Determination of sulfates and glucuronides of endogenic steroids in biofluids by high-performance liquid chromatography/orbitrap mass spectrometry

the possibility of selective determination of testosterone and epitestosterone glucuronides in urine by high-performance liquid chromatography/high-resolution mass spectrometry using solid phase microextraction on a meps cartridge was studied. the effect of the biological matrix on the spectra of conjugated steroids can be taken into account by using the spectra of conjugates recorded for urine samples after hydrolysis as reference spectra. the conditions of fragmentation in the ion source were optimized for separate analytes. this method was used for analyzing real samples with different testosterone/epitestosterone ratios. variations in conjugate contents and qualitative changes in the steroid profile of endogenic compounds were observed.     

Development and application of stir bar sorptive extraction with polyurethane foams for the determination of testosterone and methenolone in urine matrices

This work describes the development, validation, and application of a novel methodology for the determination of testosterone and methenolone in urine matrices by stir bar sorptive extraction using polyurethane foams [SBSE(PU)] followed by liquid desorption and high-performance liquid chromatography with diode array detection. The methodology was optimized in terms of extraction time, agitation speed, pH, ionic strength and organic modifier, as well as back-extraction solvent and desorption time. Under optimized experimental conditions, convenient accuracy were achieved with average recoveries of 49.7 8.6% for testosterone and 54.2 ± 4.7% for methenolone. Additionally, the methodology showed good precision (<9%), excellent linear dynamic ranges (>0.9963) and convenient detection limits (0.2-0.3 μg/L). When comparing the efficiency obtained by SBSE(PU) and with the conventional polydimethylsiloxane phase [SBSE(PDMS)], yields up to four-fold higher are attained for the former, under the same experimental conditions. The application of the proposed methodology for the analysis of testosterone and methenolone in urine matrices showed negligible matrix effects and good analytical performance.     

Behaviour of plasmalogens during high-performance liquid chromatography on a silica column with a mobile phase containing phosphoric acid

Silica high-performance liquid chromatographic separation of phospho- and sphingolipids of biological origin using a mobile phase containing phosphoric acid leads to gradual hydrolysis of plasmalogens during their passage through the column. The resulting 2-acyl lyso analogues give rise to peaks that tail in the direction of the parent intact plasmalogen. Tailing can be prevented by previous complete acid hydrolysis of plasmalogens. Direct high-performance liquid chromatographic profiling of phospholipids, their plasmalogens (as 2-acyl lyso analogues) and sphingolipids is probably the method of choice for the diagnosis of patients with deficient plasmalogen biosynthesis caused by peroxisomal abnormalities.     

Urinary testosterone measurement by gas chromatography after solid-phase extraction and high-performance liquid chromatography.

A method was developed for the rapid determination of testosterone in urine. The procedure consists of solid-phase extraction (SPE) followed by high-performance liquid chromatographic (HPLC) clean-up before gas chromatographic determination. Recovery was evaluated by adding [3H]testosterone (10(4) cpm) to urine samples; the mean recovery of radioactivity after SPE and HPLC was 82%. Precision was estimated by repeated measurement of testosterone in four different urine samples; the coefficient of variation was 7.9% (95% confidence limits 6.1-11.4%). Accuracy was evaluated by standard addition and dilution assays; a linear relationship was found between the expected and observed values (r2 = 0.982). The method is rapid, effective and suitable for routine analysis.     

Automated direct assay system for the measurement of sex steroid hormones in serum using high-performance liquid chromatography

An automated direct assay system using high-performance liquid chromatography was developed for the simultaneous measurement of estradiol, estrone, progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxyprogesterone, testosterone and androstenedione in biological fluids. A comparison between the values measured by this method and by radioimmunoassay revealed good correlation for estradiol (r = 0.938, p less than 0.001) and progesterone (r = 0.903, p less than 0.001). Estradiol and estrone could be analysed above the level of 250 pg/ml, and progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxyprogesterone, testosterone and androstenedione could be analysed above the level of 5.0-7.5 ng/ml. The method was applied to the clinical appraisal of placental function and maturation of ovarian follicles.     

Improved sample preparation for the testosterone hydroxylation assay using disposable extraction columns.

