The content of endogenous anabolic steroids is extremely low in biological matrices.Its chemical structure contains polar functional groups such as hydroxyl and carbonyl, which limits their applicability in GC-MS. The lack of ionized groups in the chemical structure leads to poor sensitivity in LC-MS, which plays a significant role in various physiological activities analysis. It is an effective way to enhance the response of mass spectrometer by modifying the chemical structure of endogenous anabolic steroids through derivatization technology.This review summarizes various derivatization reagents and corresponding derivatization processes of endogenous anabolic steroids analysis in the methods based on different testing instruments and methodologies. The advantage and disadvantage of all kinds of derivatiaztion methods and the prospect of the endogenous anabolic steroids derivatiaztion techniques are also discussed.
SHIL, MASER-GLUTHC, REMERT.Daily urinary free cortisol and cortisone excretion is associated with urine volume in healthy children[J]. Steroids, 2008, 73(10): 1446-1450.
ABSTRACT In experimental studies, a high fluid intake and a corresponding high urine volume have been shown to increase renal excretion rates of urinary free cortisol (UFF) and cortisone (UFE) in adults. We aimed to examine whether 24-h UFF and UFE excretion rates are also affected by urine volume in children. In 24-h urine samples of 100 pre-pubertal and 100 pubertal healthy children UFF, UFE, tetrahydrocortisol (THF), 5alpha-tetrahydrocortisol (5alpha-THF), and tetrahydrocortisone (THE) were quantified by RIA. The sum of THF, 5alpha-THF, and THE, the 3 primarily glucuronidated tetrahydrometabolites (GC3), reflects daily cortisol secretion. Associations of urine volume with outcome variables UFF, UFE, and GC3 were examined in both developmental groups using multiple regression models adjusted for sex, body weight and height. Significant positive associations were observed between 24-h urine volume and UFF and UFE in both groups with the highest explained variation for UFE [partial R(2)=0.11 in pre-pubertal group (P<0.005); partial R(2)=0.15 in pubertal group (P<0.0001)]. However, for outcome GC3, urine volume was not significant in either of the groups. Urinary 24-h excretion rates of UFF and UFE but not of the marker of glucocorticoid secretion are affected by daily urine volume in healthy free-living children. For a specific assessment of associations of UFF and UFE with (patho)physiologically relevant factors, urine volume should be considered as a confounder.
HSING AW, STANCZYK FZ, BELANGERA, et al.Reproducibility of serum sex steroid assays in men by RIA and mass spectrometry[J]. Cancer Epidemiol Biomarkers Prev, 2007,16(5): 1004-1008.
There is an increasing trend to apply gas chromatography combined with mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS) assay methods to large-scale epidemiologic studies for the measurement of serum sex steroids. These methods are generally considered the gold standard for sex steroid measurements because of their accuracy, sensitivity, turnaround time, and ability to assess a more complete panel of steroid metabolites in the same run. In this report, we evaluated the precision, including within-batch (intra) and between-batch (inter) reproducibility, of steroid hormone measurements determined by GC-MS and LC-MS/MS assays and RIA and compared measurements among these methods. Specifically, 282 overnight fasting serum samples from 20 male volunteers were analyzed for 12 steroid metabolites by GC-MS or LC-MS/MS in one lab over a 4-month period. Six of the analytes were also measured by RIA in another lab. Unconjugated hormones, including testosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione, androst-5-ene-3beta,17beta-diol, estrone, and estradiol, were measured by GC-MS, whereas conjugated hormones, including DHEA sulfate, androsterone glucuronide, 5alpha-androstane-3alpha,17beta-diol 3-glucuronide, 5alpha-androstane-3alpha,17beta-diol 17-glucuronide, and estrone sulfate, were measured by LC-MS/MS. A subset of these hormones, including testosterone, dihydrotestosterone, androstenedione, 5alpha-androstane-3alpha,17beta-diol 17-glucuronide, estrone, and estradiol, were also measured by RIA following extraction and chromatography. We used the coefficient of variation (CV) and the intraclass correlation coefficient (ICC) to assess within- and between-batch assay variations. For the 12 analytes measured by GC-MS or LC-MS/MS, CVs and ICCs for within- and between-batch measurements were similar, with CVs ranging from 6.1% to 21.4% and ICCs ranging from 87.6% to 99.2%. The six analytes measured by RIA had good CVs and ICCs, with CVs <10% and ICCs >70% (range, 71.7-99.7%). For the six metabolites that were measured by both methods, the CVs were similar, whereas the ICCs were generally higher with the GC-MS method. The absolute values for each analyte measured by RIA and GC-MS differed, with RIAs usually yielding markedly higher levels than GC-MS, although the Pearson and Spearman correlation coefficients for these six analytes were near one and all were significant (P < 0.001). Our results show that RIA, GC-MS, and LC-MS/MS assays for androgens and estrogens in the two labs included in the study have good reproducibility, as measured by small CVs (<15%) and high ICCs (>80%), with the exception of estradiol (71.7%) when measured by RIA. Despite substantial differences in absolute measurements of sex steroid hormones by RIA and MS methods, correlations between the two assays for the six sex steroids measured in the two labs were high (>0.9). However, it is important for future large epidemiologic studies to incorporate MS with high reproducibility and specificity to measure a more complete profile of androgen and estrogen metabolites to clarify the role of sex steroids in prostate cancer.
