 , 17a-diacetate; estradiol-3 , 17P-diacetate; 2-hydroxy estradiol- 17-acetate; 6-ketoestradiol-3 , 17-diacetate; 6- ketoestriol triacetate; 6-dehydroestradiol diacetate; estriol-3 -acetate; estriol- 16-acetate; estriol- 16,17-diacetate; 17-epies triol-triacetate; estriol triacetate; 16,17-epieslriol-triacetate; 16-epiestriol -triacetate; equilinacetate; estradiol-3-hemisuccinate; estrone-3-hemisuccinate; estradiol- 17-hemisuceinate; estradiol-3 , 17-dihemisuccinate; estriol-3-hernisuccinate; estriol-

16-hernisiiCcmate; an analog [hereof; and a derivative thereof.

[0051] In other embodiments, the steroids measured in a biological sample include one or more of the following: pregnenolone; 17-hydroxypregnenolone; androstenolone (DHEA); 5- androstenediol; progesterone; 17-h.ydroxyprogest.erone; eortexolone; Cortisol; eortexone; corticosterone; aldosterone; androstenedione; testosterone; allodihydrotestosterone; estrone El; estradiol E2; 1 la-hydroxyestrone; 1 lb-hydroxyestrone; I la-hydroxyestradiol; l ib- hydroxyestradiol; 9(1 l )-dehydroestradiol; 9(1 l)-dehydroestrone; 1 1 -ketoestrone; estradiol-3- SO4; estrone-S0₄; estradiol-17- S0₄; estradiol-3, 17-di- SO4; 3-OM estrone; estradiol-3-Glu; estrone-Giu; estradiol- 17-Glu; estradiol-3, 17-di-Glu; 4-OH estrone; 4-OH estradiol; 4-OMe estone; 4-OMe estradiol; 4-OMe estriol; 2-OH estrone; 2-hydroxyestradiol; 2-GH eslril; 3- methoxy-2-OH-estrone; 2-meth.oxy-2~QH~estrone; 2-methoxy-3-OH-estradiol ; 2,3- dimethoxyestrone; 2,3-dimethox estradiol; 6a-hydroxyestradiol; 6b-hydroxyestradiol; 6- ketostrone; 6-ketoestradiol; 6-dehydroestradio]; 6-dehydroestrone; 16a-hydroxyestrone; 17- epiestriol; estriol E3; 16,17-epiestriol; 16-epiestriol; estriol-3 -sulfate; estrioI-17-Ghi; 3- methoxystriol; 16-keto-l.7b-estrad.iol; 6-ketoestrioi; 7-dehydro-l 7b~estradiol; equilin; dihydroequi!in-3-S04; equilin-3-S0₄; 3a-hydroxy-5a-pregnan-20-one; 5 a- diiiydroprogesterone; Allopregnanediol; 1 la-hydroxy-4-pregnene-3,20-dione; 1 lb-hydroxy- 4-pregnene-3,20-dione; 1.7-hydroxypregnenolone; 17-hydroxyprogesterone; 2.1 - hydroxypregnanolone; 20-hydroxypregneiiolone; 7a-hydroxypregnenoione; 2-GH- testosterone; 20a.-h.ydroxy-5a.-preg.na.n-3 -one; 20a-dihydroprogesterone; 17a,20a- dihydroxyprogesterone; 3b-hydroxy-5-pregnen-20-one-3-SO₄; eltanolone; pregnanediol; 5b- dihydro progesterone; 5a-dihydrotestosterone; 17b-di.hydroandrost.erone; 17b- dihydroepiandrosterone; 7a-hydroxytestosterone; 7a-hydroxyandrostenedione; 6a-OH- testosterone; 6b-OH -testosterone; etiocholanolone glucuronide; DHEA GLUC; 16a-hydroxy DHEA; testosterone sulfate; desoxytestosterone; 4-androstene-30L, Ϊ 7-one; 2a- liydroxyandrostenedione; 1 la-hydroxyandrostenedione; 6-ketotesteroiie; Nl 1- ketotestosterone; 9-dehydrotestosterone; allotetrahydrocortexone; adrenosterone; etiocholanolone; 4-androstenediol; 16a-ketotestosterone; 19-hydrox androstendione; 4- androsteti-3 ,6- 17-trione; 7b-hydroxy DHEA; 7a-hydroxy DHEA; 7-ketoDHEA; 9- deiiydroepiaiidrosterone; aliochoiesterol; 9-dehydroprogesterone; 11- dehydrotetrahydrocorticosterone; tetrahydrocorticosterone; 6b-h.ydroxy DOC; urocortisol; pregnanetriol; 16a-hydroxyprogesterone; 17a-hydroxypregnenolone; 6-dehydrotestosterone; 9(l l)-dehydro DHEA; 1 1 b-hydroxyepiandrosterone; 16a-hydroxyepiandrosterone; androsterone; epiandrosterone; 7a~hydroxyandrostenediol (DHEA); 7b- hydroxyandrostenediol (DHEA); 4-cholestene-3a-OL; 4-pregnen-3b-ol-20-one; 5-androsten- 3b,17-diol-16-one; 19-hydroxy DHEA; 16a-OH-pregnenolone; 5a-dihydrocortexone (5a-DH- DOC); epitestosterone; 5b-androstandione; 1 1 b-OH-androstenedione; 1 1 -keto- etiocholanolone; 5a-pregnan-l la-OH-3,20-dione; 5a-epoxypregnenolone; 5a-dihydro-l I- keto-progesterone; 1 1 -ketoprogesterone; cholesterol; 4b-OH cholesterol; 7a-OH cholesterol; 7b-OH cholesterol; 25 -OH cholesterol; 5 a,6a-epoxy cholesterol; 7-keto-cholesterol; 7-keto- 25-OH cholesterol; 6,7-dehydrocholesterol; 6-dehydroch.olestenone; 6-ketoch.olestenone; etiocholan-3-OH- 17-one; 7-dehydrocholesterol; dihydrocholesterol; choIesterol-3-S04; coprostano3-7,12-diol; 20a-OH-cholesterol; 22b-OH-cholesterol; 27-OH-cb.oiesterol; 24-keto- cholesterol; 24-hydroxycholesterol; Me-3-OH-choI-5-n-24-oate; chenodeoxycholic acid; ursodeoxycholic acid; murocliolic acid; Iivodeoxycholic acid; allolitliocholic acid; lithocholic acid; taurocholanic acid; taurohyodeoxycholic acid; cholic acid; taurochenodeoxycholic acid; desoxvcholic acid; taurolitliocliolic acid; glycolithocholic acid; 6-keto-allolithocholic acid; glycodeoxycholic acid; glycochenodeoxycholic acid; tauroursodeoxycholic acid; taurodeoxycholic acid; glycodeoxycholic acid; a-muricholic acid; b-muricholic acid; tauro-b- muricholic acid; taurocholic acid; iaurodehydrocholic acid; giyeodehydrocholic acid; androstandiol-3-Glu; 1 l-keto-etiocholanolone-3-Glu; etiocholanolone-3-Glu; debydroepiandrosterone sulfate (DHEAS); epitestosterone- 17-S0₄; testosterone- 17-Gl.u; pregnanediol-3-Glu; 17-OH-pregnanolone-3-Glu-Na; pregnanolone-3-S04; 17,20-di-OH- progesterone-20-Glu; cortisol-21 -S0₄; pregnenolone-3-Giu; cholesterol-3-Glu; allopregnanolone- S0₄; epiallopregnanolone-S0₄; estriol-3-Glu-Na; 24-dehydrocholesterol; coprosterol; campesterol; b-sitosterol; brassicateroi; stigmasterol; epialiochoiesterol; 24,25- epoxy cholesterol; zymosterol; lanosterol; lathosterol; "C-estradiol; an analog thereof; and a derivative thereof.

[0052 j In some embodiments, the presence and/or level of at least 100 steroids (e.g., at least 105, 1 10, 1 15, 120, 125, 130, 135, 140, or 145 steroids) are detected in a steroid extract. In other embodiments, at least 150 steroids (e.g., at least 155, 160, 165, 170, 175, 180, 185, 190, or 195 steroids) are detected in a steroid extract, in some embodiments, at least 200 steroids are detected in a steroid extract (e.g., at least 205, 210, 215, 217, 220, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, or more steroids). In other embodiments, about 150 steroids to about 200 steroids, about 200 steroids to about 250 steroids, or about 175 steroids to about 225 steroids are detected in a steroid extract, in particular embodiments, all of the steroids set forth in Table 1 are detected in a steroid extract. In some embodiments, all of the steroids set forth in Table 4 are detected in a steroid extract. In other embodiments, all of the steroids set forth above are detected in a steroid extract. The extract can be prepared from a sample taken from an individual.

B„ Sample preparation by liquid-liquid extractiosi

[0053] The methods provided herein include extracting steroids from an individual's biological sample. In some embodiments, the individual is suspected of having a disorder or a disease, such as cancer, osteoporosis, neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease, a Parkinson's disease related disorder, prion disease, motor neuron disease, Huntington's Disease, spinocerebellar ataxia, spinal muscular atrophy, and dementia), cognitive disorder (e.g., mild cognitive impairment, decline of learning and/or memory processes, vascular cognitive impairment, age-related cognitive decline, Alzheimer's disease, Parkinson's disease, and dementia), cardiovascular disease or obesity. In some embodiments, the individual is suspected of having or has a hormone-related cancer, e.g., breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, prostate cancer, testis cancer, thyroid cancer, adrenal cancer, osteosarcoma endocrine cancer, a neuroendocrine tumor, an adrenal tumor, a carcinoid tumor, a parathyroid tumor, and a pituitary gland tumor. Non-limiting examples of cancers include bladder, brain, breast, bone, cervical, colon, colorectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, rectal, skin, stomach, uterine, ovarian, testicular, or hematologic cancer. The hematologic cancers include multiple myeloma, B-eell lymphoma, Hodgkin lymphoma/priniary mediastinal B-eell lymphoma, non~Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lyrnphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, precursor T-lymphoblastic lymphoma, and any combination thereof. Additional cancers include solid tumor cancers, e.g., non-small cell lung cancer, renal cell carcinoma and triple negative breast cancer, cancers of the head and neck, cutaneous or intraocular malignant melanoma, cancer of the fal lopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers, and any combination thereof. [0054] The biological sample can be whole blood, plasma, serum, saliva, mucus, urine, feces, cerebrospinal fluid, amniotic fluid, fine needle aspirate, cancer cells, cell culture media, or a tissue biopsy sample. In some embodiments, the tissue sample is obtained by, e.g., biopsy, from tissue suspected of being cancerous. In some instances, the tissue samples is from, for example, breast, ovary, vagina, vulva, cervix, endometrium, uterus, prostate, testis, adipose, bone, lung, thyroid, adrenal gland, salivary gland, gallbladder, bladder, urethra, kidney, liver, lymph, brain, other tissue of the body, tumor, or cancerous tissue.

[0055] The biological sample can be processed by liquid-liquid extraction. Any known method for the liquid-liquid extraction of steroids can be used. In some embodiments, the sample is suspended in a watenmetiianol mixture (1 : 1). In other embodiments, the tissue sample is ground, suspended in the water :methanol mixture, and homogenized. The steroids in the supernatant of the homogenate can be isolated by centrifugation, evaporation, and lyophilization. The lyophilized residue can be resuspended in a watenmetbanol mixture ( 1 : 1). Optionally, the resuspended sample can be filtered through a 5kD filter or a submicron filter, e.g., 0.2 μιπ filter. In yet other embodiments, the sample undergoes chloroform extraction prior to methanol extraction. The resulting aqueous phase can be lyophilized and subject to methanol extraction (e.g., methanol treatment, sonication, and centrifugation, and evaporation) and the organic phase can undergo multiple rounds of chloroform extraction and drying prior to methanol extraction.

C. Ultra-performance liquid chroma tography-tandem mass spectrometry (MS/MS)

[0056] The underivatized steroids in the extract can be purified under conditions suitable to produce one or more ions detectable by mass spectrometry. For instance, analytical separation of the extracted steroids of the sample can be performed by ultra-performance liquid chromatography (UPLC). In some embodiments, an UPLC analytical column with < 2 μηι particles, e.g., 1.9 μηι, 1.8 iim, 1.7 μηι, 1.6 μηι, 1.5 μηι particles is used. Exemplary UPLC columns include Acquity^*1 UPLC HSS T3 columns, e.g., 1.8 μτη, 1 x 150mm columns (Waters Corp.), Cortecs-C18^® columns, e.g., 1.6 μηι, 1 x 150mm columns (Waters Corp.) a d the like.

[0057] In some embodiments, during chromatography the temperature of the colum is maintained from about 40 °C to 55 °C, e.g., 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, or 55 °C. In some embodiments, the UPLC column is maintained at 45 °C. In other embodiments, the UPLC column is maintained at 50 °C.

[0058] In some embodiments, the flow rate of the column is at about 0,10 ml/min to about 0.20 ml/min, e.g., 0.10 ml/min, 0.11 ml/min, 0.12 ml/min, 0.13 ml/min, 0.14 ml/min, 0.15 ml/min, 0.16 ml/min, 0.17 ml/min, 0.18 ml/min, 0.19 ml/min, or 0.20 ml/min. In some embodiments, the flow rate is about 0.15 ml/min.

[0059] The gradient elution can be made using two solvents {e.g., A and B solvents). In some embodiments, the A solvent is 0.1% formic acid in water and the B solvent is 0.1% formic acid in acetonitrile. In some embodiments, the gradient is produced by the following method: 100% A and 0% B for 2 minutes, changed to 80% A and 20%o B over 2 minutes, 35% A and 65% B over 5 minutes, 20% A and 80% B over 2 minutes, and 100% A over 1 minute. In other embodiments, the gradient is produced by the following method: applying 100% A and 0% B for 1 minute, changing to 45% A and 55%> B at minute 6, 2Q%> A and 80%> B at minute 8, 0% A and 100% at minute 8.5, 0% A and 100% at minute 13, and 100% A at minute 14 and 15. Alternatively, the gradient is formed by: applying 100% A and 0% B to the column at time 0 and 2 minutes, changing to 80% A and 20% B at minute 4, 45% A and 55% B at minute 9, 20% A and 80% B at minute 1 1 , 100% B at minute 12, and 100% A at minutes 13 and 15 of the chromatography.

[0060] After the steroids are separated by UPLC, they are quantified using a mass spectrometer, e.g., a triple quadrupole mass spectrometer. The steroids can be delivered into the inlet of the mass spectrometer, preferably by electrospray ionization (ESI). The underivatized steroids in the extract can be ionized under conditions to produce one or more ions that are detectable by mass spectrometry. In some embodiments, the flow of liquid solvent from the chromatographic column enters the heated nebulizer interface of an MS/MS analyzer; and the solvent/analyte mixture is converted to vapor in the heated tubing of the interface. The steroid analytes contained in the nebulized solvent are ionized by the discharge needle of the interface, which applies a large voltage to the nebulized solvent analyte mixture, in some embodiments, electrospray ionization is performed in either positive or negative ion mode. Alternatively, ionization of the sample may also be performed by electron ionization, chemical ionization, photon ionization, atmospheric pressure chemical ionization (APCI), photoiomzation, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), liquid secondar ionization (LSI), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray/^'piasmaspray ionization, surface enhanced laser desorption ionization (SELDI), inductive!)' coupled plasma (ICP) and particle beam ionization. The skilled artisan will understand that the choice of ionization method may be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.

[0061] The ions, e.g. parent or precursor ions, can pass through the orifice of the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (Ql and Q3) are mass filters, allowing selection of ions (i.e., selection of "parent" and "daughter" ions in Ql and Q3, respectively) based on their mass to charge ratio (m/z). Quadrupole 2 (Q2) is the collision cel l, where ions are fragmented. The first quadrupole of the mass spectrometer (Ql) selects for molecules with the mass to charge ratios of one of the steroid analytes of interest. Parent ions with the correct mass/charge ratios are allowed to pass into the collision chamber (Q2), while unwanted ions with any other mass/charge ratio collide with the sides of the quadrupole and are eliminated. Parent ions entering Q2 collide with neutral collision gas molecules and fragment to generate daughter ions. The collision energy of Q2 can be varied to optimize the generation of daughter ions. The daughter ions generated are passed into quadrupole 3 (Q3), where the selected daughter ions of the steroid analytes of interest are detected. The parent- daughter ion transition is monitored.