The preparation of samples for injection into a high-performance liquid chromatograph from assay mixtures for the determination of cytochrome P-450-dependent testosterone hydroxylation has been substantially facilitated. By replacing the multiple cumbersome extraction steps of the conventional method with a single column extraction the time for sample preparation was reduced from hours to minutes. The new procedure also yields better recoveries for most of the testosterone metabolites than the original protocol. The use of extraction columns for sample preparation allows the simultaneous treatment of a large number of samples or even the automation of the whole assay procedure. The modified procedure is a straightforward, easy-to-perform method that should greatly facilitate the implementation of the testosterone hydroxylation assay for sharply discriminating between many individual cytochrome P-450 species in routine enzyme diagnostics.     

Measurement of enzyme activities of cytochrome P-450 isoenzymes by high-performance liquid chromatographic analysis of products.

A method is described for the qualitative and quantitative determination of the isoenzymes of cytochrome P-450 from rat liver microsomes. Microsomes are incubated with the endogenous steroid 17 beta-testosterone, which results in the formation of a number of stereo-specific hydroxylation products of testosterone. The hydroxylated products were identified using standards or by comparison with data from the literature. The products can be analysed by reversed-phase gradient high-performance liquid chromatography. The assay has been optimised for pH, linearity and time of incubation. An evaluation of the assay was performed for different kinds of microsomes, microsomal dilution and specificity for particular cytochrome P-450 isoenzymes.     

Separation and purification of uridine diphosphate-glucuronosyltransferases by chromatofocusing on a high-performance liquid chromatograph

A rapid method for the separation and purification of uridine diphosphate-glucuronosyltransferases (GT) was developed with the use of chromatofocusing on a high-performance liquid chromatograph. GT isoenzymes solubilized from hepatic microsomes of Wistar rats were separated on a Mono P column, a pre-packed column for chromatofocusing. Using 4-nitrophenol, testosterone and androsterone as substrates, four fractions with different GT activities were separated in a pH gradient from 9.5 to 7.0. Two isoenzymes, testosterone GT and androsterone GT were purified to apparent homogeneity. They were eluted at pH 8.9 and 8.0 and had subunit molecular weight values of 50000 and 52000, respectively. Approximately 10 mg of solubilized microsomal proteins was applied and the elution was completed within 2 h. Addition of N-nitrodiethylamine, an in vitro activator of GT activity, enhanced the GT activity toward 4-nitrophenol in the three fractions. This chromatographic analysis confirmed the absence of androsterone GT isoenzyme in LA Wistar rats, a mutant strain in terms of androsterone glucuronidation.     

Development and validation of a method for measuring the glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography

We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C18 reversed-phase column using an isocratic solvent system of acidified methanol--potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas-liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile.     

The isolation, identification and determination of dehydrotumulosic acid in Poria cocos.

Poria cocos (Fuling), a popular Chinese medicinal (CM) herb of fungal origin, has been included in many combinations with other CM herbs for its traditionally claimed activities of inducing diuresis, excreting dampness, invigorating the spleen and tranquilizing the mind and its modern pharmacological use of modulating the immune system of the body. Dehydrotumulosic acid, one of the effective constituents of Fuling, was isolated from the chloroform-soluble material of ethanol extract of the fungus. After further purification by a high-performance liquid chromatographic method on a C18 column, the purified constituent was identified using modern analytical techniques, such as UV, 13C-NMR and EI-MS. A reversed-phase high-performance liquid chromatographic method has been developed for the determination of dehydrotumulosic acid in Poria cocos. The determination can be accomplished in less than 50 min using methanol-acetonitrile-2% glacial acetic acid as the mobile phase at a flow rate of 1.0 mL/min, with a UV detector setting at 242 nm and testosterone propionate used as an internal standard. This assay for dehydrotumulosic acid is simple, rapid and with good reproducibility.     

