Phospholipid capture combined with non-linear chromatographic correction for improved serum metabolite profiling

Serum analysis with LC/MS can yield thousands of potential metabolites. However, in metabolomics, biomarkers of interest will often be of low abundance, and ionization suppression from high abundance endogenous metabolites such as phospholipids may prevent the detection of these metabolites. Here a cerium-modified column and methyl-tert-butyl-ether (MTBE) liquid–liquid extraction were employed to remove phospholipids from serum in order to obtain a more comprehensive metabolite profile. XCMS, an in-house developed data analysis software platform, showed that the intensity of existing endogenous metabolites increased, and that new metabolites were observed. This application of phospholipid capture in combination with XCMS non-linear data processing has enormous potential in metabolite profiling, for biomarker detection and quantitation.     

Detection of doxorubicin and metabolites in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis with laser-induced fluorescence detection was used to separate and detect doxorubicin and at least five metabolites from NS-1 cells that were treated with 25 microM doxorubicin for 8 h. Using 10 mM borate, 10 mM sodium dodecyl sulfate (pH 9.3) as separation buffer, the 488-nm argon-ion laser line for fluorescence excitation, and a 635 +/- 27.5 nm bandpass filter for detection, the limit of detection (S/N=3) for doxorubicin is 61 +/- 13 zmol. This low limit of detection allows for the detection of a larger number of metabolites than previously reported. Two extraction procedures were performed: a bulk liquid-liquid extraction and an in-capillary single-cell lysis. While in the bulk liquid-liquid extraction procedure, recovery for doxorubicin range from 50 to 99%, in single cell analysis the recovery is expected to be complete. Furthermore performing lysis of a single cell inside the separation capillary prevents doxorubicin or metabolite loss or degradation during handling. Based on the bulk method the calculated metabolite abundance is in the sub-amol per cell range while it varies from 0.1 to 1.1 fmol per cell in single cell analysis confirming metabolite loss during handling. Each metabolite was found at a level less than 0.1% of the doxorubicin content in either method, suggesting a slow metabolism in the NS-1 cell system or effective removal of metabolites by the cell.     

Inhibitory effect of nicotine and its metabolites on tolbutamide hydroxylation in rat liver microsomes

A simple HPLC/fluorescence method to detect hydroxytolbutamide (a major metabolite of the anti-diabetic drug tolbutamide) has been developed. The effects of nicotine and some of its metabolites on tolbutamide hydroxylation is described. An extraction procedure with diethyl ether was followed by isocratic HPLC analysis of tolbutamide hydroxylation with a binary mobile phase composed of 10 mM monobasic sodium phosphate in methanol (45:55, v/v, apparent pH 2.28). A detection limit of sub-nanogram amounts (0.353 ng) of hydroxytolbutamide was obtained with fluorescence detection at 226 nm for excitation and 318 nm for emission. Overall precision values for hydroxytolbutamide was determined with coefficients of variation of 1.4–4.6% when nanogram levels of the metabolite were analyzed. Differential inhibitory responses were demonstrated for tolbutamide hydroxylation to nicotine and its metabolites. Tolbutamide hydroxylation was apparently inhibited by cotinine and relatively less inhibited by nicotine. Nornicotine, however, caused very little inhibition of tolbutamide hydroxylation. The implication is that nornicotine may not share similar affinity for the substrate binding site for tolbutamide. The results also suggest that heavy smokers may experience reduction in tolbutamide metabolism. The assay system itself will be useful for future studies of tolbutamide, and possibly related sulfonylureas.     

Enzymatic synthesis of 3H-labeled Ring-A reduced metabolites of aldosterone and their separation by high pressure liquid chromatography

Specific methods are described for the enzymatic synthesis of each of the six possible 3H-labeled Ring-A reduced metabolites of aldosterone (5 alpha- and 5 beta-DHAldo; 3 alpha,5 alpha-THAldo; 3 beta,5 alpha-THAldo; 3 alpha,5 beta-THAldo; and 3 beta,5 beta-THAldo; see footnote 1 for full names). Use of heated jacketed columns (C8-reverse phase) and two HPLC solvent systems, with isocratic aqueous methanol or acetonitrile, respectively, have been developed which resolve all six Ring-A reduced metabolites of aldosterone. The relative retention times and elution order of each reduced metabolite are different with each solvent system and hence help confirm the identities of Ring-A reduced metabolites made in vivo from physiological quantities of [3H]aldosterone. The use of an on-line beta-radioactivity detector (Berthold LB-504) enhanced the sensitivity of detection and markedly improved the resolution of these metabolites, compared with that obtained by off-line scintillation counting. Thus, the use of increased temperature with these two solvent systems, together with an on-line radioactivity detector, provide a useful and efficient analytical tool for the separation and identification of each reduced metabolite of aldosterone.     