[0062 J As ions collide with the detector they produce a pulse of electrons that are converted to a digital signal. The acquired data is relayed to a computer, which plots counts of the ions collected versus time. The areas under the peaks corresponding to particular ions, or the amplitude of such peaks, may be measured and correlated to the amount of the steroid of interest. In certain embodiments, the area under the curves, or amplitude of the peaks, for daughter ion(s) and/or parent ions are measured to determine the amount of each steroid detected. The relative abundance of a given ion may be converted into an absolute amount of the steroid in the sample using calibration standard curves based on peaks of one or more ions of an internal molecular standard . [0063] In some embodiments, a UPLC-MS/MS method with multiple reaction monitoring (MRM) is used to obtain high sensitivity and high resolution of the steroids of interest. In some embodiments, at least 5 MRM functions are performed. In other embodiments, about 5 to about 50 MRM functions, e.g., about 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 ,25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 MRM functions are performed. In some embodiments, about 31 MRM functions are performed. In yet other embodiments, about 30 MR M functions are performed. Optionally, selected ion recording (SIR) is also performed. In some embodiments, the plurality of MRM functions is set based on the parent-daughter ion transition(s) for the steroids of interest. In some instances, the parent-daughter ion transition(s) for the steroid(s) detected by the method described herein are provided in Table 1 or Table 4.

[0064] In some embodiments, the steroids detected in a SIR function include one or more, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26, of the following steroids: 7-dehydrocholesterol; 7a-QH cholesterol; 7b-OH cholesterol; 25- OH cholesterol; coprostanol-7,12-diol; 24-hydrocholesterol; 24-dehydrocholesterol; 22b-OH- cholesterol; zymosterol; 20a~QH~cholesterol; cholesterol; dihydrocholesterol; coprosterol; campesterol; 6-dehydrocholestenone; 24-keto-cholesterol; 4b-OH cholesterol; 6,7- dehydrocholesterol; 27-QH-cholesterol; 6-ketocholestenone; brassicaterol; 7-keto- cholesterol; lanosterol; stigmasterol; b-sitosterol; and 7~keto~25~QH cholesterol. In other embodiments, two or more of the following steroids: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 of the following steroids: 7- dehydrocholesterol; 7a-OH cholesterol; 7b-OH cholesterol; 25-OH cholesterol; coprostanol- 7,12-diol; 24-hydrocholesterol; 24-dehydrocholesterol; 22b-OH-cholesterol; zymosterol; 20a- OH-choIesterol; cholesterol; dihydrocholesterol; coprosterol; campesterol; 6- dehydrocholestenone ; 24-keto-cholesterol; 4b-OH cholesterol; 6, 7-dehydrocholesterol; 27- OH -cholesterol; 6-ketocholestenone; brassicaterol; 7-keto-cholesterol; lanosterol; stigmasterol; b-sitosterol; and 7-keto-25-OH cholesterol are detected in a SIR function. In yet other embodiments, 7-dehydrocholesterol; 7a-OH cholesterol; 7b-OH cholesterol; 25-OH cholesterol; coprostanol-7, 12-diol; 24-hydrocholesterol; 24-dehydrocholesterol; 22b-OH- cholesterol; zymosterol; 20a-OH-cholesterol; cholesterol; dihydrocholesterol; coprosterol; campesterol; 6-dehydrocholestenone ; 24-keto-cholesterol; 4b-OH cholesterol; 6,7- dehydrocholesterol; 27-OH-cholesterol; 6-ketocholestenone; brassicaterol; 7-keto- cholesterol; lanosterol; stigmasterol; b-sitosterol; and 7-keto-25-OH cholesterol are detected in a SIR function, in some instances, the SIR function described above is one function (e.g., MRM 1) of a total of 31 separate functions in MRM mode. In some cases, the SIR function can measure an oxysterol.

[0065] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, or 17 of the following steroids: estradiol E2; 13C labeled estradiol; 4-androstenediol; 1 1-ketoestrone; 6-dehydro testosterone; testosterone; adrenosterone; 1 1 -keto-etiocholanolone; 16a-hydroxy DHEA; 7b-hydroxy DHEA; 7a-hydroxy DHEA; 5-androsten-3b,17-diol-16-one; l la-hydroxy-4-pregnene-3,20- dione; 17a,20a-dihydroxyprogesterone; 20-hydroxypregnenolone; 11- dehydrotetrahydrocorticosterone; and b-muricholic acid. In other embodiments, the steroids detected in a MRM function include 2 or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14. 15, 16, or 17 of the following steroids: estradiol E2; 13C labeled estradiol; 4-androstenediol; 11- ketoestrone; 6-dehydrotestosterone; testosterone; adrenosterone; 1 1 -keto-etiocholanolone; 16a-hydroxy DHEA; 7b-hydroxy DHEA, 7a-bydroxy DHEA; 5-androsten-3b, 17-diol- 16-one; 1 la-hydroxy-4-pregnene-3,20-dione; 17a,20a-dihydroxyprogesterone; 20- hydroxypregnenolone; 1 l-dehydrotetrahydrocorticosterone; and b-muricholic acid. In yet other embodiments, estradiol E2; 13C labeled estradiol; 4-androstenediol; 11-ketoestrone; 6- dehydrotestosterone; testosterone; adrenosterone; I I -keto-etiocholanolone; 16a-hydroxy DHEA; 7b-hydroxy DHEA, 7a-hydroxy DHEA; 5-androsten-3b, 17-diol- 16-one; 1 1a- hydroxy-4-pregnene-3,20-dione; 17a,20a-dmydroxyprogesterone; 20-hydroxypregnenolone; 11-dehydrotetrahydrocorticosterone; and b-muricholic acid are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 2) of a total of 31 separate functions in MRM mode. n some cases, the MRM 2 function can measure an androgen, corticosteroid, progestogen, estrogen, bile acid, neurosteroid, or any combination thereof.

[0066] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, 3, or 4, of the following steroids: 3-methoxystriol; eltanolone; 3a-hydroxy- 5a-pregnan-20-one; 7-keto-25-OH cholesterol. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, or 4, of the following steroids: 3- methoxystriol; eltanolone; 3a-hydroxy-5a-pregnan-20-one; 7-keto-25-OH cholesterol. In yet other embodiments, 3-methoxystriol; eltanolone; 3a-hydroxy-5a-pregnan-20-one; 7-keto-25- OH cholesterol are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 3) of a total of 31 separate functions in MRM mode. In some cases, the MRM 3 function can measure an androgen, corticosteroid, progestogen, estrogen, oxysterol, neurosteroid, or any combination thereof.

[0067] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 or 23, of the following steroids: 6-dehydroestrone; 9(l l)~dehydroestrone; equilin; 6~dehydroestradiol; estrone El; DHEA GLUC; 17b-dihydroepiandrosterone; androstenedione; 9(1 l)-dehydro DHEA; epi testosterone, androstenolone (DHEA); 2-memoxy-3-QH-estradiol; 17a- hydroxy pregnenolone; 1 1 -ketoprogesterone; 1 lb-hydroxy-4-pregnene-3,20-dione; 17- hydroxyprogesterone; cortexone; 17-hydroxyprogesterone; 5a-pregnan-l Ia-QH-3,20-dione; 5a-dihydrocortexone (5a-DH-DOC); 17-hydroxypregnenolone; murocholic acid and ursodeoxycholic acid. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 or 23, of the following steroids: 6-dehydroestrone; 9(1 l)-dehydroestrone; equilin; 6- dehydroestradioi; estrone El ; DHEA GLUC; 17b-dihydroepiandrosterone; androstenedione; 9(1 l)-dehydro DHEA; epitestosterone, androstenolone (DHEA); 2-methoxy-3-OH-estradiol; 17a-hydroxypregnenolone; 11-ketoprogesterone; 1 lb~hydroxy~4~pregnene~3,20~dione; 17- hydroxyprogesterone; cortexone; 17-hydroxyprogesterone; 5a~pregnan-l l a-()H-3,20-dione; 5a-dihydrocortexone (5a-DH-DOC); 17-hydroxypregnenolone; murocholic acid and ursodeoxycholic acid. In yet other embodiments, 6-dehydroestrone; 9(1 l)-dehydroestrone; equilin; 6-dehydroestradiol; estrone El; DHEA GLUC; 17b-dihydroepiandrosterone; androstenedione; 9(1 l)-dehydro DHEA; epitestosterone, androstenolone (DHEA); 2- methoxy-3-OH-estradiol; 17a-hydroxypregnenolone; 1 1-ketoprogesterone; 1 lb-hydroxy-4- pregnene-3,20-dione; 17-hydroxyprogesterone; cortexone; 17-hydroxyprogesterone; 5a- pregnan-1 la-OH-3,20-dione; 5a-dihydroeortexone (5a-DH-DOC); 17-hydroxypregnenolone; murocholic acid and ursodeoxycholic acid are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 4) of a total of 31 separate functions in MRM mode. In some cases, the MRM 4 function can measure an androgen, corticosteroid, progestogen, estrogen, bile acid, neurosteroid, or any combination thereof. [0068] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, of the following steroids: 16-epiestriol; estriol E3; 17-epiestriol; 16,17-epiestriol; l la-hydroxyestradiol; 11b- hydroxyestradioi; 6b-hydroxyestradiol; 16-keto-l 7b-estradiol; 1 la-hydroxyestrone; 19- hydroxyandrostendione; 7a-hydroxyandrostenedione; 6b-OH-testosterone; 19-hydroxy DHEA; 6b-hydroxy DOC; Cortisol; giycodehydrocholic acid; testosterone- 17-Glu and 17,20- di-OH-progesterone-20-Glu. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. or 18, of the following steroids; 16-epiestriol; estriol E3; 17-epiestri.ol; 16,17-epiestriol; 1 1 a- hydroxyestradiol; 1 lb-hydroxy estradiol; 6b-hydroxyestradiol; 16-keto-17b-estradiol; 11a- hydroxyestrone; 19-hydroxy androstendione; 7a-hydroxyatidrostenedione; 6b~OH~ testosterone; 19-hydroxy DHEA; 6b-hydroxy DOC; Cortisol; giycodehydrocholic acid; testosterone-17-Glu and 17,20-di-OH-progesterone-20-G3.u. In yet other embodiments, 16- epiestriol; estriol E3; 17-epiestriol; 16,17-epiestriol; 1 la-hydroxyestradiol; 11b- hydroxyestradiol; 6b-hydroxyestradiol; 16-keto-17b-estradiol; 1 1 a-hydroxyestrone; 19- hydroxyandrostendione; 7a-hydroxyandrostenedione; 6b-OH-testosterone; 19-hydroxy DHEA; 6b-hydroxy DOC; Cortisol; giycodehydrocholic acid; testosterone- 17-Glu and 17,20- di-OH-progesterone-20-Gl are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 5) of a total of 31 separate functions in MRM mode. In some cases, the MRM 5 function can measure an androgen, corticosteroid, progestogen, estrogen, bile acid, neurosteroid, steroid conjugate, or any combination thereof.

[0069] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, or 14, of the following steroids: 6a- hydroxyestradiol; 2-hydroxyestradiol; 4-OH estradiol; 6-ketostrone; 16a-hydroxyestrone; 1 lb-OH-androstenedione; 7-ketoDHEA; 16a~ketotestosterone; Nl 1-ketotestosterone; 11a- hydroxyandrostenedione; 2-OH-testosterone; 17-hydroxypregnenolone; urocortisol and taurolithochoiic acid. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, or 14, of the following steroids: 6a-hydroxyestradiol; 2-hydroxyestradiol; 4-OH estradiol; 6-ketostrone; 16a-hydroxyestrone; 1 lb-OH-androstenedione; 7-ketoDHEA; 16a-ketotestosterone; N1 1-ketotestosterone; 11 a- hydroxyandrostenedione; 2-OH-testosterone; 17-hydroxypregnenolone; urocortisol and taurolithochoiic acid. In yet other embodiments, 6a-hydroxyestradiol; 2-hydroxyestradioi; 4- OH estradiol; 6-ketostrone; I6a-hydroxyestrone; 1 Ib-OH-androstenedione; 7-ketoDHEA; 16a-ketotestosterone; Nl 1-ketotestosterone; 1 1 a-hydroxyandrostenedione; 2-OH- testosterone; 17-hydroxypregnenolone; urocortisol and taurolithochoiic acid are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 6) of a total of 31 separate functions in MRM mode. In some cases, the MRM 6 function can measure an androgen, corticosteroid, progestogen, estrogen, bile acid, neurosteroid, or any combination thereof.

[0070] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 , of the following steroids: 9(11 Vdehydroestradiol; 5-androstenediol; 9-dehydroepiandrosterone; etiocholan-3-OH-17-one; 4-OMe estradiol; 2,3- dimethox estradiol; pregnelone; 20a-dihydroprogesterone; pregnanetriol; 5 a- dihydrocortexone (5a-DH-DOC) and allotetrahydrocortexone. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 , of the following steroids: 9(1 l)-dehydroestradiol; 5-androstenediol; 9- dehydroepiandrosterone; etiocholan-3-OH-17-one; 4-OMe estradiol; 2,3-dimethox estradiol; pregnelone; 20a-dihydroprogesterone; pregnanetriol; Sa-dihydrocortexone (5a-DH-DOC) and allotetrahydrocortexone. In yet other embodiments, 9(l l)-dehydroestradiol; 5- androstenediol; 9-dehydroepiandrosterone; etiocholan-3-OH- 17-one; 4-OMe estradiol; 2,3- dimethox estradiol; pregnelone; 20a-dihydroprogesterone; pregnanetriol; 5a- dihydrocortexone (5a-DH-DOC) and allotetrahydrocortexone are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 7) of a total of 31 separate functions in MRM mode. In some cases, the MRM 7 function can measure an androgen, corticosteroid, progestogen, estrogen, oxysterol, neurosteroid, or any combination thereof. [0071 J In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, or 18, of the following steroids: 7-dehydro-17b-estradiol; 4-0 H estrone; 6-ketoestradiol; 2-OH estril; 1 1b- hydroxy estrone; 2-OH estrone; 4-androsten-3,6-17-trione; 6-ketotesterone; 2a- hydroxyandrostenedione; 16a-hydroxyepiandrosterone; 1 1 b-hydroxyepiandrosterone; 7a- hydroxypregnenolone; 16a-hydroxyprogesterone; 5a-epoxypregnenolone; eortexolone; corticosterone; tetrahydrocorticosterone; and glycodeoxycholic acid. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, or 18, of the following steroids: 7-dehydro-17b-estradiol; 4-OH estrone; 6-ketoestradiol; 2-OH estril; 1 lb-hydroxyestrone; 2-OH estrone; 4~androsten-3,6-17~ trione; 6-ketotesterone; 2a-hydroxyandrostenedioiie; 16a-hydroxyepiandrosterone; 11b- hydroxyepiandrosterone; 7a-hydroxypregnenolone; 16a-hydroxyprogesterone; 5a- epoxypregnenolone; eortexolone; corticosterone; tetrahydrocorticosterone; and glycodeoxycholic acid. In yet other embodiments, 7-dehydro- 17b-estradio f; 4-OH estrone; 6- ketoestradiol; 2-OH estril; 1 lb-hydroxyestrone; 2-OH estrone; 4-androsten-3 ,6- 17-trione; 6- ketotesterone; 2a-hydroxyandrostenedione; 16a-hydroxyepiandrosterone; 1 lb- hydroxyepiandrosterone; 7a-hydroxypregnenolone; 16a-hydroxyprogesterone; 5 a- epoxypregnenolone; cortexolone; corticosterone; tetrahydrocorticosterone; and glycodeoxyc olic acid are detected in a MRM function. In some instances, the MRM function described above is one function (e.g. , MRM 8) of a total of 31 separate functions in MRM mode. In some cases, the MRM 8 function can measure an androgen, corticosteroid, progestogen, estrogen, bile acid, neurosteroid, or any combination thereof.

