Improved detection of steroids on NH2 layers

Summary Thin-layer chromatography (TLC) using NH₂-modified TLC plates is a reproducible method for the measurement of steroids. The sensitivity of determination of steroids (cortisol, cortisone, testosterone, progesterone) by measuring fluoresence in situ can be increased twofold if chromatograms are dipped into a mixture of hexane-paraffin. Fluorescence is further increased if filters are used, which cut emission wavelengths below 400 nm. Using the modifications described, steroid concentrations of about 5 ng per spot could be accurately determined. The increased sensitivity allows measurement of the plasma levels of cortisol in small laboratory animals such as guinea pigs.     

Determination of cortisol in human plasma by thin-layer chromatography and fluorescence derivatization with isonicotinic acid hydrazide

The present work describes a specific and rapid determination of cortisol in human plasma. The method includes liquid-liquid extraction of plasma samples, thin-layer chromatography (TLC) of ethanolic extracts on aluminium foil-backed silica gel 60 TLC plates, derivatization of cortisol with isonicotinic acid hydrazide, and densitometric measurement of the fluorescence intensity of cortisol hydrazone. The fluorescence was linearly related to cortisol amounts; the correlation coefficients of standard curve plots were r>0.99. The coefficient of variation ranged between 2.8-7.9% (20 ng, within-assay/between assay variation) and 1.6-6.8% (80 ng, within-assay/between assay variation). The recovery of cortisol from plasma spiked with 21-deoxycortisol was 85%+/-4%. Cortisol concentration in the plasma was 66+/-32 ng/mL (mean+/-standard deviation, n=24). The advantage of this method is its simplicity to separate cortisol from other steroids by TLC, its specificity (formation of cortisol hydrazone), and the rapid quantitation of cortisol by densitometry.     

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.     

Determination of cortisol in guinea pig urine by high performance thin-layer chromatography, high performance liquid chromatography, and thin-layer chromatography/radioimmunoassay

Summary In order to collect urinary samples from unrestrained guinea pigs, animals were kept in their familiar home cages with wood shavings for bedding. Cortisol was removed from shavings by a simple washing step, and an attempt was made to measure its concentrations by high performance thin-layer chromatography (HPTLC), high performance liquid chromatography (HPLC), or thin layer chromatography/radioimmunoassay (TLC-RIA). After intramuscular administration of 25 mg cortisol, cortisol excretion increased from about 20–30 μg/day to 400–500 μg/day (HPTLC: 531 μg/day, HPLC: 493 μg/day; TLC-RIA: 394 μg/day). Similarly, the treatment of the animals with 20 IU ACTH resulted in an augmented cortisol excretion, with mean values of 294 μg/day (HPTLC), 256 μg/day (HPLC) and 143 μg/day (TLC-RIA), respectively. The present study shows, for the first time, that cortisol excretion in unrestrained laboratory animals can be determined. Whilst the cortisol values measured by HPTLC and HPLC agree, the amounts measured by TLC-RIA were significantly lower. These differences are probably due to the presence of substances in urine or shavings which interfere with the radioimmunological determination. Hence, cortisol should be determined either by HPTLC or HPLC. Beside having a desirable specificity, these methods are more suited than TLC/RIA for steroid analysis since they confer the possibility of measuring additional steroids (e.g. precursors and/or metabolites of cortisol) in a single urine extract. This is especially the case for the HPTLC method since substances can be transformed into fluorescent derivatives.     

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.     

Thin Layer Chromatographic Method for Rapid Identification and Quantification of Corticosteroid Sodium Phosphates in Pharmaceutical Preparations

Abstract

A rapid and inexpensive thin layer chromatography (TLC) procedure for the assay of dexamethasone sodium phosphate (DSP) and betamethasone sodium phosphate (BSP) persent in pharmaceutical preparations is described. Free steroids liberated after alkaline phosphatase reaction is isolated by TLC on silica gel layer and estimated after elution with ethanol. Assay of unesterified steroids and identification of the preservatives like methyl or propyl parabenzoates, phenol, benzyl alcohol etc. may also be carried out by this method without any additional cost. Possibility of using this method for other corticosteroid sodium phosphates (CSP) are discussed.     

