An LC/MS/MS method for analyzing the steroid metabolome with high accuracy and from small serum samples

Abstract

Analyzing global steroid metabolism in humans can shed light on the etiologies of steroid-related diseases. However, existing methods require large amounts of serum and lack the evaluation of accuracy. Here, we developed an LC/MS/MS method for the simultaneous quantification of 12 steroid hormones: testosterone, pregnenolone, progesterone, androstenedione, corticosterone, 11-deoxycortisol, cortisol, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, estriol, and estradiol. Steroids and spiked internal standards in 100 μl serum were extracted by protein precipitation and liquid-liquid extraction. The organic phase was dried by evaporation, and isonicotinoyl chloride was added for steroid derivatization, followed by evaporation under nitrogen and redissolution in 50% methanol. Chromatographic separation was performed on a reverse-phase PFP column, and analytes were detected on a triple quadrupole mass spectrometer with ESI. The lower limits of quantification ranged from 0.005 ng/ml for estradiol to 1 ng/ml for cortisol. Apparent recoveries of steroids at high, medium, and low concentrations in quality control samples were between 86.4% and 115.0%. There were limited biases (−10.7% to 10.5%) between the measured values and the authentic values, indicating that the method has excellent reliability. An analysis of the steroid metabolome in pregnant women highlighted the applicability of the method in clinical serum samples. We conclude that the LC/MS/MS method reported here enables steroid metabolome analysis with high accuracy and reduced serum consumption, indicating that it may be a useful tool in both clinical and scientific laboratory research. Analyzing global steroid metabolism in humans can shed light on the etiologies of steroid-related diseases. However, existing methods require large amounts of serum and lack the evaluation of accuracy. Here, we developed an LC/MS/MS method for the simultaneous quantification of 12 steroid hormones: testosterone, pregnenolone, progesterone, androstenedione, corticosterone, 11-deoxycortisol, cortisol, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, estriol, and estradiol. Steroids and spiked internal standards in 100 μl serum were extracted by protein precipitation and liquid-liquid extraction. The organic phase was dried by evaporation, and isonicotinoyl chloride was added for steroid derivatization, followed by evaporation under nitrogen and redissolution in 50% methanol. Chromatographic separation was performed on a reverse-phase PFP column, and analytes were detected on a triple quadrupole mass spectrometer with ESI. The lower limits of quantification ranged from 0.005 ng/ml for estradiol to 1 ng/ml for cortisol. Apparent recoveries of steroids at high, medium, and low concentrations in quality control samples were between 86.4% and 115.0%. There were limited biases (−10.7% to 10.5%) between the measured values and the authentic values, indicating that the method has excellent reliability. An analysis of the steroid metabolome in pregnant women highlighted the applicability of the method in clinical serum samples. We conclude that the LC/MS/MS method reported here enables steroid metabolome analysis with high accuracy and reduced serum consumption, indicating that it may be a useful tool in both clinical and scientific laboratory research. Steroid hormones are endogenous compounds that are synthesized from cholesterol by multiple enzymes in the gonads, adrenal glands, and placenta (1Keski-Rahkonen P. Huhtinen K. Poutanen M. Auriola S. Fast and sensitive liquid chromatography–mass spectrometry assay for seven androgenic and progestagenic steroids in human serum.J. Steroid Biochem. Mol. Biol. 2011; 127: 396-404Crossref PubMed Scopus (95) Google Scholar, 2Soldin S.J. Soldin O.P. Steroid hormone analysis by tandem mass