[0072] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15, of the following steroids: 4- androstene-30L, 17-oiie; 17b-diiiydroandrosterone; allodihydrotestosterone; etiocholanolone glueurottide; epiandrosterone; etiocholanolone; 5a-dihydrotestosterone;3~methoxy~2~OH- estrone; 4-OMe estone; androstandiol-3-Ghi; 5a-dihydro-l 1-keto-progesterone; 21- hydroxypregnanolon; allocholesterol; 6~keto-allolithocholic acid; and glycodeoxycholic acid. In some embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, of the following steroids: 4-androstene-3QL,17~ one; 17b-dihydroandrosterone; allodihydrotestosterone; etiocholanolone glucuronide; epiandrosterone; etiocholanolone; 5a-dihydiOtestosterone;3-methoxy-2-OH-estiOne; 4-QMe estone; androstandiol-3-Giu; 5a~dihydro-l 1 -keto-progesterone; 21 -hvdroxypregnanolon; allocholesterol; 6-keto-alloIitliocliolic acid; and glycodeoxycholic acid. In yet other embodiments, 4~androstene-3QL, 17-one; 17b-dihydroandrosterone; allodihydrotestosterone; etiocholanolone glucuronide; epiandrosterone; etiocholanolone; 5a-dihydrotestosterone;3- methoxy-2-OH-estrone; 4-OMe estone; androstandiol-3-Glu; 5a~dihydro-l 1 -keto- progesterone; 21-hydroxypregnanolon; allocholesterol; 6-keto-allolithocholic acid; and glycodeoxycholic acid are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 9) of a total of 31 separate functions in MRM mode. In some cases, the MRM 9 function can measure an androgen, corticosteroid, progestogen, estrogen, bile acid, neurosteroid, steroid conjugate, or any combination thereof.

[0073] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, of the following steroids: desoxytestosterone; 25-OH cholesterol; zymosterol; 20a-OH-cholesterol; 24-hydroxycholesterol; Me-3-()H-chol-5-n~24- oate; 27-OH-cholesterol; coprostanol-7,12-diol and 22b-QH-cholesteroI. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, 6, 7, 8, or 9, of the following steroids: desoxytestosterone; 25-OH cholesterol; zymosterol; 20a- OH -cho lesterol; 24-hydrox.y cho 1 esterol; Me-3 -O H-chol-5 -n-24-oate; 27-0 H-choles terol; coprostanol-7,12-diol and 22b-OH-cholesterol. In yet other embodiments, desoxytestosterone; 25-OH cholesterol; zymosterol; 20a-OH-cholesterol; 24- hydroxycholesterol; Me-3-OH-chol-5-n-24-oate; 27-OH-cholesterol; coprostanol-7, 12-diol and 22b-OH-cholesterol are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 1 0) of a total of 31 separate functions in MRM mode. In some cases, the MRM 10 function can measure an androgen, corticosteroid, progestogen, estrogen, oxysterol or any combination thereof. [0074 J In some embodiments, the steroids detected in a M RM function include one or more, e.g. , 1 , 2, 3, 4, 5, or 6, of the following steroids: 3-OMe strone; 2-methoxy-2-OH- estrone; 5b-dihydroprogesterone; 5a-dihydroprogesterone; eampesterol; and allolithocholic acid. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, or 6, of the following steroids: 3-OMe strone; 2-methoxy-2-OH-estrone; 5b- dihydroprogesterone; 5a-dihydroprogesterone; eampesterol; and allolithocholic acid. In yet other embodiments, 3-OMe strone; 2-methoxy-2-OH-estrone; 5b-dihydroprogesterone; 5 a- dihydroprogesterone; eampesterol; and al lolithocholic acid are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 1 1) of a total of 31 separate functions in MRM mode. In some cases, the MRM 1 1 function can measure an androgen, corticosteroid, progestogen, estrogen, oxysterol, neurosteroid, or any combination thereof.

[0075] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or I I , of the following steroids: 5b-androstandione; androsterone; 4-pregnen-3b-ol-20-one; allopregnanediol; pregnanediol; 9- dehydroprogesterone; 2,3-dimethoxyestrone; progesterone; 20a-hydroxy-5a-pregnan-3-one; hyodeoxycholic acid; and chenodeoxycholic acid. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 , of the following steroids: 5b~androstandione; androsterone; 4-pregnen-3b-ol-20-one; al lopregnanediol; pregnanediol; 9-dehydroprogesterone; 2,3-dimethoxyestrone; progesterone; 20a-hydroxy-5a-pregnan-3-one; hyodeoxycholic acid; and chenodeoxycholic acid. In yet other embodiments, 5b-androstandione; androsterone; 4-pregnen-3b-oi-20-one; allopregnanediol; pregnanediol; 9-dehydroprogesterone; 2,3-dimethoxyestrone; progesterone;

20a-hydroxy-5a-pregnan-3-one; hyodeoxycholic acid; and chenodeoxycholic acid are detected in a MRM function. In some instances, the MRM function described above is one function {e.g., MRM 12) of a total of 31 separate functions in MRM mode. In some cases, the MRM 12 function can measure an androgen, corticosteroid, progestogen, estrogen, bile acid, neurosteroid, or any combination thereof. [0076] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, 3, 4, 5, or 6, of the following steroids: 7a-hydroxyandrostenediol (DHEA); 7b-hydroxyandrostenediol (DHEA); 4-OMe estriol; 6a-OH-testosterone; 7a- hydroxytestosterone; and aldosterone. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 1 , 2, 3, 4, 5, or 6, of the following steroids: 7a- hydroxyandrostenediol (DHEA); 7b-hydroxyandrostenedio f (DHEA ); 4-OMe estriol; 6a-OH- testosterone; 7a-hydroxytestosterone; and aldosterone. In yet other embodiments, 7a- hydroxyandrostenediol (DHEA); 7b~hydroxyandrostenediol (DHEA); 4-OMe estriol; 6a-OH- testosterone; 7a-hydroxytestosterone; and aldosterone are detected in a MRM function. In some instances, the MRM function described above is one function {e.g., MRM 13) of a total of 31 separate functions in MRM mode. In some cases, the MRM 13 function can measure an androgen, corticosteroid, progestogen, estrogen, neurosteroid, or any combination thereof.

[0077] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 or 2, of the following steroids: 6-ketoestriol and estriol- 17-Glu. In other embodiments, 6-ketoestriol and estriol- 17-Glu are detected in a MRM function. In some instances, the MRM function described above is one function {e.g., MRM 14) of a total of 31 separate functions in MRM mode. In some cases, the MRM 14 function can measure an androgen, corticosteroid, progestogen, estrogen, or any combination thereof.

[0078] In some embodiments, the steroids detected in a MRM function includes lithocholic acid. In other embodiments, lithocholic acid is detected in a MRM function. In some instances, the MRM function described above is one function {e.g., MRM 15) of a total of 31 separate functions in MRM mode. In some cases, the MRM 15 function can measure a bile acid.

[0079] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, 3, or 4, of the following steroids: 7-dehydrocholesterol; 24- dehydrocholesterol; 6-dehydrocholestenone; and 24-keto-cholesterol. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, or 4, of the following steroids: 7-dehydrocholesterol; 24-dehydrocboiesterol; 6-dehydrochoiestenon.e; and 24-keto- cholesterol, in yet other embodiments, 7-dehydrocholesterol; 24-dehydrocholesterol; 6- dehydrocholestenone; and 24-keto-cholesterol are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 16) of a total of 31 separate functions in MRM mode. In some cases, the MRM 16 function can measure an oxysterol.

[0080] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, 3, 4, 5, 6, or 7, of the following steroids: 25 -Of I cholesterol; lathosterol; 4- ehoiestene-3a-()L; cholesterol; lanosterol; dihydrocholesterol; and coprosterol. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, 6, or 7, of the following steroids: 25-OH cholesterol; lathosterol; 4~eho!estene-3a-()L; cholesterol; lanosterol; dihydrocholesterol; and coprosterol. In yet other embodiments, 25- OH cholesterol; lathosterol; 4-cholestene-3a-OL; cholesterol; lanosterol; dihydrocholesterol; and coprosterol are detected in a MRM function. In other embodiments, 6-ketoestriol and estriol-17-Glu are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 17) of a total of 31 separate functions in MRM mode. In some cases, the MRM 17 function can measure an androgen, progestogen, oxysterol, or any combination thereof.

[0081] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, 3, 4, or 5, of the following steroids: 7a-OH cholesterol; 7b-OH cholesterol; 22b-OH -cholesterol; stigmasterol; and 7-keto-cholesterol. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, or 5, of the following steroids: 7a-OH cholesterol; 7b-OH cholesterol; 22b-OH -cholesterol; stigmasterol; and 7-keto- cholesterol. In yet other embodiments, 7a-OH cholesterol; 7b-OH cholesterol; 22b-OH- cholesterol; stigmasterol; and 7-keto-cholesterol are detected in a MRM function. In other embodiments, 6-ketoestriol and estrioi-17-Glu are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 18) of a total of 31 separate functions in MRM mode. In some cases, the MRM 18 function can measure an oxysterol.

[0082] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, or 3, of the following steroids: 22b-QH-eholesterol; 7b-OH cholesterol; and brassicaterol. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, or 3, of the following steroids: 22b-OH-cholesterol; 7b-OH cholesterol; and brassicaterol. In yet other embodiments, 22b-OH-cholesterol; 7b-OH cholesterol; and brassicaterol are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 19) of a total of 31 separate functions in MRM mode. In some cases, the MRM 19 function can measure an oxysterol. [0083] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, 3, or 4, of the following steroids: epiailocholesterol; 4-cholestene-3a-OL; 6,7-dehydrocholesteroi; and 4b-OH cholesterol. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, or 4, of the following steroids: epiailocholesterol; 4-cholestene-3a-OL; 6,7-dehydrocholesteroi; and 4b-OH cholesterol. In yet other embodiments, epiailocholesterol; 4-cholestene-3a-OL; 6,7-dehydrocholesterol; and 4b-OH cholesterol are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 20) of a total of 31 separate functions in MRM mode. In some cases, the MRM 20 function can measure an oxysterol, androgen, progestogen, or any combination thereof. [0084 J In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, or 2, of the following steroids: 6-ketocholestenone and b-sitosterol. In yet other embodiments, 6-ketocholestenone and b-sitosterol are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 21) of a total of 31 separate functions in MRM mode. In some cases, the MRM 21 function can measure an oxysterol.

[0085] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, 3, 4, 5, or 6, of the following steroids: estradio3-3,17-di-S04; 2-OH estril; estradiol-3-Glu; estradiol- 17-Glu; taurodehydrocholic acid and tauro-b-muricholic acid. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, or 6, of the following steroids: estradiol -3, 17-di-S0₄; 2-OH estril; estradiol -3 -Giu; estradiol- 17-Glu; taurodehydrocholic acid and tauro-b-muricholic acid. In yet other embodiments, estradiol-3,17-di-S0₄; 2-OH estril; estradiol-3-Glu; estradiol- 17-Glu; taurodehydrocholic acid and tauro-b-muricholic acid are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 22) of a total of 31 separate functions in MRM mode. In some cases, the MRM 22 function can measure an androgen, corticosteroid, progestogen, estrogen, bile acid, or any combination thereof. [0086] In some embodiments, the steroids detected in a MRM function include one or more, e.g. , 1, 2, 3, 4, or 5, of the following steroids: estradiol E2; 4-OH estrone; pregnenolone-3-Glu; pregnanediol-3-Giu; and taurodeoxycholic acid. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, or 5, of the following steroids: estradiol E2; 4-OH estrone; pregnenolone-3-G i; pregnanediol-3-Glu; and taurodeoxycholic acid. In yet other embodiments, estradiol E2; 4-OH estrone; pregnenolone- 3-Glu; pregnanediol-3-Glu; and taurodeoxycholic acid are detected in a M RM function. In some instances, the MRM function described above is one function (e.g., MRM 23) of a total of 31 separate functions in MRM mode. In some cases, the MRM 23 function can measure an androgen, corticosteroid, progestogen, estrogen, bile acid, steroid conjugate, or any combination thereof.

[0087] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, 3, 4, or 5, of the following steroids: 2-OH estrone; epitestosterone- 17- S04; testosterone sulfate; a-muricholic acid; and etiocholanolone-3-Glu. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, or 5, of the following steroids: 2-OH estrone; epitestosterone- 17-S0₄; testosterone sulfate; a-muricholic acid; and etiocholanolone-3-Glu. In yet other embodiments, 2-OH estrone; epitestosterone- 17-SQ₄; testosterone sulfate; a-muricholic acid; and etiocholanolone-3-Glu are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 24) of a total of 31 separate functions in MRM mode. In some cases, the MRM 24 function can measure an androgen, corticosteroid, progestogen, estrogen, bile acid, steroid conjugate, or any combination thereof.

[0088] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, 3, 4, 5, 6, 7, or 8, of the following steroids: equifm-3-S0₄; dihydroequilin-3- S0₄; estradiol- I7-SO4; estradiol-3-S0₄; cortisol-21-S0₄; estrone-Glu; testosterone- 17-Glu and 17,20-di-OH-progesterone-20-G3.u. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, 6, 7, or 8, of the following steroids: equilin-3-S0₄; dihydroequilin-3-S0₄; estradiol- 1.7-S0₄; estradiol-3-S04; cortisol-21 -S0₄; estrone-Glu; testosterone- 17-Glu and 17,20-di-OH-progesterone-20-Glu. In yet other embodiments, equilin-3-S0₄; dihydroequilin-3-S04; estradiol- 17-S0₄; estradiol-3-S04; cortisol-21-S0₄; estrone-Glu; testosterone- 17-Glu and 17,20-di-OH-progesterone-2G-Glu are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 25) of a total of 31 separate functions in MRM mode. In some cases, the MRM 25 function can measure an androgen, corticosteroid, progestogen, estrogen, steroid conjugate, or any combination thereof.

[0089] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, 3, 4, 5, 6, or 7, of the following steroids: estrone-S0₄; dehydroepiandrosterone sulfate (DHEAS); 1 l-keto-etiocholanolone-3-Glu; tauroursodeoxycholic acid; taurohyodeoxyeholic acid; iaurochenodeoxycholic acid; and taurochoiic acid. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, 5, 6, or 7, of the following steroids: estrone-S0₄; dehydroepiandrosterone sulfate (DHEAS); l l-keto-etiocholanolone-3-Glu; tauroursodeoxycholic acid; iaurohyodeoxycholic acid; iaurochenodeoxycholic acid; and taurochoiic acid. In yet other embodiments, estrone-S0₄; dehydroepiandrosterone sulfate (DHEAS); 1 l-ket.o-etiocholanokme-3-Glu; tauroursodeoxycholic acid; taurohyodeoxyeholic acid; taurochenodeoxyeholic acid; and taurochoiic acid are detected in a MRM function. In some instances, the MRM function described above is one function {e.g., MRM 26) of a total of 31 separate functions in MRM mode. In some cases, the MRM 26 function can measure an androgen, corticosteroid, progestogen, estrogen, steroid conjugate, bile acid, or any combination thereof.

[0090] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, or 3, of the following steroids: estrioI-3 -sulfate; estriol-3-Glu~Na and estradiol-3,17-di-Glu. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, or 3, of the following steroids: estriol-3-sulfate; estriol-3-Glu-Na and estradiol-3,17-di-Giu. In yet other embodiments, estriol-3 -sulfate; estrioi-3-Gl -Na and estradiol-3,17-di-Glu are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 27) of a total of 31 separate functions in MRM mode. In some cases, the MRM 27 function can measure an androgen, corticosteroid, progestogen, estrogen, or any combination thereof.

[0091] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, or 2, of the following steroids: desoxycholic acid and glycolithoeholic acid. In yet other embodiments, desoxycholic acid and glycolithoeholic acid are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 28) of a total of 31 separate functions in MRM mode. In some cases, the MRM 28 function can measure a bile acid. [0092] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, 3 or 4 of the following steroids: 3b-hydroxy-5-pregnen~2Q~Qne~3-SQ₄; cholic acid; glycochenodeoxycholic acid; and 17-OH-pregnanolone-3-Glu-Na. In some embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3 or 4 of the fo! lowing steroids: 3b-hydroxy-5-pregnen-2()-one-3-S04; cholic acid; glycochenodeoxycholic acid; and 17-OH~pregnanolone-3-Glu-Na, In yet other embodiments, 3b-hydroxy-5-pregnen-20-one-3-SO4; cholic acid; glycochenodeoxycholic acid; and 17-OH- pregnanolone-3-Glu-Na are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 29) of a total of 31 separate functions in MRM mode. In some cases, the MRM 29 function can measure a neurosteroid, bile acid, steroid conjugate or any combination thereof.

[0093] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1, 2, or 3, of the following steroids: pregnan.Glone-3-S04; allopregnanolone-S04; and epiallopregnanolone-S0₄. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, or 3, of the following steroids: pregnanolone-3-80₄; allopregnanolone-S0₄; and epialiopregnanolone~SQ₄. In yet other embodiments, pregnanolone-3-S04; allopregnanolone-S()4; and epiallopregnanoione-804 are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 30) of a total of 31 separate functions in MRM mode. In some cases, the MRM 30 function can measure a steroid conjugate.