Detection of manipulation in doping control urine sample collection: a multidisciplinary approach to determine identical urine samples

Manipulation of urine sampling in sports drug testing is considered a violation of anti-doping rules and is consequently sanctioned by regulatory authorities. In 2003, three identical urine specimens were provided by three different athletes, and the identity of all urine samples was detected and substantiated using numerous analytical strategies including gas chromatography–mass spectrometry with steroid and metabolite profiling, gas chromatography–nitrogen/phosphorus detector analysis, high-performance liquid chromatography–UV fingerprinting, and DNA-STR (short tandem repeat) analysis. None of the respective athletes was the donor of the urine provided for doping analysis, which proved to be a urine sample collected from other unidentified individual(s). Samples were considered suspicious based on identical steroid profiles, one of the most important parameters for specimen individualization in sports drug testing. A database containing 14,224 urinary steroid profiles of athletes was screened for specific values of 4 characteristic parameters (ratios of testosterone/epitestosterone, androsterone/etiocholanolone, androsterone/testosterone, and 5α-androstane-3α,17β-diol/5β-androstane-3α,17β-diol) and only the three suspicious samples matched all criteria. Further metabolite profiling regarding indicated medications and high-performance liquid chromatography–UV fingerprinting substantiated the assumption of manipulation. DNA-STR analyses unequivocally confirmed that the 3 urine samples were from the same individual and not from the athletes who provided DNA from either buccal cell material or blood specimens. This supportive evidence led to punishment of all three athletes according to the rules of the World Anti-Doping Agency. Application of a new multidisciplinary strategy employing common and new doping control assays enables the detection of urine substitution in sports drug testing. Figure Identical GC-MS/NPD profiles of three urine specimens collected from three different individuals for doping control purposes     

Determination of a guanosine-malonaldehyde adduct in urine by high-performance liquid chromatography with a thiobarbituric acid reaction detector.

A new method for the determination of a guanosine-malonaldehyde adduct, beta-D-ribofuranosylpyrimido[1,2-a]purin-10(3H)-one (GMA), in rat and human urine is described. The method involves rapid pretreatment using, in sequence, polyamide, ion-exchange and reversed-phase cartridges; determination is by means of high-performance liquid chromatography with a thiobarbituric acid reactor in series with a fluorescence detector. This device can quantitatively determine the adduct at the sub-picomole level. This rapid, selective and sensitive method is suitable for the determination of guanine-malonaldehyde adducts in biological samples, such as human and rat urine. A semi-preparative method for the extraction and purification of these adducts from rat urine and for their identification by mass spectrometry and high-performance liquid chromatography with ultraviolet detection is also reported.     

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.     

Isolation from fetal bovine serum of a peptide similar to the alpha chain of thrombin by reversed-phase high-performance liquid chromatography

During the preparation of erythrotropin from fetal bovine serum, a group of peptides co-eluted with this erythroid cell stimulating factor on semi-preparative reversed-phase high-performance liquid chromatography. They could be subsequently separated by a combination of reversed-phase high-performance liquid chromatography in the presence of heptafluorobutyric acid as ion-pairing reagent and gel permeation high-performance liquid chromatography. One of these peptides has been extensively purified. Partial amino acid sequence analysis indicated that fourteen of the seventeen N-terminal amino acids are identical with the N-terminal sequence of the alpha chain of bovine thrombin. The same isolation procedure could be useful for the identification of other major peptides of fetal bovine serum.     

Peptide maps at picomolar levels obtained by reversed-phase high-performance liquid chromatography and pre-column derivatization with phenyl isothiocyanate. Microsequencing of Phenylthiocarbamyl Peptides

A new reversed-phase high-performance liquid chromatography approach to the production of analytical peptide maps by pre-column derivatization using phenylisothiocyanate is described. Tryptic peptide digests were derivatized with phenyl isothiocyanate to form the phenylthiocarbamyl peptides followed by reversed-phase high-performance liquid chromatographic analysis. The phenylthiocarbamyl peptides were separated by reversed-phase high-performance liquid chromatography with the conventional gradient elution system of water-acetonitrile containing trifluoroacetic acid. The sensitivity of detection of these peptide derivatives was within the range 5-10 pmol with a constant baseline at 254-260 nm. The isolated phenylthiocarbamyl peptides can be subjected to automatic Edman degradation. The effectiveness of this method was exemplified by microsequencing of phenylthiocarbamyl peptides isolated from tryptic digests of three different proteins: alpha-lactalbumin, beta-lactoglobulin and a lambda light-chain immunoglobulin.     