spectrometry.Clin. Chem. 2009; 55: 1061-1066Crossref PubMed Scopus (207) Google Scholar). Although steroids are present at low concentrations, they play a vital role in human health and have been reported to be associated with many diseases, including type 2 diabetes (3Ding E.L. Song Y.Q. Malik V.S. Liu S.M. Sex differences of endogenous sex hormones and risk of type 2 diabetes: a systematic review and meta-analysis.JAMA. 2006; 295: 1288-1299Crossref PubMed Scopus (1035) Google Scholar), polycystic ovary syndrome (4Handelsman D.J. Teede H.J. Desai R. Norman R.J. Moran L.J. Performance of mass spectrometry steroid profiling for diagnosis of polycystic ovary syndrome.Hum. Reprod. 2017; 32: 418-422Crossref PubMed Scopus (28) Google Scholar), and depression (5Girdler S.S. Lindgren M. Porcu P. Rubinow D.R. Johnson J.L. Morrow A.L. A history of depression in women is associated with an altered GABAergic neuroactive steroid profile.Psychoneuroendocrinology. 2012; 37: 543-553Crossref PubMed Scopus (53) Google Scholar). In steroid-related research, steroid metabolome analysis including steroids and their metabolites seems to be more critical than assaying a single steroid to obtain more comprehensive information (1Keski-Rahkonen P. Huhtinen K. Poutanen M. Auriola S. Fast and sensitive liquid chromatography–mass spectrometry assay for seven androgenic and progestagenic steroids in human serum.J. Steroid Biochem. Mol. Biol. 2011; 127: 396-404Crossref PubMed Scopus (95) Google Scholar, 6Eisenhofer G. Fassnacht M. Steroid profiling for adrenocortical disorders: a pathway for omics-based diagnostics.Clin. Chem. 2017; 63: 1787-1789Crossref PubMed Scopus (8) Google Scholar, 7Jeanneret F. Tonoli D. Rossier M.F. Saugy M. Boccard J. Rudaz S. Evaluation of steroidomics by liquid chromatography hyphenated to mass spectrometry as a powerful analytical strategy for measuring human steroid perturbations.J. Chromatogr. A. 2016; 1430: 97-112Crossref PubMed Scopus (67) Google Scholar). Thus, accurate steroid metabolome analysis is considered very important for exploring the mechanism of steroid-related diseases. Traditional methods for steroid analysis are based on immunoassays (IAs) due to their accessibility and ease of use (8Fahlbusch F.B. Heussner K. Schmid M. Schild R. Ruebner M. Huebner H. Rascher W. Doerr H.G. Rauh M. Measurement of amniotic fluid steroids of midgestation via LC-MS/MS.J. Steroid Biochem. Mol. Biol. 2015; 152: 155-160Crossref PubMed Scopus (23) Google Scholar). However, IA techniques often lack sufficient specificity, especially when steroids are present at very low concentrations (9Yuan T-F. Le J. Cui Y. Peng R. Wang S-T. Li Y. An LC-MS/MS analysis for seven sex hormones in serum.J. Pharm. Biomed. Anal. 2019; 162: 34-40Crossref PubMed Scopus (30) Google Scholar). The use of IAs can be also compromised by their matrix interferences, limited dynamic range, and single analyte measurement at a time (10Koal T. Schmiederer D. Pham-Tuan H. Röhring C. Rauh M. Standardized LC-MS/MS based steroid hormone profile-analysis.J. Steroid Biochem. Mol. Biol. 2012; 129: 129-138Crossref PubMed Scopus (156) Google Scholar). More MS methods have been developed for steroid detection due to their high specificity and sensitivity. GC/MS has a long history and remains an effective tool for steroid analysis; however, extremely high analytical and biochemical knowledge is needed for GC/MS steroid analysis, which is performed in a few highly specialized laboratories (11Krone N. Hughes B.A. Lavery G.G. Stewart P.M. Arlt W. Shackleton C.H.L. Gas chromatography/mass spectrometry (GC/MS) remains a pre-eminent discovery tool in clinical steroid investigations even in the era of fast liquid chromatography tandem mass spectrometry (LC/MS/MS).J. Steroid Biochem. Mol. Biol. 2010; 121: 496-504Crossref PubMed Scopus (295) Google Scholar, 12Travers S. Martinerie L. Bouvattier C. Boileau P. Lombès M. Pussard E. Multiplexed steroid