Preparation of sterol-imprinted polymers with the use of 2-(methacryloyloxy)ethyl phosphate.

Steroid-selective polymers were prepared by the molecular imprinting technique, using 2-(methacryloyloxy)ethyl phosphate as functional monomer. The retentivity and selectivity of the obtained imprinted polymers were evaluated by liquid chromatography. The cholesterol-imprinted polymer showed higher affinity for cholesterol than that for cholesterol derivatives. The selectivity of the imprinted polymer was superior to the imprinted polymer prepared with the conventional functional monomer, 2-(trifluoromethyl)acrylic acid. Estradiol was also imprinted and gave similar results, demonstrating that 2-(methacryloyloxy)ethyl phosphate would be suitable for imprinted polymers of cholesterol and related compounds.     

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.     

Cell-based assay for screening 11beta-hydroxysteroid dehydrogenase inhibitors using liquid chromatography/tandem mass spectrometry detection

Cortisol is an important glucocorticoid that regulates many physiological pathways by activating various intracellular receptors. The type 1 isozyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) functions in vivo predominantly as a reductase by converting cortisone into cortisol. A high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to screen for inhibitors of 11beta-HSD1 by monitoring cortisol and cortisone simultaneously. The injection cycle time can be as fast as 1 min/sample, making it amenable to the analysis of large numbers of the cell-assay samples in the screening of 11beta-HSD inhibitors. The reductase and dehydrogenase activities of 11beta-HSD1 are assessed separately.     

Recognition of Sialic Acid Using Molecularly Imprinted Polymer

Abstract

The molecular imprinting technique was applied for the preparation of a polymer selective for sialic acid. To evaluate its binding ability the molecularly imprinted polymer obtained was used as a stationary phase in liquid chromatography. The polymer showed pH-dependent characteristics for binding: an optimum specificity to sialic acid at pH 8.1 and a higher affinity with group selectivity for cis-diol containing sugars at higher pH.     

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.     

Micro determination of cortisol and cortisone in umbilical cord blood by chemiluminescent high‐performance liquid chromatography

A simple, sensitive and specific chemiluminescent high‐performance liquid chromatography method, based on the luminol reaction, for determination of serum cortisol and cortisone, was established. In infants, placental 11β‐hydroxysteroid dehydrogenase type 2 enzyme (11β‐HSD2) activity may affect adrenal function early after birth. The cortisol–cortisone ratio of serum concentrations in umbilical cord blood is an indicator of placental 11β‐HSD2 activity. The optimum conditions for the luminol reaction were determined to be 1.5 mM luminol, 0.6 M sodium hydroxide, 0.15 mm potassium hexacyanoferrate(III) and 200 mM potassium hexacyanoferrate (II). The calibration curves for cortisol and cortisone exhibited good linearity. The correlation coefficients of the calibration curves were 0.996. The intra‐ and inter‐day precisions were in the ranges: cortisol 7.0–12.2 and 4.4–9.2%, cortisone 5.3–7.0 and 6.2–9.9%. The recoveries of these steroids were in the ranges: cortisol 97–105%, cortisone 94–102%. The limits of detection were as follows: cortisol, 0.17 μg/dl; cortisone 0.15 μg/dl. This assay could be successfully applied to determination of the cortisol–cortiosone ratio of serum concentrations in umbilical cord bloods. Copyright © 2009 John Wiley & Sons, Ltd.     

腈菌唑分子印迹聚合物的制备及识别性能研究

以腈菌唑为模板分子，采用原位分子印迹技术，制备具有特定识别性能的连续棒状分子印迹聚合物。考察了流动相中酸量对分离的影响，研究了几种结构类似物在所得分子印迹柱上的保留特性。结果表明，这种棒状分子印迹聚合物比相应的空白聚合物有高的识别性能和选择性。     

分子印迹聚合物拆分消旋体酮洛芬

以(S) 酮洛芬为印迹分子利用分子印迹技术合成能识别(S) 酮洛芬的聚合物。聚合物作为高效液相色谱的固定相,消旋体酮洛芬在固定相能分开,同时聚合物还能将酮洛芬和布洛芬的混合物分开。     

Simultaneous Determination of Free Cortisol,Cortisone and their Tetrahydrometabolites in Urine by Single Solvent Extraction and Liquid Chromatography–Tandem Mass Spectrometry

