Separation cortisol

The first liquid-solid separation of the corticosteroids was carried out using a silica stationary phase with a mobile phase of chloroform-dioxane (100 5) to separate cortisol, cortisone and 11-de-oxycortisol (Touchstone and Wortmann, 1973). Most separations using silica stationary phases have used variations of the mobile phase originally described by Hesse and Hovermann (1973). Thus, a mobile phase of dichloromethane-ethanol-water (93.6 4.7 1.7) has been used to resolve prednisolone, cortisol, prednisone, cortisone, corticosterone, deoxycortisol and 17-a-hydroxy-progesterone on a silica stationary phase (Trefz et al., 1975). Other mobile phases which have been used include hexane-methylene chloride-ethanol-acetic acid (63.8 30 6 0.2) for the determination of plasma prednisolone (Loo et al., 1977), a mixture of 1.5% methanol and 0.2% water in chloro-... 

The steroids aldosterone, cortisone, cortisol, 11-P-hydroxyandrostenedione, corticosterone, and rostenedione, 11-desoxycorticosterone, 17-hydroxy-progesterone, and progesterone have been performed on Ultrasphere ODS using methanokwater.19 Ranitidine N-[2-[[[5-[(dimethylamino)methyl]-2-furanyl]-methyl]thio]ethyl]-N1-methyl-2-nitro-l,l-ethenediamine has been separated using a p-Bondapak C18 column operated with acetoni-trile methanol water buffered with triethylamine phosphate.117 Pyridoxal-5 -phosphate and other B6 vitamers, including pyridoxamine phosphate, pyri-doxal, pyridoxine, and 4-pyridoxic acid, were separated as bisulfite adducts... 

The application from van der Hoeven et al. (1997) used an ADS cartridge online SPE to measure cortisol and prednisolone in plasma and arachidonic acid in urine. A precolumn packed with a C18 alkyl-diol support (LiChrosphere RP-18 ADS, 25 /an, Merck) was used. To reduce run time, column switching was programmed as heart-cut , diverting only the analyte fraction into the analytical column. Another LiChrosphere column (125 x 4 mm inner diameter, Merck) handled separation. After the injection of 100 fiL plasma, the lower limit of detection for prednisolone was 1 ng/mL while cortisol was readily quantitated at its endogenous level of 100 ng/mL. The run time was 5 min. For arachidonic acid, a Hypersil ODS column (200 x 3.0 mm inner diameter, 5 /.an) was used. The injection volume was 200 //I. and run time was 9.5 min. The detection limit was 1 ng/mL and recovery was 77%. 

An account of the principles which help to understand how hormones achieve their roles in the body is given in Chapter 12. The understanding is based on separation of the effects of hormones into three components the action, the effects (biochemical and physiological) and the function. A steroid hormone binds to a cytosolic intracellular receptor, which then moves into the nucleus where it binds to DNA at a specific site (the steroid response element) and activates genes which result in the formation of proteins that elicit biochemical and physiological effects. This is discussed for cortisol in Chapter 12 and aldosterone in Chapter 22. Much of the interest in the reproductive steroid hormones is in the physiological effects and how these account for their functions. 

Fig. 8 Capillary EKC separations of (A) acetophenone (1), propiophenone (2), butyrophenone (3), valerophenone (4), and hexanophenone (5) on vesicles composed of sodium dodecylsulfate and ra-dodecyltrimethylammoniumbromide at pH 7.2, (B,C) of 1-dehydroaldosterone (1), cortisone (2), cortisol (3), 21-deoxycortisol (4), 11-deoxycortisol (5), and dexamethasone (6) on liposomes composed of 1-palmitoyl-2-oleoyl-sra-glycero-3-phosphocholine (80%) and (B) phosphatidylserine or (C) car-diolipin at pH 9, lipid concentrations (B) 2.4 mM and 0.6 mM, (C) 3.6 and 0.9 mM. (Part A is reprinted with permission from Ref. 59, copyright 1998 American Chemical Society Parts B and C are reprinted with permission from Ref. 33, copyright 2000 Wiley-VCH Verlag, all with slight modifications.)...

