高效液相色谱-二极管阵列/荧光检测器串联法同时测定精油中的七种性激素

建立了高效液相色谱法(HPLC)-二极管阵列检测器(DAD)/荧光检测器(FLD)串联技术同时测定精油中7种性激素(雌二醇、雌三醇、雌酮、睾酮、甲基睾酮、孕酮、己烯雌酚)的方法。样品先用正己烷溶解后,用90%的甲醇水溶液提取,弃去正己烷层,下层清液再用正己烷脱脂、净化2次,目标化合物以水-甲醇-乙腈(体积比为50:30:20)为流动相,经XTerraRP18色谱柱(250 mm×4.6 mm, 5 μm )分离,用DAD-FLD串联法进行检测。雌二醇、雌三醇、雌酮、己烯雌酚的DAD检测波长为197 nm,睾酮、甲基睾酮、孕酮的DAD检测波长为240 nm。雌二醇、雌三醇、雌酮同时用FLD定性定量,激发波长为280 nm,发射波长为310 nm。7种性激素分离效果良好并消除了样品中杂质峰的干扰。7种性激素除孕酮的回收率为79.5%以外,其余组分的平均回收率均在93%以上;相对标准偏差为0.90%～1.89%;检出限为0.010 ～1.0 mg/L。该方法简便、准确,可用于同时测定精油中的7种性激素。     

高效液相色谱法同时测定化妆品中七种性激素

建立了化妆品中7种性激素(雌二醇、雌三醇、雌酮、睾酮、甲基睾酮、孕酮、己烯雌酚)同时测定的高效液相色谱法。先在试样中加入20 g/L氢氧化钠溶液与油脂进行皂化反应,然后用二氯甲烷-乙酸乙酯(体积比为40∶1)混合液在酸性条件下(pH 3)萃取,选择XTerraTMRP18色谱柱,以水-甲醇-乙腈(体积比为50∶32∶18)混合液为流动相,在波长230 nm处检测。7种性激素分离良好并排除了样品中杂质峰的干扰,低、高浓度平均回收率范围为75.6%~97.8%;相对标准偏差为1.9%~7.2%;检出限为3.7     

Isocratic reversed phase high performance liquid chromatography determination of twelve natural corticosteroids in serum with on-line ultraviolet and fluorescence detection

An isocratic reversed phase high performance liquid chromatography procedure utilizing ultraviolet and fluorescence detectors linked in series is described for the analysis of cortisone (E), cortisol (F), corticosterone (B), 11-deoxycortisol (S), 11-deoxycorticosterone (DOC), androstenedione (A), testosterone (T), 17-hydroxyprogesterone (17-OHP), progesterone (P), estriol, estradiol, estrone, prednisone acetate and dexamethasone acetate in serum. Serum specimens were extracted with ethyl ether. The optimized mobile phase was methanol + tetrahydrofuran + water (26:18:56, v/v/v). A Shim-pack ODS column was used. The recoveries were 80 to 103%. Intra- and inter-day coefficient of variance were less than 8%. The detection limit is 0.5 pmol per injection volume for estriol, estradiol, E, F and B; 1 pmol for S, A, DOC and estrone; 2 pmol for T and 17-OHP; and 4 pmol for P. Serum from normal subjects and patients with congenital adrenal hyperplasia due to 21- or 17-hydroxylase deficiency were measured, as well as samples of maternal and umbilical cord serum.     

Optimization of an isocratic reversed phase liquid chromatographic system for the separation of fourteen steroids using factorial design and computer simulation

An optimization strategy for an isocratic reversed phase high performance liquid chromatographic system (RP-HPLC) is described. Factorial design and a computer program are used to predict the retention time and resolution of fourteen steroids. An optimized rapid (less than 25 min) isocratic RP-HPLC system for the satisfactory separation of cortisone, cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17 alpha-hydroxyprogesterone, progesterone, androstenedione, testosterone, estrone, estradiol, estriol, prednisone acetate and dexamethasone acetate has been developed using this strategy through eight experiments.     

