Studies on steroids. CCXVI. Separation of bile acid 3-glucuronides by high-performance liquid chromatography

The separation of 3-glucuronides of cholate, chenodeoxycholate, deoxycholate, ursodeoxycholate and lithocholate, and their glyco- and tauro-conjugates, has been carried out by high-performance liquid chromatography on a reversed-phase column. The chromatographic behaviour of bile acid 3-glucuronides was dependent on the type of conjugation. An effect of the pH of the mobile phase on the capacity ratio was observed at higher pH for chenodeoxycholate 3-glucuronide, probably owing to steric interaction of the 7 alpha-hydroxy group with the carboxy group in the glucuronyl moiety. Conversion of the alpha-hydroxy function on the steroid nucleus into an oxo group resulted in a 50% decrease in the capacity ratio. Bile acid 3-glucuronides were efficiently separated on Shodex ODS Pak F-411 using three kinds of ammonium phosphate buffer-acetonitrile systems.     

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.     

High-performance liquid chromatographic analysis of steroid hormones

In the present work, a practical, rapid, reliable and isocratic reversed-phase high-performance liquid chromatographic (HPLC) method is described for the qualitative and quantitative analysis of estriol, estradiol-17 beta, estrone, testosterone, and progesterone. Chromatographic separation is complete in 16 min using a mobile phase of 50% acetonitrile (v/v) in water. The order of elution is estriol, testosterone, estradiol-17 beta, estrone, and progesterone; retention times are 2.5, 5.5, 5.6, 6.9, 16.3 min, respectively. Absorbance maxima of individual steroids is the limiting factor in quantitative determination. The recommended wavelengths for UV monitoring are E3 214, E2 280, T 254, E1 214, and P4 254 nm.     

On-line high-performance liquid chromatographic diode array spectrophotometric analysis of steroidal hormones in illegal preparations

An on-line high-performance liquid chromatographic diode array spectroscopic analytical method for the identification of more than 60 different steroidal compounds is described. For the chromatographic separation, a gradient elution that could distinguish the esterified and non-esterified steroids in the same run on a reversed-phase C18 column, using methanol as modifier, was developed. For both types of compound an internal standard was chosen to establish reproducible relative retention times that could be used as one element of the identification; the second element is the UV spectrum, which is recorded on-line during the separation. The combination of chromatographic and UV spectroscopic recordings selects only a few probable steroids, which could be the unknown. This approach has been applied to forensic analysis of illicit preparations used in cattle-breeding, some examples of which are shown. For those steroids that are very difficult to distinguish using this procedure, because of their chromatographic and spectroscopic similarity on this system, other solvent mixtures are used in place of methanol as modifier, namely acetonitrile or tetrahydrofuran, or both, with the same solvent strength, as proposed by Snyder. In this way totally different elution patterns and separations are obtained, providing complementary information for identification, as shown by the systematic analysis on two other isoeluotropic solvent systems.     

Reversed phase high performance liquid chromatographic separation of hydroxy steroidal unsaturated esters and their hemisuccinates.

The high performance liquid chromatographic (HPLC) separation and identification of 12 isomeric and/or highly chemically related steroids with an unsaturated ester moiety at position 17 beta has been achieved. The main stereochemical features of the steroid skeleton cover 3 alpha/beta, 5 alpha/beta or delta, and 20 E/Z, bearing the alcohol or hemisuccinate group at the 3 position. The isocratic reversed phase C18 HPLC separation employed ethanol, methanol and its mixtures with water or 0.01 M phosphoric acid as the mobile phase. The best separation of the respective alcohols from their hemisuccinates has been achieved with 20% of aqueous phase content. The best separation among isomeric or related steroids has been achieved with methanol:water 8:2 and 85:15 and similar systems containing phosphoric acid.     