CARTER GD, CARTERR, JONESJ, et al.How accurate are assays for 25-hydroxyvitamin D-data from the international vitamin D external quality assessment[J].Clin Chem,2004,50(1):2195-2197.
Elsevier’s Scopus, the largest abstract and citation database of peer-reviewed literature. Search and access research from the science, technology, medicine, social sciences and arts and humanities fields.
GITONF, FIETJ, CORNU JN, et al.Serum sex steroids measured in middle-aged European and African Caribbean men by gas chromatography mass spectrometry[J]. Eur J Endocrinol, 2011, 165(5): 917-924.
Differences in circulating steroid hormone levels have been hypothesized to explain ethnic differences in steroid-related diseases. The aim of this study was to determine the serum levels of a wide panel of steroid hormones, both androgens and estrogens, in healthy middle-aged African-Caribbean and European men.Serum steroid hormone levels were determined in men participating in a systematic public health study funded by the French National Health Insurance system. Blood was collected in the morning from 304 healthy African-Caribbean and European men aged between 40 and 69 years. Serum steroids were measured by mass spectrometry-gas chromatography, except for DHEAS and sex hormone-binding globulin, which were determined by RIA. Data were analyzed in 10-year age intervals by analysis of covariance, with adjustment for age, body mass index, waist-to-hip ratio, tobacco and alcohol consumption, and season of sampling.Compared with Europeans, African-Caribbean men presented significantly higher serum levels of measured bioavailable testosterone, 4-androstenedione (4-dione), and estrone (E1) regardless of the age group, of 5-androstenediol (5-diol) in those aged 40-49 and 50-59 years, and of testosterone (TT) and dihydrotestosterone in those aged 40-49 years. In contrast, European men aged 40-69 years showed significantly higher serum levels of DHEA and DHEAS.Significant differences in serum steroid hormone levels were observed in middle-aged African-Caribbean and European men. Whether such differences could contribute to ethnic differences in disease risk in adult men remains to be investigated. Some steroids, such as bioavailable TT, 4-dione, 5-diol, and E1, deserve particular attention.
WANGY, GAY GD, BOTELHO JC, et al.Total testosterone quantitative measurement in serum by LC-MS/MS[J].Clin Chim Acta, 2014, 436(25): 263-267.
Abstract Reliable measurement of total testosterone is essential for the diagnosis, treatment and prevention of a number of hormone-related diseases affecting adults and children. A mass spectrometric method for testosterone determination in human serum was carefully developed and thoroughly validated. Total testosterone from 100 L serum is released from proteins with acidic buffer and isolated by two serial liquid-liquid extraction steps. The first extraction step isolates the lipid fractions from an acidic buffer solution using ethyl acetate and hexane. The organic phase is dried down and reconstituted in a basic buffer solution. The second extraction step removes the phospholipids and other components by hexane extraction. Liquid chromatography-isotopic dilution tandem mass spectrometry is used to quantify the total testosterone. The sample preparation is automatically conducted in a liquid-handling system with 96-deepwell plates. The method limit of detection is 9.71 pmol/L (0.280 ng/dL) and the method average percent bias is not significantly different from reference methods. The performance of this method has proven to be consistent with the method precision over a 2-year period ranging from 3.7 to 4.8% for quality control pools at the concentrations 0.527, 7.90 and 30.7 nmol/L (15.2, 228, and 886 ng/dL), respectively. This method provides consistently high accuracy and excellent precision for testosterone determination in human serum across all clinical relevant concentrations. Published by Elsevier B.V.
NIESSENW.Current practice of gas chromatography-masss spectrometry[M]. New York: Marcel Dekker, Inc, 2001:309-326.
[本文引用:1]
[7]
CEGLAREKU, WERNERM, KORTZR, et al.Preclinical challenges in steroid analysis of human samples[J].J Steroid Biochem Mol Biol, 2010, 121(3/5):505 512.
ABSTRACT Preclinical challenges in the analysis of steroid hormones are primarily determined by biological factors involved in the physiology and pathophysiology of hormone secretion. Major biologically influencing factors like age, sex, pubertal stage, pregnancy, phase of the menstruation, and diurnal rhythm have to be considered in the definition of reference ranges for steroids and their clinical interpretation. Hitherto, in clinical routine laboratories steroids were mainly determined by direct immunoassays applied on automated platforms, which are simple, rapid and cheap if a high number of samples are measured. However, technical factors like cross-reactivity of related steroid metabolites or limited analytical ranges have to be taken in account and may impair accuracy and precision of these direct methods. The actual development of mass spectrometry based analytical platforms for the determination of single steroid or steroid patterns seems to be an alternative analytical approach combining multi-parametric analysis, high sensitivity and specificity as well simple sample pre-treatment, robustness and low running costs for steroid analysis. This short review will give an overview about biological influencing factors and technical disturbing factors of routinely used immunoassay for the analysis of steroids. The application of LC-MS/MS as an alternative routine high-throughput platform for steroid analysis and its perspective role in the standardization and harmonisation of steroid measurements in clinical routine application will be discussed.
KURTD, WOODIN KD, PENNELL PB.Quantification of neurosteroids during pregnancy using selective ion monitoring mass spectrometry[J]. Steroids, 2015, 95(1): 24-31.