Simultaneous determination of nine endogenous steroids in human urine by polymeric-mixed micelle capillary electrophoresis

A new CE system based on the use of polymeric-mixed micelles (cholic acid, SDS and the poloxamine Tetronic(?) 1107) was developed for the simultaneous determination of nine steroids in human urine. This method allows the baseline separation and quantitation of cortisol, androstenedione, estriol, dehydroepiandrosterone sulfate, testosterone, dehydroepiandrosterone, estrone, progesterone and estradiol in less than 25 min showing to be sensitive enough to detect low concentrations of these steroids in urine samples (5-45 ng/mL). The optimized electrophoretic conditions were performed using a 50 cm × 75 μm capillary, 18 kV, 25°C, with 44 mM cholic acid, 10 mM SDS, 0.05% w/v tetronic(?) 1107, 2.5% v/v methanol, 2.5% v/v tetrahydrofuran in 5 mM borate - 5 mM phosphate buffer (pH=8.0) as a background electrolyte and a dual 210/254 UV-detection. The method can simultaneously determine 0.1-120 μg/mL, which corresponds to 5-6000 ng/mL of steroids in 2 mL urine. The recoveries ranged between 82.4 and 101.5%. Due to its simplicity, speed, accuracy and reliability, the proposed method could be a potential alternative to the traditional methodologies used with clinical purposes.     

Application of chemiluminescence immunoassays for steroid hormones in clinical endocrinological investigations in women

Immunoassays based on chemiluminescence for the measurement of serum and plasma steroids (estradiol, estriol, progesterone, testosterone, and cortisol), urinary steroid conjugates (estrone-3-glucuronide, estriol-16α-glucuronide and pregnanediol-3α-glucuronide) and peptide hormones (choriogonadotropin and luteinizing hormone) are surveyed briefly. These immunoassays are simple, robust and valid alternatives to radioimmunoassay. Homogeneous procedures and recent solid-phase assays based on purified specific antibodies, covalently coupled to polymer beads are discussed. Some new results are presented for solid-phase chemiluminescence immunoassays: estradiol is quantified in extracts of serum by using a monoclonal antibody to estradiol with estradiol-6-carboxymethyloxime-aminobutylethyl isoluminol as the marker ligand, and progesterone is quantified in unextracted serum by using a polyclonal antibody to progesterone, progesterone-11-hemisuccinyl-aminobutylethyl isoluminol as the marker ligand, and danazol (17α-pregna-2,4-dien-20-yno[2,3-d]-isoxazol-17-ol) to displace progesterone from serum binding-proteins. Their clinical utility is demonstrated.     

Analysis and chromatographic separation of some steroid hormones on NH2 layers

Summary The previously described analytical method for carbohydrates, catecholamines, uric acid, creatine and creatinine using thin-layer chromatography on aminomodified HPTLC plates and subsequent thermal activation of the chromatogram zones is expanded to include several steroid hormones. Specifically, they are the pharmacologically relevant compounds cortisone and hydrocortisone, estradiol and estradiol benzoate, estriol, estrone, methyltestosterone, testosterone and testosterone propionate, prednisolone, pregnandiol and triol, progesterone and Reichstein's S.     

Automated direct assay system for the measurement of sex steroid hormones in serum using high-performance liquid chromatography

An automated direct assay system using high-performance liquid chromatography was developed for the simultaneous measurement of estradiol, estrone, progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxyprogesterone, testosterone and androstenedione in biological fluids. A comparison between the values measured by this method and by radioimmunoassay revealed good correlation for estradiol (r = 0.938, p less than 0.001) and progesterone (r = 0.903, p less than 0.001). Estradiol and estrone could be analysed above the level of 250 pg/ml, and progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxyprogesterone, testosterone and androstenedione could be analysed above the level of 5.0-7.5 ng/ml. The method was applied to the clinical appraisal of placental function and maturation of ovarian follicles.     