[0094] In some embodiments, the steroids detected in a MRM function include one or more, e.g., 1 , 2, 3, 4, or 5, of the following steroids: choiesterol-3-S0₄; taurocholanic acid; 5a,6a-epoxycho1esterol; epiallocholesterol; and cholesterol-3-Glu. In other embodiments, the steroids detected in a MRM function include two or more, e.g., 2, 3, 4, or 5, of the following steroids: cholesterol-3-S0₄; taurocholanic acid; 5a,6a-epoxycholesterol; epiallocholesterol; and cholesteroi-3-Glu. In yet other embodiments, cholesteroi-S-SC ; taurocholanic acid; 5a,6a-epoxycholesterol; epiallocholesterol; and cholesteroI-3-Glu are detected in a MRM function. In some instances, the MRM function described above is one function (e.g., MRM 31 ) of a total of 31 separate functions in MRM mode. In some cases, the MRM 31 function can measure a steroid conjugate, oxy sterol, bile acid, or any combination thereof.

[0095] In some embodiments, the detection time window for the MRM function is one of the following ranges: time 7.00 to 15.00 minutes, time 4.50 to 6.50 minutes, time 7.00 to 9.00 minutes, time 5.00 to 7.00 minutes, time 3.00 to 5.00 minutes, time 3.50 to 5.50 minutes, time 6.00 to 8.00 mmutes, time 4.00 to 6.00 minutes, time 5.50 to 7.50 minutes, time 9.00 to 11.00 minutes, time 7.50 to 9.50 mmutes, time 6.50 to 8.00 mmutes, time 2.50 to 4.50 minutes, time 8.00 to 10.00 minutes, time 11.00 to 13.00 minutes, time 12.00 to 15.00 mmutes, time 9.50 to 1 1.50 minutes, time 10.50 to 12.50 minutes, time 11.50 to 13.50 mmutes, time 10.00 to 12.00 minutes, time 2.50 to 4.50 minutes, time 4.50 to 6.50 minutes, time 4.00 to 6.00 minutes, time 3.00 to 5.00 minutes, time 3.50 to 5.50 minutes, time 0.00 to 3.50 mmutes, time 6.50 to 8.50 mmutes, time 5.00 to 7.00 mmutes, time 5.50 to 7.50 minutes, and time 9.5 to 15.00 mmutes. In other embodiments, the detection time window is based on the selected parent-daughter ion transition(s) for the steroid(s) of interest and/or the selected MRM function. In some embodiments, a MRM function and its corresponding detection time window provided in Table 6 are used to measure the steroid(s) listed in Table 7 corresponding to the MRM function.

[0096] in some embodiments, the at least 200 steroids in the sample are quantified according to the method described herein, wherein the detection time windows in MRM mode include 7.00 to 15.00 minutes, 4.50 to 6.50 minutes, 7.00 to 9.00 mmutes, 5.00 to 7.00 minutes, 3.00 to 5.00 minutes, 3.50 to 5.50 minutes, 6.00 to 8.00 minutes, 4.00 to 6.00 mmutes, 5.50 to 7.50 mmutes, 9.00 to 11.00 minutes, 7.50 to 9.50 minutes, 6.50 to 8.00 minutes, 2.50 to 4.50 minutes, 8.00 to 10.00 minutes, 1 1 .00 to 13.00 minutes, 12.00 to 15.00 minutes, 9.50 to 11.50 minutes, 10.50 to 12.50 mmutes, 11.50 to 13.50 minutes, 10.00 to 12.00 minutes, 2.50 to 4.50 minutes, 4.50 to 6.50 minutes, 4.00 to 6.00 mmutes, 3.00 to 5.00 minutes, 3.50 to 5.50 minutes, 0.00 to 3.50 minutes, 6.50 to 8.50 minutes, 5.00 to 7.00 minutes, 5.50 to 7.50 minutes, 9.5 to 15.00 minutes, and any combination thereof, in some cases, the useful detection time windows for the method described herein are provided in Table 6. In some embodiments, the selected MRM functions utilize at least 5, e.g., 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more, detection time windows to profile the at least 100 or at least 200 steroids. In other embodiments, the selected MRM functions utilize at least 10, e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more, detection time windows to profile the at least 100 or at least 200 steroids. In yet other embodiments, at least 30, e.g., 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more, detection time windows are used to measure the at least 100 or the at least 200 steroids in a sample.

3 [0097] The number of MRM functions can correspond to the number of detection time windows used to detect the steroids of interest, e.g., the at least 100 steroids of interest. In some embodiments, the method described herein utilizes at least 30 (e.g., 31) MRM functions and at least 30 (e.g., 31) detection time windows as set forth in Table 6 to detect and measure the steroids set forth in Table 7. In some instances, each of the steroid(s) detected in a specific MRM function of Table 6 is set forth in Table 7. For example, the method provided herein can utilize a MRM function of 5 mass pairs with a detection time window' of 2.50 to 4.50 minutes (see, MRM 13 of Table 6) and detect 6 steroids, such as 7a- hydroxyandrostenediol (DHEA); 7b-hydroxyandrostenedioI (DHEA); 4-OMe estriol; 6a-OH- testosterone; 7a-hydroxytestosterone; and aldosterone (see, MRM 13 of Table 7).

[0098] In some embodiments, the presence and/or level of the steroids (e.g., the at least 100 steroids) in the biological sample are detected in about 5 minutes to about 25 minutes. In other embodiments, the presence and/or levels of the steroids (e.g., the at least 100 steroids) are detected in about 10 minutes to about 20 minutes. In yet other embodiments, the presence and/or levels of the steroids (e.g., the at least 100 steroids) are detected in about 10 minutes to about 15 minutes (e.g., about 10, 11, 12, 13, 14, or 15 minutes).

[0099] The results of a mass spectrum may be related to the amount of the steroid in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion may be compared to a table that converts that relative abundance to an absolute amount of the original steroid molecule. Alternatively, external standards may be run with the samples, and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion may be converted into an absolute amount of the steroid molecule present in the biological sample. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the quantity of one or more steroids. Methods of generating and using such standard curves are well known in the art and one of ordinary skill is capable of selecting an appropriate internal standard. Numerous other methods for relating the amount of an ion to the amount of the corresponding steroid in the biological sample are well known to those of ordinary skill in the art. D. Steroid nietabolome profiling

[0100] A. profile of an individual's steroid rnetabolorne can be determined based on the presence and/or level of the steroids detected by the method described herein. The steroid rnetabolorne profile can be used to determine whether the individual has an imbalance in steroid metabolism (e.g., an alteration in steroid metabolism), or a disease associated with such an imbalance. In some embodiments, the steroid rnetabolorne profile includes the presence and/or level of one or more neurosteroids.

[0101] For instance, a reference steroid rnetabolorne profile associated with an individual having normal steroid metabolism can be compared to that of a test subject. If there are differences between the reference profi le and the test subject's profile (e.g., less than 30%, 25%, 20%), 15%, 10%, 5%), or 1% overlap between the reference and test steroid rnetabolorne profiles) based on the presence of the same steroids and/or the levels (e.g., normalized or relative amounts) of the same steroids, then the test subject has a steroid imbalance. If there are similarities between the reference profile and the test subject's profile {e.g., greater than 70%, 75%), 80%, 85%, 90%), or 95% overlap between the reference and test steroid rnetabolorne profiles based on the presence of the same steroids and/or the levels (e.g., normalized or relative amounts) of the same steroids, then the test subject does not have a steroid imbalance. In some embodiments, a reference steroid rnetabolorne profile is determined from one or more subjects having a normal steroid metabolism. In some cases, the profile is based on the average level of steroids detected in a population of subjects with a normal steroid metabolism .

[0102] In other embodiments, the reference steroid rnetabolorne profile is of an individual with a steroid imbalance or a disease/disorder associated with a steroid imbalance. If there are similarities between a reference profile from an individual with a steroid imbalance or an associated disease/disorder and the test subject's profile (e.g., greater than 70%), 75%), 80%, 85%, 90%, or 95% overlap between the reference and test steroid rnetabolorne profiles) based on the presence of the same steroids and/or the levels (e.g., normalized or relative amounts) of the same steroids, then the test subject has a steroid imbalance or the associated disease/disorder. If there are differences between the reference steroid imbalance profile and the test subject's profile (e.g., less than 30%, 25%, 20%, 15%, 10%, 5%, or 1% overlap between the reference and test steroid rnetabolorne profiles) based on the presence of the same steroids and/or the levels (e.g., normalized or relative amounts) of the same steroids, then the test subject does not have a steroid imbalance or a disease/disorder associated with a steroid imbalance. The reference steroid imbalance profile can be derived from one or more subjects having a steroid imbalance or a disease/disorder associated with a steroid imbalance. The profile can be based on the average level of steroids detected in a population of subjects with impaired steroid metabolism or a disease/disorder associated with such an impairment. [0103] The determination of a steroid imbalance may indicate that the individual has cancer, e.g., lung cancer, colorectal cancer, esophageal cancer, head and neck cancer, brain cancer, liver cancer, or hormone-related cancer, e.g., breast cancer, endometrial cancer, ovarian cancer, pancreatic cancer, prostate cancer, testis cancer, thyroid cancer, adrenal cancer, osteosarcoma endocrine cancer, a neuroendocrine tumor, an adrenal tumor, a carcinoid tumor, a parathyroid tumor, and a pituitary gland tumor; osteoporosis; a neurodegenerative disease; a cognitive disorder, a psychiatric disorder, multiple sclerosis; cardiovascular disease; infertility; Gushing 's syndrome; endometriosis; or obesity. Examples of neurodegenerative diseases include, but are not limited to, Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ATS), Niemann- Pick type C disease, Smith-Lemli Optiz syndrome, a Parkinson's disease-related disorder, prion disease, motor neuron disease, Huntington's Disease, spinocerebellar ataxia, spinal muscular atrophy, and dementia. Examples of cognitive disorders include, but are not limited to, mild cognitive impairment, vascular cognitive impairment, a decline of learning and/or memory processes, age-related cognitive decline, Alzheimer's disease, Parkinson's disease, delirium, amnesia, and dementia. Non-limiting examples of a psychiatric disorder include depression, schizophrenia, an anxiety disorder, such as generalized social phobia, generalized anxiety disorder, panic disorder, and posttraumatic stress disorder, acute stress, chronic stress, and a mood disorder. Non-limiting examples of cardiovascular diseases include coronary disease, cardiac hypertrophy, heart failure, myocardial infarction, stroke, atherosclerosis, and arrhythmias. Other conditions associated with alterations in steroid metabolism include cortiso i-induced immune suppression, hyperglycemia, hyperlipidemia, insulin resistance, obesity, osteoporosis, memory impairment, and altered thyroid function,

[0104] In some embodiments, the steroid metabolome profile generated according to the methods described herein can be used to determine whether a subject has a particular type of cancer. For instance, a steroid metabolome profile associated with a cancer such as breast cancer (e.g., breast cancer reference profile) can be compared to that of a test subject. For example, if there are similarities between the reference profile and the test subject's profile (e.g., greater than 70%, 75%, 80%. 85%, 90%, or 95% overlap between the reference and test steroid metabolome profiles) based on the presence of the same steroids and/or the levels (e.g., normalized or relative amounts) of the same steroids, then the test subject has that type of cancer (e.g., breast cancer). If there are few or no similarities between the profiles (e.g., less than 30%, 25%, 20%, 15%, 10%, 5%, 1% or 0% overlap between the reference and test steroid metabolome profiles), then the test subject does not have that type of cancer (e.g., breast cancer), A comparison can be performed with reference profiles of other cancers, such as lung cancer, prostate cancer, adrenal cancer, thyroid cancer, pancreatic cancer, gastrointestinal cancer, pituitary cancer, colorectal cancer, ovarian cancer, liver cancer, kidney cancer, endometrial cancer, neuroendocrine cancer, bone cancer, hematological cancer, and a combination thereof. In some instances, the test subject's profile may be compared to a plurality of reference profiles until a similarity between steroid metabolome profiles is determined. The methods described herein can be used to rule -out and/or rule-in the presence of specific cancers in an individual.

[0105] In some embodiments, provided herein is a method for diagnosing a cognitive disorder, such as mild cognitive impairment, vascular cognitive impairment, decline of learning and/or memory processes, age-related cognitive decline, A lzheimer's disease, Parkinson's disease, delirium, amnesia, and dementia. The method includes profiling a steroid metabolome (e.g., at least 100 steroids) comprising one or more neurosteroids in a biological sample obtained from the individual according to the method described herein to form a steroid metabolome profile for the individual. For instance, the level of one or more neurosteroids, metabolites of a neurosteroid, neurosteroi variants and/or neurosteroi derivatives can be measured. The individual's steroid metabolome profile can be compared to a reference steroid metabolome profile for a specific cognitive disorder (e.g., age-related cognitive decline). If the individual's steroid profile is substantially similar to the reference profile for a specific cognitive disorder (e.g., greater than 70%, 75%, 80%, 85%, 90%, or 95% overlap between the reference and test steroid metabolome profiles) based on the presence of the same steroids and/or the levels (e.g., normalized or relative amounts) of the same steroids, it is determined that the individual has the cognitive disorder (e.g., age-related cognitive decline). If there are few or no similarities between the profiles (e.g., less than 30%, 25%, 20%), 15%, 10%, 5%, 1 % or 0% overlap between the reference and test steroid metabolome profiles), the individual does not have the specific cognitive disorder. In some instances, the individual's steroid profile is compared to two or more reference profiles for different cognitive disorders. [0106] Similarly, the method described herein can be used to diagnose a neurodegenerative disease in an individual. In some embodiments, the level of a plurality of steroids {e.g., at least 100 steroids) including one or more neurosteroids is profiled in a biological sample obtained from the individual using the UPLC-tandem mass spectrometry method described herein. For instance, the level of one or more neurosteroids, metabolites of a neurosteroid, neurosteroid variants and/or neurosteroid derivatives can be measured. The level of the steroids including one or more neurosteroids is used to form the individual's steroid profile, which is compared to a reference steroid profile for a specific neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease, a Parkinson's disease-related disorder, prion disease, motor neuron disease, Huntington's Disease, spinocerebellar ataxia, spinal muscular atrophy, and dementia. If the individual's steroid profile is substantial ly similar to the reference profile for a specific neurodegenerative disorder (e.g., greater than 70%, 75%, 80%, 85%, 90%, or 95% overlap between the reference and test steroid metabolome profiles) based on the presence of the same steroids and/or the levels (e.g., normalized or relative amounts) of the same steroids, it is determined that the individual has the neurodegenerative disorder. If there are few or no similarities between the profiles (e.g., less than 30%, 25%, 20%, 15%, 10%, 5%, 1% or 0% overlap between the reference and test steroid metabolome profiles), the individual does not have the specific neurodegenerative disorder. In some instances, the individual's steroid profile is compared to two or more reference profiles for different neurodegenerative disorders.

[0107] In yet other embodiments, the method described herein can be used for diagnosing a psychiatric disorder, e.g., depression, schizophrenia, an anxiety disorder, such as generalized social phobia, generalized anxiety disorder, panic disorder, and posttraumatic stress disorder, acute stress, chronic stress, and mood disorder in an individual. In some embodiments, the level of a plurality of steroids (e.g., at least 100 steroids) including one or more neurosteroids is profiled in a biological sample, e.g., whole blood, plasma, serum or cerebral spinal fluid, obtained from the individual using the UPLC-tandem mass spectrometry method described herein. For instance, the level of one or more neurosteroids, metabolites of a neurosteroid, neurosteroid variants and/or neurosteroid derivatives can be measured. The level of the steroids including one or more neurosteroids is used to form the individual's steroid profile, which is compared to a reference steroid profile for a specific psychiatric disorder. If the individual's steroid profile is substantially similar to the reference profile for a specific psychiatric disorder, such as schizophrenia (e.g., greater than 70%), 75%, 80%, 85%, 90%, or 95% overlap between the reference and test steroid metabolome profiles) based on the presence of the same steroids and/or the levels (e.g., normalized or relative amounts) of the same steroids, it is determined that the individual has the psychiatric disorder (e.g., schizophrenia). If there are few or no similarities between the profiles (e.g., less than 30%, 25%, 20%, 15%, 10%), 5%, 1% or 0% overlap between the reference and test steroid metabolome profiles), the individual does not have the specific psychiatric disorder. In some instances, the individual 's steroid profi le is compared to two or more reference profiles for different psychiatric disorders.