Abstract

A fast, efficient and low-cost high performance liquid chromatography–tandem mass spectrometry methodology was developed and validated for the simultaneous determination of free urinary cortisone, cortisol and their tetrahydro-metabolites. The developed method comprises a simple liquid-liquid extraction with CH₂Cl_2, followed by reversed-phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI) in positive mode. The baseline chromatographic separation of the analytes, including the stereoisomers tetrahydrocortisol (THF) and allo-THF, was achieved on a Hypersil Gold C₁₈ column with a mobile phase consisting of 0.05%v/v formic acid in water—acetonitrile, using a gradient elution program. The influence of the mobile phase composition and the ESI parameters on the sensitivity of the method was extensively studied. Sample preparation was also optimized, testing two techniques: solid phase extraction (SPE) and liquid-liquid extraction (LLE). Recoveries ranged from 74.7% (a-THF) to 93.5% (cortisol) and the method limits of detection (MLD) ranged from 0.34?ng mL^?1 (cortisol) to 1.37?ng mL^?1 (THF). Intra- and inter-day coefficient of variation of the assay varied from1.5% (allo-THF) to 13% (tetrahydrocortisone) and from 3.6% (allo-THF) to 14.9% (tetrahydrocortisone), respectively. The method was applied for the analysis of urine samples from 53 healthy individuals with a mean age of 13.96?years in order to estimate the concentration of the five corticosteroids and the ratio of the metabolites. Associations between urinary cortisol/cortisone and serum cortisol/cortisone values were also characterized.     

分子烙印亲和色谱与质谱联用实现中草药活性成分分离鉴定一体化

以骆驼蓬种籽中抗肿瘤活性化合物哈尔明及哈马灵的结构类似物哈尔满作为模 板,用非共价键法制备了对哈尔明及哈马灵具有强亲和性的分子烙印聚合物。此分 子烙印聚合物作为液相色谱固定相与大气压电离飞行时间质谱联用,直接分离鉴定 了草药骆驼蓬种籽甲醇粗提物中所含的哈尔明及哈灵两种抗肿瘤活性成分。实验结 果证明了通过分子烙印亲和色谱与质谱联用方法,快速有效地对中草药活性成分分 离鉴定是可能是。     

Thin-Layer Chromatographic Competitive Protein-Binding Assay for Cortisol and Cortisone, and its Application to Urine Samples from Healthy Men Undergoing Water Diuresis

Specific and rapid determination of free cortisol (compound F) and cortisone (compound E) in human urine has been achieved by concentration of the urine samples, liquid–liquid extraction of the concentrated samples, thin-layer chromatography of ethanolic extracts on aluminium foil-backed silica gel 60 TLC plates, location of the steroids under UV light, elution of cortisol and cortisone from sections of the plates with phosphate buffer, and measurement by competitive protein-binding assay. Chicken serum was used as the source of corticosteroid binding globulin, because it binds cortisol and cortisone with similar high affinity. The method combining thin-layer chromatography and competitive protein-binding assay is easy to perform, specific, sensitive, and quite rapid. Free cortisol and cortisone were measured in the urine of male individuals who abstained from water intake for 2 h or who ingested 1 L of water. The results show, for the first time, that short-term water diuresis markedly increases urinary free cortisone excretion, supporting our previous hypothesis that its excretion is positively correlated with urine volume, i.e. with the volume of 24-h urine samples.     

The application of pseudo template molecularly imprinted polymer to the solid-phase extraction of cyromazine and its metabolic melamine from egg and milk

The combination of molecularly imprinted polymer with high performance liquid chromatography has been developed to determine cyromazine and its metabolic melamine in some samples. However, the potential risk of template leakage used in molecularly imprinted polymer is a major disadvantage. To solve this problem, 2-(4,6-diamino-1,3,5-triazin-2-ylamino) ethanethiol disulfide, a molecule that shares the similar imprinting sites with cyromazine and melamine, was selected as pseudo template to prepare molecularly imprinted polymer. Methacrylic acid, ethylene glycol dimethyl acrylate and toluene were selected as functional monomer, crosslinker and porogen, respectively. The molecular recognition property and binding capability of cyromazine and melamine were evaluated by adsorption test and Scatchard analysis. The results showed that the molecularly imprinted polymer based on pseudo template had more excellent affinity and selectivity for cyromazine and melamine. The resulting molecularly imprinted polymer was used as a solid-phase extraction material to enrich cyromazine and melamine in egg and milk samples for high performance liquid chromatography analysis. The solid-phase extraction process was carefully optimized. It was found that when different concentration of cyromazine and melamine standards were spiked into samples, satisfactory recovery rate of cyromazine and melamine were obtained as 85.6-98.8% with relative standard deviation <5.5%.     