ABSTRACT Analytical techniques used to quantify neurosteroids in biological samples are often compromised by non-specificity and limited dynamic range which can result in erroneous results. A relatively rapid and inexpensive gas chromatography-mass spectrometry (GC-MS) was developed to simultaneously measure nine neurosteroids, including allopregnanolone, estradiol, and progesterone, as well as 25-hydroxy-vitamin D3 in plasma samples collected from adult women subjects during and after pregnancy. Sample preparation involved solid-phase extraction and derivatization, followed by automated injection on a GC equipped with a mass selective detector (MSD) operated in single ion monitoring (SIM) mode to yield a run time of less than 11 minutes. Method detection limits for all neurosteroids ranged from 30 to 200 pg/mL (parts per trillion), with coefficients of variation that ranged from 3 to 5% based on intra-assay comparisons run in triplicate. Although concentrations of estradiol measured by chemiluminescent immunoassay (CIA) were consistent with values determined by GC-MS values, CIA yielded considerable higher values of progesterone, suggesting antibody cross reactions resulting from low specificity. Mean neurosteroid levels and representative time-course data demonstrate the ability of the method to quantify changes in multiple neurosteroids during pregnancy, including rapid declines in neurosteroid levels associated with delivery. This simplified GC-MS method holds particular promise for research and clinical laboratories that require simultaneous quantification of multiple neurosteroids, but lack the resources and expertise to support advanced liquid chromatography-tandem mass spectrometry facilities. Copyright 2014. Published by Elsevier Inc.
WANGQ, BOTTALICOL, MESAROSC, et al.Analysis of estrogens and androgens in postmenopausal serum and plasma by liquid chromatography mass spectrometry[J]. Steroids, 2015, 99(1): 76-83.
Liquid chromatography-selected reaction monitoring/mass spectrometry-based methodology has evolved to the point where accurate analyses of trace levels of estrogens and androgens in postmenopausal serum and plasma can be accomplished with high precision and accuracy. A suite of derivatization procedures has been developed, which together with modern mass spectrometry instrumentation provide investigators with robust and sensitive methodology. Pre-ionized derivatives are proving to be useful as they are not subject to suppression of the electrospray signal. Postmenopausal women with elevated plasma or serum estrogens are thought to be at increased risk for breast and endometrial cancer. Therefore, significant advances in risk assessment should be possible now that reliable methodology is available. It is also possible to conduct analyses of multiple estrogens in plasma or serum. Laboratories that are currently employing liquid chromatography/mass spectrometry methodology can now readily implement this strategy. This will help conserve important plasma and serum samples available in Biobanks, as it will be possible to conduct high sensitivity analyses using low initial sample volumes. Reported levels of both conjugated and non-conjugated estrogen metabolites are close to the limits of sensitivity of many assays to date, urging caution in the interpretation of these low values. The analysis of serum androgen precursors in postmenopausal women has not been conducted routinely in the past using liquid chromatography/mass spectrometry methodology. Integration of serum androgen levels into the panel of metabolites analyzed could provide additional information for assessing cancer risk and should be included in the future.
MCWHINNEY BC, BRISCOE SE, UNGERER JP, et al.Measurement of cortisol, cortisone, prednisolone, dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography tandem mass spectrometry: application for plasma, plasma ultrafiltrate, urine and saliva in a routine laboratory[J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2010, 878(28): 2863-2869.
[本文引用:1]
[11]
KRONEN, HUGHES BA, LAVERYG, et al.Gas chromatography/mass spectrometry (GC/MS) remains a pre-eminent discovery tool in clinical chemistry investigations even in the era of fast liquid chromatography tandem mass spectrometry (LC/MS/MS)[J]. Steroid Biochem Mol Bio, 2010, 121(2): 496-504.
[本文引用:1]
[12]
CABANM, CZERWICKAM, UKASZECICAP, et al.A new silylation reagent dimethyl(3,3,3-trifluoropropyl)silyldiethylamine for the analysis of estrogenic compounds by gas chromatography-mass spectrometry[J]. J Chromatogr A, 2013,1301(2): 215-224.
ABSTRACT In this study we applied DIMETRIS (dimethyl(3,3,3-trifluoropropyl)silyldiethylamine), a new silylating reagent, to derivative natural estrogens such as estrone (E1), 17尾-estradiol (E2) and estriol (E3), as well as the synthetic 17伪-ethinylestradiol (EE2) and the non-steroid diethylstilbestrol (DES). Its derivatizing properties were compared with those of the commonly used mixture of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide)+1% trimethylchlorosilane (TMCS) and with MTBSTFA (N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide). The use of DIMETRIS for the silylation all of them is reported for the first time. The nucleophilic properties of DIMETRIS were found to be superior to those of MTBSTFA, but slightly inferior to those of BSTFA. It was used to derivatize steroid (E1, E2, E3 and EE2) and non-steroid (DES) estrogens at 30 C prior to GC/MS analysis. These DMTFPS-derivatives exhibited good separation (low retention times despite the high molecular masses) and ionization properties in GC/MS analyses (the highest relative response factors for DMTFPS-derivatives among those tested). However, DIMETRIS and MTBSTFA (which produce mono-O-silyl derivatives of EE2) should not be used for the simultaneous analysis of EE2 and E1. Only a mixture of BSTFA+1% TMCS in pyridine, which generates the fully derivatized EE2 product (stable in GC injector), permits the determination of these two estrogenic compounds during one GC-MS run. On the other hand, because DIMETRIS requires a lower derivatization temperature than BSTFA, it could be very useful for the derivatization of thermally unstable estrogenic compounds. In the next step of this study, the SPE-GC-MS method based on DIMETRIS derivatization for the analysis of DES, E2 and E3 in aqueous samples was evaluated and validated. The MQL values: 1.4, 1.6 and 1.5ngL(-1) for DES, E2 and E3, respectively, proved its suitability to determine target compounds in environmental samples. Finally, the proposed method was successfully applied to the analysis of selected estrogenic compounds in real seawater and wastewater samples in Poland.