Analysis of urinary estrogens,their oxidized metabolites,and other endogenous steroids by benchtop orbitrap LCMS versus traditional quadrupole GCMS

Estrogens and other endogenous steroids are known risk markers for cancer. Gas chromatography (GC) with mass spectrometry (MS) has traditionally predominated the analysis of estrogens and other endogenous steroids, but liquid chromatography (LC) MS is increasingly favored. Direct comparisons of the two technologies have hitherto not been performed. Steroids were analyzed from 232 urine samples of 78 premenopausal women in a blinded fashion by benchtop orbitrap LCMS and single quadrupole GCMS. Sixteen steroidal estrogens including oxidized metabolites could be analyzed by LCMS. LCMS–GCMS Spearman rank correlations of the major estrogens E₁, E₂, E₃, 16α-OHE₁, and 2-OHE₁ were very high (r = 0.72–0.91), and absolute concentrations also agreed (<5% difference for E₁, E₂, E₃, 16α-OHE₁). LCMS allowed reinterrogation of the acquired data due to orbitrap technology, which permitted post-analysis quantitation of progesterone, cortisol, and cortisone (LCMS–GCMS Spearman rank correlations = 0.80–0.84; absolute difference, <7%; n = 137). GCMS allows the measurement of a wide range of steroids including non-polar analytes that escape the presented LCMS assay. In contrast, orbitrap-based LCMS can detect more estrogens, is faster, less costly, allows post-data acquisition reinterrogation of certain analytes that had not been targeted a priori, and requires much less urine.     

Matrix‐free thin‐layer chromatography/laser desorption ionization mass spectrometry for facile separation and identification of medicinal alkaloids

Quaternary protoberberine alkaloids belong to a pharmaceutically important class of isoquinoline alkaloids associated with bactericidal, fungicidal, insecticidal and antiviral activities. As traditional medicine gains wider acceptance, quick and robust analytical methods for the screening and analysis of plants containing these compounds attract considerable interest. Thin‐layer chromatography (TLC) combined with matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) is a powerful technique but suffers from dilution of the TLC bands resulting in decreased sensitivity and masking of signals in the low‐mass region both due to addition of matrix. This study integrates for the first time conventional silica gel TLC and laser desorption ionization mass spectrometry (LDI‐MS) thus eliminating the need for any external matrix. Successful separation of berberine (R_f = 0.56) and palmatine (R_f = 0.46) from Berberis barandana including their identification by MS are demonstrated. Furthermore, a robust electrospray ionization (ESI)‐MS method utilizing residual sample from TLC for quantification of berberine applying selected reaction monitoring and standard addition method is presented. The amount of berberine in the plant root prepared for the study was determined to be 0.70% (w/w). Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of steroids by liquid chromatography/mass spectrometry

On-line atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) liquid chromatography/mass spectrometry (LC/MS) were evaluated for the analysis of a variety of steroids. Steroids were classified into three major groups based on the spectra and the sensitivities observed: (I) those containing a 3-one, 4-ene functional group, (II) those containing at least one ketone group without conjugation, and (III) those containing hydroxy group(s) only. In the APCI mode, the best sensitivity and the lowest detection limit for all three groups were obtained by using a mobile phase consisting of methanol and 1%–2% acetic acid in water. The APCI spectra were characterized by MH⁺, MH⁺-H₂O, MH⁺-2H₂O, etc., with the degree of H₂O loss being compound dependent: group I steroids produced stable MH⁺ and group III steroids showed extensive water loss. In the electrospray mode the best sensitivity and the lowest detection limit for the first two groups were obtained when pure methanol and water were used as the mobile phase. This condition produced abundant stable MNa⁺ due to ubiquitous sodium. Detection limits in the 5–15 pg range can be easily achieved using ESI LC/MS. Addition of ammonium acetate or use of acetonitrile in the mobile phase, common in the LC/MS analysis of steroids, decreased the sensitivity for the group I and II steroids and thus should be avoided. For group III steroids, the detection limit can be improved by the addition of acetic acid to the mobile phase.     