Chromatographic characterization of molecularly imprinted polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid

Two polymers binding the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were prepared by utilising the technique of the non-covalent molecular imprinting polymerisation in an aqueous medium. The polymers obtained were packed in HPLC columns and the effects of the mobile phase composition on the retention of the imprinting molecule and the selectivity of the stationary phases towards several analogous structures were studied by liquid chromatography. The columns showed a good level of selectivity towards the template and strictly related molecules. It was found that the molecular recognition mechanism acting on the columns was dependent on a combination of ion pair and hydrophobic interactions.     

Steroidogenesis in Patients with Various Adrenal Cortex Diseases as Studied by Reversed-Phase High-Performance Liquid Chromatography

A method is proposed for the quantitative determination of diagnostically important corticosteroids (cortisol, cortisone, corticosterone, 11-deoxycorticosterone, and 11-deoxycortisol) in blood serum and urine (cortisol and cortisone) in an isocratic mode of reversed-phase high-performance liquid chromatography (RP HPLC) with the use of -cyclodextrin as a component of the mobile phase (CH₃CN : H₂O). Biological fluids (blood serum and urine) from a group of healthy donors and patients with various endocrine diseases (Cushing's syndrome, congenital adrenal hyperplasia, aldosteroma, and adrenal cortex carcinoma) were examined, and characteristic chromatographic steroid profiles were obtained for these disturbances.     

Method development for cortisol and cortisone by micellar liquid chromatography using sodium dodecyl sulphate: application to urine samples of rugby players

The chromatographic behavior of cortisol and cortisone using a micellar medium of sodium dodecyl sulphate (SDS) as surfactant, a Hypersil C18 (150- x 3.2-mm i.d., 5 microm) column, a flow rate of 0.5 mL/min, and UV absorbance detection at 245 nm is described. The effect of several organic modifiers and the surfactant concentration on the separation is studied. A mobile phase of 18 mM SDS and 8.3% tetrahydrofuran allows for the separation of cortisol and cortisone up to baseline. These results are also achieved by applying a bivariant optimization method. The proposed method is sensitive, reproducible, and selective. In addition, it is less expensive than conventional high-performance liquid chromatography methods for cortisol and cortisone. The method is applied to the determination of cortisol and cortisone in urine samples of rugby players before and after stress for doping control purposes.     

Automated direct high-performance liquid chromatographic assay for estetrol, estriol, cortisone and cortisol in serum and amniotic fluid.

An automated direct assay for the simultaneous determination of unconjugated estetrol, estriol, cortisone and cortisol in serum and amniotic fluid, using high-performance liquid chromatography with electrochemical detection and ultraviolet detection, has been developed. The analysis time is ca. 1 h. This system offers good reproducibility with low coefficients of variation (estetrol, 2.3%; estriol, 2.3%; cortisone, 2.6%; cortisol, 1.9%). Detection limits are low enough for routine determinations (estetrol and estriol, 150 pg; cortisone and cortisol, 5 ng). Comparison of the values measured by the present method and by radioimmunoassay revealed significant correlations for estetrol (r = 0.787, p less than 0.01), estriol (r = 0.957, p less than 0.01), cortisone (r = 0.956, p less than 0.01) and cortisol (r = 0.865, p less than 0.01). This system proved to be valuable in monitoring feto-placental function.     