HILLM, HAVLIKOVAH, VRBIKOVAJ, et al.The identification and simultaneous quantification of 7-hydroxylated metabolites of pregnenolone, dehydroepiandrosterone, 3,17-androstenediol, and testosterone in human serum using gas chromatography mass spectrometry[J]. J Steroid Biochem Mol Biol, 2005, 96(2): 187-200.
[本文引用:1]
[16]
AHMIDA HS, BERTUCCIP, FRANZOL, et al.Simultaneous determination of plasmatic phytosterols and cholesterol precursors using gas chromatography-mass spectrometry (GC-MS) with selective ion monitoring(SIM)[J]. J Chromatogr B, 2006, 842(1): 43-47.
Phytosterols (P-sitosterol, cholestanol and campesterol) and cholesterol precursors (desmosterol and lathosterol), have been suggested as important biochemical markers of intestinal cholesterol absorption and liver biosynthesis, respectively, and as useful clinical parameters in the study of hypercholesterolemia, β-sitosterolemia, atherosclerosis and cardiovascular disease, including pharmacological response to hypolipidemic agents. We developed an optimised analytical method for the simultaneous analysis of cholestanol, desmosterol, lathosterol, campesterol and β-sitosterol in plasma using capillary gas chromatography coupled to mass spectrometry (GC-MS) with multiple selected ion monitoring (SIM). This method is based on the alkaline hydrolysis of sterol esters, extraction of free sterols and derivatization. The recovery of all sterols was in the range 76-101%. Within-day relative standard deviations (R.S.Ds.) and the between-day R.S.Ds. of cholestanol, desmosterol, lathosterol, campesterol and P-sitosterol were less than 8%, and their plasma levels in 161 normal subjects were (mean±S.D.) 4.73±2.57, 2.37 ±1.04, 6.23±3.14, 3.67 ± 1.95 and 5.92 ± 3.62 μmol/l, respectively.
SARAIVAD, SEMEDOR, CASTILHO MC, et al.Selection of the derivatization reagent-the case of human blood cholesterol its precursors and phytosterols GC-MS analyses[J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2011, 879(32): 3806-3811.
Phytosterols (PS; β-sitosterol and campesterol) and cholesterol precursors (CP; desmosterol and lathosterol) have been suggested as important biochemical markers of cholesterol intestinal absorption and liver biosynthesis, respectively. Given that these compounds appear in human blood in low amounts, sensitive and accurate methodology is required, such as gas chromatography–mass spectrometry (GC–MS) the most frequently used. One of the most critical factors of the GC analytical determination is the step of derivatization. Thus, the main objective of the present study was compare and select the better (one out of three) silylation mixtures as follows: N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide/ammonium iodide (MTBSTFA:NH 4 I), N-O-bis-(trimethylsilyl) trifluoroacetamide/trimethylchlorosilane (BSTFA:TMCS), and N-methyl-N-(trimethylsilyl)-trifluoroacetamide/1,4-dithioerythritol/trimethyliodosilane (MSTFA:DTE:TMIS). The results of this study are discussed and accompanied by a brief review on the importance and principles of derivatization process, specifically in silylation reactions in GC–MS sterols analyses. Furthermore, a scrutiny of some published results is presented, along with additional information about mass spectral data of these potentially useful compounds. Interestingly, the results of the study showed that from the three validated methodologies, the selected one, based on the best relation specificity/sensibility, is MSTFA:DTE:TMIS. With this silylation procedure for simultaneous determination of PS and CP in human serum by GC–MS in selected ion monitoring (SIM) mode, good linearity ( r 2 02≥020.931), precision (repeatability ranging from 0.92 to 3.91 CV and intermediate precision ranging from 5.12 to 6.33) and recoveries (≥93.6%) were obtained. Thus, it proved to be a helpful methodology in the quantification of predominant serum cholesterol origin in each patient.
TSAKALOF AK, GKAGTZIS DC, KOUKOULIS GN, et al.Development of GC-MS/MS method with programmable temperature vaporization large volume injection for monitoring of 17-estradiol and 2-methoxyestradiol in plasma[J]. Anal Chim Acta, 2012, 709(1): 73-80.
Under optimized GC–MS/MS conditions in product ion mode, the Limit of Detection (LOD) of the analyzed steroids ranged from 18.4pgmL 611 for 17-BE to 5.5pgmL 611 for 2-MEOE (S/N=3). The instrument response was linear in the investigated concentration range from 0.1 to 10ngmL 611 with R 2 >0.99 for 17-BE and 2-MEOE. The intra-batch accuracy obtained for quality control samples at the concentration levels of 0.1, 1, 3, 7ngmL 611 ranged from 94.9% to 109.9% for 17-BE and from 99.9% to 104.5% for 2-MEOE.