[0108] Non-limiting examples of neurosteroids include pregnenolone, pregnenolone sulfate, al loprenanolone, 17-hydropregenoione, androstenolone (dehydroepiandrosterone or DHEA), allotetrahydrodeoxycorticosterone, 3 dehydroepiandrosterone sulfate, a-hydroxy- 5a-pregnan-20-one, 5a~dihydroprogesterone, allopregnanediol, 1 la-hydroxy-4-pregnene- 3,20-dione, 1 lb-hydroxy-4-pregnene-3,20-dione, 17-hydroxypregnenolone, 17- hydroxyprogesterone, 21 -hydroxypregnanolone, 20-hydroxypregnenolone, 7a- hydroxypregnenolone, 2-OH-testosterone, 20a-hydroxy-5a-pregnan-3-one, 20a- dibydroprogesterone, 17a,20a~dihydroxyprogesterone, 3b-hydroxy-5-pregnen~20-one-3-SO₄, eltanolone, pregnanediol, 5b-dihydroprogesterone, 5a-dihydrotestosterone, 17b- dihydroandrosterone, 17b-dihydroepiandrosteroiie, 7a-hydroxytestosterone, 7 a- hydroxyandrostenedione, 6a-OH-testosterone, 6b-OH -testosterone, metabolites thereof, variants thereof and derivatives thereof. In some instances, the methods of steroid metabolome profiling include determining the level of one or more neurosteroids in a biological sample using the UPLC-tandem mass spectrometry described herein.

IV. Examples

[0109] The following examples are offered to illustrate, but not to limit, the claimed invention.

Example 1. Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Profiling of Steroid Metabolome in Hitmasi Tissue,

[0110] This example illustrates a method for profiling steroid hormone levels in human breast tissue. In particular, the method uses a liquid-liquid extraction (LLE) followed by ultra performane liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) for simultaneous measurement of over 100 steroids in about 12 minutes without the need of steroid derivatization during sample preparation. [0111] Measuring steroid levels in individuals may be useful for monitoring human health, e.g., diagnosing or prognosing disease. Methods for measuring steroids in biological samples include radioimmunoassay, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). However, these methods require elaborate sample preparation procedures and have issues related to inadequate reproducibi lity, a limited dynamic range, time, high costs, and insufficient coverage of steroids.

[ΘΠ 2] Currently there is no available method for comprehensive steroid profiling of androgens, corticosteroids, progestogens, estrogens, estrogen metabolites, estrogen conjugates, estrogen-DNA adducts as well as exogenous steroid derivatives in a single LC- MS r n. Provided herein is a method for global steroid metabolic profiling based on liquid- liquid extraction (LLE) followed by UPLC-MS/MS. The method allows one to simultaneously measure over 100 endogenous as well, as exogenous steroids in about 12 min, without steroid derivatization. This example illustrates the use of the method to measure steroid hormone levels in the breast tissue samples from healthy women. Notably, all major classes of steroids as well as estrogen derivatives were detected.

Experimental Section

[0113] Steroid reference standards (Table 1) #1-31 , 42, 43, 52-77, 80-101 were purchased from Steraloids (Newport, RJ), and #32-41 , 44-51 were synthesized by using published procedures (Li et al., Carcinogenesis, 2004, 25 :289-297; Cao et al, Chem. Res. Toxicol, 1998, l i ;909-916; Stack et al, Chem. Res. Toxicol , 1996, 9:851-859). All solvents were HPLC grade and all other chemicals used were of the highest grade available.

Table 1. Mass spectrometry parameters for 101 steroids.

Linear

LOD

Compound Mode Parent Daughter R_t R² range pmole

(pg/ul)

Cortexolone PI 347,3 97.2 8.5 0,007 0.9904 5-20000

Cortisol Pi 363.4 121.1 7.5 0.068^a 0.9701 1-20000

Cortexone PI ji l .J 97.1 9.9 0.015^a 0.9974 5-20000

Corticosterone PI 347.3 121.0 8.4 0.072^a 0.9925 5-20000

Aldosterone I 361.3 299.1 7.1 0.693 0.9629 25-20000

Androstenedione PI 287.3 97.0 9.8 0.009" 0.9906 0.5-20000

Testosterone PI 289.3 96.9 9.4 0.009" 0.9888 0.5-20000

Allodihydrotestosterone PI 291.3 159.0 10.2 0.344 0.8795 10-20000

Estrone (E l) PI 271.2 253.2 9.6 0.009" 0.9596 0.5-20000

Estradiol (E2) I 255.3 159.0 9.4 0.009" 0.9903 2.5-20000

11 a- Hydroxy El I 287.3 251.2 7.5 0.087" 0.9854 2.5-20000 l i -Hydroxy_El PI 287.3 1 1 1.1 11.1 0.873 0.9569 0.5-20000 l la-Hydroxy E2 PI 289.3 253.1 9.7 0.173 0.9942 25-20000 l l j3~Hydroxv _E2 PI 289.3 106.0 9.4 1.734 0.6273 0.5-20000

9,l l-Dehydro_El PI 271.3 159.3 9.6 0.092" 0.9758 1-20000

9,11-Dehydro _E2 PI 269.3 209.0 9.4 0.093" 0.9884 2.5-20000

11- etoestrone I 285.3 267.1 8.2 0.088" 0.9869 25-20000

3 -Methoxy estrone I 285.3 147.1 11.7 0.009" 0.9935 1-20000

Es tradiol-3-glucuronate NI 471.3 198.9 7.4 1.062 0.9789 50-20000

Estradiol- 17-giucuronate NI 447.3 84.7 7.4 0.111 0.9909 5-20000

4-Hydroxyestrone PI 287.3 269.0 8.8 0.002" 0.9274 0.5-20000

4-Hydroxyestradiol I 271.3 174.8 8.2 0.347 0.9179 10-20000

4-Methoxyes trone PI 301.3 163.2 10.1 0.083" 0.9740 2.5-20000

4-Methoxyestradio ί PI 303.3 136.8 9.2 3.307 0.8696 100-20000

4-Methoxyestriol PI 319.3 137.3 8.3 15.703 0.9449 250-20000

4-Hydroxy-El-2- glutatione PI 592.3 317.1 6.6 0.042" 0.9662 10-20000

4-Hydroxy-E2-2- glutatione PI 594.4 318.9 6.5 0.169 0.9591 100-20000

4-Hydroxy-El-2- cysteine PI 406.2 284.3 8.1 0.025" 0.8798 25-20000

4-Hydroxy-E2-2- cysteine PI 408.2 286.5 7.9 0.614 0.8465 100-20000

4-Hydroxy-El-2-N- acetylcysteine PI 448.3 161.8 7.9 0.224 0.9625 25-20000

4-Hydroxy-E2-2-N- acetylcysteine PI 450.1 162.1 7.3 0.223 0.9769 25-20000

4-Hydroxy-El-l-N-3- adenine PI 420.3 135.9 6.2 0.012" 0.9748 2.5-20000 Linear

LOD

Compound Mode Parent Daughter Rt R² range pmole

(pg/ul)

4-Hydroxy-E2-l-N-3- adenine PI 422.3 136.0 6.0 0.024^a 0.9796 5-20000

4-Hydroxy-El-l-N-7- guanine PI 436.2 152.0 6.3 0.230 0.9634 25-20000

4-Hydroxy-E2-l-N-7- guanine PI 438.1 272.0 6.3 0.229 0.9606 10-20000

2-Hydroxyestrone PI 287.3 268.9 8.7 0.349 0.9182 1-20000

2-Hydroxyestradiol PI 271.2 175.1 8.3 0.347 0.9692 5-20000

2-Hydroxy-E 1 - 1 +4- glutatione PI 592.2 463.2 6.6 0.423 0.9594 25-20000

2-Hydroxy-E2- 1 +4- giutatione PI 594.1 465.4 6.6 0.421 0.9028 100-20000

2-Hydroxy-E 1 - 1 +4-c

ysteine PI 406.0 316.9 8.2 0.247 0.9640 25-20000

2-Hydroxy-E2- 1 +4-c

ysteine PI 408.3 319.0 7 2 0.614 0.9495 50-20000

2-Hydroxy-El -l+4-N- ac etyleysteme PI 448.3 161.9 7.7 0.1 12 0.9705 10-20000

2-Hydroxy-E2-l+4-N- ac etylcysteine PI 450.1 162.0 7.3 0.557 0.9829 50-20000

2-Hydroxy-E 1-6-N-3- adenine PI 420.0 135.9 6.1 0.1 19 0.9742 25-20000

2-Hydroxy-E2-6-N-3- adenine PI 422.2 136.0 5.9 0.1 19 0.8714 50-20000

2-Hydroxyestriol PI 287.3 269.0 8.6 0.008^a 0.9691 5-20000

3-Methoxy-2-OH- estrone PI 301 .3 137.0 10.1 0.166 0.9767 10-20000

2-Methoxy-3-OH- estrone PI 301 .3 1 89.2 10.1 0.008^a 0.9819 0.5-20000

2-Methoxy-3-OH- estradiol PI 303.3 137.2 9.5 0.331 0.9827 25-20000

2,3-Dimethoxyestrone PI 315.3 201.0 1 1.0 0.032^a 0.9987 2.5-20000

2,3-Dimethoxyestradiol PI 317.3 302.2 10.7 0.079^a 0.9819 2.5-20000

6a-H y droxyestradiol PI 271.2 156.9 6.9 0.173 0.9412 5-20000

6P-Hydroxyestradiol Pi 271.2 156.9 6.6 0. 173 0.9861 25-20000

6- etoestrone PI 285.3 133.0 8.1 0.088^a 0.9469 2.5-20000

6- etoestradiol I 287.3 121.2 7.9 0.087^a 0.9743 10-20000

6-Dehydroestradiol I 271 .2 137.2 8.3 0.092^a 0.7277 50-20000

6-Dehydroestrone PI 269.3 156.9 9.4 0.019^a 0.9384 2.5-20000

16a-Hydroxyestrone PI 287.3 251.0 9.8 0.175 0.9916 25-20000

17-Epiestriol PI 289.3 271.2 9.4 0. 173 0.9776 50-20000 Linear

LOD

Compound Mode Parent Daughter R_t R² range pmole

(pg/ul)

Estriol PI 289,3 225.4 9 7 0.347 0.9103 25-20000

16, 17-Epi estriol Pi 289.3 106.1 9.4 3.468 0.8397 5-20000

16-Epiestriol PI 289.4 106.1 9.4 3.468 0.8312 10-20000

Estriol-3-suifate NI 367.2 287.3 6.5 12.806 0.7324 500-20000

Estriol-3 -glucuronate PI 487.3 198.9 5.5 2.153 0.9784 250-20000

3 -Methoxy estriol PI 285.3 266.9 9.1 0.002^a 0.9653 0.5-20000

16-Keto-17 -estradiol PI 287.2 251.4 7.6 3.492 0.7780 100-20000

6-Ketoestriol PI 303.3 107.0 6.2 0.331 0.8905 10-20000

7-Dsehydroestradiol PI 271.3 229.8 12.4 3.699 0.9847 2.5-20000

Equilin I 269.3 21 1.1 9.4 0.019^a 0.9530 2.5-20000

Dihydroequilin-3-S0₄ I 373.2 332.0 9.7 1.342 0.9602 50-20000

Equilin-3-S0₄ PI 347.2 267.3 8.4 6.749 0.9566 250-20000

Estrone-9-N₃-Ade PI 404.3 269.0 6.4 0.124 0.9661 10-20000

Estradiol-9-N₃-Ade PI 406.3 271.0 6.0 0.247 0.9336 50-20000

Estradiol-3-acetate PI 315.3 107.1 1 1.1 0.08^a 0.9960 10-20000

Estrone-3 -acetate PI 313.3 253.1 10.5 0.160 0.9973 10-20000

Estradiol -3, 17a- diacetate PI 297.3 255.1 11.1 0.07^a 0.9960 2.5-20000

Estradiol -3,17β- diacetate PI 357.3 135.2 10.2 1.402 0.9722 100-20000

2-Hydroxy estradiol -17- acetate PI 329.2 269.2 10.6 0.003^a 0.9444 1-20000

6- etoestradiol-3 ,17- diacetate PI 371.3 329.2 1 1.3 0.006^a 0.9967 0.5-20000

6-Ketoestriol triacetate I 429.3 267.3 10.6 0.023^a 0.9997 2.5-20000

6-Dehydroestradiol

diacetate I 355.3 313.4 12.4 0.141 0.9819 25-20000

Estriol-3-acetate I 331 .3 289.0 8.7 0.303 0.9825 50-20000

Estriol- 16-acetate PI 331.3 253.1 8.9 0.303 0.9415 25-20000

Estriol- 16,17-diacetate PI 373.3 313.2 10.4 0.268 0.9958 10-20000

17-Epies triol-tri acetate PI 415.3 295.4 1 1 .9 0.241 0.8913 10-20000

Estriol triacetate PI 415.3 355.2 1 1.9 0.006^a 0.9650 1-20000

16, 17-Epies triol- triacetate PI 415.3 295.0 1 1.9 0.06^a 0.9639 5-20000

16-Epies triol-triacetate PI 415.3 355.1 11.9 0.121 0.9373 25-20000

Equilinacetate I 31 1 .3 210.9 11.1 0.08 Γ 0.9458 5-20000

Estradiol-3- hemi succinate NI 255.3 159.1 9.9 0.00 Γ 0.9339 0.5-20000

Estrone-3 -hemisuccmate I 371.2 177.2 11.6 0.007^a 0.9863 2.5-20000 Linear

LOD

No. Compound Mode Parent Daughter L i R₇ range pmole

(pg/ul)

Estradiol- 17-

98 hemisuccmate PI 373.3 255.1 9.9 0.268 0.9467 25-20000

Estradiol-3,.17-

99 diheniisuccinate PI 255.3 158.9 6.5 0.00 i " 0.9608 1-20000

100 Estriol-3-h.emisuccinate PI 271.2 252.9 7.0 0.064·' 10.8435 2.5-20000

Estriol-16-

101 hemisuccmate PI 389.3 253.1 8.0 0.257 0.9587 25-20000

List of the 101 steroids and estrogen-related compounds wi th the masses of parent and daughter ions thai were used for MRM method optimization. Compound #86 through #101 are ester derivatives. Pi denotes positive ionization mode. NI denotes negative ionization mode. ^aAnaiyies that are detected in the low range, LOD < 0.300 (arbitrary).

Table 2. Method validation parameters for 101 steroids.