The chromatographic spectra of the monomeric components of ribonucleic acids

Conclusions The chromatographic mobilities of the monomeric components of ribonucleic acids-adenine, guanine, uracil, and cytosine- and their nucleosides and nucleotides have been studied in two sets of solvent systems. The chromatographic spectra permitting the optimum choice of solvent systems in the solution of various problems on the separation and identification of the components of nucleic acids have been compared.Khimiya Prirodnykh Soedinenii, Vol. 5, No. 4, pp. 291–296, 1969     

Chromatographic identification of pesticides

The chromatographic properties of 51 common pesticides have been measured using seven different chromatographic systems involving gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC) with diode-array spectrophotometric detection and thin-layer chromatography (TLC) with different spray reagents. Correlation coefficients were calculated for combinations of all systems. The best combination of the chromatographic systems examined for the identification of an unknown compound is GLC on OV-17, HPLC on ODS-Hypersil with acetonitrile-water as eluent and TLC using an isooctane-ethyl acetate solvent system.     

Separation of thiazide diuretics and antihypertensive drugs by thin-layer chromatography.

A procedure is reported for the thin-layer chromatographic (TLC) separation and identification of thiazide diuretics and other antihypertensive drugs. Several new solvent systems and a variety of possible detection reagents were examined. Twenty thiazides used routinely in therapy were successfully separated and identified. Use of the TLC systems and combinations of the detecting methods described should be useful for the identification of these drugs in biological fluids and in various dosage forms.     

Experimental choices for the determination of carbonyl compounds in air

The analysis of carbonyls in ambient air has received a great deal of scientific attention with the advancement of analytical techniques and increased demand for the build-up of its data base. In this review article, we have attempted to provide some insight into the relative performance of different instrumental approaches available for the analysis of ambient carbonyls with a major emphasis on high performance liquid chromatographic and gas chromatographic methods. Reported in several international standard procedures, derivatization of carbonyls with 2,4-dinitrophenylhydrazine (2,4-DNPH) with either an impinger or cartridges is the most commonly used method of HPLC detection. In this respect, a number of alternative hydrazine reagents have also been discussed for use with HPLC. In contrast, GC methods based on the combined application of adsorptive enrichment on solid sorbents and thermal desorption are examined with regard to their suitability for carbonyl analysis in air. Particular emphasis has been directed towards the advantages and drawbacks of these different instrumental techniques for ambient carbonyls. Based on this comparative approach, we discuss the suitability for each method for carbonyl analysis.     

Column-switching reversed phase-hydrophilic interaction liquid chromatography/tandem mass spectrometry method for determination of free estrogens and their conjugates in river water

We report a column-switching liquid chromatography (LC) tandem mass spectrometry (MS/MS) method for highly sensitive determination of both free estrogens (estrone, estradiol, and estriol) and their conjugates (estrone-3-sulfate, estradiol-3-sulfate, estriol-3-sulfate, estrone-3-glucuronide, estradiol-3-glucuronide, estriol-16-glucuronide, and estriol-3-glucuronide) in river water. This technique combines reversed phase (RP) chromatographic separation of the dansyl chloride derivatized free estrogens and hydrophilic interaction liquid chromatographic (HILIC) separation of the estrogen conjugates with multiple reaction monitoring (MRM). Using this new method, sensitivity increases 100- to 1000-fold for free estrogens and 2- to 10-fold for estrogen conjugates over RPLC-MS/MS alone. Method detection limits (MDL) range from 0.038 to 6.9 ng L⁻¹ with accuracy of 68-105% and precision of 1.7-17%. We successfully used this method to analyze river water samples collected from the North Saskatchewan River at the same location and detected trace concentrations of estrone (0.042 ng L⁻¹) and estrone-3-sulfate (0.84 ng L⁻¹), demonstrating the application of this method for environmental analysis.     

Thin-layer chromatographic enantiomeric resolution via ligand exchange

A thin-layer chromatographic technique for the separation of proteinogenic and non-proteinogenic amino acids, dipeptides and alpha-hydroxy acids is described. Other examples are given from the field of alpha-methyl, N-alkyl and halogenated amino acids. The separation of the enantiomers is achieved, without derivatization, by means of ligand exchange on a reversed-phase silica gel as stationary phase, which is covered with a chiral selector (proline derivative). The resolution is so good that the respective enantiomers can be determined at trace levels (greater than or equal to 0.25%). The proposed method is simple, inexpensive and needs no sophisticated instruments.     