Molecularly imprinted polymer film grafted from porous silica for efficient enrichment of steroid hormones in water samples

Steroid hormones as endocrine disrupting compounds can interfere with the functioning of hormonal systems of organisms and thus affect the health and reproduction of humans and wildlife. Unfortunately, these types of harmful endocrine disrupting compounds have been found in a variety of environmental samples at very low concentrations. Therefore, a simple, fast, and efficient method for enrichment of water samples is needed. A molecularly imprinted solid‐phase extraction combined with high performance liquid chromatography coupled with diode array detection was developed for the determination of six steroid hormones, such as estrone, 17‐β‐estradiol, estriol, 17‐α‐ethinylestradiol, progesterone, and testosterone in water samples. The recoveries obtained in the proposed method were in the range of 78.7–101.3%. Matrix effect below 20% suggests that the quantitative and qualitative results of the analysis were not significantly affected by the matrix. The results show that molecularly imprinted polymers based on spherical silica gel had the potential to be a highly innovative and selective sorbent. The proposed method was proved to be applicable for molecularly imprinted solid‐phase extraction in selective and reliable extraction and enrichment of steroid hormones in environmental water samples.     

Simultaneous determination of 15 steroidal oral contraceptives in water using solid-phase disk extraction followed by high performance liquid chromatography–tandem mass spectrometry

A rapid, accurate and sensitive method for simultaneous determination of 15 steroidal hormones including four estrogens (estrone, 17β-estradiol, 17α-ethynylestradiol, estriol) and eleven progestogens (17β-estradiol-3-benzoate, 19-norethindrone, gestodene, levonorgestrel, medroxyprogesterone, cyproterone acetate, megestrol-17-acetate, progesterone, norethindrone acetate, chlormadinone-17-acetate, and hydroxy progesterone caproate) in environmental waters was developed by coupling solid-phase disk extraction (SPDE) to ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) with electrospray ionization. Among three types of extraction tested (C₈ SPDE, C₁₈ SPDE and C₁₈ SPE), the most satisfactory result was achieved using C₁₈ SPDE for its satisfactory recovery (75.6 to 101.4%) and short extraction time (15 min for 1 L deionised water). The validity of this method was investigated and good analytical performance for all the analytes was obtained, including low limits of method detection (0.5–3.4 ng/L) and excellent linear dynamic range (1.0–50.0 ng/L). The method was applied to determine the steroidal hormones in 10 environmental waters including tap water, river water, lake water and waste water in Beijing. No progestogen was detected in all samples and estrone, estriol, 17α-ethynylestradiol were found in most samples at levels between 1.8 and 127.9 ng/L.     

Multiple monolithic fiber solid‐phase microextraction based on a polymeric ionic liquid with high‐performance liquid chromatography for the determination of steroid sex hormones in water and urine

The development of a simple and sensitive analytical approach that combines multiple monolithic fiber solid‐phase microextraction with liquid desorption followed by high‐performance liquid chromatography with diode array detection is proposed for the determination of trace levels of seven steroid sex hormones (estriol, 17β‐estradiol, testosterone, ethinylestradiol, estrone, progesterone and mestranol) in water and urine matrices. To extract the target analytes effectively, multiple monolithic fiber solid‐phase microextraction based on a polymeric ionic liquid was used to concentrate hormones. Several key extraction parameters including desorption solvent, extraction and desorption time, pH value and ionic strength in sample matrix were investigated in detail. Under the optimal experimental conditions, the limits of detection were found to be in the range of 0.027–0.12 μg/L. The linear range was 0.10–200 μg/L for 17β‐estradiol, 0.25–200 μg/L estriol, ethinylestradiol and estrone, and 0.50–200 μg/L for the other hormones. Satisfactory linearities were achieved for analytes with the correlation coefficients above 0.99. Acceptable method reproducibility was achieved by evaluating the repeatability and intermediate precision with relative standard deviations of both less than 8%. The enrichment factors ranged from 54‐ to 74‐fold. Finally, the proposed method was successfully applied to the analysis of steroid sex hormones in environmental water samples and human urines with spiking recoveries ranged from 75.6 to 116%.     