5000 pg nL 2500 pg/nL 1000 pg nL 500 pg/μΕ 250 pg/ L

Compound % cv %RE % cv %RE % CV %RE % cv;%RE % cv %RE

(P) (A) (P) (A) (P) (A) CP) (A) (P) (A)

3 -Metb oxy estrone 6.8 97 3.0 99 6.6 97 12.2 94 6.1 97

Es tradiol - 3 -giucur onate 13.1 93 18.1 91 33.4 83 31.2 84 67.0 53

Estradiol- 17-glucuronate 4.7 98 4.9 98 7.2 96 4.8 98 8.2 96

4-Hydroxyestrone 6.8 97 4.4 98 21.0 89 17.7 91 19.9 90

4-Hydroxyestradiol 5.6 97 6.3 97 7,3 96 9.7 95 10.7 95

4-Methoxyes trone 6.1 97 5.1 97 1 1.3 94 7.4 96 11.5 94

4-Methoxyestradiol 9.6 95 25.0 86 25.7 85 44.2 74 123.3 13

4-Methoxyestrioi 23.7 83 / / . / 45 51.9 63 18.3 87 50.4 64

4-Hydroxy-El -2- glutatione 8.8 96 5.5 97 9.7 95 8.6 96 35.0 82

4-Hydroxy-E2-2- glutatione 13.4 92 12.3 94 28.8 86 15.3 92 59.5 70

4-Hydroxy-El-2- cysteine 9.9 95 7.3 96 4.7 98 17.1 91 14.0 93

4-Hydroxy-E2-2- cysteine 10.4 95 1 1.4 93 31.0 84 38.6 78 1.0 99

4-Hydroxy-El-2-N- acetylcysteine 7,5 96 6.0 97 18.2 91 1 1.5 93 23.0 89

4-Hydroxy-E2-2-N- acetylcysteine 9.7 95 7.2 96 12.6 94 5.6 97 39.5 80

4-Hydroxy-El -l-N-3- adenine 4.2 98 5.1 97 5.2 97 9.5 95 9.0 95

4-Hydroxy-E2-l-N-3- adenine 3.8 98 6.8 97 1 1.1 94 17.2 91 18.2 91

4-Hydroxy-El-l -N-7- guanine 14.8 93 12.5 94 9.2 95 16.4 92 40.0 80

4-Hydroxy-E2-l-N-7- guanine 3.3 98 9.9 95 8.5 96 27,3 86 62.7 69

2-Hydroxyestrone 10.2 95 13.5 93 19.1 90 18.4 91 17.4 91

2-Hydroxyestradiol 1 1.8 94 1 1.7 94 15.4 92 21 .3 89 13.8 93

2-Hydroxy-E l -H-4- glutatione 13.6 93 8.6 96 5.6 97 20.7 90 17.8 91

2-Hydroxy-E2-l+4- glutatione 14.9 93 6.9 97 14.2 93 18.4 91 35.3 82

2-Hydroxy-El-l +4-c

ysteine 16.3 92 9.4 95 10.9 95 26.9 87 25.0 87

2-Hydroxy-E2-H-4-c

ysteine 10.4 95 12.2 94 13.3 93 12.5 94 6.9 97

2-Hydroxy-E 1^■· 1 +4-N-ac

etyicysteine 8.2 96 3.6 98 16.6 92 13.5 93 17.1 91

2-Hydroxy-E2 - +4-N-ac

etyicysteine 12.3 94 7.8 96 10.4 95 8.7 96 9.4 95

2-Hydroxy-E 1 -6-N-3- 4.1 98 10.0 95 6.4 97 9.7 95 30.8 85

5000 pg nL 2500 pg nL 1000 pg nL 500 pg/μΕ 250 pg/ L

No. Compound % cv %RE % cv %RE % CV %RE % cv;%RE % cv %RE

(P) (A) (P) (A) (P) (A) (P) 1(A) (P) (A) acetate

6-Ketoestradiol-3, 17-

85 diacetate 5.9 97 8.6 96 4.9 98 10.8 95 13.1 93

86 6-Ketoestriol triacetate 7.4 96 6.7 97 10.1 95 13.9 93 4.0 98

6-Dehydroestradiol

87 diacetate 13.0 93 19.0 90 14.2 92 15.6 92 21.6 89

88 Estriol- 3 -acetate 12.3 94 18.3 89 14.1 92 42.3 76 67.6 66

89 Estriol- 16-acetate 3.8 98 2.6 99 16.9 92 20.4 90 20.9 90

90 Estriol- 16,17-diacetate 9.3 95 4.4 98 10.7 95 22.2 89 45.5 77

91 17-Epies triol-triacetate 4.0 98 10.7 95 7.6 96 7.0 96 1 1.2 94

92 Estriol triacetate 3.1 98 5.1 97 4.3 98 9.2. 95 13.8 93

16, 17-Epies triol-

93 triacetate 1.4 99 2.4 99 3.4 98 8.0 96 4.1 98

94 16-Epies triol-triacetate 4.1 98 3.5 98 4.1 98 15.1 92 16.0 92

95 Equilinacetate 5.7 97 1 1.4 94 1 1.1 94 28.1 86 22.9 89

Estradiol -3-

96 hemisuccinate 5.2 97 2.7 99 6.2 97 7.5 96 8.9 96

97 Estrone-3 -hemisuccinate 1 1.7 94 8.9 96 6.6 97 9 7 95 18.8 91

Estradiol- 17-

98 hemisuccinate 6.2 97 4.2 98 3.9 98 ^*71 96 10.2 95

Estradiol-3, 17-

99 dihemisuccinate 0.9 100 2.2 99 7.2 96 5.0 98 7.6 96

100 Estriol-3-hemisuccinate 10.5 95 18.2 91 12.2 94 17.5 91 16.8 92

101 Estriol- 16-hemisuccinate 10.1 95 5.6 97 14.0 93 2.8 99 27.4 86

%CV refers to percentage coefficient of variation. %RE refers to the relative error of the measurement of five replicates of each of the validation standard concentrations analyzed on the same day.

[0114] For each steroid analyte, stock solutions of lmg/ml concentrations were prepared in methanol: water (1 : 1 v/v) mixture. In some cases, when solubility was an issue, either water or methanol was used for preparing the stock solution. Stock steroid mixtures were prepared by mixing 20μ1 of lmg/ml solution of each steroid and adjusting final volume to 1ml by using methanol: water (1 : 1 v/v). All the stock solutions were stored at -80°C.

MS, MS/MS and UPLC-MS/MS analysis of estrogen metabolites [0115] Xevo^®-TQ triple quadrupole mass spectrometer (Waters Corp, Milford, MA) recorded MS and MS/MS spectra using Electro Spray Ionization (ESI) in positive ion (PI) and negative ion (Νί) mode, capillary voltage of 3,0 kV, extractor cone voltage of 3 V and detector voltage of 650 V. Cone gas flow was set at 50 L/h and desolvation gas flow was maintained at 600 L/h. Source temperature and desolvation temperatures were set at 150 and 350 °C, respectively. The collision energy was varied between to optimize daughter ions. The acquisition range was 20-500 Da. The test samples (compounds #1-101) at 5iig/mL were introduced to the source at a flow rate of 5 μΐ/^'min by using acetonitrile: water (1 : 1) and 0.1 % formic acid mixture as the carrier soiution and mass spectra were recorded. The masses of parent ion and daughter ions were obtained in the MS and MS/MS operations. MS/MS parameters were further used in multiple reaction monitoring (MRM) method for UPLC MS/MS operation.

[0116] Analytical separations of the mixture of 101 standards was conducted on the UPLC system using an Aequity* UPLC HSS T3 1 .8μιη 1 X 150 mm analytical column (Waters Corp, Milford, MA) maintained at 50°C and at a flow rate of 0.15 ml/^'min. The gradient started with 100% A (0.1% formic acid in H₂0) and 0% B (0.1% formic acid in CH CN), after 2min, it was changed to 80% A over 2 min, then 45% A over 5min, followed by 20%) A in 2min. Finally over 1 min it was changed to original 100% A, resulting in a total separation time of 12 min. The elutions from the UPLC column were introduced to the mass spectrometer and the resulting data were analyzed and processed using MassLynx 4.1 software (Waters Corp., Milford, MA).

M ethod Validation

[0117] Human breast tissues were used for method validation. Recovery and linearity as well as accuracy, precision and limit of detection were determined for al l 101 anaiytes. To calculate limits of detection, various concentrations, 0.05 pg/^'μΐ, 0.1 pg/μΐ, 0.25 pg/μΐ, 0.5 pg/μΐ, 1.0 pg/μί, 2.5 pg/μΐ, 5.0 pg/μΐ, 10 pg/μΐ, 25 pg/μΐ, 50 pg/μΐ, 100 pg/μΐ, 250 pg/μΐ, 500 pg/iii, and 1000 pg/μΐ of the anaiytes were injected to UPLC/MS-MS. The injected amount that resulted in a peak with a height at least 3 times as high as the baseline noise level was used as the limit of detection. The intra-day precision and accuracy were determined by calculating the percent coefficient of variation (%CV) and relative error (RE %) of the measurement of five replicates of each of the validation standard concentrations, e.g., 250 pg/iii, 500 pg/μΐ, 1000 pg/iil, 2500 pg/μΐ, and 5000 pg/μΐ, analyzed on the same day.

Sample Preparation and Analysis [0118] Ten breast tissue samples of women (26-53 yrs range, median age 47 yrs; 9-white and 1 -asian) were obtained from UC Davis, Cancer Center Bio-specimen Repository, which were approved for use by the Internal Review Board. Weighed (100 mg) breast tissue samples were ground and suspended in 4 ml of watenmethanol (1 :1) mixture. The suspension was homogenized and the resulting homogenate was cooled on ice. The precipitated material was removed by centrifuging at high speed for 5 min, and the supernatant was removed and evaporated in a SpeedVac (Labconco Inc) followed by lyophilizer (Labconco Inc, Kansas City, MO). The residue was suspended in 150 μΐ of C¾OH:H₂0 (1 :1), filtered through a 0.2 μπι ultracentrifuge filter (EMD MiUipore, Billerica, MA).

[0119] Ten samples were mn in duplicate during UPLC-MS/MS analysis. Samples were placed in a Acquity sample manager which was cooled to 5°C to preserve the analytes. Pure standards were used to optimize the UPLC-MS MS conditions prior to sample analysis. The standard mixture was run before the first sample, after the 5th sample and after the last (10th) sample to prevent errors due to matrix effect and day-to-day instrument variations. In addition, immediately after the initial standard and before the first sample, two spiked samples were run to calibrate for the drift in the retention time of all analytes due to the matrix effect. After standard and spiked sample runs, blank was injected to wash injector and remove carry over effect.

Results and Discussion

[0120] Small molecule metabolites such as steroids have immense effect on human health. Estrogens and androgens estrogens are involved in growth and function of the reproductive organs, development of secondary sexual characteristics, and behavioral patterns in humans. Progestogens serve as a precursor to other steroids. Corticosteroids are involved in the regulation of many aspects of metabolism, stress and immune response, inflammation, electrolyte, and water levels. In addition, the balance of various steroid metabolic pathways has been shown to be associated with human health. Thus, measurement of steroid metabolome in biological samples can play a pivotal role in disease diagnosis and/or prognosis. The data presented here shows a simple, low cost, liquid-liquid extraction (LLE) method for extracting 101 endogenous and exogenous steroids from tissue samples followed by UPLC-MS/MS analysis.

[0121] In spite of recent advances, the development of analytical methods for global analysis of steroids has been challenging due to their low expression levels, diversity in their chemical and physical properties, e.g., polarity and stability, dynamic range of their physiological concentrations and variation in physiological conditions. Current solid phase extraction methods are not efficient in extracting various classes of steroids together. Some methods, especially GC-MS, require a derivatization step. Moreover, although there are existing LC-MS/MS-based methods, these cannot be employed for high-throughput analysis due to extended analytical times, high costs per analysis, and/or inadequate coverage of steroids. To overcome these problems LLE strategies were employed for tissue analysis.

[0122] Extraction of tissue homogenate samples with methanol provided broad steroid coverage (FIG, 2), Average extraction recovery of 101 steroids was 89,2 % with standard deviation ±22 ( FIG. 2) and the average % CV for recover was 1 8.7±18.2 (Table 3).

Table 3. Accuracy and Precision of LLE

25 23 86 76 8 95

26 17 90 77 69 60

27 4 98 78 30 83

28 9 95 79 70 60

29 13 93 80 25 85

30 24 86 81 10 94

31 4 98 82 16 91

32 34 80 83 32 81

33 19 89 84 14 92

34 20 88 85 3 98

35 47 73 86 11 94

36 10 94 87 21 88

37 7 96 88 9 95

38 13 92 89 23 87

39 11 93 90 29 83

4Θ 6 96 91 10 94

41 19 89 92 7 96

42 98 93 26 85

43 21 88 94 1 1 94

44 31 82 95 12 93

45 13 93 96 6 96

46 66 62 97 16 91

47 47 73 98 17 90

48 3 98 99 5 97

49 25 85 100 4 98

50 4 98 101 18 90

51 4 98

[0123J In general, the average recover}' of androgens (#3, 4, 12, 13, 14), corticosterones (#7,8,10,11), catecholestrogens (#27, 28, 42, 43), methoxyestrogens (#24, 29, 30, 31, 53-57, 71), hydroxyestrogetis (#17-20, 52, 58, 59, 67, 68), 2-catechol adducts (#50, 51), acetates (#80, 81 , 84, 88, 89, 95), triacetates (#86, 91-94), and hemisuccinates (#96-101) was above 90%, Estrogens (#15, 16, 66), ketoestrogens (#23, 60, 61, 72), estrogen 4-catechol adducts (#38-41) and estrogen diacetates (#82, 83, 85, 87, 90) had an average recovery between 80- 90%. Progestogens, (#1, 2, 5, 6, 9), dehydroestrogens (#21, 22, 62, 63, 74), 2-catechol conjugates (#44-49) and 4-catechol conjugates (#32-37) recover was between 70-80%. Moderate recovery in progestogens and dehydroestrogens suggests that the water: methanol (1 :1) mixture may not be a good extraction solvent system for these analytes, whereas 2- eateeho! conjugates and 4-catechol conjugates may have undergone oxidation.

[0124] Mass spectrometry methods were optimized to detect the majority of steroids (total 97) under positive ionization mode, only 4 analytes were detected in negative ionization mode resulting in improved sensitivity due to fewer polarity switches (Table 1). This was achieved by adding 0.1% formic acid to the carrier solution when MRM method was developed and then 0.1 % formic acid to both, A and B, mobile phases. MRM transitions were then optimized to ensure optimal detection responses and specificity. Use of UPLC, equipped with 1 x 150 mm 1.8μτη analytical column significantly reduced chromatography time for 101 steroids to just 12 min resulting in reduction of potential costs per sample. In recent years UPLC or U-HPLC has been demonstrated to outperform traditional HPLC separations resulting in higher efficiencies and sensitivity.

[0125] The method described herein has many advantages over the current LC-MS methods that focus on estrogens and estrogen metabolites. For example, a stable isotope dilution liquid chromatography/selected reaction monitoring/mass spectrometry assay for measuring serum estrone, 16a-hydroxyestrone, 4-methoxyestrone, and 2-methoxyestrone has been reported. This method requires a three step sample preparation, in particular, LLE followed by derivatization followed by SPE. The Girard P reagent used for derealization only forms Schiff s base with estrones (ketones) leaving all the estradiol related metabolites unmeasured. Another method reported is capable of quantifying 15 estrogens and estrogen metabolites simultaneously in a single HPLC run for about 75 min (Xu et a!,, Anal. Chem,, 2007, 79, 7813-7821). This method uses a two/three step sample preparation. First the serum samples are extracted with dichloromethane and then are derivatized with dansyl chloride. The method described herein does not require derivatization and uses a single step LLE followed by UPLC-MS/MS to measure over 100 endogenous as well as exogenous steroids in 12 min.

[0126] The UPLC-MS/MS method was validated by determining limit of quantification, linear dynamic range, recovery, precision, and accuracy (Tables 1 and 2). The analytical parameter determinations of the targeted endogenous as well as exogenous steroids were performed using standard mixtures. The results indicated that the LOD was in the range of 0.001-15.7 pmole, suggesting that the method is highly sensitive for simultaneous quantification of a complete panel of steroids. The linearity of the method was determined from the calibration curves constructed for each analyte in human breast tissue matrices. Regression analysis showed that the correlation coefficients (R²) were above 0.90 for most of the analytes in breast tissue matrices (Table 1). [0127] The data for accuracy, precision and recovery are presented in Table 2, The median value for intraday variance were below 20% at 250 pg/μΐ and 500 pg/^'μΐ concentration, whereas it was 11.1 %, 7.7% and 8.2% at 1000 pg μΐ, 2500 pg/μΐ and 5000 pg/μΐ concentrations, respectively, indicating moderate!)' good precision of the method. Sample analysis also showed good accuracy at all five concentration levels (RE < 10%), and the average extraction recovery was greater than 90%. These results shows that the UPLC- MS/MS method is dependable and reproducible for the simultaneous analysis of 101 endogenous and exogenous steroids from various metabolic networks.

[0128] Finally, targeted UPLC- S/MS based metaboiomic analysis was performed on human breast tissues to investigate total steroid anabolic/catabolic pathways in normal human breast. The multiple reaction monitoring (MRM) chromatograms of representative steroids and estrogen metabolites, which were obtained in a single 12 min injection of breast tissue extract, are shown in FIG. 3. The average levels of steroids and estrogen related compounds measured from ten breast tissue samples are presented in Table 1. The presence of all major classes of steroids, e.g., androgens, corticosteroids, progestogens and estrogens, were detected in breast tissue (Table 1 and FIG. 3). Analysis of the metabolic profile shows that estrogens are further metabolized to various classes of derivatives, such as glucuroiiates, hydroxyestrogens, methoxyestrogens, conjugates and adducts.