STANCZYK FZ, CLARKE NJ.Advantages and challenges of mass spectrometry assays for steroid hormones[J]. Steroid Biochem Mol Biol, 2010, 121(3/5):491-495.
Although steroid hormones have been measured, primarily in urine, by gas chromatography–mass spectrometry (GC–MS) assays for many years, in the past decade both clinical and research laboratories have dramatically increased usage of liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays for measuring circulating levels of steroid hormones. Because of their high validity and throughput, mass spectrometry (MS) assays have replaced conventional radioimmunoassays (RIAs) and direct immunoassays for steroid hormones in larger reference laboratories, and they are touted to become the “gold standard” for steroid hormone quantitation. These advances in MS assays present several major challenges, which include the affordability of smaller laboratories to purchase MS instruments and pay for related operating costs; improving assay sensitivity, especially for measuring low estradiol levels in postmenopausal women and women treated with aromatase inhibitors; developing assays for quantitating profiles of steroid hormone metabolites in serum and tissues; standardizing steroid MS assays; and obtaining reliable reference intervals. The present review discusses the advantages of MS assays over conventional RIAs and direct immunoassays in steroid hormone measurements, and points out some of the important challenges facing the rapid increase in usage of MS assays.
BERTINJ, DURY AY, KEY, et al.Accurate and sensitive liquid chromatography/tandem mass spectrometry simultaneous assay of seven steroids in monkey brain[J]. Steroids, 2015, 98(1): 37-48.
ABSTRACT Following its secretion mainly by the adrenal glands, dehydroepiandrosterone (DHEA) acts primarily in the cells/tissues which express the enzymes catalyzing its intracellular conversion into sex steroids by the mechanisms of intracrinology. Although reliable assays of endogenous serum steroids are now available using mass spectrometry (MS)-based technology, sample preparation from tissue matrices remains a challenge. This is especially the case with high lipid-containing tissues such as the brain. With the combination of a UPLC system with a sensitive tandem MS, it is now possible to measure endogenous unconjugated steroids in monkey brain tissue. A Shimadzu UPLC LC-30AD system coupled to a tandem MS AB Sciex Qtrap 6500 system was used. The lower limits of quantifications are achieved at 250 pg/mL for DHEA, 200 pg/mL for 5-androstenediol (5-diol), 12 pg/mL for androstenedione (4-dione), 50 pg/mL for testosterone (Testo), 10 pg/mL for dihydrotestosterone (DHT), 4 pg/mL for estrone (E1) and 1 pg/mL for estradiol (E2). The linearity and accuracy of quality controls (QCs) and endogenous quality controls (EndoQCs) are according to the guidelines of the regulatory agencies for all seven compounds. We describe a highly sensitive, specific and robust LC-MS/MS method for the simultaneous measurement of seven unconjugated steroids in monkey brain tissue. The single and small amount of sample required using a relatively simple preparation method should be useful for steroid assays in various peripheral tissues and thus help analysis of the role of locally-made sex steroids in the regulation of specific physiological functions. Copyright 漏 2015. Published by Elsevier Inc.
SHIBATAY, ASRAI, HONMAS.Methodological approach to the intracrine study and estimation of DHEA and DHEA-S using liquid chromatography tandem mass spectrometry (LC-MS/MS)[J]. J Steroid Biochem Mol Biol, 2015, 145(2): 193-199.
ABSTRACT A reliable and sensitive method for analyzing steroids using liquid chromatography tandem mass spectrometry (LC-MS/MS) is required for research concerning dehydroepiandrosterone (DHEA), which plays a central role in steroid hormone biosynthesis and metabolism. Furthermore, after the first proposal of the concept of intracrine DHEA, stable isotope tracer analysis, which is useful for structural recognition as well as determination of steroids, has been required to evaluate physiological action and hormone biosynthesis/metabolism in target organs. We describe sample processing and analysis methods for simultaneous quantification of multiple hormones, including DHEA, in serum, saliva and tissue using LC-MS/MS. A derivatization technique compatible with each functional group for measuring 3尾-hydroxy-5-enes, such as DHEA and 5伪/5尾-steroids, with high sensitivity and specificity is also described. Finally, we describe a newly developed method for intracrine research using stable isotope-labeled (13)C-steroid substrates with tracer analysis of their metabolites by LC-MS/MS.
GUOT, GUJ, SOLDIN OP, et al.Soldin, Rapid measurement of estrogens and their metabolites in human serum by liquid chromatography-tandem mass spectrometry without derivatization[J], Clin Biochem,2008, 41(9): 736-741.
OBJECTIVES: The steroids estradiol (E2), estrone (E1), and estriol (E3) are the major estrogens. E1/E2 and their metabolite 16-hydroxyestrone (16-OHE1, known to be carcinogenic) could be involved in the development of many cancers including human breast cancer. The aim of the current study was to develop a rapid and simple high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to simultaneously measure E1, E2, E3 and 16-OHE1 in human serum without the need for solid phase extraction or derivatization. METHODS: An API-5000 triple-quadrupole mass spectrometer coupled with electrospray ionization (ESI) source and Shimadzu HPLC system was used employing isotope dilution with deuterium-labeled internal standard (IS) for each analyte. Quantitation by multiple reaction monitoring (MRM) analysis was performed in negative ion mode. RESULTS: The limits of detection were 1.0 pg/mL for E1 and 16-OHE1 and 2.0 pg/mL for E2 and E3. Within-day CVs were <6.5% for all analytes tested and between-day CVs ranged from 4.5% to 9.5%. Recovery ranged from 88% to 108%. CONCLUSION: This method allows for the simultaneous measurement of four estrogens in human serum within 8 min. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing, micro sample requirement, and high throughput.
