High Performance Liquid Chromatography (HPLC) with Fluorescence Detection for Quantification of Steroids in Clinical,Pharmaceutical, and Environmental Samples: A Review

Steroids are compounds widely available in nature and synthesized for therapeutic and medical purposes. Although several analytical techniques are available for the quantification of steroids, their analysis is challenging due to their low levels and complex matrices of the samples. The efficiency and quick separation of the HPLC combined with the sensitivity, selectivity, simplicity, and cost-efficiency of fluorescence, make HPLC coupled to fluorescence detection (HPLC-FLD) an ideal tool for routine measurement and detection of steroids. In this review, we covered HPLC-FLD methods reported in the literature for the steroids quantification in clinical, pharmaceutical, and environmental applications, focusing on the various approaches of fluorescent derivatization. The aspects related to analytical methodology including sample preparation, derivatization reagents, and chromatographic conditions will be discussed.     

Selection of high-performance liquid chromatographic methods in pharmaceutical analysis. III. Method validation

The most important steps in the validation of high-performance liquid chromatographic (HPLC) methods are discussed. The establishment of system suitability data and the assessment of peak purity are demonstrated on the example of bisquaternary amino steroids. For the recognition of incomplete resolution of adjacent peak pairs, the absorbance-ratio method in which the ratio of absorbances at two preselected wavelengths are plotted as a function of time in combination with the separation of sample components subjected to various chemical and physico-chemical treatments (stress conditions) is applied. The separation power and performance of the HPLC systems are characterized by the system resolution (SR) and system selectivity (SS). The special demands of stability-indicating methods are summarized.     

Gas chromatography and high-performance liquid chromatography of natural steroids.

This review article underlines the importance of gas chromatography (GC), high-performance liquid chromatography (HPLC) and their hyphenated techniques using mass spectrometry (MS) for the determination of natural steroids, especially in human biological fluids. Steroids are divided into eight categories based on their structures and functions, and recent references using the above methodologies for the analysis of these steroids are cited. GC and GC-MS are commonly used for the determination of volatile steroids. Although HPLC is a widely used analytical method for the determination of steroids including the conjugated type in biological fluids, LC-MS is considered to be the most promising one for this purpose because of its sensitivity, specificity and versatility.     

Simultaneous determination of serum cortisol and cortisone by reversed-phase liquid chromatography with ultraviolet detection.

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cortisol and cortisone in a single extract of 1 ml of serum is described. The method employs meprednisone as the internal standard. The steroids were analysed isocratically by reversed-phase HPLC with an octadecylsilane-bonded (ODS) column using ultraviolet detection. The matrix effect was reduced by lowering the sample pH by adding glacial acetic acid to the sera. The samples were then filtered through regenerated cellulose membranes at 4 degrees C and extracted with diethyl ether. The dried eluates were redissolved in the mobile phase and injected into the column. The detection limit of the assay for both steroids was 500 ng/l. Cortisol was determined in twenty serum samples by both HPLC and radioimmunoassay (RIA). The results were similar. Interference by other steroids and certain steroid analogue drugs was also studied. The HPLC method yielded no cross-reactivity between the different steroids as may occur with the RIA technique. The HPLC method was technically easy to perform and it allowed us to quantify both cortisol and cortisone in a single serum extract with high specificity.     

Derivatization,Separation and Determination of Steroids and Triterpenoids by Reverse-Phase HPLC and UV Detection

The steroids were derivatised with aroyl chloride to yield the corresponding benzoates. Among them, their 4-methoxybenzoates were obtained in greatest yield and were analyzed by HPLC with the smallest detection limit (10 ng) by means of a UV detector (254 nm). This method of derivatization can be applied for analysis of steroids and triterpenoids from crude products with the HPLC technique.     

Characterization of inclusion complexes of betamethasone-related steroids with cyclodextrins using high-performance liquid chromatography

HPLC was used to study the inclusion complexes formed between various beta- and gamma-cyclodextrins and a series of corticosteroids related to betamethasone. Apparent association constants were measured in acetonitrile-water for a set of 13 steroids. An increase in the stability of the steroid-cyclodextrin complex is observed at lower concentrations of acetonitrile. The effects of the nature of the halide at the 9-position, the location of a double bond within the C-ring, substitution at the 9- and 11-positions, and modification of the D-ring of the steroid backbone were studied. The 11- and 17-positions were found to be critically involved in the inclusion process. Larger apparent association constants were obtained with gamma-cyclodextrin (gamma-CD) than with beta-cyclodextrin (beta-CD) due to the increased diameter of the gamma-CD cavity. Van't Hoff plots were constructed to examine the thermodynamic properties of the inclusion process. Plots constructed using retention factors were found to be nonlinear when gamma-CD was present in the mobile phase. This is due to an increase in the strength of the inclusion complex as temperature decreases. Plots constructed using apparent association constants were linear, indicating that the mechanism of inclusion does not change over the range of temperatures studied (10 to 80 degrees C). Enthalpy-entropy compensation was observed for 11 of the 13 steroids studied. The usefulness of cyclodextrins to achieve the separation of steroids in HPLC is discussed and a practical application for the analysis of a steroid and three potential impurities is described.     