[ 0129] Endogenous steroids and their derivatives have long been of interest as biomarkers of various diseases resulting from steroid imbalances and have been used in clinical and preclinical studies (Findling and Raff, J. Clin. Endocrinol. Metab., 2006, 91 :3746-3753; Nomura et al, Clin. Chim. Acta. 1996, 256: 1-11; Lee et al, Cancer Lett., 2003, 201 :121- 131). Recently steroid metabolite profiling has been used to examine potential biomarkers indicating the activity of nonfunctioning adrenal incidentalomas (Kotlowska et al., Clin. Biochem., 2009, 42:448-454), adrenal disorders and adrenal tumors (Arlt et al., J. Clin. Endocrinol. Metab., 201 1, 96:3775-3784), and the influence of gestational age on the preterm labor (Hill et al., J. Steroid Biochem. Mol. Biol , 2010,121 , 594-610). Furthermore, steroid profiling was also applied in search of biomarkers of breast cancer, prostate cancer,

5 non-Hodgkin lymphoma, Parkinsons disease, urogenital tract cancer diseases (bladder, kidney, prostate, and testis) (Konieczna et ai, J. Pharm. Biomed. Anal., 2013, 73: 108-115), stress and depressive disorders (Plenis et ai, Mol. Biosyst., 201 1 , 7: 1487-1500), and for diagnosing genetic defects in newborns (Homma et a!., J. Clin Endocrinol. Metab., 2004, 89:6087-6091 ). The ability to detect more than hundred steroids in single run in less than fifteen minutes can be useful in number of clinical, lexicological and nutritional investigations.

[0130] In summary, the UPLC-MS/MS assay method described herein provides analysis of wide range of endogenous steroids (# 1-85) as well as exogenous steroid esters (# 86-101) in 12 min. The limit of detection and linear range indicates that the method can detect and quantify steroids in tissue samples to investigate fluxes in their metabolic pathway.

Example 2. Ultra Performance Liquid Chroraatograpliy-Tasidem Mass Spectrometry Method for Global Profiling of 206 Steroid Metabolites,

[0131] This example describes a method for profiling 206 steroids and metabolites thereof (Table 4) in a biological sample using the UPLC-MS/MS method described below. The method was used to profile the steroids listed in Table 4 in various biological samples including tissue, urine and serum samples.

[0132] Mass pairs (parent -daughter) were generated by performing MS/MS analysis of each steroid standard individually . Also, retention times of each steroid were determined by nmniiig LC-MS/MS analysis of individual standards. Finally the steroids were grouped depending on their retention time. For some of the steroids, such as estradiol E2 and oxysterols more than one mass pair was used.

Table 4. Parameters for steroid measurements.

8 8 Cortisol 363.35 > 121.14 4.03

9 9 Cortexone 331.3 > 97.12 6.34

10 10 Corticosterone 347.3 > 121.01 4.86

11 11 Aldosterone 361.25 > 299.06 4.07

12 12 Androstenedione 287.3 > 97.01 6.24

13 13 Testosterone 289.3 > 96.94 5.80

14 14 Alkxlihydrotestosterone 291 .3 > 158.95 6.70

15 15 Estrone E 1 271.25 > 132.9 6.12

16 16 Estradiol E2 255.25 > 158.95 5.55

17 17 1 la-hydroxyestrone 287.25 > 251.23 4.49

18 18 1 lb-hydroxy estrone 287.3 > 111.06 5.25

19 19 1 1 a-hydroxyestradiol 271.2 > 133 3.82

20 20 11 b-hydroxy estradiol 271.2 > 133 3.82

21 21 9(1 l)-dehydroestradiol 271.25 > 159.3 6.95

-> -> 22 9(1 l)-dehydroestrone 269.25 > 208.96 6.05

23 23 11-ketoestrone 285.3 > 267.12 5.69

24 24 Estrad.iol-3~S0₄ 351.2 > 271 3.98

25 25 Estrone- SO4 349 > 269 4.22

26 26 Estradiol- 17- SO₄ 351.1 > 96.8 3.94

'">7 27 Estradiol-3,17-di- S0₄ 215 > 174.9 0.86

28 28 3-OMe stone 285.25 > 147.11 8.49

29 29 Estradiol-3-Glu 447 > 270.9 3.63

30 30 Estrone-Glu 445 > 268.8 4.10

31 31 Estradiol- 17-Glu 447.1 > 112.9 3.63

32 32 Estradio]-3,17-di-Glu 623.1 > 1 13 2.66

33 4-OH estrone 287.2 > 269.02 5.25

34 34 4-OH estradiol 271.3 > 175.1 4.71

35 35 4-OMe estone 301.25 > 163.2 6.32

36 36 4-OMe estradiol 303.25 > 136.77 6.01

37 37 4-OMe estriol 301 .2 > 251.1 3.65

38 48 2-OH estrone 287.31 > 172.99 5.51

39 49 2-hydroxyestradiol 271.2 > 175.1 4.71

40 58 2-OH estril 287.25 > 269.01 5.28

41 59 3-methoxy-2-OH-estrone 301.25 > 137.02 6.64

42 60 2-methoxy-2-OH-estrone 301 .25 > 189.17 8.46

43 61 2-Methoxy-3 -OH-estradiol 303.25 > 137.22 6.24

44 62 2, 3-dimethoxy estrone 315.25 > 200.97 7.63

45 63 2,3-dimethox estradiol 317.25 > 302.2 7.16

46 64 6a-hydroxyestrad.iol 271.2 > 156.92 4.08

47 65 6b-hydroxyestradiol 271.2 > 156.93 3.82

48 66 6-ketostrone 285.25 > 133.02 4.63 67 6-ketoestradiol 5.25

68 6-dehydroestrad.iol 271 .2 > 137.22 6.22

69 6-dehydroestrone 269.25 > 156.9 5.98

70 16a-hydroxyestrone 287.2 > 251.1 4.49

71 17-epiestriol 271 .15 > 157 4.34

72 Estriol E3 271.15 > 132.9 3.82

73 16, 17-epiestriol 271 .15 > 252.9 4.08

74 16-epiestriol 271.15 > 132.8 4.10

75 Estrioi-3-sulfate 367 > 286.9 2.55

76 Estri.ol-17-Glu 465.3 > 271.13 3.00

3-methoxystriol 267.25 > 98.82 8.39

78 16-keto- 17b-estradiol 287.2 > 251.44 4.10

79 6-ketoestriol 303.25 > 107.01 2.87

80 7-dehydro- 17b-estradiol 271 25 > 229 82 4.72

81 Equiiin 269.25 > 21 1.06 6.05

82 Dihydroequilin-3-S0₄ 349.1 > 268.9 4.24

83 Equilin-3-S0₄ 347 > 266.9 4.12

\S ! 3 a-Hydroxy-5 a-pregnan-20-one 319.35 > 301.1 8.46

NS .?. 5a-Dihydroprogesterone 317.35 > 281.1 8.59

NS__3 Ailopregnanediol 302.35 > 70 7.23

1 1 a-hydroxy-4-pregnene-3,20-

NS 4 331.3 > 313 6.09 dione

11 b-hydroxy-4-pregnene-3 ,20-

NS_5 331.2 > 313 6.10 dione

NS_6 17-hydroxypregnenolone 333.3 > 296.9 4.77

NS_7 17-hydroxyprogesterone 331.3 > 96.9 6.34

NS 8 21 -hydroxypregnanolone > ? 6.87

NS 9 20-hydroxypregnenolone 333.3 > 314.9 5.83

NS 10 7a-hydroxypregnenolone 315.3 > 297.1 4.97 S 1 1 2-OH-testosterone 305.25 269 4.66

NS 1 20a-hydroxy-5 a-pregnan-3 -one 319.35 > 283.1 7.90

NS 13 20a-dih.ydroprogesterone 317.35 > 96.9 7.00

NS_14 17a,20a-dihydroxyprogesterone 333.3 > 96.9 5.58

3b-hydroxy-5-pregnen-20-one-3-

NS_15 395.25 > 96.8 5.46

SO<

NS_16 Eltano!one 319.35 > 301.05 8.24

NS_17 Pregnanediol 303.3 > 285.1 7.80

NS 1 5b-dihydroprogesterone 317.35 > 281.05 8.58

NS 1 5 a-dihydrotestosterone 291.3 > 255.1 6.70

NS 20 17b-dihydroandrosterone 275.35 > 257 5.90

NS_21 17b-dihydroepiandrosterone 275.3 > 257 5.89

NS_22 7a-hydroxytestosterone 305.3 > 97 3.94 88 NS_23 7a-hydroxyandrostenedione 303.3 > 285 4.20

89 NS 24 6a-OH-Testosterone 305.25 > 96.9 3.68

90 XS 25 6b-OH-Testosterone 305.25 > 96.91 3.94

91 New Sic I etiocholanolone glucuronide 291.3 > 254.9 6.70

92 XevvSic 2 DHEA GLUC 271.3 96.9 6.22

93 NewSteJ? 16a-hydroxy DHEA 305.3 > 286.9 5.47

94 New8te__4 Testosterone sulfate 367.2 > 96.7 4.45

95 NewSte_5 Desoxytestosterone 273.3 > 96.9 10.00

96 NewSte __7 4-androstene-30L, 17-one 271.3 > 80.9 6.81

97 NewSte_8 2a-hydroxyandrostenedione 303.3 > 285 4.91

98 NewSte 9 11 a-hydroxyandrostenedione 303.3 > 267 4.90

99 NewSte J O 6-ketotesterone 303.3 > 266.9 4.91

100 NewSte 1 1 1 1 -ketotestosterone 303.3 > 121 4.47

101 \c Sic 1 9-de ydrotestosteroiie 287.3 > 146.9 5.61

102 NewSte_13 A! lotetrahydrocortexone 335.3 > 317 6.89

103 New Sic ! 4 Adrenosterone 301.3 > 120.9 4.95

104 NewSte 15 Etiocholanolone 291 .3 > 255 6.72

105 NewSte 1 6 4-androstenediol 273.3 > 80.9 5.58

106 NewSte_17 16a~ketotestosterone 303.3 > 97 4.28

107 NewSte__18 19-hydroxyandrostendione 303.3 > 254.9 4.06

108 NewSte 19 4-Androsten-3,6-l 7-trione 301.3 > 96.9 4.97

109 NewSte_20 7b-hydroxy DHEA 305.3 > 287 5.47

1 10 NewSte_21 7a-hydroxy DHEA 305.3 > 287.1 5.47

1 1 1 NewSte _23 7-k.etoDHEA 303.25 > 80.9 4.25

1 12 NewSte__24 9-dehydroepiandrosterone 289.3 > 271.01 6.95

1 13 NewSic 25 Allocholesterol 387.3 > 105 6.30

1 14 XevvSic 27 9-dehydroprogesterone 313.3 > 85 7.53

1 1 -

1 15 NewSte_28 349.3 > 331 5.12

dehydrotetrahydrocorticosterone

1 16 NewSic 29 Tetrahydrocorticosteroiie 351.35 > 314.9 5.08

1 17 XevvSic 30 6b-hydroxy DOC 347.2 > 329 4.86

1 18 NewSte_33 Urocortisol 367.3 > 330.9 4.45

1 19 NewSte _34 Pregnanetriol 318.4 > 256.1 7.00

120 NewSte_36 16a-hydroxyprogesterone 33 1.35 > 96.9 5.05

121 NewSic 37 17a-hydroxypregnenolone 315.35 > 297 5.84

1 XevvSic 38 6-dehydrotestosterone 287.3 > 133 5.49

123 NewSte_39 9(1 l)-dehydro DHEA 287.3 > 269 5.24

124 NewSte _40 1 1 b-by droxyepiandrosterone 307.3 > 289.1 4.88

125 NewSte_41 16a-hydroxyepiandrosterone 307.3 > 289 4.55

126 New Sic 42 AndrOuSterone 291.3 > 273 7.29

127 XevvSic 43 Epiandrosterone 291.3 > 254.9 6.70 7a-hydroxyandrostenediol

128 NewSte_44 (DHEA); 289.3 > 271 3.38 neurosteroid

7b-hydroxyandrostenediol

129 NewSte__45 (DHEA); 289.3 > 271 3.14 neurosteroid

130 NewSte_46 4-cholestene-3a-OL 369.4 > 81 12.81

131 NewSte _ 47 4-pregnen-3b-ol-20-one 299.35 > 80.9 7.71

132 ^'NewSte_49 5-androsten-3b, 17-diol- 16-one 305.3 > 287.2 5.47

133 NewSte 50 19-Hydroxy DHEA 305.3 > 257 4.13

134 NewSte_51 16a-OH-pregnenolone 333.35 > 297 5.84

5 a-dihydrocortexone (5a- DH-

135 New Sic 52 333.35 > 315.1 6.91

DOC)

136 NewSte 53 Epitestosterone 289.2 > 108.9 5.81

137 NewSte_54 5 b-androstandione 289.2 > 270.9 6.94

138 NewSte_55 1 lb-OH-androstenedione 303.2 > 266.8 4.25

139 NewSte_56 11 -keto-Etiocholanolone 305.2 > 287 5.47

140 NewSte 57 5 a-pregnan- 1 1 a-OH-3 ,20-dione 333.25 > 84.9 5.95

141 NewSte 58 5 a-epoxypregnenolone 333.25 > 315 5.11

142 NewSte_59 5a-dibydro-l 1-keto-progesterone 331 .25 > 313 6.1 1

143 NewSte_60 11 - etoprogesterone 329.2 > 120.9 5.98

144 OS Cholesterol 369.4 12.80

145 OS1 4b-OH cholesterol 385.2 1 1.68

146 OS2 7a-QH cholesterol 367.4 > 94.8 10.79

147 OS3 7b-OH cholesterol 367.45 > 159.1 10.79

148 OS4 25-OH cholesterol 367.4 > 80.93 9.88

149 OSS 5 a,6a-epoxycholestero 1 481.3 > 96.7 12.29

150 OS6 7-keto-cholesterol 401.45 > 109 10.88

151 OS7 7-keto-25-OH cholesterol 417.2 > 399 8.21

152 OS8 6,7-dehydrocholesterol 385.25 > 96.9 12.81

153 OS9 6-dehydrocholestenone 383.2 > 80.9 12.41

154 OS 10 6-ketocholestenone 399.3 10.68

155 OS 11 Etiocholan-3-OH-17-one 291.3 > 273 6.76

156 OS13 7-Dehydrochoiesterol 365.4 > 156.9 12.46

157 OS 14 Dihydrocholesterol 372.35 13.74

158 OS20 Cholesterol-3-S0₄ 465.45 > 96.9 14.24

159 OS21 Coprostanol-7,12-diol 367.2 9.51

160 OS22 20a-OH-Cholesterol 367.4 10.33

161 OS23 22b-OH-Cholesterol 367.45 > 159.1 9.88

162 OS24 27-OH-Cholesterol 385.4 > 161 9.96

163 OS25 24-keto-Cholesterol 383.2 > 80.9 12.41

164 OS28 24-Hydroxycholesterol 367.4 > 147.05 10.12 165 OS29 e-3-OH-chol-5-n-24-oate 371.3 > 160.9 9.88

166 ΒΛ ! Chenodeoxychoiic acid 375.4 > 357.1 7 ^₇

167 BA_2 Ursodeoxycholic acid 375.4 > 357.1 6.20

168 ΒΛ 3 Murocholic acid 357.4 > 134.9 5.89

169 BA_4 Hyodeoxycholic acid 357.4 > 134.9 7.37

170 BA 5 Alloiitiiociiolic acid 359.45 > 95 8.36

171 BA. 6 Lithochoiic acid 359.45 > 95.1 8.93

I /2 BA_7 Taurocholanic acid 466.45 > 79.9 10.00

173 BA _.8 Taurohyodeoxycholie acid 498.4 > 79.8 4.27

174 BA_9 C ho lie acid 407.4 > 288.8 6.01

175 BA_10 Taurochenodeoxycholic acid 498.4 > 79.8 5.08

176 BA 11 Desoxycholic acid 391.4 > 345.1 7.56

177 BA12 Taurolithocholic acid 466.4 > 125.8 4.28

178 BA13 Glycolithocholic acid 432.4 > 73.8 7.70

179 BA14 6-keto-a! iolithocholic acid 391.3 > 373 6.68

180 BA15 Glycodeoxyciiolic acid 450.3 > 414 5.17

181 BA.16 Giycochenodeoxychoiic acid 448.3 > 73.7 6.16

182 BA17 Tauroursodeoxycholic acid 498.3 > 79.7 4.27

183 BA18 Taurodeoxycholic acid 498.3 > 79.8 5.08

184 BA19 Glycodeoxyciiolic acid 450.3 > 414 6.37

185 BA20 a-Muric olic acid 407.3 > 386.8 5.26

186 BA21 b-Muric olic acid 3 1.3 > 355 5.44

187 BA22 Tauro-b-muricholic acid 514.3 > 79.7 3.67

188 BA23 Taurocholic acid 514.3 > 79.73 4.36

189 BA24 Taurodehydrocholic acid 508.2 > 79.74 3.37

190 BA25 Glycodeliydrochoiic acid 460.3 > 366.9 4.13

191 SCI Androstandiol-3-GIu 316.2 > 275 6.62

192 SC2 1 l-keto-etiocholanolone-3-Glu 479.3 > 84.9 4.24

193 SC3 Etiocboianolone-3-Glu 465.2 > 84.9 5.26

Dehydroepiandrosterone sulfate

194 SC4 367.2 > 96.87 4.19

(DHEAS)

