An HPLC method for the determination of the free cortisol/cortisone ratio in human urine

The measurement of the urinary free cortisol-cortisone ratio has been reported to be a sensitive indicator of renal 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD 2) activity. This converts biologically active cortisol to inactive cortisone. A decrease in its activity (e.g. through disease or inhibition caused by a therapeutic agent or a foodstuff) may increase cortisol levels and susceptibility towards hypertension. The method presented here uses a simple isocratic tandem column HPLC system. The method has been validated and found to be robust and reproducible. The lower limit of quantification (LLOQ) was found to be 10 ng/mL for both cortisol and cortisone. Samples of urine (n = 99) from patients, most of whom were on complex combinations of drugs, were analyzed and 92% of samples were found to give successful results with this method (cortisol and cortisone above LLOQ). The ratio ranged from 0.07 to 5.61. No interferences were noted from the drugs that the patients were taking. It was also found that a morning spot urine sample gave comparable results to 24 h collection samples, thus making sample collection much easier.     

Radioimmunoassay of Plasma Cortisol

Abstract

A radioimmunoassay method for the direct measurement of cortisol in plasma after precipitation of the proteins with ethanol is described. Utilizing a specific antiserum against cortisol, with minimal or no cross reaction with other steroids, cortisol was meas-red accurately in a volume of 0.001 ml of plasma. The range of levels that could be measured per ml of plasma varied from 10 to 500 ng. The mean value of plasma cortisol at 8 a. m. obtained in twelve subjects by this method was about half the mean levels reported by another competitive protein binding assay.     

Effect of glucocorticoids and their complexes with apolipoprotein A‐I on the secondary structure of eukaryotic DNA

The tetrahydrocortisol–apolipoprotein A‐I complex specifically interacts with eukaryotic DNA isolated from rat liver. This interaction is highly cooperative and of a saturating nature. One DNA molecule binds about 54 molecules of the complex. Small‐angle X‐ray scattering has shown that hydrogen bonds between nitrous bases are destroyed and that single‐stranded structures are formed at the interaction of the tetrahydrocortisol–apolipoprotein A‐I complex with eukaryotic DNA. The most probable site of binding the tetrahydrocortisol–apolipoprotein A‐I complex with DNA is the sequence of the CC(GCC)_n type entering the structure of many genes, among them the structure of the human apolipoprotein A‐I gene. Oligonucleotide of this type has been synthesized. The association constant (K_ass) of its complexation was shown to be 1.66 · 10⁶ M^?1. Substitution of tetrahydrocortisol for cortisol in the complex results in a considerable decrease of K_ass. IR‐spectroscopy study has shown that the interaction of tetrahydrocortisol with oligonucleotide CC(GCC)_3–5 is accompanied by the formation of hydrogen bonds via the CO‐NH, PO₂, and OH groups of desoxycytidinephosphate. The tetrahydrocortisol–apolipoprotein A‐I complex alters the DNA secondary structure formed at the interaction with the hormone, causing the structural transition “order → tangle.” It is assumed that in the GC‐pairs of the given DNA sequence, tetrahydrocortisol initiates the rupture of hydrogen bonds, while the hydrophobic interactions between nitrous bases and apoA‐I intensify this process. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

Electrochemical Determination of Cortisol by Capillary Electrophoretic Enzyme Immunoassay

In recent years, capillary electrophoretic immunoassay (CEIA) has become a primary analytical tool in clinical diagnostics. The primary detection methods in CEIA are UV absorbance detection and laser induced fluorescence detection (LIF)1. The major disadvantages of the UV detector are the lack of sensitivity and not available for many antigens which are small molecules without strong UV absorbance. LIF is a more general approach to improve sensitivity. However, it is difficult to u…     

海洋鱼类血清游离皮质醇液质分析研究

血清样品中加入地塞米松作为定量内标,用氯仿提取,氮气吹干,V(甲醇):V(水)=1:3混合溶液定容后用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)对游离皮质醇进行分析。采用电喷雾电离源负离子模式,分别取m/z407.2和437.2作为皮质醇和内标的定量目标离子进行检测。结果表明,皮质醇与内标在BEHC18色谱柱上得到良好的分离,方法在0～200μg/L浓度范围内线性相关系数为0.9993。仪器检出限为1.64pg。方法平均回收率达到93.3%～98.1%,相对标准偏差小于10%。对采自浙江宁波几批大黄鱼(Preudosciaena Crocea)和鲈鱼(Lateolabrax Japonicus)血清进行测定,游离皮质醇含量在2.11～75.84μg/L范围内。与三重四极质谱仪(Q-Q-Q-MS)进行比较,仪器检测灵敏度要优于三重四极质谱仪;在0～2000μg/L。浓度范围内线性相关不及三重四极质谱仪的分析结果,但在0～200μg/L浓度范围内相关性相近。     