Several successful attempts were done to transfer classical CEIA to a microchip-based format. This kind of miniaturization is a trend that can overcome the limitations of CE in high-throughput systems. On-chip CE offers both parallel analysis of samples and short separation times. Koutny et al. showed the use of an immunoassay on-chip (32). In this competitive approach fluorescein-labeled cortisol was used to detect unlabeled cortisol spiked to serum (Fig. 8). The system showed good reproducibility and robustness even in this problematic kind of sample matrix. Using serum cortisol standards calibration and quantification is possible in a working range of clinical interest. This example demonstrated that microchip electrophoretic systems are analytical devices suitable for immunological assays that can compete with common techniques. 

As a possible consequence of direct neurotoxic effects of sustained hypercortisolism, hippocampal atrophy has now repeatedly been reported for depressed patients (Sheline et ah, 1996 Bremner et al., 2000a). Hippocampal atrophy may be associated with disinhi-bition of CRF secretion and further increases in cortisol secretion, which in turn may further damage the hippocampus. Impaired inhibition of the HPA axis is also evidenced by nonsuppression of cortisol by dexame-thasone and decreased GR numbers in depressed patients both findings parallel those in maternally separated rats. 

Effects of early environmental adversity on HPA mediation of neurodevelopment have also been demonstrated in non-human primates (Coplan et al., 1995). Corticotropin-releasing hormone (CRH) intracerebro-ventricular administration in rhesus monkeys that had been separated from their mothers produced behavioral inhibition and increases in ACTH and cortisol. Coplan et al (1995) presented evidence for persistently elevated cerebrospinal fluid concentrations of corticotropin-releasing factor (CRF) in grown macaques that had been reared by mothers in unpredictable environmental conditions. Further studies in adversely reared adult monkeys demonstrated an inverse relationship between mean CRF concentrations and GH response to clonidine (Coplan et al., 2000). In light of evidence that reduced GH response to clonidine has been shown in other anxiety disorders (Charney and Bremner, 1999), Coplan et al. (2000) hypothesize that GH response to clonidine may inversely reflect trait-like increases of central nervous system CRF activity. Data linking childhood anxiety to growth deficits are consistent with this view (Pine et al., 1996). Activity, of the HPA axis, as related to early environmental... 

People suffering from PTSD frequently have abnormal levels of the hormones that are involved in the body s response to stress. Studies have shown that baseline cortisol levels in people with PTSD are lower than normal, and epinephrine and norepinephrine levels are higher than normal. However, it is not known whether these differences in hormones and neurotransmitters involved in the bod/s stress response precede or follow the development of the disorder. It is important to note that the neurotransmitter and hormone changes seen with PTSD are separate from, and actually opposite to, those seen in major depression. In major depression, cortisol levels are elevated and epinephrine and norepinephrine levels are low. The distinctive profile associated with PTSD is also seen in individuals who have both PTSD and depression. One hypothesis is that people who cannot mount a robust stress... 

Taylor and colleagues [98] at the Mayo Clinic published a method for the simultaneous analysis of urinary cortisol and cortisone. They used 2H4 cortisol as an internal standard and took a 0.5-ml urine sample. An API 2000 with Turboion-spray source was used in the positive-ion mode. Chromatography was conducted on a standard-bore C18 column with Q8 precolumn filter. MRM was conducted in the positive-ion mode monitoring m/z 363—>121 for cortisol, 367—>121 for 2TL, cortisol, and 361— -121 for cortisone. Cortisol and cortisone were separated and both were eluted within 2 min. Inter- and intra-assay variation for both compounds was < 9% for amounts above 2 pig/dl. The values obtained agree well with those of other studies, such as ours (Table 5.3.2) [62]. They found a range for cortisol for adult males of 4.2-60 pg/24 h and for adult females 3.0-43 pg/24 h. In summary, the 3-min run time of their method has allowed the Mayo group to completely transfer their cortisol and cortisone workload from RIA and HPLC to MS/MS. 