HARWOOD DT, HANDELSMAN DJ.Development and validation of a sensitive liquid chromatography-tandem mass spectrometry assay to simultaneously measure androgens and estrogens in serum without derivatization[J]. Clin Chim Acta ,2009, 409(1/2): 78-84.
Elsevier’s Scopus, the largest abstract and citation database of peer-reviewed literature. Search and access research from the science, technology, medicine, social sciences and arts and humanities fields.
CHENY, YAZDANPANAHM, WANG XY, et al.Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry[J]. Clin Biochem, 2010, 43(4/5): 490-496.
ABSTRACT Currently there is no reliable method suitable for routine measurement of serum free testosterone (FT). To develop such a method involving liquid chromatography tandem mass spectrometry (LC-IDMS/MS) that directly detects and quantifies the FT present in serum. Ultrafiltrate testosterone obtained from 0.5 mL of serum was partially purified by liquid/liquid extraction and quantified using an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source. Using split samples serum free testosterone was compared between direct ultrafiltration (UF) coupled LC-MS/MS, analogue FT immunoassay, free testosterone calculated from mass action equations (cFT) and with equilibrium dialysis (ED) coupled LC-MS/MS. Total imprecision determined over twenty runs was <6% at 67 pmol/L and 158 pmol/L FT. The dynamic response was linear up to at least 2500 pmol/L while physical LLOQ (18 % CV) equaled 16 pmol/L. The UF method agreed poorly with analogue immunoassay (correlation coefficient 0.667; bias -81%), somewhat better against cFT when total testosterone was determined by immunoassay (correlation coefficient 0.816, bias 21% ) and still better yet against cFT when total testosterone was determined by LC-MS/MS (correlation coefficient 0.8996, bias 10%). Agreement was closest with ED method (correlation coefficient 0.9779, bias 2.4%). We present a relatively simple UF coupled LC-MS/MS definitive method that measures serum free testosterone. The method is relatively fast, reliable and is suitable for the routine clinical laboratory practice.
LIEN GW, CHEN CY, WANG GS.Comparison of electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization for determining estrogenic chemicals in water by liquid chromatography tandem mass spectrometry with chemical derivatizations[J]. J Chromatogr A, 2009, 1216(6):956-966.
[本文引用:1]
[31]
KUSHNIR MM, ROCKWOOD AL, ROBERTS WL, et al.Performance characteristics of a novel tandem mass spectrometry assay for serum testosterone[J]. Clin Chem, 2006, 52(1): 120-128.
Commercial immunoassays for testosterone (Te) may give inaccurate results for samples from women and children, leading to misdiagnosis and inappropriate treatment. We developed a sensitive and specific tandem mass spectrometric assay for measurement of Te at the concentrations encountered in women and children.Te was extracted with methyl tert-butyl ether from 100 microL of serum or plasma, derivatized to form an oxime, and reextracted by solid-phase extraction. Instrumental analysis was performed on an API 4000 HPLC tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode. The MRM transitions (m/z) were 304-->124 and 304-->112 for Te and 307-->124 and 307-->112 for d3-Te.Within- and between-run CVs were <12% and 7.9%, respectively. The limit of quantification was 0.0346 nmol/L (1 ng/dL). Reference intervals for sex hormone-binding globulin and total, free, and bioavailable Te were established for children of Tanner stages 1 through 5 and adult males and females.The sensitivity and specificity of the method are adequate for analysis of Te in samples from women and children. The method requires small sample volumes, has adequate precision, and is not subject to interferences.
XUL, SPINK DC.Analys is of steroidal estrogens as pyridine-3-sulfonyl derivatives by liquid chromatography electrospray tandem mass spectrometry[J]. Anal Biochem, 2008, 375(1): 105-114.
Sulfonyl chlorides substituted with functional groups having high proton affinity can serve as derivatization reagents to enhance the sensitivity for steroidal estrogens in liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The most commonly used reagent for derivatization of estrogens for LC-ESI-MS/MS is dansyl chloride. In this study, we compared dansyl chloride, 1,2-dimethylimidazole-4-sulfonyl (DMIS) chloride, pyridine-3-sulfonyl (PS) chloride, and 4-(1H-pyrazol-1-yl)benzenesulfonyl (PBS) chloride for derivatization of 17beta-estradiol (E2) prior to LC-ESI-MS/MS. The product ion spectra of the dansyl and DMIS derivatives were dominated by ions representing derivatization reagent moieties. In contrast, the product ion spectrum of the PS derivative of E2 and, to a lesser extent, the PBS derivative, showed analyte-specific fragment ions. Derivatization with PS chloride was therefore chosen for further investigation. The product ion spectrum of the PS derivative of E2 showed intense ions at m/z 272, assigned to the radical E2 cation, and at m/z 350, attributed to the loss of SO(2) from the [M+H](+) ion. Third-stage mass spectrometry of the PS derivative of E2 with isolation and collisional activation of the m/z 272 ion resulted in steroid C and D ring cleavages analogous to those observed in electron ionization mass spectrometry. The product ion spectra of the PS derivatives of estrone, 17alpha-ethinylestradiol, equilin, and equilenin showed similar estrogen-specific ions. Using derivatization with PS chloride, we developed an LC-ESI-MS/MS method with multiple reaction monitoring of primary and confirmatory precursor-to-product ion transitions for the determination of E2 in serum.