profiling of gluco- and mineralocorticoids pathways using a liquid chromatography tandem mass spectrometry method.J. Steroid Biochem. Mol. Biol. 2017; 165: 202-211Crossref PubMed Scopus (46) Google Scholar, 13Wudy S.A. Schuler G. Sánchez-Guijo A. Hartmann M.F. The art of measuring steroids: principles and practice of current hormonal steroid analysis.J. Steroid Biochem. Mol. Biol. 2018; 179: 88-103Crossref PubMed Scopus (112) Google Scholar). An alternative and complementary analytical technique that has been used for decades is LC/MS/MS. LC/MS/MS methods can provide high analytical sensitivity and specificity and simultaneously detect multiple analytes, which exerts important roles on metabolomics studies (2Soldin S.J. Soldin O.P. Steroid hormone analysis by tandem mass spectrometry.Clin. Chem. 2009; 55: 1061-1066Crossref PubMed Scopus (207) Google Scholar, 14Peitzsch M. Dekkers T. Haase M. Sweep F. Quack I. Antoch G. Siegert G. Lenders J.W.M. Deinum J. Willenberg H.S. et al.An LC-MS/MS method for steroid profiling during adrenal venous sampling for investigation of primary aldosteronism.J. Steroid Biochem. Mol. Biol. 2015; 145: 75-84Crossref PubMed Scopus (102) Google Scholar, 15Rauh M. Steroid measurement with LC–MS/MS. Application examples in pediatrics.J. Steroid Biochem. Mol. Biol. 2010; 121: 520-527Crossref PubMed Scopus (83) Google Scholar). Considering these superiorities, many LC/MS/MS methods have been developed to measure steroid profiling or steroid metabolome. However, there are some problems that remain unresolved. One of the shortcomings of the existing methods is a lack of traceability for the steroid metabolome. Such deficient traceability inevitably will reduce the recognition of the results from these methods. Another problem is that these methods for steroid metabolome analysis cannot detect important estrogens such as estradiol and estriol in a single analysis (14Peitzsch M. Dekkers T. Haase M. Sweep F. Quack I. Antoch G. Siegert G. Lenders J.W.M. Deinum J. Willenberg H.S. et al.An LC-MS/MS method for steroid profiling during adrenal venous sampling for investigation of primary aldosteronism.J. Steroid Biochem. Mol. Biol. 2015; 145: 75-84Crossref PubMed Scopus (102) Google Scholar, 16Häkkinen M.R. Heinosalo T. Saarinen N. Linnanen T. Voutilainen R. Lakka T. Jääskeläinen J. Poutanen M. Auriola S. Analysis by LC-MS/MS of endogenous steroids from human serum, plasma, endometrium and endometriotic tissue.J. Pharm. Biomed. Anal. 2018; 152: 165-172Crossref PubMed Scopus (44) Google Scholar) because the estrogens are often detected under negative mode, which is incompatible with the other steroids (1Keski-Rahkonen P. Huhtinen K. Poutanen M. Auriola S. Fast and sensitive liquid chromatography–mass spectrometry assay for seven androgenic and progestagenic steroids in human serum.J. Steroid Biochem. Mol. Biol. 2011; 127: 396-404Crossref PubMed Scopus (95) Google Scholar). In addition, these methods require a large sample volume (10Koal T. Schmiederer D. Pham-Tuan H. Röhring C. Rauh M. Standardized LC-MS/MS based steroid hormone profile-analysis.J. Steroid Biochem. Mol. Biol. 2012; 129: 129-138Crossref PubMed Scopus (156) Google Scholar, 14Peitzsch M. Dekkers T. Haase M. Sweep F. Quack I. Antoch G. Siegert G. Lenders J.W.M. Deinum J. Willenberg H.S. et al.An LC-MS/MS method for steroid profiling during adrenal venous sampling for investigation of primary aldosteronism.J. Steroid Biochem. Mol. Biol. 2015; 145: 75-84Crossref PubMed Scopus (102) Google Scholar, 17Fanelli F. Belluomo I. Di Lallo V.D. Cuomo G. De Iasio R. Baccini M. Casadio E. Casetta B. Vicennati V. Gambineri A. et al.Serum steroid profiling by isotopic dilution-liquid chromatography–mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults.Steroids. 