Comprehensive chromatographic separations in proteomics

In such a complicated field as proteomic analysis, scientists are more and more challenged in implementing separation systems capable to provide enhanced separation power, as well as sensitivity of detection for adequate identification and, to a lesser extent, quantification of the separated compounds. To address such issues, several combinations of different separation modes have been investigated in comprehensive liquid chromatographic platforms, in which the entire sample eluted from the first dimension is subjected to a secondary chromatographic separation. The different applications exploited for comprehensive LC analysis of intact or digested proteins are the focus of this review, in which advantages and disadvantages of the different columns combinations, interfaces, and operating modes are pointed out. The combination with mass spectrometry as part of the total system is stressed, and illustrated in more detail. Theoretical concerns and practical requirements will be briefly discussed, as well.     

Blue natural organic dyestuffs--from textile dyeing to mural painting. Separation and characterization of coloring matters present in elderberry, logwood and indigo

Natural dyestuffs used for painting or dyeing of textiles are complex mixtures of compounds of various chemical properties. Proper identification of the dye used by a painter and, even better, its origin is possible only when its compositional 'fingerprint' can be evaluated. For this reason gradient program for liquid chromatographic separation of 16 color compounds--components of natural blue dyes: elderberry, logwood and indigo--has been developed. Two detector systems were used simultaneously: UV-Vis spectrophotometry (at 280, 445, 520 and 600 nm) and ESI mass spectrometry (positive and negative SIM mode). It was found that fragmentation observed in ESI-MS is affected not only by ion source parameters, but also by chromatographic conditions, especially in case of the less stable substances: cyanidin glucosides, tannic acid, rutin and hematoxylin. Examination of characteristic dissociation pathways of the compounds under investigation after direct admission into ion source or after chromatographic separation allowed to select proper ions for SIM detection and to develop novel and efficient reversed phase high performance liquid chromatographic (RP-HPLC)-UV-Vis/ESI-MS method for the analysis of natural blue dyes. The procedure was successfully applied for identification of indigotin and carminic acid-main colorants extracted from a fiber taken from the blue-red 'Italian' tapestry (the collection of the National Museum in Warsaw, Poland).     

Impact of adsorption isotherm parameters on the performance of enantioseparation using simulated moving bed chromatography

Often there are several chromatographic systems, i.e., combinations of mobile and stationary phases, available to solve a certain separation problem. Essential differences of these chromatographic systems are the separation factors and the efficiencies. For preparative applications in addition also the column saturation capacities and solubility limits are of importance. The impact of all these parameters appears to be rather well understood for conventional overloaded elution chromatography using a single column. In the last years the continuous simulated moving bed (SMB) process was increasingly used as a powerful alternative to batch elution since increased productivities and reduced solvent consumptions could be realised. However, the selection of suitable chromatographic systems is more sophisticated for this process. In this paper five different chromatographic systems capable of separating the enantiomers of mandelic acid are compared based on the achievable productivities using SMB chromatography. For these five systems the adsorption isotherms have been determined experimentally. Subsequently, an analysis of the SMB process was performed numerically using a well-established model.     

Analysis of steroid hormones in effluents of wastewater treatment plants by liquid chromatography-tandem mass spectrometry

This paper presents the development of an analytical procedure for the determination of two sexual steroid hormones: 17beta-estradiol and estrone, and the synthetic contraceptive estrogen, 17alpha-ethynylestradiol in effluents of wastewater treatment plants. Samples are extracted via solid-phase extraction using C18 cartridges. Extracts in ethyl acetate are then purified with a liquid-liquid separation with aqueous sodium chloride, then with a clean-up on a Florisil cartridge. Steroids are analyzed using an LC-MS-MS ion trap system. Internal quantification with the corresponding deuterated steroids leads to limits of quantification at 5 ng/l for estrone and 10 ng/l for estradiol and ethynylestradiol. In mineral spiked water, recoveries are 91% for 17beta-estradiol, 97% for estrone and 87% for 17alpha-ethynylestradiol and RSDs are 15% for 17beta-estradiol, 11% for estrone and 23% for 17alpha-ethynylestradiol.     

Application of molecular statistical calculations to the prediction of chromatographic separation of isomeric difluorobiphenyls

Molecular statistical calculations have been used for estimation of the possibility of chromatographic separation of difluorobiphenyl mixtures. GC-MS investigation of the mixture of difluorobiphenyls has been carried out. It is impossible to identify each isomer with only mass spectrometric data due to the similarity of their mass spectra. A chromato graphic column that makes adequate chromatographic separation possible has been selected based on the molecular statistical calculations. The identification of each isomer resulted from combination of the mass spectrometric investigation results and molecular statistical calculations.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 623–626, March, 1996.     