NGUYEN HP, LIL, GATSON JW, et al.Simultaneous quantification of four native estrogen hormones at trace levels in human cerebrospinal fluid using liquid chromatography tandem mass spectrometry[J]. J Pharm Biomed Anal, 2011, 54(4): 830-837.
Estrogens are known to exhibit neuroprotective effects on the brain. Their importance in this regard and in others has been emphasized in many recent studies, which increases the need to develop reliable analytical methods for the measurement of estrogen hormones. A heart-cutting two-dimensional liquid chromatography separation method coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been developed for simultaneous measurement of four estrogens, including estriol (E3), estrone (E1), 17β-estradiol (17β-E2), and 17α-estradiol (17α-E2), in human cerebrospinal fluid (CSF). The method was based on liquid-liquid extraction and derivatization of estrogens with dansyl chloride to enhance the sensitivity of ESI-based detection in conjunction with tandem mass spectrometry. Dansylated estriol and estrone were separated in the first dimension by an amide-C18 column, while dansylated 17β- and 17α-estradiol were resolved on the second dimension by two C18 columns (175 mm total length) connected in series. This is the first report of a method for simultaneous quantification of all four endogenous estrogen compounds in their dansylated form. The detection limits for E1, 17α-E2, 17β-E2, and E3 were 19, 35, 26, and 61pg/mL, respectively. Due to matrix effects, validation and calibration was carried out in charcoal-stripped CSF. The precision and accuracy were more than 86% for the two E2 compounds and 79% for E1 and E3 while the extraction recovery ranged from 91% to 104%. The method was applied to measure estrogens obtained in a clinical setting, from the CSF of ischemic trauma patients. While 17β-estradiol was present at a significant level in the CSF of some samples, other estrogens were present at lower levels or were undetectable.
LIW, LI YH, LI AC, et al.Simultaneous determination of norethindrone and ethinyl estradiol in human plasma by high performance liquid chromatography with tandem mass spectrometry-experiences on developing a highly selective method using derivatization reagent for enhancing sensitivity[J]. J Chromatogra B Analyt Technol Biomed Life Sci, 2005, 825(2): 223-232.
[本文引用:0]
[35]
KUSHNIR MM, ROCKWOOD AL, BERGQUISTJ, et al.High-sensitivity tandem mass spectrometry assay for serum estrone and estradiol[J]. Am J Clin Pathol, 2008, 129(4): 530-539.
High-sensitivity measurement of serum estrogens is important in adult and pediatric endocrinology and oncology. We developed a high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of estrone (E1) and estradiol (E2). Aliquots of 200 muL of serum were spiked with internal standard, extracted, derivatized with dansyl chloride, and analyzed by LC-MS/MS using 2-dimensional chromatographic separation. Total imprecision for the method was less than 11%; the limit of quantitation was 1 pg/mL. Reference intervals were established with samples from more than 900 healthy postmenopausal women, men, girls, and boys. Concentrations of estrogens in children reached adult levels by Tanner stage 3. In men and postmenopausal women, the median concentrations of total estrogens (E1 + E2) were 39 and 22 pg/mL, and the median E2/E1 ratios were 0.98 and 0.55, respectively. The method requires a small sample volume and has adequate sensitivity and specificity for analyzing estrogens in samples from postmenopausal women, men, and children.
HIGASHIT, TAKAYAMAA, KYUTOKUM, et al.Liquid chromatography mass spectrometric assay of androstenediol in prostatic tissue: influence of androgen deprivation therapy on its level[J]. Steroids, 2006, 71(11/12): 1007-1013.
Androstenediol (Adiol, androst-5-ene-3β,17β-diol) is suspected of being an endogenous proliferation agent of prostate cancer (PCa) even after androgen deprivation therapy (ADT). A liquid chromatography-electron capture atmospheric pressure chemical ionization-mass spectrometric (LC-ECAPCI-MS) method for the determination of Adiol in prostatic tissue was developed and validated for evaluating the influence of ADT on the prostatic Adiol level. After derivatization of Adiol with 4-nitrobenzoyl chloride, the detection response of the derivative was increased 150 times more than that of intact Adiol. The LC-MS method was specific and reliable for the measurement of a trace amount of Adiol in 30 mg of tissue. The clinical study using the developed method showed that the prostatic Adiol level was not changed by ADT. That is, the prostatic Adiol levels of PCa patients with ADT (n = 12), benign prostate hypertrophy patients without ADT (n = 8) and bladder cancer patients (without prostatic disease) (n=6) were 0.83±0.28, 0.62±0.31 and 0.71±0.28 ng g
STICKNEY DR, GROOTHUIS JR, AHLEMC, et al.Preliminary clinical findings on NEUMUNE as a potential treatment for acute radiation syndrome[J]. J Radiol Prot, 2010, 30(4): 687-698.