2011; 76: 244-253Crossref PubMed Scopus (160) Google Scholar), which reduces the applicability of the method. Therefore, it is necessary to develop a new LC/MS/MS method for comprehensive steroid metabolome analysis to solve these problems. In this study, we established a novel LC/MS/MS method based on acylation derivatization with alcoholic hydroxyl groups for the steroid metabolome, including progestogens, androgens, estrogens, mineralocorticoids, and glucocorticoids (Fig. 1), and this proposed method was successfully applied to detect the steroid metabolome in second-trimester pregnant women. The proposed method shows a number of advantages. First, the method was proven to exhibit high accuracy by certified reference materials covering most steroids except for pregnenolone, 17-hydroxypregnenolone, and estriol. Second, only 100 μl serum was required in this method, and the serum consumption was very low among the reported methods analyzing the steroid metabolome (1Keski-Rahkonen P. Huhtinen K. Poutanen M. Auriola S. Fast and sensitive liquid chromatography–mass spectrometry assay for seven androgenic and progestagenic steroids in human serum.J. Steroid Biochem. Mol. Biol. 2011; 127: 396-404Crossref PubMed Scopus (95) Google Scholar, 12Travers S. Martinerie L. Bouvattier C. Boileau P. Lombès M. Pussard E. Multiplexed steroid profiling of gluco- and mineralocorticoids pathways using a liquid chromatography tandem mass spectrometry method.J. Steroid Biochem. Mol. Biol. 2017; 165: 202-211Crossref PubMed Scopus (46) Google Scholar, 14Peitzsch M. Dekkers T. Haase M. Sweep F. Quack I. Antoch G. Siegert G. Lenders J.W.M. Deinum J. Willenberg H.S. et al.An LC-MS/MS method for steroid profiling during adrenal venous sampling for investigation of primary aldosteronism.J. Steroid Biochem. Mol. Biol. 2015; 145: 75-84Crossref PubMed Scopus (102) Google Scholar, 17Fanelli F. Belluomo I. Di Lallo V.D. Cuomo G. De Iasio R. Baccini M. Casadio E. Casetta B. Vicennati V. Gambineri A. et al.Serum steroid profiling by isotopic dilution-liquid chromatography–mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults.Steroids. 2011; 76: 244-253Crossref PubMed Scopus (160) Google Scholar, 18Karvaly G. Kovács K. Mészáros K. Kocsis I. Patócs A. Vásárhelyi B. The comprehensive characterization of adrenocortical steroidogenesis using two-dimensional ultra-performance liquid chromatography – electrospray ionization tandem mass spectrometry.J. Pharm. Biomed. Anal. 2018; 153: 274-283Crossref PubMed Scopus (12) Google Scholar). The minimized serum consumption will increase the applicability of method. Third, the important estrogens (estradiol and estriol) were included in steroid profiling. In this proposed method, similar to other steroids, estrogens could react to generate derivative products that could be detected in the ESI positive mode. Therefore, we achieved the simultaneous quantification for estrogens and other steroids in a single analysis. In this study, we enrolled 30 singleton second-trimester pregnant women and analyzed their steroid metabolome using the proposed LC/MS/MS method. Informed consent was obtained from the subjects and there was protocol approval from Ethics Committee of Renmin Hospital of Wuhan University. The study abides by the Declaration of Helsinki principles. Pregnenolone (Preg), pregnenolone-17,21,21,21-d4 (Preg-d4), 17-hydroxypregnenolone (17OHPreg), 17-hydroxypregnenolone-21,21,21-d3 (17OHPreg-d3), 17-hydroxyprogesterone (17OHP), 17-hydroxyprogesterone-2,2,4,6,6,21,21,21-d8 (17OHP-d8), corticosterone (CORT), corticosterone-2,2,4,6,6,17,21,21-d8 (CORT-d8), cortisol (COR), 11-deoxycortisol (DOC), estradiol (E2), estriol (E3), estriol-2,4,17-d3 (E3-d3), and progesterone-2,2,4,6,6,17,21,21,21-d9 (P-d9) were purchased from Toronto Research Chemicals (Toronto, Canada). Cortisol-9,11,12,12-d4 (COR-d4), 11-deoxycortisol-2,2,4,6,6-d5 (DOC-d5), progesterone, estradiol-2,4-d2 (E2-d2), androstenedione-2,3,4-13C3 (AD-13C3), dehydroepiandrosterone-2,2,3,4,4,6-d6 (DHEA-d6), testosterone-16,16,17-d3 (T-d3), methyl tert-butyl ether (MTBE), and isonicotinoyl