Separation of oestrogen conjugates in urine and synthetic mixtures by high-performance liquid chromatographic methods

The analytical and clinical advantages that would be expected to follow the adoption by clinical laboratories of a routine HPLC method for the partial oestriol conjugate profiling of human pregnancy urine are outlined in the Introduction. In order to ascertain if a candidate method for this assay has yet been devised, a complete survey of the published HPLC separations of oestrogen conjugate mixtures is presented, in tabular form, and discussed. From this survey it is concluded that a number of good separations of these steroids from synthetic mixtures have already been published. The third and final section of the paper contains the results of a detailed examination of those papers in which separation of oestriol conjugates present in pregnancy urine specimens have been reported. The paper is concluded with the recommendation that the method of Dixon, Lukha and Scott should be further investigated as a candidate method for adoption by clinical laboratories for the purpose of oestriol conjugate profiling.     

Fluorimetric detection of serum corticosterone using high-performance liquid chromatography.

A simple, sensitive, specific and reproducible method for the determination of corticosterone concentrations in rat serum using high-performance liquid chromatography (HPLC) with fluorimetric detection is described. Corticosterone is detectable down to 0.1 ng injected onto the HPLC column. Cortisol is used as an internal standard. Ethyl acetate was used for both initial serum corticosteroid extraction and the subsequent fluorophore extraction after sulfuric acid hydrolysis; thus sulfuric acid does not enter the HPLC system. The resultant fluorophores for both corticosterone and cortisol are stable for at least two weeks at ambient temperature not requiring storage at -20 degrees C. The procedure is highly suitable for use with HPLC systems utilising automatic sample injectors. The method is specific for corticosterone; dexamethasone, cortisone and gonadal steroids are not detectable and do not interfere in this assay.     

Reversed-phase high-performance liquid chromatography separation of adrenal steroids prior to radioimmunoassay: application in congenital adrenal hyperplasia

21-Hydroxylase deficiency (21-OHD) is the most common form of congenital adrenal hyperplasia (CAH), followed by 11beta-hydroxylase deficiency (11beta-OHD). Diagnostic serum markers for these conditions are 17-hydroxyprogesterone (17-OHP) and 11-desoxycortisol (S), respectively. In 21-OHD, the large amounts of 17-OHP are further 11beta-hydroxylated to form 21-deoxycortisol (21-DF), making it also an excellent marker of this disease. These steroids can be measured in blood by radioimmunoassay (RIA). In this paper, we report the use of high-performance liquid chromatography (HPLC) for steroid purification, prior to RIA determinations of 21-DF, S, 17-OHP, and testosterone (T) in ether-extracted serum. The chromatographic separation is developed in a BDS-Hypersil column using water-methanol (53:47, v/v) as the mobile phase. The method is applied to 35 patients with the classic form of 21-OHD (18 females, 17 males, 5.1-14.2 years old) and 2 with 11beta-OHD (1 female, 1 male, 9.5 and 12.6 years old). Thirteen control children (5 females and 8 males, 5.2-15.2 years) are also studied. The results obtained for all measured steroids are compatible with those reported in the literature. The method is precise, and recovery is adequate. The HPLC technique proved to be of value for the purification of several steroids from single serum samples prior to RIA in patients with CAH.     

Isotope effects of steroids during analytical separations by paper- and high-pressure liquid chromatography

Conclusions During HPLC (reversed phase), tritiated steroids elute earlier than the unlabeled and the [4-¹⁴C]-labeled analogues. On formamide impregnated paper [4-¹⁴C]-labeled steroids move faster than the tritiated analogues. Thus, in both systems the ³H-labeled compounds behave as if they were more polar [1]. In the case of sixfold labeled estradiol the highly efficient resolution of the paperchromatographic and HPLC-systems (Fig. 1 A) may be applied to obtain carrier-free steroids with high specific activities. On the other hand the analytical application of these systems may lead to great systematic errors if the isotope effects of tritiated steroids are not taken into consideration.