195 SC5 Epitestosterone-17- SO4 367.1 > 96.8 4.19

196 SC6 Testosterone- 17-Gl.u 465.2 > 289.1 4.20

197 SC7 Pregnanediol-3-Glu 495.2 > 75 5.68

198 SC8 17-OH-pregnanolone-3-Glu-Na 493.2 > 84.9 5.96

199 SC9 Pregnanolone-3-S0₄ 397.1 > 96.8 5.63

17,20-di-OH-progesterone-20-

200 SC10 509.2 > 314.9 4.18

Glu

201 sen. Cortisol-21- SO₄ 441.1 > 96.7 3.60

202 SCI 2 Pregneno lone-3 -Glu 491.3 > 1 12.8 5.46

203 SC13 Cholesterol-3-Glu 561.3 > 74.9 10.78 204 SCI 4 Allopregnanolone- S0₄ 397.2 > 96.6 5.63

205 SC I 5 Epiallopregnanokme- S0₄ 397.2 > 96.7 5.66

206 88 Estriol-3-Glu-Na 463.1 > 287 2.36

207 OS 12 24-dehydrocholesterol 367.4 > 147 1 1.93

208 OS 15 Coprosterol 383.2 > 95 1 1.65

209 OS 16 Campesterol 359.4 > 341.1 10.97

210 OS 17 b-sitosterol 415.45 > 1 19 10.92

21 1 OS 18 Brassicaterol 381.4 > 340.4 1 1.57

212 OS 19 Stigmasterol 394.4 > 353 10.66

213 OS26 Epia! locholesterol 369.4 > 81 12.99

214 OS30 Zymosterol 367.4 > 80.93 1 1.46

215 OS31 Lanosierol 409 > 80.9 14.22

216 OS32 Lathosterol 369.3 > 147 13.00

217 3C-16 ^l C labeled estradiol 257.3 > 160.89 5.55 33] The following procedure was used to extract steroids from the samples:

1. Aliquot 0.025-1 ml/0.025-1 g of biological sample (e.g., tissue or biofluid)

2. Add 1ml of chloroform to the sample

3. Homogenize/ vortex the sample 2 -times for 30 sec.

4. Centrifuge for 5 minutes

5. Remove chloroform layer (bottom) and put into speed-vac tube

6. Place the tubes in speed- ac (it takes ~ 30min to evaporate 1ml chloroform)

7. After 15 minutes check on samples

8. Repeat steps 1 -4 and add chloroform to speed- vac tube from Step 5

9. Store speed-vac tubes with chloroform extract in -80C immediately.

10. Freeze the aqueous layer/tissue-pellet (top and interphase) in liquid nitrogen

1 1. Freeze dry (it takes -6-10 h)

12. Once the sample is completely dry, remove from the freeze dryer

13. Extract the residue with 1 ml of methanol

14. Vortex 3 -times for 30 seconds each

15. Sonicate in sonicator bath for 1 minute

16. Vortex 3-times for 30 seconds each

17. Centrifuge for 5 minutes

18. Remove the methanol and add to new speed- vac tube.

19. Place the tubes in speed-vac (it takes ~ I h to evaporate 1 ml methanol)

20. Repeat steps 13-17 and add methanol to speed-vac tube from Step 19 21. Place the tubes in speed- vac and evaporate to dryness. ( about 1 hour).

22. Add chloroform extract from Step 9 to methanol extract tubes in Step 21.

23. Speed- vac the samples until just dry (~30min).

24. Add 125ul of methanol to dry pellet from Step 23.

25. Vortex 3-times for 30 seconds each

26. Sonicate for 1 minute

27. Vortex 3 times for 30 seconds each

28. Centrifuge for 5 minutes

29. Aliquot sample into 5kD filter tubes

30. Centrifuge for 5 minutes

31 . Transfer sample into labelled LCMS vial with inserts

32. Samples are ready to be am on the LCMS.

[0134] Analytical separations of the mixture was conducted on the UPLC system using a Cortecs*-C18 UPLC, 1.6 μηι, 2.1 X 150 mm analytical column (Waters Corp, Milford, MA) maintained at 45°C and at a flow rate of 0.15 ml/min. The gradient started with 100% A (0.1% formic acid in H₂0) and 0% B (0.1% formic acid in CH₃CN) and changed as set forth in Table 5.

Table 5. Chromatography gradient

[0135] The elutions from the UPLC column were introduced to the mass spectrometer,

[0136] Xevo^-TQ triple quadruple mass spectrometer (Waters Corp, Milford, MA) recorded MS and MS/MS spectra using Electro Spray Ionization (ESI) in positive ion (PI) and negative ion (NI) mode, capillar}' voltage of 3.0 kV, extractor cone voltage of 3 V and detector voltage of 490 V. Cone gas flow was set at 10 L/h and desolvation gas flow was maintained at 600 L/h. Source temperature and desolvation temperatures were set at 150 and 350 °C, respective!)'.

[ 0137] The masses of parent ion and daughter ions measured are described in Table 4. The MR.M method used is described in Table 6. The steroids detected in each MRM function set forth in Table 6 (e.g., MRM 1-31) are provided in Table 7. The resulting data were analyzed and processed using MassLynx 4.1 software (Waters Corp., Milford, MA).

Table 6. MRM Analysis

Detector

MRM

Monitoring Method Time ESI Mode Function

Window

1 SIR of 20 masses 7.00-15.00 Positive ion

2 MRM of 18 mass pairs 4.50-6.50 Positive ion

3 M RM of 4 mass pairs 7.00-9.00 Positive ion

4 M RM of 23 mass pairs 5.00-7.00 Positive ion

5 MRM of 17 mass pairs 3.00-5.00 Positive ion

6 MRM of 14 mass pairs 3.50-5.50 Positive ion

7 MRM of 1 1 mass pairs 6.00-8.00 Positive ion

8 MRM of 18 mass pairs 4.00-6.00 Positive ion

9 MRM of 14 mass pairs 5.50-7.50 Positive ion

10 MRM of 6 mass pairs 9.00-1 1.00 Positive ion

1 1 MRM of 6 mass pairs 7.50-9.50 Positive ion

12 MRM of 1 1 mass pairs 6.50-8.50 Positive ion

13 MRM of 5 mass pairs 2,50-4.50 Positive ion

14 MRM of 2 mass pairs 2.00-4.00 Positive ion

MRM of 1 mass pair 8.00-10.00 Positive ion

16 MRM of 3 mass pairs 1 1.00-13.00 Positive ion

17 MRM of 8 mass pairs 12.00-15.00 Positive ion

1 8 MRM of 4 mass pairs 9.50-1 1.50 Positive ion

19 MRM of 2 mass pairs 10.50-12.50 Positive ion

20 MRM of 3 mass pairs 1 1 .50-13.50 Positive ion

21 MRM of 2 mass pairs 10.00-12.00 Positive ion

-> -> MRM of 6 mass pairs 2,50-4.50 Negative ion

23 MRM of 5 mass pairs 4.50-6.50 Negative ion

24 MRM of 5 mass pairs 4.00-6.00 Negative ion

25 MRM of 8 mass pairs 3.00-5.00 Negative ion

26 MRM of 6 mass pairs 3.50-5.50 Negative ion

27 MRM of 3 mass pairs 0.00-3.50 Negative ion

28 MRM of 2 mass pairs 6.50-8.50 Negative ion

29 MRM of 4 mass pairs 5.00-7.00 Negative ion

30 M RM of 3 mass pairs 5.50-7.50 Negative ion

31 MRM of 4 mass pairs 9.50-15.00 Negative ion Table 7. MRM Functions

JA25

SC6

SC10

MRM 6 64

49

34

66

70

NewSte 55

NewSte 23

NewSte 17

NewSte 1 1

NewSte 9

NS 1 1

NS 6

NewSte 33

BA 12

MRM 7 21

4

NewSte 24

OS 1 1

36

NS 13

NewSte 34

NewSte_52 NewSte_13

MRM 8 80

67

18

48

NewSte 19

NewSte 10

NewSte 8

NewSte 41

NewSte 40

5

NS 12

BA4

BA1

MRM 13 New Sic 44 and NewSte _45

37

8J24

NS_22

11

MRM 14 79

76

MRM: 15 BA6

MRM: 16 OS 13

OS12

OS9, OS25

MRM 17 OS4

OS 32

New8te__46

OS, OS31

OS 14, OS 15

OS 15

MRM 18 OS31

OS2

OS3, OS23

OS19

OS6

MRM 19 OS23, OS3

OS 18

MRM 20 OS26, NewSte_46

OSS

OS1

MRM: 21 OS 10

OS 17

MRM: 22 27

58

29

31

B.A24

BA22 MRM 23 16

33

SC12

SC7

BA18

MRM 24 48

SC5

NewSte_4

BA20

SC3

MRM: 25 83

82

26

24 sen

30

SC6

SC10

MRM 26 25

SC4

SC2

BA17

BA8. BA10

BA23

MRM 27 75

88

32

MRM 28 BA J 1

BA13

MRM 29 NS_15

BA_9

BA16

SC8

MRM: 30 SC9

SC14

SCI 5

MRM 31 OS20

BA_7

OS5, OS26

SCI 3 [0138] This example describes an exemplary embodiment of the present invention. In particular, this example provides a method for the comprehensive steroid metabolome profiling of 206 steroids from a biological sample, e.g., a tissue, urine and serum sample. Without wishing to be bound by any particular theory, the extraction, UPLC and MS parameters used in the method described in this example improve peak resolution, provide a better signal-to-noise ratio, and provide better coverage of the steroid metabolome compared to the method described in Example 1 above.

[0139] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that cextain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was indi vidually incorporated by reference.

Claims

WHAT IS CLAIMED IS: L A method for profiling a steroid metabolome in a biological sample, the method comprising:

(a) extracting at least 100 steroids from the biological sample using liquid- liquid extraction (LLE) to form a steroid extract, wherein the at least 100 steroids in the steroid extract are not derivatized; and

(b) detecting the presence and/or level of the at least 100 steroids in the steroid extract using ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS).

2. The method of claim 1 , wherein the biological sample is obtained from an individual.

3. The method of claim 2, wherein the individual is a human.

4. The method of any one of claims 1 to 3, wherein the individual is suspected of having cancer, osteoporosis, a neurodegenerative disease, a cognitive disorder, a psychiatric disorder, cardiovascular disease, or obesity.

5. The method of claim 4, wherein the cancer is a hormone-related cancer selected from the group consisting of breast cancer, prostate cancer, adrenal cancer, thyroid cancer, pancreatic cancer, gastromtestinal cancer, pituitary cancer, colorectal cancer, ovarian cancer, endometrial cancer, and osteosarcoma.

6. The method of any one of claims 1 to 5, wherein the biological sample is a whole blood, serum, plasma, saliva, urine, cerebrospinal fluid, amniotic fluid, fine needle aspirate, cancer cells, cultured cells, cell culture media, or tissue biopsy sample.

7. The method of any one of claims 1 to 6, wherein the at least 100 steroids comprises at least 150 steroids.

8. The method of any one of claims 1 to 7, wherein the at least 100 steroids comprises from about 150 to about 200 steroids.

9. The method of any one of claims I to 8, wherein the at least 100 steroids comprises at least 200 steroids.

10. The method of any one of claims 1 to 9, wherein the at least 100 steroids are selected from the group consisting of androgens, corticosteroids, progestogens, estrogens, bile acids, oxysterois, neurosteroids, exogenous steroids, steroid metabolites, steroid derivatives, steroid conjugates, steroid adducts, and combinations thereof.

1 1. The method of claim 10, wherein the steroid adducts comprise estrogen-DNA adducts.

12. The method of any one of claims 1 to 11, wherein the at least 100 steroids comprise 50 or more steroids from Table 1 or 4.

13. The method of any one of claims 1 to 12, wherein the presence and/or level of the at least 100 steroids is detected in about 10 to about 15 minutes.

14. The method of any one of claims 1 to 13, wherein the UPLC comprises a chromatography column at about 45 °C to about 50 °C.

15. The method of any one of claims 1 to 14, wherein step (b) further comprises using multiple reaction monitoring (MRM),

16. The method of any one of claims 1 to 15, wherein step (b) further comprises selecting parent-daughter ion transitions for each of the at least 100 steroids.

17. The method of claim 16, wherein the selected parent-daughter ion transitions comprise 100 or more parent-daughter ion transitions from Table 1 or 4.

18. The method of claim 15, wherein at least 5 MRM functions are selected and at least 5 detection time windows are selected.

19. The method of claim 18, wherein the at least 5 MRM functions and the at least 5 detection time windows are set forth in Table 6.

20. The method of claim 19, wherein the steroids detected in each of the at least 5 MRM functions are set forth in Table 7.

21 . The method of claim 15, wherein at least 30 MRM functions are selected and at least 30 detection time windows are selected.

22. The method of claim 21, wherein the at least 30 MRM functions and the at least 30 detection time windows are set forth in Table 6.

23. The method of claim 22, wherein the steroids detected in each of the at least 30 MRM functions are set forth in Table 7.

24. A method for determining an imbalance in steroid metabolism in an individual, the method comprising:

(a) profiling a steroid metabolome in a biological sample obtained from the individual in accordance with the method of any one of claims 1 to 23 to form a steroid metabolome profile for the individual;

(b) comparing the steroid metabolome profile for the individual to a steroid metabolome profile for a control sample; and

(c) determining an imbalance in steroid metabolism in the individual when a difference between the steroid metabolome profile for the individual and the steroid metabolome profile for the control sample is detected.

25. The method of claim 24, wherein the control sample is a sample from an individual not having an imbalance in steroid metabolism.

26. The method of claim 24 or 25, wherein the steroid metabolome comprises the presence and/or level of one or more neurosteroids.

27. The method of any one of claims 24 to 26, wherein the imbalance in steroid metabolism is associated with cancer, osteoporosis, a neurodegenerative disease, a cognitive disorder, a psychiatric disorder, cardiovascular disease, or obesity.

28. The method of claim 27, wherein the cancer is a hormone-related cancer selected from the group consisting of breast cancer, prostate cancer, adrenal cancer, thyroid cancer, pancreatic cancer, gastrointestinal cancer, pituitary cancer, colorectal cancer, ovarian cancer, endometrial cancer, and osteosarcoma.

29. A method for diagnosing cancer in an individual, the method comprising: (a) profiling a steroid metabolome in a biological sample obtained from the individual in accordance with the method of any one of claims 1 to 23 to form a steroid metabolome profile for the individual;

(b) comparing the steroid metabolome profile for the individual to at least one steroid metabolome profile for at least one type of cancer; and

(c) determining the type of cancer in the individual when a similarity between the steroid metabolome profile for the individual and one of the at least one steroid metabolome profiles for the at least one type of cancer is detected.

30. The method of claim 29, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, prostate cancer, adrenal cancer, thyroid cancer, pancreatic cancer, gastrointestinal cancer, pituitary cancer, colorectal cancer, ovarian cancer, liver cancer, kidney cancer, endometrial cancer, neuroendocrine cancer, bone cancer, hematological cancer, and a combination thereof.

31 . The method of claim 29 or 30, wherein the at least one steroid metabolome profile for the at least one type of cancer comprises a plurality of steroid metabolome profiles for different types of cancer.

32. The method of claim 31 , wherein the different types of cancer comprise at least two cancer types selected from the group consisting of breast cancer, lung cancer, prostate cancer, adrenal cancer, thyroid cancer, pancreatic cancer, gastrointestinal cancer, pituitary cancer, colorectal cancer, ovarian cancer, liver cancer, kidney cancer, endometrial cancer, neuroendocrine cancer, bone cancer, hematological cancer, and a combination thereof.