Direct measurement of urinary testosterone and epitestosterone conjugates using high-performance liquid chromatography/tandem mass spectrometry

Measurement of the ratio of testosterone (T) and epitestosterone (E) in urine has been used as an indication of 'natural' steroid supplementation for a decade. The direct measurement of the glucuronide and sulfate conjugates of testosterone and epitestosterone by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) should resolve a number of issues regarding unusual metabolism due to either genetic disposition or attempts to avoid detection of abuse. Determination of nanomoles per liter (0.1 ppb) concentrations of analytes in a complex biological matrix by HPLC/MS/MS is complicated by sample matrix-specific ion suppression during ESI. Deuterated internal standards of all compounds were used to overcome the effects of suppression. Comparison of the HPLC/MS/MS method with a two-part gas chromatographic/mass spectrometric method showed statistical equivalence in urine samples. Analysis of urine samples with elevated T-glucuronide to E-glucuronide ratios did not show that a significant number could be explained by an elevated excretion of epitestosterone sulfate. The HPLC/MS/MS method was also used further to characterize genetic and metabolic factors that give rise to unusual T/E ratios.     

Computer simulation as a tool for the rapid optimization of the high-performance liquid chromatographic separation of a tryptic digest of human growth hormone.

Computer simulation was used to optimize the separation of a tryptic digest of recombinant human growth hormone using reversed-phase high-performance liquid chromatography in a gradient mode. DryLab G/plus software modelled the retention behavior of the complex tryptic digest mixture as a function of gradient conditions, based on data from two experimental gradient runs. The theoretical optimum separation conditions were rapidly obtained and reproduced experimentally. Resolution did not simply increase as gradient steepness was decreased, rather, an intermediate gradient time provided maximum sample resolution. The simulation results also indicate that the method is reasonably rugged, with little change in the separation expected for different high-performance liquid chromatography systems, and changes in the separation can be compensated by a change in the gradient steepness. Computer simulation can also be useful to quickly reoptimize conditions for a new column, if it fails to provide the same separation.     

Identification of ergot alkaloids with a photochemical reaction detector in liquid chromatography

An on-line photochemical reaction detector is described for the identification of ergot alkaloids by high-performance liquid chromatography. The fluorescence signal of alkaloids decreases within about 20 sec of irradiation and disappears selectively from complex chromatograms. The application of this principle to urine samples is described.     

Application of reversed-phase high-performance liquid chromatography for structural characterization of abnormal hemoglobins

The various steps for the structural analysis of a hemoglobin molecule (protein isolation, peptide purification, amino acid composition) can be achieved by the unique use of high-performance liquid chromatography. Reversal-phase high-performance liquid chromatography is of special interest for the identification of neutral variants of hemoglobin.     

Comparison of different analytical methods for speciation of seven gadolinium-based magnetic resonance imaging contrast agents and the applications in wastewater and whole blood

Three methods, high-performance liquid chromatography hyphenated with inductively coupled plasma mass spectrometry, high-performance liquid chromatography-tandem mass spectrometry, and ion chromatography, were compared for simultaneous speciation of seven commercial gadolinium-based contrast agents for magnetic resonance imaging. Optimizations of experimental conditions for individual method were conducted, respectively. Methods of high-performance liquid chromatography hyphenated with inductively coupled plasma mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry showed the capability of speciation for all seven target compounds, whereas ion chromatography was only suitable for three of them when using electronic conductivity detector. The limits of detection and limits of qualification by the three methods were compared, and high-performance liquid chromatography hyphenated with inductively coupled plasma mass spectrometry was found to be the most sensitive one. The limits of detection for seven target compounds by high-performance liquid chromatography hyphenated with inductively coupled plasma mass spectrometry were in the range of 0.15–0.55 pg. Thus, high-performance liquid chromatography hyphenated with inductively coupled plasma mass spectrometry was chosen as the final method and successfully applied to speciation analysis of seven gadolinium-based contrast agents in wastewater and whole blood. Compounds of gadoxetic acid disodium, gadobenate dimeglumine, gadodiamide, and gadobentetate dimeglumine were found in wastewater.