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Isotopieeffekte von Steroiden bei der Papier- und Hochdruckflüssigkeits-Chromatographie

     

High-performance liquid chromatography of seized drugs at elevated pressure with 1.7 microm hybrid C18 stationary phase columns

High-performance liquid chromatography (HPLC) separation of drugs at elevated pressure with 1.7 microm hybrid C18 stationary phase columns was investigated. This technique, which uses instrumentation engineered to handle the narrow peaks and high back pressures generated by 1.7 microm particle columns, provided significantly better resolution and/or faster analysis than conventional HPLC and capillary electrophoresis (CE). The use of 2mm internal diameter (i.d.) columns of 3-10 cm length has been evaluated for the separation of basic and neutral drugs, drug profiling, and general screening (including acidic drugs). For these applications, compared to conventional HPLC and CE, it provided up to 12x and 3x faster analyses, respectively. Precision was excellent for both isocratic and gradient analyses. For retention time and peak area, RSDs of < or =0.1% were obtainable. Fifteen anabolic steroids and esters were well separated in a 2.5 min gradient. For drug profiling, compared to HPLC and CE, approximately twice as many peaks were resolved. HPLC at elevated pressure is also well suited as a general screening technique. Twenty-four solutes of varying drug classes including narcotic analgesics, stimulants, depressants, hallucinogens, and anabolic steroids were fully separated in a 13.5 min gradient.     

Fluorimetrische Reaktionsdetektoren zur HPLC-Analyse von Steroiden

Zusammenfassung Acht bekannte Umsetzungsmöglichkeiten von Steroiden zu fluoreszierenden Derivaten wurden auf automatische, luftsegmentierte Reaktionssysteme übertragen. Die Reaktionen mit Isonicotinoylhydrazid sowie mit Periodat/Acetylaceton lassen sich auch in Verbindung mit HPLC-Trennungen in einem Reaktionsdetektor unter Verwendung von Teflonschläuchen einsetzen. Die Nachweisgrenzen für das Corticosteron liegen bei 7 bzw. 100 ng im Reaktionssystem. Die fluorimetrische Bestimmung mehrerer Steroide in Urinextrakten nach Trennung an einer chemisch-gebundenen Nitrophase mit dem Isonicotinoylhydrazid-Reaktionsdetektor wird gezeigt.

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Fluorimetric reaction detectors for the HPLC analysis of steroids

Summary Eight known reaction possibilities for steroids to fluorescent derivatives were transferred to automatic, air-segmented reaction systems. The reactions with isonicotinoylhydrazide and with periodate/acetylacetone under application of teflon tubes are suitable for application in a reaction detector in connection with HPLC separations. The determination limits for corticosterone are 7 resp. 100 ng in the reaction system. The fluorimetric determination of some steroids in urine extracts after separation on a chemical-bonded nitrophase by the isonicotinoylhydrazide reaction detector is demonstrated.

     

Chromatographic behaviour of selected steroids and their inclusion complexes with beta-cyclodextrin on octadecylsilica stationary phases with different carbon loads

Retention and separation studies of selected estrogens, progestogens and their inclusion complexes with beta-cyclodextrin were conducted using two C18 HPLC columns with different carbon loads. The difference in carbon load between investigated octadecylsilica packing materials was about 50%. The mobile phases were composed of a 30% v/v acetonitrile-water mixture without and with addition of beta-cyclodextrin at a concentration of 12 mM. The experimental data revealed that retention of the steroids was significantly reduced on the column with the lower carbon load. Moreover, it was found that this column offers better separation power and shorter analysis time at the temperatures studied. However, the calculated values of the retention factor ratios (k0(mMCD))/k(12mMCD)) of the steroids were similar for both columns investigated. This observation suggests that the stationary phase structure appears to have little effect on the formation of host-guest complexes if the complexation process is localised to the chromatographic mobile phase. From a practical point of view, when the mobile phase is modified with beta-cyclodextrin, the separation of the steroids is strongly influenced by temperature. The best chromatographic conditions were determined for the separation of multicomponent samples on the column with lower carbon load. A possible retention mechanism for components of interest in the presence of macrocyclic additives is discussed.     

Optimization of the mobile phase composition in gradient elution reversed-phase HPLC by stochastic prediction

Summary Quantitative analysis of more than ten compounds in a sample generally requires complex mobile phases to optimize the separation of the analytes by gradient elution reversed-phase HPLC. For this purpose, CHEOPS, a software package has been developed from the fully stochastic Computer Chromatogram Simulation Method. Calculation principles and optimization criteria are described. Experimental validation is presented with amino acids and steroids.     