Metabolite analyses of single cells

Single-cell analysis is a promising method for understanding not only cellular physiology but also biological mechanisms of multicellular organisms. Although neighboring cells in multicellular organisms originate from the same genomic information, different circumstances around cells or epigenetic differences have different influences on each cell, leading to differing expression of genes, and thus differing levels and dynamics of metabolites, in single cells. However, single-cell analysis is a tough challenge, even with recent technologies, because of the small size of single cells. Unlike genes, metabolites cannot be amplified, and therefore metabolite analysis is another issue. To analyze such a tiny quantity of metabolites in a single cell, various techniques have been tried and developed. Especially in mass spectrometry, marked improvements in both detection sensitivity and ionization techniques have opened up the challenge for the analysis of metabolites in single cells. In this review, we discuss the method for metabolite detection at the level of single cells and recent advancements in technology.     

An estimation of the exposure to organophosphorus pesticides through the simultaneous determination of their main metabolites in urine by liquid chromatography-tandem mass spectrometry

A rapid method has been developed for the determination of 4-nitrophenol (PNP) (parathion and methyl-parathion metabolite) and 3-methyl-4-nitrophenol (3-Me-PNP) (fenitrothion metabolite) in human urine by coupled-column liquid chromatography combined with tandem mass spectrometry (LC-LC-MS/MS). The LC-LC-MS/MS approach allows the determination at sub-ppb level of free metabolites by injecting the urine directly into the system and the total metabolites after a simple enzymatic hydrolysis. The method has been validated, obtaining limits of detection of 0.1 and 0.2 microg/L for 4-nitrophenol and 3-methyl-4-nitrophenol, respectively. Additionally, a multi-residue LC-MS/MS method is proposed in order to evaluate the levels of other parathion and methyl parathion metabolites. This approach allows the simultaneous determination of dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP), 4-nitrophenolsulphate and 4-nitrophenolglucuronide without tedious sample treatments. The applicability of both methods is demonstrated by applying them to various urine samples from an unexposed population and a grower who applied methyl parathion. The combination of both methods allows a general overview on the presence of different metabolites (free and conjugated) and the concentration ratios between them, giving useful information on organophosphorus pesticides metabolism and excretion.     

Monitoring defensive responses in macroalgae – limitations and perspectives

As part of an ongoing research program aiming at monitoring molecular changes in the tissues and metabolite trafficking in the hydrosphere of algae subjected to chemical stresses, we are discussing the various analytical techniques that have been employed to characterize, and sometimes to quantity these metabolites. High-field multinuclear and solid-state nuclear magnetic resonance (NMR) spectroscopies are powerful tools for metabolite characterization from extracts and in vivo, but quantification and kinetic aspects show some limitations. Modern MS (mass spectrometry) is extremely useful for fingerprinting samples against databases and when dealing with very low concentrations of metabolites, the limitations being set by the type of chromatographic separation and mode of detection coupled with the mass spectrometer. Regarding chemical communication, optimization in terms of resolution and efficiency of hydrosphere chemical analysis can theoretically be achieved in a system which integrates (i) a multiparametric incubation chamber, (ii) a gasphase or a liquid-phase separation system and (iii) mass spectrometer(s) equipped with one or two detectors responding to the analytical and quantitative needs. This text reviews some of the techniques that have been employed in various types of plant metabolic studies, which may serve as a basis towards an integrative analytical strategy directly applicable to the metabolomics of selected marine macrophytes.     

Gas—liquid chromatographic determination of flurazepam and its major metabolites in plasma with electron-capture detection

A sensitive and selective gas—liquid chromatographic method, using the electron-capture detector for the quantitative determination of flurazepam and its major blood metabolites is described. After extraction and back-extraction steps, flurazepam (I) is well separated from its main metabolites, N-1-hydroxyethylflurazepam (metabolite II) and N-1-desalkylflurazepam (metabolite III). Metabolite II is quantitated after forming its stable tert-butyldimethylsilyl derivative by reaction with tert-butyldimethylchlorosilane—imidazole reagent. The procedure permits the rapid and selective routine determination of flurazepam and its metabolites (II and III) in plasma with a detection limit of 3 ng/ml for flurazepam (I), 1 ng/ml for metabolite II and 0.6 ng/ml for metabolite III. The procedure is linear over the range of concentrations encountered after administration of a single oral therapeutic dose. No interference from the biological matrix is apparent. The suitability of the method for the analysis of biological samples was tested by studying the variation with time of flurazepam and its metabolites' plasma concentrations in normal human volunteers after a single, therapeutic 30-mg oral dose of flurazepam.     