5-(5-AED) has been advanced as a possible countermeasure for treating the haematological component of acute (). It has been used in animal models to stimulate both innate and adaptive immunity and treat and radiation-induced . We here report on the safety, tolerability and haematologic activity of 5-AED in four double-blinded, randomized, placebo-controlled studies on healthy adults including elderly subjects. A 5-AED injectable suspension formulation (NEUMUNE) or placebo was administered intramuscularly as either a single injection, or once daily for five consecutive days at doses of 50, 100, 200 or 400 mg. Subjects (n = 129) were randomized to receive NEUMUNE (n = 95) or the placebo (n = 34). NEUMUNE was generally well-tolerated; the most frequent adverse events were local (n = 104, 81%) that were transient, dose-volume dependent, mild to moderate in severity, and that resolved over the course of the study. Blood chemistries revealed a transient increase (up to 28%) in phosphokinase and levels consistent with intramuscular injection and . The blood concentration profile of 5-AED is consistent with a depot formulation that increases in disproportionate increments following each dose. NEUMUNE significantly increased circulating neutrophils (p < 0.001) and platelets (p < 0.001) in the peripheral blood of adult and elderly subjects. A dose-response relationship was identified. Findings suggest that parenteral administration of 5-AED in aqueous suspension may be a safe and effective means to stimulate and alleviate and thrombocytopenia associated with .
KESKI-RAHKONENP, HUHTINENK, POUTANENM, et al.Fast and sensitive liquid chromatography mass spectrometry assay for seven androgenic and progestagenic steroids in human serum[J]. J Steroid Biochem Mol Biol, 2011, 127(3/5): 396-404.
A fast and sensitive LC–MS/MS method for the quantitative analysis of seven steroid hormones in 15002μl of human serum was developed and validated. The following compounds were included: 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, testosterone, pregnenolone, and progesterone. Individual stable isotope-labeled analogues were used as internal standards. Sample preparation was performed by liquid–liquid extraction, followed by oxime derivatization to improve the ionization efficiency of the analytes. In contrast to the common derivatization-based methods, the reaction was incorporated into the sample preparation process and the only additional step due to the derivatization was a short heating of the autosampler vials before the sample injection. Chromatographic separation was achieved on a reversed-phase column using a methanol–water gradient. For the analyte detection, a triple quadrupole instrument with electrospray ionization was used. Total run time was 7.002min and the lower limits of quantification were in the range of 0.03–0.3402nM (0.01–0.1002ng/ml), depending on the analyte. The method was validated using human serum samples from both sexes and applied for the serum steroid profiling of endometriosis patients.
KUSHNIR MM, ROCKWOOD AL, ROBERTS WL, et al.Performance characteristics of a novel tandem mass spectrometry assay for serum testosterone[J].Clin Chem, 2006, 52(1): 120-128.
Commercial immunoassays for testosterone (Te) may give inaccurate results for samples from women and children, leading to misdiagnosis and inappropriate treatment. We developed a sensitive and specific tandem mass spectrometric assay for measurement of Te at the concentrations encountered in women and children.Te was extracted with methyl tert-butyl ether from 100 microL of serum or plasma, derivatized to form an oxime, and reextracted by solid-phase extraction. Instrumental analysis was performed on an API 4000 HPLC tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode. The MRM transitions (m/z) were 304-->124 and 304-->112 for Te and 307-->124 and 307-->112 for d3-Te.Within- and between-run CVs were <12% and 7.9%, respectively. The limit of quantification was 0.0346 nmol/L (1 ng/dL). Reference intervals for sex hormone-binding globulin and total, free, and bioavailable Te were established for children of Tanner stages 1 through 5 and adult males and females.The sensitivity and specificity of the method are adequate for analysis of Te in samples from women and children. The method requires small sample volumes, has adequate precision, and is not subject to interferences.
Analysis of estrogens and androgens in postmenopausal serum and plasma by liquid chromatography mass spectrometry
2015
Measurement of cortisol, cortisone, prednisolone, dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography tandem mass spectrometry: application for plasma, plasma ultrafiltrate, urine and saliva in a routine laboratory
Gas chromatography/mass spectrometry (GC/MS) remains a pre-eminent discovery tool in clinical chemistry investigations even in the era of fast liquid chromatography tandem mass spectrometry (LC/MS/MS)
A new silylation reagent dimethyl(3,3,3-trifluoropropyl)silyldiethylamine for the analysis of estrogenic compounds by gas chromatography-mass spectrometry
The identification and simultaneous quantification of 7-hydroxylated metabolites of pregnenolone, dehydroepiandrosterone, 3,17-androstenediol, and testosterone in human serum using gas chromatography mass spectrometry
Simultaneous determination of plasmatic phytosterols and cholesterol precursors using gas chromatography-mass spectrometry (GC-MS) with selective ion monitoring(SIM)
Development of GC-MS/MS method with programmable temperature vaporization large volume injection for monitoring of 17-estradiol and 2-methoxyestradiol in plasma
Development and validation of a sensitive liquid chromatography-tandem mass spectrometry assay to simultaneously measure androgens and estrogens in serum without derivatization
Comparison of electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization for determining estrogenic chemicals in water by liquid chromatography tandem mass spectrometry with chemical derivatizations
Simultaneous quantification of four native estrogen hormones at trace levels in human cerebrospinal fluid using liquid chromatography tandem mass spectrometry
2011
Simultaneous determination of norethindrone and ethinyl estradiol in human plasma by high performance liquid chromatography with tandem mass spectrometry-experiences on developing a highly selective method using derivatization reagent for enhancing sensitivity
2005
High-sensitivity tandem mass spectrometry assay for serum estrone and estradiol
, 杨建云