High-pressure liquid chromatography of free and conjugated 17-ketosteroids (author's transl)]

High-pressure liquid chromatography (HPLC) should be used more frequently in biomedical research because heavy molecules can be analysed at ambient temperature without derivatisation to volatile compounds. In this paper the separation of four free 17-ketosteroids, their ester sulfates and glucuronides by reversed-phase HPLC on a Micropak CH column is described. In order to improve the separation several parameters have been studied. Retention data allow comparison of the hydrophilic behaviour of free and conjugated steroids.     

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.     

Normal-phase high-performance liquid chromatographic separations using ethoxynonafluorobutane as hexane alternative. I. Analytical and chiral applications.

A novel, environmentally friendly, fluorinated solvent--ethoxynonafluorobutane--has been used to replace n-hexane in normal-phase HPLC applications. Fast gradients of methanol in ethoxynonafluorobutane on a cyano column have been successfully applied to the separation of steroids, benzodiazepines, NSAIDs, tricyclic antidepressants, beta-adrenergic blocking agents and mixtures of purines and pyrimidines. Small amounts of triethylamine and trifluoroacetic acid added to such gradients significantly improved peak shape and column performance for basic and acidic solutes. Ethoxynonafluorobutane and its mixtures with methanol have also been demonstrated to have a unique selectivity in chiral HPLC applications.     

Heterocyclic steroids: Synthetic routes and biological characteristics of steroidal fused bicyclic pyrimidines

Natural steroids are characterized as a vital class of compounds, a type of secondary metabolites and components of cell membranes that widely accessible in plants or animals displayed significant pharmacological and varied biological properties. The present study aims to highlight the conveyed researches of synthetic routes adopted to obtain the various structures of steroidal fused bicyclic pyrimidines with substantial biological and pharmaceutical importance. The topic was discussed in light of the synthesis of fused [6 + 5] bicyclic systems, fused [6 + 6] bicyclic systems, binary heterocycles, and biological applications. In detail, the various synthetic strategies for the construction of steroids fused to bicyclic pyrazolopyrimidines, thiazolopyrimidines, triazolopyrimidines, pyridopyrimidines, pyranopyrimidines, and binary pyrimidines were discussed. Heterocyclic steroids of this class of compounds demonstrated potent anticancer, anti-proliferative, anti-neuro-inflammatory, anti-inflammatory, and anti-ulcer activities. It was perceived that the synthetic steroids of such bicyclic pyrimidine scaffolds are fused into the steroid basic skeleton is essential for the potent bioactivities.     

Synthesis and binding properties of a noncovalent molecularly imprinted testosterone-specific polymer

In this study, molecular imprinting was used to develop a method based on noncovalent interactions for synthesis of a testosterone-specific polymer. The effect of the different template–monomer ratios, the particle sizes of polymers, and chromatographic mobile phases on steroid–polymer interactions are discussed. The polymer obtained was found to interact specifically with testosterone, while other steroids under study were eluted close to the void volume in the HPLC experiments. Batch rebinding studies in acetonitrile were undertaken to quantitatively evaluate the affinity of the polymer for testosterone. During this experiment, the testosterone concentration was measured in two ways: spectrophotometrically and by HPLC on a column with testosterone-specific imprinted polymer synthesized by us. Both methods resulted in similar values of association constants and the number of binding sites. However, the second method has obvious advantages when the analyzed solution contains a mixture of optically dense compounds. The results obtained focus on the two-point binding nature of the imprinted polymer–testosterone interaction and the significant role of hydrogen bonds between the OH group of testosterone and carboxy group of methacrylic acid residues inside specific recognition sites of the imprinted polymer. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 1725–1732, 1998     

Determination of 13C/12C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary steroids is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography (HPLC) of underivatized and acetylated steroids, which allows the isolation of ten analytes (11beta-hydroxyandrosterone, 5alpha-androst-16-en-3beta-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5alpha-androstane-3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol and dehydroepiandrosterone) from a single urine specimen. These steroids are of particular importance to doping controls as they enable the sensitive and retrospective detection of steroid abuse by athletes.Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers repeatability and reproducibility. Specificity was further demonstrated by gas chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring (13)C/(12)C ratios of all implemented steroids, a reference population of n = 61 subjects was measured to enable the calculation of reference limits for all relevant steroidal Delta values.