Differences in the metabolite profiles of spinach (Spinacia oleracea L.) leaf in different concentrations of nitrate in the culture solution

The nitrogen (N) status of a plant determines the composition of its major components (amino acids, proteins, carbohydrates and organic acids) and, directly or indirectly, affects the quality of agricultural products in terms of their calorific value and taste. Although these effects are guided by changes in metabolic pathways, no overall metabolic analysis has previously been conducted to demonstrate such effects. Here, metabolite profiling using gas chromatography-mass spectrometry (GC-MS) was used to evaluate the effect of N levels on spinach tissue, comparing two cultivars that differed in their ability to use N. Wide variation in N content was observed without any distinct inhibition of growth in either cultivar. Principal component analysis (PCA) and self-organizing mapping (SOM) were undertaken to describe changes in the metabolites of mature spinach leaves. In PCA, the first component accounted for 44.5% of the total variance, the scores of which was positively correlated with the plant's N content, and a close relationship between metabolite profiles and N status was observed. Both PCA and SOM revealed that metabolites could be broadly divided into two types, correlating either positively or negatively with plant N content. The simple and co-coordinated metabolic stream, containing both general and spinach-specific aspects of plant N content, will be useful in future research on such topics as the detection of environmental effects on spinach through comprehensive metabolic profiling.     

Determination of two mebeverine metabolites, mebeverine alcohol and desmethylmebeverine alcohol, in human plasma by a dual stable isotope-based gas chromatographic-mass spectrometric method

A dual stable isotope-based GC-MS method was developed for the simultaneous determination of two metabolites of mebeverine, mebeverine alcohol and desmethylmebeverine alcohol, in human plasma. Plasma samples were treated with β-glucuronidase to cleave the glucuronide conjugates of both compounds prior to analysis. The treated plasma was prepared for analysis by solid-phase extraction using octadecylsilane cartridges. The isolated metabolites were derivatized and analyzed by GC-MS using selected-ion monitoring. Plots of peak-area ratio were linear with metabolite concentration from 2 to 200 ng/ml and the limit of detection for both metabolites was 0.5 ng/ml. The GC-MS methodology was applied to the analysis of plasma from human subjects following peroral administration of mebeverine. Pharmacokinetic parameters for both metabolites were determined and suggest that relative systemic mebeverine exposure may potentially be assessed using metabolite kinetics, if the latter subsequently are demonstrated to be linear with mebeverine dose.     

Detection of fenspiride and identification of in vivo metabolites in horse body fluids by capillary gas chromatography-mass spectrometry: administration, biotransformation and urinary excretion after a single oral dose

Studies related to the in vivo biotransforrmation and urinary excretion of fenspiride hydrochloride in the horse are described. After oral administration, the drug is metabolised by both phase I functionalisation and phase II conjugation pathways. Following enzymatic deconjugation, fenspiride and its phase I metabolites were isolated from post-administration biofluids using bonded co-polymeric mixed mode solid-phase extraction cartridges to isolate the basic compounds. Following trimethylsilylation (TMS), the parent drug and metabolites were identified by capillary gas chromatography-mass spectrometry (GC-MS). Fenspiride (A) and seven metabolites (B-->G) arising from oxidation on both the aromatic and heterocyclic substructures were detected in urine. The positive ion electron ionisation mass spectra of the TMS derivatives of fenspiride and its metabolites provided useful information on its metabolism. Positive ion methane chemical ionisation-GC-MS of the derivatives provided both derivatised molecular mass and structural information. Unchanged fenspiride can be detected in post-administration plasma and urine samples for up to 24 h. Maximum urinary levels of 100-200 ng ml(-1) were observed between 3 and 5 h after administration. After enzymatic deconjugation, the major phenolic metabolite (G) can be detected in urine for up to 72 h. This metabolite is the analyte of choice in the GC-MS screening of post-race equine urine samples for detection of fenspiride use. However, a distinct difference was observed in the urinary excretion of this metabolite between the thoroughbred horses used in UK study and the quarterbred and standardbred horses used for the USA administrations.     