Guo and co-workers [24,25] have spearheaded the development of MS/MS serum steroid profiles. Their most recent report describes profiling in 11 min of 12 steroids in 200 pi serum with minimal work-up, comprising acetonitrile protein precipitation. The steroids analyzed were as follows DHEA sulfate, DHEA, aldosterone, cortisol, corticosterone, 11-deoxycortisol, androstenedione, estradiol, testosterone, 17-hy-droxyprogesterone, progesterone, and 25-hydroxyvitamin D3. Stable-isotope-labeled internal standards were incorporated for each steroid. An API-5000 instrument was used with the APPI source in positive-ion mode, with the exception of aldosterone, which had greater sensitivity in negative-ion mode. Separation was carried out on a C8 column, which allowed more rapid separation than the more commonly utilized C18. The MRM transitions utilized are shown in Table 5.3.1. The lower level of sensitivity was between 1.5 and 10 pg/ml, dependent on the steroid. The authors were exhaustive in addressing issues of accuracy, recovery (90-110%) and reproducibility (< 12.2% for same-day and between-day). 

An interesting application of microchip CE is the analysis of serum cortisol immunoassays that have been performed off-chip prior to their injection [64]. Separation and quantitation of the free and bound labeled antigen could be achieved in less than 30 s and allowed the determination of serum cortisol in the clinical range of interest. 

In competitive homogeneous immunoassay, separation and quantitation of free and bound labeled antigen (cortisol) were carried out in a fused silica chip. Since the antibody-antigen complex was not detected, an internal standard (fluorescein) was added to aid quantitation. In addition, since most of the total cortisol was bound in the serum, a releasing agent, 8-anilino-l-naphthalenesulfonic acid (ANS), should be added [1006]. In other reports, competitive immunoassay for BSA was demonstrated after performing a CE separation on-chip [105,1005]. 

In the on-chip theophylline homogeneous immunoassay, the antibody-antigen complex can be separated from the antigen. Why can such a separation be successful, but not in the case of the cortisol assay [330,1006] (2 marks)... 

The following protocol was proposed and consisted of 4 measuring days. Each day, four (or six at day 1) standards and four samples are analyzed. The calibration curves are constructed by least squares regression analysis and statistically tested for nonlinearity by means of an F-test on the residuals. The amount of cortisol in the serum samples is obtained by linear interpolation on the daily calibration curve. Preliminary experiments were also set up to determine the influence of the use of peak height or peak area ratios. For the cortisol measurement, some separation takes place between syn and anti isomers, therefore the use of peak heights is less favorable. 

A sensitive enzyme-immunoassay for plasma or saliva testosterone uses testosterone-lla-hemisuccinate antiserum, bound to cellulose to provide a solid phase for convenient separation. Testosterone-horseradish peroxidase conjugate was employed as the label.Another enzyme-immunoassay for testosterone employs a testosterone-3-(0-carboxymethyloxime)-pencillinase conjugate for competitive binding to the testosterone antiserum. 21-Amino-ll/3,17o -dihydroxypregn-4-ene-3,20-dione ( Cortisol 21-amine ) linked to alkaline phosphatase has been used ° in an effective enzyme-immunoassay for cortisol. 

One of the earliest efforts of qualitative measurement of a protein (human serum albumin) in a microchip-based device was based on bead agglutination in a microchamber (approximately lOjaL). Subsequently, several quantitative immunoassays have been performed using microchip electrophoretic systems that permit separation and quantitation of free- and bound-labeled antigens in competitive assays (see Chapter 5). Most are carried out in channels micro-machined into fused silica substrates. Early work on quantitative assays achieved measurement of cortisol in serum.The assay used cortisol labeled with fluorescein and an argon laser detector at 488 nm and required only 80 pL of a 40x dilution of serum as the sample. Other capillary electrophoresis-based assays for a variety of antibodies have also been developed that include immunoglobulins (IgG, IgA, and IgM), antibovine serum albumin, and antiestradiol. ... 

There is no experimental evidence that steroid hormone concentrations in serum are different from those in plasma. However, rapid separation of red blood cells in the specimen is important because red blood cells at room temperature can alter plasma concentrations of active steroid hormones red blood cells degrade estradiol to estrone and cortisol to cortisone, and they can adsorb testosterone. 

A fluorescence-quenching immunoassay for cortisol, based upon the use of a tracer comprising fluorescein linked to the steroid via a 21-amino-group, claims a good correlation with radioimmunoassay results. Separation of free and bound fractions is unnecessary with this system.141... 

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