Use of biotin-streptavidin system for developing a viable, sensitive and specific antigen heterologous assay for hapten

The present study demonstrates improvement in sensitivity and specificity of hapten assay by using antigen heterology in conjunction with low molecular weight biotin label as compared to high molecular weight horseradish peroxidase (HRP) label. For generation of antiserum, cortisol-3-O-carboxylmethyl-oxime-bovine serum albumin (F-3-CMO-BSA) was used as immunogen whereas, for the preparation of primary label, corticosterone-3-carboxymethyl oxime (B-3-CMO) was coupled with biotinylcaproylhydrazide and HRP by employing N-hydroxysuccinimide mediated carbodiimide reaction. The data of the present study revealed that the antigen heterologous assay which employed high molecular weight HRP label showed 100% cross-reaction with corticosterone. On the contrary, when HRP was replaced with low molecular weight biotin label, less than 0.1% cross-reaction was observed with all analogous C₁₈, C₁₉, C₂₁ and C₂₇ steroids including corticosterone (0.2%). Moreover, the sensitivity of the later assay was 0.09 μg/dL, which is appreciable as compared to previously reported enzyme based assays. The recovery of the exogenously spiked serum pools lies in the range of 90.3-104.2%. The intra-assay and inter-assay coefficient of variation (CVs) ranged from 3.3% to 7.8% and 2.3% to 7.7%, respectively. The serum cortisol values obtained by this method correlated well with those obtained by radioimmunoassay; r = 0.9 (n = 50). The use of much stable biotin label in place of HRP has made the antigen heterologous enzyme linked immunosorbent assay (ELISA) of cortisol assay highly specific and sensitive.     

Comparison of the use of anionic and cationic surfactants for the separation of steroids based on MEKC and sweeping-MEKC modes

In attempts to improve the selectivity and sensitivity of steroid separation and to determine their migration order, a comparison of the use of anionic and cationic surfactants based on the MEKC and sweeping-MEKC modes was made. A mixture of six steroids (progesterone, 17-hydroxy progesterone, 11-deoxycortisol, corticosterone, cortisone, and cortisol) could be separated and detected by means of the CE/UV-absorption method. The order of migration time for these steroids was compared under various conditions, including acidic/alkaline buffers, anionic/cationic surfactants, and positive/negative applied voltage, causing the direction of the EOF and the migration of micelles to change. The major rules for generally predicting the migration order of steroids are summarized. The detection limits were significantly improved when the sweeping-MEKC mode was applied.     

Noninvasive determination of human cortisol and dehydroepiandrosterone sulfate using liquid chromatography-tandem mass spectrometry

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurements of steroids in human saliva has garnered increased interest in the area of clinical psychoneuroendocrinological research. However, performance characteristics of LC-MS/MS methods for the analysis of steroids in saliva are limited. Human saliva samples were collected via passive drool. Cortisol and dehydroepiandrosterone sulfate (DHEA-S) in the samples were extracted together, resolved on a C18-A column, and analyzed using tandem mass spectrometry. The LC-MS/MS method had limits of quantitation of 0.03 and 0.06 ng/mL for DHEA-S and cortisol, respectively. Method evaluations showed coefficient variation (%CV) of inter-assay ranging 4.6–17.9% for DHEA-S and cortisol, recoveries of 102.4–109.5% for DHEA-S and 94.6–98.3% for cortisol, and assay linearity with R² = 0.9964 for DHEA-S (1.0–25.0 ng/mL) and R² = 0.997 (1.0–25.0 ng/mL) for cortisol. No cross contamination among samples was observed. Human saliva showed 20% and 18% ion enhancement effect for DHEA-S and cortisol assay, respectively. No interference by ten common steroids was detected. Regression analysis of method comparisons with laboratory-developed test (LDT) method revealed R² = 0.9688 (LC-MS/MS = 0.9665 LDT-LC-MS/MS − 0.7355) for cortisol, and R² = 0.9039 (LC-MS/MS = 1.0173 LDT-LC-MS/MS + 3.6797) for DHEA-S. Reference ranges for young adults were determined to be 0.3–5.9 ng/mL for females and 0.1–5.6 ng/mL for males for salivary cortisol, and 0.6–7.4 ng/mL for females and 0.6–10.1 ng/mL for males for salivary DHEA-S. An LC-MS/MS method for quantifying cortisol and DHEA-S in human saliva was developed and validated for clinical and psychoneuroendocrinological research that require noninvasive means of measuring these hormones.