基于便携拉曼光谱仪和双层纸芯片的头发皮质醇超高灵敏检测

人头发中的皮质醇是反映现代人长期精神压力累积的主要临床标记物.建立了一种人发皮质醇的超高灵敏检测方法.方法基于表面增强拉曼光谱、免疫分析和双层纸芯片.纸芯片的第一层用于滤除人发提取液中的头发残渣（样品前处理）,第二层用于实施竞争性免疫反应和拉曼检测.固定在纸表面的皮质醇抗原和游离的头发皮质醇竞争结合能产生拉曼信号的皮质醇单克隆抗体,通过检测结合在纸表面的皮质醇抗原-抗体复合物的拉曼信号进行游离头发皮质醇的定量.在便携式拉曼光谱仪上,皮质醇抗体的拉曼信号经过优化,方法的检测限可以达到1 pg/mL,平行测定相对标准偏差为8.38%（n=6）.进行了两例实际样品检测,液质检测结果分别为0.771和0.153 ng/mL,本方法检测结果分别为0.63和0.247 ng/mL,两种方案的结果在一个数量级,证明了方法的实用性.利用此方法,一块芯片可以同时测定48个样品,专属性和准确性很好,特别适合于人群精神压力的普查.     

Sensitive and simultaneous quantitation of 6β‐hydroxycortisol and cortisol in human plasma by LC‐MS/MS coupled with stable isotope dilution method

CYP3A phenotyping provides a means for personalized drug therapy. We focused our attention on the plasma 6β‐hydroxycortisol (6β‐OHF) to cortisol ratio as an index for CYP3A phenotyping. In the present study, we developed a sensitive and reliable method for the simultaneous determination of 6β‐OHF and cortisol in human plasma using high‐performance liquid chromatography/tandem mass spectrometry together with picolinylester derivatization or nonderivatization methods and 6β‐[9,11,12,12‐²H₄]hydroxycortisol and [1,2,4,19‐¹³C₄]cortisol as internal standards for in vivo CYP3A phenotyping in humans. The lower limits of quantification were 38.513 pg/mL for 6β‐OHF and 38.100 pg/mL for cortisol. The relative error and relative standard deviation of the lower limits of quantification were <5% for both methods. The intra‐day and inter‐day assay reproducibilities of the determined 6β‐OHF and cortisol concentrations were consistent with the actual amounts added as relative errors and relative standard deviations for both methods, which were <5.4% and <3.9%, respectively. Both methods were applied for the quantification of plasma 6β‐OHF and cortisol concentrations in healthy subjects taking oral contraceptives. The absolute concentrations and time course of 6β‐OHF and cortisol were found to be consistent when measured using the 2 methods. The ratio as an index for in vivo CYP3A activity decreased after 21 days of taking oral contraceptives for both methods. To the best of our knowledge, this is the first study reporting the detailed investigation of accuracy and precision in the simultaneous measurement of 6β‐OHF and cortisol in human plasma using liquid chromatography/tandem mass spectrometry coupled with stable isotope dilution method, which can be applied to CYP3A phenotyping.     

A Simple,sensitive, and selective electrochemical aptasensor for cortisol based on rGO-AuNPs

Cortisol is a steroid hormone naturally produced by the adrenal glands. It participates in and controls several processes in the body and is considered an important physiological biomarker. Due to its very low concentrations in body fluids, its detection requires high sensitivity and specificity. Here, we present a simple electrochemical biosensor based on reduced graphene oxide (rGO) modified with gold nanoparticles (AuNPs) for the immobilization of the cortisol-specific aptamer (Ap) (MCH-Ap-AurGO/GCE). Important analytical parameters for identifying the target analyte were optimized, such as conditions and amount of immobilized Ap, the influence of the concentration and nature of the supporting electrolyte, pH of the medium, and incubation time. The optimized conditions for the aptasensor were: concentration of Ap 1.0 × 10⁻⁶ mol L⁻¹, support electrolyte Tris/HCl 50 mmol L⁻¹, MgCl₂ 10 mmol L⁻¹, and NaCl 10 mmol L⁻¹, at pH 5.0 and incubation time of 15 min. A linear response range was obtained from 1 × 10⁻¹⁸ up to 1 × 10⁻¹¹ mol L⁻¹ of cortisol with a detection limit (LOD) of 1.0 × 10⁻¹⁸ mol L⁻¹. A curve adjusted for operational purposes in the saliva sample was fitted for the concentration range between 0.5 × 10⁻¹⁴ and 1 × 10⁻¹¹ mol L⁻¹, with a linear regression equation ΔRtc/Rtc₁ = 2.70 + 0.17 × log([Cortisol]). The aptasensor demonstrated a great potential for detecting cortisol in a simple, fast, and highly sensitive way, opening its path for application in real samples, which present levels below the concentration in which cortisol is commonly found in body fluids.