表面活性剂存在下雌激素的直接电化学分析

雌二醇（E2）、雌三醇（E3）和雌酮（E1）是人体内3种重要的雌激素,它们在妇女的生殖生育方面发挥重要的作用。人体雌激素缺乏或过乘会导致诸多疾病。雌激素的检测方法已有不少报道,如气相色谱－质谱（GC－MS）、液相色谱（LC）以及化学发光免疫法等。E2,E1和E3的结构相似,且均具有疏水性及非电活性,用电化学直接测定十分困难,一般只能间接测定。本文报道了一种新的电化学分析方法。该方法对三种雌激素有十分灵敏的伏安响应,可用于微量测定。     

Determination of Estrogens by High Performance Liquid Chromatography with Pre-Label Derivatization with 2-(4-Carboxy)-5,6-Dimethylbenzimidazole (II): Shortening of Analysis Time and Application to Monitoring Estrogen in Serum From In-Vitro Fertilization Embryo Transfer (IVF-ET) Patients

Abstract

We report a sensitive high performance liquid chromatography (HPLC) method for determination of free and conjugated estrogens (estrone, estradiol and estriol) by a fluorescent pre-labeling regent, 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole, with modification of previous work. The modified method was also tried, in preliminary work, for diagnosis of the in-vitro fertilization embryo transfer (IVF-ET) process. The reagent volumes were changed to one-tenth, derivatization conditions were changed to mild conditions at 40 C, and a solid-phase extraction process by SEP-PAK could be omitted after restudy of reaction conditions. As a result analysis time could be shorted within 40 min. The proposed HPLC method was applied to monitoring of free and conjugated estrogens in the patients who attend in-vitro fertilization embryo transfer (IVF-ET). The subsequent increase of free and conjugated estrone, estradiol and estriol was observed with the progress of follicle growth following ovulation stage in the IVF-ET process. We tried to plot estrogens for assist of clinical diagnosis of IVF-ET. The free estrone: 200-600 pg/ml, estradiol: 200-600 pg/ml and estriol: 100-300 pg/ml, conjugate estrone: 1000-5000 pg/ml, estradiol: 3000-8000 pg/ml and estriol: 2000-7000 pg/ml) in the patients without hormone disease were observed before human chorionic gonadotropin stimulation (hCG) on IVF-ET process. It was expected that free estrogen values, especially E1 and E3 could be use as validation products for diagnosis of hormone disease in IVF-ET process.     

Interaction of native α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin and their hydroxypropyl derivatives with selected organic low molecular mass compounds at elevated and subambient temperature under RP-HPLC conditions

The main focus of this study was to explore the capability of native α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin and their hydroxypropyl derivatives for host-guest interaction with 7,8-dimethoxyflavone, selected steroids (estetrol, estriol, estradiol, estrone, testosterone, cortisone, hydrocortisone, progesterone and 17α-hydroxyprogesterone) and polycyclic aromatic hydrocarbons (toluene, naphthalene, 1,8-dimethylnaphthalene, 1-acenaphthenol, acenaphthylene and acenaphthene) under reversed-phase liquid-chromatography conditions. The study revealed that native cyclodextrins interact more efficiently with the analytes investigated than do their hydroxypropyl counterparts. In the low-temperature region, enormously high ratios were observed for polycyclic aromatic hydrocarbons, particularly 1,8-dimethylnaphthalene, acenaphthene and acenaphthylene chromatographed on a β-cyclodextrin-modified mobile phase. In such a case, the retention times of the polycyclic aromatic hydrocarbons were strongly reduced (e.g. from 127 to 1.2 min for 1,8-dimethylnaphthalene) and were close to the hold-up time of the high-performance liquid chromatography (HPLC) system (0.7 min). Moreover, chiral separation of 1-acenaphthenol optical isomers was observed and the elution order of the enantiomers was determined. Within the steroids group, strong interaction was observed for estradiol and testosterone. The results of cluster analysis indicate that β-cyclodextrin as well as γ-cyclodextrin and its hydroxypropyl derivative can be most effective mobile-phase additives under reversed-phase HPLC conditions for 3D-shape-recognition-driven separation, performed at subambient and elevated temperatures, respectively.     