chloride (INC) were purchased from Sigma-Aldrich (Beijing, China). We obtained dehydroepiandrosterone (DHEA), androstenedione (AD), and testosterone from Aladdin Industrial Corporation (Shanghai, China). Dichloromethane was purchased from J&K Scientific (Beijing, China). HPLC-grade methanol and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA). Purified water was produced using a Milli-Q apparatus (Millipore, Bedford, MA). DC Mass Spect Gold from Golden West Diagnostics (Temecula, CA) was used as a blank matrix, which was prepared by treating human serum with charcoal to get rid of steroids. We obtained SRM 971 from the National Institute of Standards and Technology and BCR 576, 577, and 578 from the Institute for Reference Materials and Measurements. MassCheck® Steroid Panel 1 Serum Control and MassCheck Steroid Panel 2 Serum Control were purchased from Chromsystems Instruments and Chemicals (Munich, Germany). We prepared an INC solution by dissolving 10 mg INC in 1 ml acetonitrile. After ultrasonic treatment and centrifugation, the supernatant was used for derivatization. We prepared stock solutions of each analyte and internal standard (IS) in acetonitrile at 1 mg/ml. Working solutions were prepared in acetonitrile containing the following steroid concentrations: E2, 40 ng/ml; E3, 2 ng/ml; testosterone, 800 ng/ml; Preg, 400 ng/ml; 17OHPreg, 800 ng/ml; 17OHP, 400 ng/ml; CORT, 2,000 ng/ml; COR, 40,000 ng/ml; DOC, 200 ng/ml; DHEA, 2,000 ng/ml; AD, 400 ng/ml; and progesterone, 400 ng/ml. We prepared IS working solutions containing the following concentrations: E2-d2, 4 ng/ml; E3-d3, 200 ng/ml; T-d3, 80 ng/ml; Preg-d4, 40 ng/ml; 17OHPreg-d3, 80 ng/ml; 17OHP-d8, 40 ng/ml; CORT-d8, 200 ng/ml; COR-d4, 2,000 ng/ml; DOC-d5, 20 ng/ml; DHEA-d6, 200 ng/ml; AD-13C3, 40 ng/ml; and P-d9, 40 ng/ml. We prepared six calibrators at different concentrations (C1–C6) covering physiological ranges by adding working solutions into the blank matrix followed by stepwise dilution using the blank matrix. The concentrations of steroids in calibrators are listed in supplemental Table S1. The quality control (QC) samples were prepared at three levels by adding working solutions into the blank matrix (high, medium, and low QC, respectively). We transferred 100 μl of calibrators, QC samples, and serum samples to polypropylene tubes. Then, 5 μl of IS working solution and 200 μl acetonitrile were added, followed by for protein 1 ml was added, and the was for 5 for liquid-liquid extraction. After 5 the organic was transferred to a and under nitrogen at The dried was in 100 μl and 10 μl The was under nitrogen at and in 100 μl 50% methanol and transferred to an The was an and the tandem mass spectrometer was an mass We the separation of steroids on a PFP 100 with a of The was to and the volume was 20 were water and methanol The was as to and B. The analysis required and more compounds were to for the to the mass spectrometer The mass spectrometer was in positive using an ESI The analytes were by multiple and mass are listed in Table In addition, positive ESI mass of steroids are in supplemental The and of analytes are in supplemental Table and were using for steroids using in a new the LC/MS/MS method, we the matrix and in with the and and and Standards we recoveries by the measured steroid concentrations to the spiked values in the blank matrix, and the values of recoveries be between and The were by measuring the six calibrators in using a and the of was used to the The lower limits of quantification were as the in serum that could be measured with and accuracy we spiked high concentrations of 12 steroids into and samples to serum, and we the recoveries of steroids except for the and the recoveries be between and was using QC samples at three The measurement was performed six in and in six The of measurement values was used to the and the be than were with the the of steroids to from liquid-liquid of the matrix, and