     

Quantitative Bestimmung von Sterigmatocystin in verschimmelten pflanzlichen Nahrungsmitteln

Zusammenfassung Ein Verfahren für die quantitative Bestimmung von Sterigmatocystin in pflanzlichen Nahrungsmitteln durch zweidimensionale DC-Trennung eines Extraktes und Fluorescenzintensitätsmessung der mit AlCl₃ behandelten DC-Platte wird beschrieben. Durch die hier vorgeschlagene Auftrageweise auf der DC-Platte ist ein rasches Identifizieren und Auswerten von Probe- und Eichflecken möglich. Als Extraktionsmittel hat sich Acetonitril-KCl-Lösung am besten bewährt. Versuche mit zugesetztem Toxin zeigten Verluste von weniger als 5 %.

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Quantitative determination of sterigmatocystin in mouldy vegetable foods

Summary The quantitative determination of sterigmatocystin is achieved by two-dimensional thin-layer chromatography (TLC) of extracts from vegetable foods and measurement of the fluorescence of the spots on AlCl₃-treated TLC plates. Samples and controls (references) are applied to the TLC plates in a way that permits their quick identification and assay. Acetonitrile-KCl solution has proved to be the most advantageous extracting agent. Losses of less than 5 % were observed in tests with added toxin.

     

Kinetic analysis of PI3K reactions with fluorescent PIP<Subscript>2</Subscript> derivatives

Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP₂ derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.     

Phosphatidylcholines and -ethanolamines can be easily mistaken in phospholipid mixtures: a negative ion MALDI-TOF MS study with 9-aminoacridine as matrix and egg yolk as selected example

Phospholipids (PL) are increasingly analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). As in the case of polar molecules, however, the careful selection of the matrix is crucial for optimum results. 9-Aminoacridine (9-AA) was recently suggested as the matrix of choice to analyze PL mixtures because of (a) the improved sensitivity and (b) the reduction of suppression effects compared to other matrices. However, the distinction of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the negative ion mode is obscured as PC is also detectable as –CH₃⁺ ion if 9-AA is used as matrix. This may result in the erroneous assignment of PC as a PE species. Using an organic extract from hen egg yolk as example it will be shown that the contribution of PC must be taken into consideration if the negative ion mass spectra are used to evaluate the fatty acyl compositions of PE mixtures. 9-AA can as well be used in hyphenated thin-layer chromatography (TLC)-MALDI-TOF MS where PC and PE are chromatographically well separated for unequivocal assignments.      

Advantages and limitations of HPLC in environmental analysis

Summary The application of HPLC to environmental analysis is often hindered by difficulty not experienced in other areas of analysis. Usually the components being determined are at parts per million levels or less and are usually in sample matrices that can yield many interferences. In order to develop successful methodology the prime requirements for an HPLC system are column efficiency and the sensitivity and selectivity of the detection system. In this presentation, examples are given to illustrate how HPLC can be used to advantage by comparing it to gas chromatographic (GC) methods and even TLC methods. For many classes of compounds, such as halogenated hydrocarbons (pesticides, PCB’s, dioxins), the GC methods may be preferred. However, for polycyclic aromatic hydrocarbons (PAH) HPLC with fluorescence detection has proven to be excellent for trace environmental analysis. Comparisons of HPLC with TLC for aflatoxins and with bioassays for paralytic shellfish toxins are made. Novel combinations such as headspace-HPLC analyses for SO₂ and HPLC-AA for organometallic compounds are discussed.     