超高效液相色谱法同时测定化妆品中的15种激素

建立了采用超高效液相色谱同时测定化妆品中糖皮质激素、雌激素、雄激素、孕激素等共15种激素(曲安西龙、氢化可的松、泼尼松、可的松、甲基泼尼松龙、倍他米松、地塞米松、醋酸泼尼松龙、醋酸氢化可的松、雌三醇、雌二醇、雌酮、己烯雌酚、睾酮、孕酮)的分析方法。不同形态的化妆品样品均以甲醇为提取溶剂进行超声提取,经Oasis HLB固相萃取柱富集净化,以Waters ACQUITY UPLC BEH C18色谱柱(1.7 μm,2.1 mm×50 mm)分离,乙腈和水为流动相梯度洗脱,6 min 内完成了15种激素的分离及检测。在1～25 ng进样范围内,15种激素的工作曲线的线性相关系数r均高于0.9995。在低、中、高（2,10,20 mg/kg）3个添加水平下15种激素的平均回收率为88.2%～102.4%,相对标准偏差为1.6%～7.4%。     

Determination of hormones in human urine by ultra‐high‐performance liquid chromatography/triple‐quadrupole mass spectrometry

Steroid hormones play a critical role in maintaining the homeostasis of human metabolism. Urine as a noninvasive sample has been extensively used in clinical diagnosis for hormones homeostasis. In this study, the simultaneous characterization of fourteen hormones in urine was performed based on ultra‐high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPHLC/ESI(+)‐MS/MS) with multiple reaction monitoring in the positive ionization mode. The target hormones were cortisone, cortisol, 11‐deoxycortisol, aldosterone, corticosterone, 11‐deoxycorticosterone, progesterone, 17‐OH‐progesterone, pregnenolone, estrone, estradiol, estriol, testosterone and dehydreopiandrosterone. β‐Glucuronidase/sulfatase deconjugation and liquid–liquid extraction (LLE) were conducted for the determination of urinary hormones (free + conjugated forms). The limits of detection (LODs) ranged from 0.2 ng/mL (11‐deoxycortisol and testosterone) to 1 ng/mL (cortisone). The extraction recovery of the targeted compounds ranged from 87% to 127%, indicating sufficient extraction efficiency for the LLE process. Intraday precision was below 10% and the accuracy ranged from 84% to 122%. The profiling analysis of hormones in urine samples helps to understand the metabolic state of biological systems and can be employed as a diagnostic tool in diseases developed by endocrine‐disrupted systems.     

Isolation and determination of estrogens in water samples by solid‐phase extraction using molecularly imprinted polymers and HPLC

Advanced SPE with molecularly imprinted polymers (MIP) was used in this study. A noncovalent imprinting approach was applied to separate 17β‐estradiol, estriol, and estrone from water samples. Polymer material was prepared by bulk polymerization with methacrylic acid as a functional monomer, divinylbenzene and ethyleneglycol dimethacrylate as crosslinkers, and acetonitrile, acetonitrile/toluene (3:1, v/v) or isooctane/toluene (1:99, v/v) as a porogen. We also prepared an MIP film on a silica gel surface with methacrylic acid and ethyleneglycol dimethacrylate as monomers and acetonitrile as a solvent. Qualitative and quantitative hormone analyses were carried out by HPLC with various detection techniques, including UV/visible spectroscopic detection (diode array detection) and electrochemical detection (CoulArray). The results of the study indicate that MIP technology is an excellent method for the quality control of estrogens in environmental analyses with a low quantification limit for 17β‐estradiol of around 26 (diode array detection) and 0.25 ng/mL (electrochemical detection). The proposed method was found to be suitable for routine determinations of the analyzed compound in environmental laboratories.     