A to the of steroids to from liquid-liquid of The values be between and were performed using QC samples at three First, was at and and and and Second, was three of and Third, was at and for 4 and we the traceability of this method using certified reference The MassCheck Steroid Panel included testosterone, E2, progesterone, AD, DHEA, 17OHP, COR, CORT, and at three The SRM included testosterone, progesterone, and at concentrations, and the BCR included at three reference materials were measured in using the proposed method, and the measured values were with the certified After the protein liquid-liquid was In to obtain we the recoveries of organic including and recoveries were on the of a standard solution of steroids was added to blank serum and the IS was added to the organic extraction. both the standard and IS were added extraction. recoveries were by the of steroids to the IS in A to the in B. in produced the for most steroids. Therefore, was as the for liquid-liquid in this method. The derivatization is in the were including and We that was the most to the with and evaporation by nitrogen adding and INC was an for the The may be that evaporation could to the of and the evaporation, time and could have on the Considering the of steroids at high and evaporation and INC and a of were The evaporation required 2 the was to 2 There are of isonicotinoyl derivatization with other derivatization methods. First, this derivatization is and 2 Second, and other or be may the ionization or the and mass The and for steroids are listed in Table Such sensitivity was even for groups of such as and and the values for the of steroids were than Although and were detected derivatization due to the lack of alcoholic hydroxyl their sensitivity was compromised by the of and for in a new to the similar of steroids, we the specificity by steroids as of other steroids. After interferences, we for recoveries from to indicating of this method. accuracy and were on three QC high, medium, and Apparent recoveries of steroids at and concentrations are listed in Table steroids, the recoveries were between 86.4% and at different After method we that and were between and for each steroid results accuracy and of steroids in and samples and recoveries of in a new and of steroid metabolome in a new The ranges of steroids for levels were between and indicating that there was or from the matrix, and the quantification of steroids be by the serum matrix during steroid analysis. Although the blank matrix is used for matrix we the that the could many other that may matrix After we that steroids could be in serum for at at and at and the in steroid concentrations were of concentrations three that the products were for at and After measuring the certified reference materials and the measured values with certified values at different we that there were limited biases (−10.7% to 10.5%) between the measured values of developed method and the certified values in the SRM and BCR reference materials and the MassCheck Steroid Panel Serum Control results the high accuracy of and measured values of steroids in the SRM and BCR reference in a new and measured values of steroids in the MassCheck® Steroid Panel Serum in a new is a for women. this steroids are of for and the concentrations of many steroids In this study, we enrolled 30 singleton second-trimester pregnant women and analyzed their steroid metabolome using the proposed LC/MS/MS method. The separation excellent during sample and of steroids in serum samples of pregnant women are in The and steroid concentrations are listed in Table and steroid concentrations of pregnant in a new In this LC/MS/MS method a and sensitive assay based on acylation derivatization for steroid metabolome that high accuracy and very low serum method was successfully applied in a steroid metabolome analysis of pregnant women and is a for on steroid

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