A Novel Liquid-Gel Chromatographic Method for Extraction of Unconjugated Steroids from Aqueous Solutions

Abstract

Unconjugated steroids of low and medium polarity can be essentially quantitatively extracted from aqueous solutions by rapid filtration through a small column of Lipidex 1000. Forty ml of urine can be extracted by a 4 ml column bed at a flow rate of 5 ml/ min. Less polar steroids (progesterone, testosterone) can be extracted directly, whereas 5% pentylamine has to be added to the aqueous solution to give a quantitative extraction of cortisol. The same method can be used for extraction of steroids in plasma. It is suggested that this method may be generally applicable to the extraction from biological fluids of compounds with a polarity similar to or lower than that of steroids.     

SPE–TLC Profiling of Impurities in 1-(3,4-Methylenedioxyphenyl)-2-nitropropene, an Intermediate in 3,4-Methylenedioxy- methamphetamine (MDMA) Synthesis

Impurity profiling of 1–(3,4-methylenedioxyphenyl)-2-nitropropene, an intermediate in the synthesis of 3,4-methylenedioxymethamphetamine (MDMA), has been studied by SPE combined with TLC. Extraction of impurities was performed on C₁₈ columns. TLC separation was performed on 0.2 mm silica gel 60 plates, with fluorescent indicator F₂₅₄, in a horizontal developing chamber. To improve the quality of the profile (increase the TLC sensitivity) SPE extracts were concentrated by evaporation in a stream of nitrogen. The usefulness of methanol, ethanol, ethyl acetate, chloroform, acetonitrile, and their mixtures as mobile phases was tested by use of Gibbss triangle. Spots of the separated impurities were observed under UV light (_exc=254 and 366 nm). The proposed characteristics of profile quality (optimization criteria) are based on matrix presentation of the TLC pattern. They take into account, simultaneously, the number of spots revealed, differences between R_F values, and the intensity of fluorescence.     

LC–MS/MS Method for the Simultaneous Determination of Free Urinary Steroids

Cortisol homeostasis is implicated in hypertension and metabolic syndrome. Two enzymes modulate cortisol availability; 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) preferentially converts inactive cortisone to cortisol, whereas 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) converts cortisol to cortisone. In contrast, 5α and 5β reductases inactivate cortisol by conversion to its tetrahydrometabolites: tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone. A subtle local increase in cortisol can be detected by measuring 24-h urine metabolites, LC–MS/MS being the reference method. The 11β-HSD2 activity is assessed based on the cortisol/cortisone ratio, and the 11β-HSD1 activity on the (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ratio. To better understand hypertension and/or metabolic syndrome pathogenesis a method for simultaneous determination of cortisol, cortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone was developed and validated in an LC coupled with the new detector AB Sciex QTrap^® 4500 tandem mass spectrometer. The steroids were extracted from 1 mL urine, using cortisol-D4 as internal standard. The quantification range was 0.1–120 ng/mL for cortisol and cortisone, and 1–120 ng/mL for tetrahydrometabolites, with >89 % recovery for all analytes. The coefficient of variation and accuracy was <10 %, and 85–105 %, respectively. Our LC–MS/MS method is accurate and reproducible in accordance with Food and Drug Administration guidelines, showing good sensitivity and recovery. This method allows the assessment of 11β-HSD2 and 11β-HSD1 activities in a single analytical run providing an innovative tool to explain etiology of misclassified essential hypertension and/or metabolic syndrome.     

Thin-layer chromatography (TLC) as pilot technique for HPLC. Utilization of retention database (R F) vs. eluent composition of pesticides

Summary TLC and HPLC are frequently unjustly regarded as competitive methods. Using the advantages of both methods, a combination of TLC and HPLC leads to a considerable saving of time and expense in analysis. Experimental data are presented aslog k (HPLC) vs.R _M (TLC) correlations; high correlation coefficients indicate that TLC using octadecyl silica, wettable with water (RP-18W) can be applied as a pilot technique for HPLC (RP-18).