New Discoveries of Heating Effect on Trimethylsilyl Derivatization for Simultaneous Determination of Steroid Endocrine Disrupting Chemicals by GC–MS

Most previously described derivatization procedures with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) or N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) for the GC–MS analysis of steroids, such as estrone (E1), 17β-estradiol (E2), estriol (E3) and 17α-ethynylestradoil (EE2), used a heating process of 45–80 °C (typically 70 °C) for 25–60 min, usually in combination with a catalyst. However, we found that it is not necessary to heat and add catalyst for the derivatization with BSTFA. Best reaction conditions for MSTFA are heating at 70 °C for 10 min. Derivatization of EE2 using MSTFA without heating results in three products: TMS-E1, mono-TMS-EE2 and di-TMS-EE2.

     

Development of a Solid‐Phase Extraction‐Enzyme‐Linked Immunosorbent Assay Method with a New Sorbent of Multiwall Carbon Nanotube for the Determination of Estrone in Water

Abstract

A sensitive solid‐phase extraction‐enzyme‐linked immunosorbent assay (SPE‐ELISA) method was developed to analyze the estrone in environmental water. A new SPE sorbent of the multiwall carbon nanotube was tested and proved to have similar adsorbability for estrone comparing to the commercial C₁₈ SPE. A specific polyclonal antibody for estrone (A‐E1) and a broad‐spectrum antibody for estrone, estradiol and estriol (A‐E2) were produced. For A‐E1, the limit detection of estrone was 0.04 µg/l and for A‐E2 were 0.07, 0.04 and 0.2 µg/l of estrone, estradiol and estriol, respectively. Different river water samples were analyzed by ELISA and HPLC method.     

Solid-phase extraction using molecularly imprinted polymer for selective extraction of natural and synthetic estrogens from aqueous samples

A method is proposed for the clean-up and preconcentration of natural and synthetic estrogens from aqueous samples employing molecularly imprinted polymer (MIP) as selective sorbent for solid-phase extraction (SPE). The selectivity of the MIP was checked toward several selected natural and synthetic estrogens such as estrone (E1), 17β-estradiol (β-E2), 17α-estradiol (α-E2), estriol (E3), 17α-ethinylestradiol (EE2), dienestrol (DIES) and diethylstilbestrol (DES). Ultrahigh pressure liquid chromatography (UHPLC) coupled to a TSQ triple quadrupole mass spectrometry (QqQ) was used for analysis of target analytes. The chromatographic separation of the selected compounds was performed in less than 2 min under isocratic conditions. The method was applied to the analysis of estrogens in spiked river and tap water samples. High recoveries (>82%) for estrone, 17β-estradiol, 17α-estradiol, estriol and 17α-ethinylestradiol were obtained. Lower but still satisfactory recoveries (>48%) were achieved for dienestrol and diethylstilbestrol. The method was validated and found to be linear in the range 50-500 ng L(-1) with correlation coefficients (R(2)) greater than 0.995 and repeatability relative standard deviation (RSD) below 8% in all cases. For analysis of 100-mL sample, the method detection limits (LOD) ranged from 4.5 to 9.8 ng L(-1) and the limit of quantitation (LOQ) from 14.9 to 32.6 ng L(-1). To demonstrate the potential of the MIP obtained, a comparison with commercially available C(18) SPE was performed. Molecularly imprinted SPE showed higher recoveries than commercially available C(18) SPE for most of the compounds. These results showed the suitability of the MIP-SPE method for the selective extraction of a class of structurally related compounds such as natural and synthetic estrogens.