Determination of M+4 stable isotope labeled cortisone and cortisol in human plasma by microElution solid-phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive microElution solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of M+4 stable isotope labeled cortisone and cortisol in human plasma. In this method, M+4 cortisone and M+4 cortisol were extracted from 0.3 mL of human plasma samples using a Waters Oasis HLB 96-well microElution SPE plate using 70 microL methanol as the elution solvent, and chromatographed on a Waters Symmetry C18 column (4.6 x 50 mm, 3.5 microm). M+9 cortisone and M+9 cortisol were used as the internal standards. A PE Sciex API 4000 tandem mass spectrometer interfaced with the liquid chromatograph via a turboionspray source was used for mass analysis and detection. The selected reaction monitoring (SRM) of precursor --> product ion transitions were monitored at m/z 365.2 [M+H](+) --> 167.0 and at m/z 367.3 [M+H](+) --> 125.1 for M+4 cortisone and M+4 cortisol, respectively. The lower limit of quantitation was 0.1 ng mL(-1) and the linear calibration range was from 0.1 to 100 ng mL(-1) for both analytes. This method demonstrated to be very reproducible and reliable.     

橙皮苷印迹聚合物的识别性能及机理研究

为了制备对橙皮苷(HES)具有特定识别能力的吸附材料,以HES为模板分子,丙烯酰胺(AM)为功能单体,甲基丙烯酸乙二醇酯(EDMA)为交联剂,在甲醇中制备了HES印迹聚合物(MIP),采用平衡吸附实验方法研究了聚合物的吸附性能和选择性能,探讨了聚合物的印迹机理和识别机理.结果表明,MIP对HES具有较高的亲和性和选择性.当HES浓度为0.048 mmol/L时,MIP及相应NMIP对HES的分配系数KD分别为10.17 和2.973,印迹因子α达到3.421.MIP对结构相似物芦丁及柚皮苷的选择因子β分别为2.446和1.246.机理研究表明识别位点来自AM与HES苯甲酰系统的氢键作用,吸附溶液中水含量的增加对MIP的识别能力有较大的影响.最后,以高效液相色谱研究了MIP在样品中的分离富集能力,表明该印迹聚合物具有一定的应用潜能.     

Water‐compatible molecularly imprinted polymer as a sorbent for the selective extraction and purification of adefovir from human serum and urine

A molecularly imprinted polymer has been synthesized to specifically extract adefovir, an antiviral drug, from serum and urine by dispersive solid‐phase extraction before high‐performance liquid chromatography with UV analysis. The imprinted polymers were prepared by bulk polymerization by a noncovalent imprinting method that involved the use of adefovir (template molecule) and functional monomer (methacrylic acid) complex prior to polymerization, ethylene glycol dimethacrylate as cross‐linker, and chloroform as porogen. Molecular recognition properties, binding capacity, and selectivity of the molecularly imprinted polymers were evaluated and the results show that the obtained polymers have high specific retention and enrichment for adefovir in aqueous medium. The new imprinted polymer was utilized as a molecular sorbent for the separation of adefovir from human serum and urine. The serum and urine extraction of adefovir by the molecularly imprinted polymer followed by high‐performance liquid chromatography showed a linear calibration curve in the range of 20–100 μg/L with excellent precisions (2.5 and 2.8% for 50 μg/L), respectively. The limit of detection and limit of quantization were determined in serum (7.62 and 15.1 μg/L), and urine (5.45 and 16 μg/L). The recoveries for serum and urine samples were found to be 88.2–93.5 and 84.3–90.2%, respectively.     

Simultaneous Determination of Cortisol,Cortisone, and 6β-Hydroxycortisol in Human Urine by UPLC with UV Detector and Application to Determine Diurnal Variations

In the present studies, a simple rapid ultra performance liquid chromatography (UPLC) method with UV detection for the simultaneous determination of cortisol, cortisone and 6β-hydroxycortisol in human urine was developed. The three analytes and the internal standard dexamethasone were separated on a Waters Acquity UPLC-Tunable UV system with an Acquity UPLC BEH C18 column (50 × 2.1 mm ID, 1.7 μm) using a gradient elution of methanol and water (containing 0.01% formic acid) with a run time of 7 min. The method was accurate and precise, over the ranges of 5–200 ng mL⁻¹ for cortisol, and 10–1,000 ng mL⁻¹ for both cortisone and 6β-hydroxycortisol, and showed good linearity (r ² > 0.999). This method was applied for the measurement of cortisol, cortisone and 6β-hydroxycortisol in samples collected over different periods as a tool to assess the activity of CYP3A and 11β-hydroxysteroid dehydrogenase type 2 enzymes.