Synthesis and analysis of conjugates of the major vitamin E metabolite,alpha-CEHC

Glucuronide and sulphate conjugates of 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the major metabolite of alpha-tocopherol (vitamin E), have been synthesized and used for the first direct analysis of conjugated urinary vitamin E metabolites. The metabolites of vitamin E (alpha-tocopherol) could be useful as markers of the function(s) of vitamin E in vivo. A number of methods have been described for the analysis of urinary vitamin E metabolites but these have relied on either acid or enzymatic deconjugation of the metabolites prior to analysis by high performance liquid chromatography or gas chromatography/mass spectrometry. These methods have provided useful information about the amount and types of metabolites excreted in the urine but suffer from a number of disadvantages. Deconjugation has been shown to produce artifacts as a result of the conversion of alpha-CEHC to alpha-tocopheronolactone and the efficiency of deconjugation is also difficult to assess. Methods that allow the direct measurement of the conjugated metabolites would overcome these problems and would also substantially reduce the preparation and analysis time. Here we describe the use of conjugated standards to characterize alpha-CEHC conjugates in human urine by tandem mass spectrometry (MS-MS). The future use of MS-MS to measure urinary vitamin E metabolites is also discussed.     

Late-onset 21-hydroxylase deficiency: reliable diagnosis by steroid analysis of random urine collections

The feasibility of performing steroid analysis by capillary gas chromatography on random urine samples for the detection of mild late-onset 21-hydroxylase deficiency was evaluated. Comparisons were made of basal excretions of androgen and 17 alpha-hydroxyprogesterone metabolites with plasma levels (basal and stimulated) of 17 alpha-hydroxyprogesterone and testosterone in six patients with the disorder. The following steroid metabolite excretion ratios were determined for normal controls and affected individuals. 1) 17 alpha-hydroxypregnanolone/tetrahydrocortisone + tetrahydrocortisol + 5 alpha-tetrahydrocortisol (cortisol metabolites) (normal 0.017-0.10, affected 0.17-0.42); 2) pregnanetriol/cortisol metabolites (normal 0.03-0.15, affected 0.17-0.99); 3) pregnanetriolone/cortisol metabolites (normal 0.02-0.014, affected 0.08-0.20); 4) androsterone + etiocholanolone/cortisol metabolites (normal 0.26-1.02, affected 0.34-1.47). Among the 21-deoxy steroid ratios, there was no overlap between affected and unaffected individuals. Two of six affected individuals had androsterone + etiocholanolone/cortisol metabolite ratios in the normal range. This method provides excellent discrimination between normal and affected individuals, precluding the need for an ACTH-stimulation test. It is anticipated that it will be increasingly used for diagnosis of the condition.     

Simultaneous determination of tricarboxylic acid cycle metabolites by high-performance liquid chromatography with ultraviolet detection

Here we describe a simple high-performance liquid chromatography (HPLC) procedure for the simultaneous detection and quantitation in standard solutions of 13 important metabolites of cellular energy metabolism, including 9 tricarboxylic acid (TCA) cycle components and 4 additional metabolites. The metabolites are detected by their absorbance at 210 nm. The procedure does not require prior derivatization, and an analysis can be carried out at ambient temperature within 15 min. The significance of the current work is that the current HPLC procedure should motivate the development of simplified TCA cycle enzyme assays, isotopomer analysis, and determination of selected TCA metabolite levels in plasma/tissues.     

Differences in metabolite profiles between blood matrices,ages, and sexes among Caucasian individuals and their inter-individual variations

Endobiotic metabolites are associated with biological processes in the body and therefore may serve as biomarkers for disease states or therapeutic efficacy and toxicity. However, information is limited regarding how differences between blood matrices, patient backgrounds, and sample handling affect human metabolite profiles. Our objective was to obtain metabolite profiles from Caucasian individuals, based on different matrices (plasma and serum), subject backgrounds (male/female and young/old), and storage conditions (2 or 10 freeze–thaw cycles). In total, 297 metabolites were detected by LC/MS and GC/MS, and more than 75 % of them were highly represented in all sample groups. The multivariate discriminant analysis (OPLS-DA as a model) singled out the matrix type as the most important variable influencing global metabolic profiles; that is, more than 100 metabolites were significantly different based on the matrix type. The influence of subject backgrounds on global metabolic profiles was consistent between plasma and serum. Age-associated differences were more predominant in females than males, whereas gender-associated differences were more prevalent in young subjects than old individuals were. The relative standard deviation of metabolite levels in subjects with the same background ranked from 0.1 to 1.5. Moreover, the changes of metabolite levels caused by freeze–thaw cycles were limited, and the effect was more prominent in plasma than serum. These data demonstrate the impact of matrix, age, gender, and freeze–thaw cycles on the metabolite profiles and reveal metabolites affected by these factors. Thus, our results provide would useful fundamental information for exploring and qualifying biomarkers for clinical applications.     