An automated food protein isolation approach on preparative scale by two‐dimensional liquid chromatography with active modulation interface

In this work, an automated 2D‐LC approach for protein isolation from egg samples on preparative scale is proposed. The method is based on the use of a C18 guard column installed in a switching valve to focus the proteins coming from the first dimension column, before their elution in the second column. For the first dimension separation, a size‐exclusion column, packed with 3 μm ultrapure silica particles was used. An RP column based on core‐shell technology was used for the second dimension separation. A standard mixture of BSA, β‐lactoglobulin, and glucose oxidase, chosen as a protein model system, was used to optimize the chromatographic separation conditions. The fully automated workflow allowed to isolate, in a single‐chromatographic analysis, a protein amount of 50 μg for each peak fraction, with a total time of 15 min for the first separation and additional 30 min of the second separation for each trapped protein. The final aim was the development of proper analytical tools for protein isolation from foodstuffs to be used for the molecular identification by MS, as well as for biotherapeutic uses, allergy testing, and large‐scale investigations in biological systems.     

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.     

Rapid purification yielding highly active 17 beta-hydroxysteroid dehydrogenase: application of hydrophobic interaction and affinity fast protein liquid chromatography.

Homogeneous human placental 17 beta-hydroxysteroid dehydrogenase was obtained by a procedure consisting of two fast protein liquid chromatographic (FPLC) steps using Phenyl-Sepharose hydrophobic interaction and Blue-Sepharose affinity columns. In the first chromatography, the enzyme eluted only when an additional decrease in ionic strength was inserted after the ammonium sulphate concentration had reached zero, thus enhancing the separation. In the affinity chromatography, separation of contaminating proteins occurred at different stages of loading and washing. The specific elution of the enzyme by the co-factor NADP+ is very efficient in obtaining a homogeneous preparation in high yield. The rapidity of FPLC was further increased by a maximum simplification of the intermediate steps, and the whole procedure lasted only two days. This preparation has a yield of more than 50% and a high specific activity, catalysing the formation of 7.9 mumol of estrone from estradiol per minute at pH 9.2 and 23 degrees C. It has an apparent molecular mass of 35,000. This provides an efficient candidate for the purification of other membrane-associated proteins.     

Fast gas chromatography-negative-ion chemical ionization mass spectrometry with microscale volume sample preparation for the determination of benzodiazepines and alpha-hydroxy metabolites, zaleplon and zopiclone in whole blood

Fast gas chromatography/negative-ion chemical ionization mass spectrometric (GC/NICI-MS) assay combined with rapid and nonlaborious sample preparation is presented for the simultaneous determination of benzodiazepines and alpha-hydroxy metabolites, zaleplon and zopiclone in whole blood. The compounds were extracted from 100 microl of whole blood by simultaneous multitube, microscale liquid-liquid extraction (LLE) and derivatized by N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), without the need for the time-consuming concentration stage. In the analytical separation, various parameters of fast GC/NICI-MS were applied, e.g. the use of hydrogen as a GC carrier gas, a high carrier gas velocity, a small film thickness of the analytical column, fast MS data acquisition, fast temperature ramping, and high initial and final temperatures of GC column. Sensitive identification, screening and quantitation of 18 compounds of interest were achieved in chromatographic separation in only 4.40 min. Accurate and reproducible results were obtained by using five different and carefully selected deuterated analogues on the basis of the chemical properties of the target analytes. Nevertheless, for alpha-OH-midazolam, and for bromazepam and flunitrazepam at low concentrations, the results can be considered only semiquantitative on the basis of the validation data. The extraction efficiencies ranged from 74.3 to 105.7% and the limits of quantitation (LOQ) from 1 to 100 ng ml(-1). Rapid sample preparation and fast chromatographic separation allowed cost-efficient, reliable and high sample-throughput analyses with a low amount of manual work. The method was fully validated and accredited according to EN ISO/IEC 17025 standards and is applicable for sensitive, reliable and quantitative determination of benzodiazepines, zaleplon and zopiclone, e.g. in clinical and forensic toxicology.