Rice-<Emphasis Type="Italic">Arabidopsis</Emphasis> FOX line screening with FT-NIR-based fingerprinting for GC-TOF/MS-based metabolite profiling

The full-length cDNA over-expressing (FOX) gene hunting system is useful for genome-wide gain-of-function analysis. The screening of FOX lines requires a high-throughput metabolomic method that can detect a wide range of metabolites. Fourier transform-near-infrared (FT-NIR) spectroscopy in combination with the chemometric approach has been used to analyze metabolite fingerprints. Since FT-NIR spectroscopy can be used to analyze a solid sample without destructive extraction, this technique enables untargeted analysis and high-throughput screening focusing on the alteration of metabolite composition. We performed non-destructive FT-NIR-based fingerprinting to screen seed samples of 3000 rice-Arabidopsis FOX lines; the samples were obtained from transgenic Arabidopsis thaliana lines that overexpressed rice full-length cDNA. Subsequently, the candidate lines exhibiting alteration in their metabolite fingerprints were analyzed by gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) in order to assess their metabolite profiles. Finally, multivariate regression using orthogonal projections to latent structures (O2PLS) was used to elucidate the predictive metabolites obtained in FT-NIR analysis by integration of the datasets obtained from FT-NIR and GC-TOF/MS analyses. FT-NIR-based fingerprinting is a technically efficient method in that it facilitates non-destructive analysis in a high-throughput manner. Furthermore, with the integrated analysis used here, we were able to discover unique metabotypes in rice-Arabidopsis FOX lines; thus, this approach is beneficial for investigating the function of rice genes related to metabolism.     

Dissection of genotype-phenotype associations in rice grains using metabolome quantitative trait loci analysis

A comprehensive and large‐scale metabolome quantitative trait loci (mQTL) analysis was performed to investigate the genetic backgrounds associated with metabolic phenotypes in rice grains. The metabolome dataset consisted of 759 metabolite signals obtained from the grains of 85 lines of rice (Oryza sativa, Sasanishiki × Habataki back‐crossed inbred lines). Metabolome analysis was performed using four mass spectrometry pipelines to enhance detection of different classes of metabolites. This mQTL analysis of a wide range of metabolites highlighted an uneven distribution of 802 mQTLs on the rice genome, as well as different modes of metabolic trait (m‐trait) control among various types of metabolites. The levels of most metabolites within rice grains were highly sensitive to environmental factors, but only weakly associated with mQTLs. Coordinated control was observed for several groups of metabolites, such as amino acids linked to the mQTL hotspot on chromosome 3. For flavonoids, m‐trait variation among the experimental lines was tightly governed by genetic factors that alter the glycosylation of flavones. Many loci affecting levels of metabolites were detected by QTL analysis, and plausible gene candidates were evaluated by in silico analysis. Several mQTLs profoundly influenced metabolite levels, providing insight into the control of rice metabolism. The genomic region and genes potentially responsible for the biosynthesis of apigenin‐6,8‐di‐C‐α‐l‐ arabinoside are presented as an example of a critical mQTL identified by the analysis.     

Detection of new urinary exemestane metabolites by gas chromatography coupled to mass spectrometry

Exemestane is an aromatase enzyme complex inhibitor. Its metabolism in humans is not fully described and there is only one known metabolite: 17β-hydroxyexemestane. In this work, excretion studies were performed with four volunteers aiming at the detection of new exemestane metabolites in human urine by gas chromatography coupled to mass spectrometry (GC-MS) after enzymatic hydrolysis and liquid-liquid extraction. Urine samples collected from four volunteers were analyzed separately. The targets of the study were mainly the 6-exomethylene oxidized metabolites. Two unreported metabolites were identified in both free and glucuconjugated urine fractions from all four volunteers, both of them were the result of the 6-exomethylene moiety oxidation: 6ξ-hydroxy-6ξ-hydroxymethylandrosta-1,4-diene-3,17-dione (metabolite 1) and 6ξ-hydroxyandrosta-1,4-diene-3,17-dione (metabolite 2). Furthermore, only in glucoconjugated fractions from all volunteers, one metabolite arising from the A-ring reduction was identified as well, 3ξ-hydroxy-5ξ-androst-1-ene-6-methylene-17-one (metabolite 3). The molecular formulae of all these metabolites were ascertained by the determination of exact masses using gas chromatography coupled to high resolution mass spectrometry (GC-HRMS). Moreover, all metabolites were confirmed using an alternative derivatization with methoxyamine and MSTFA/TMS-imidazole.     

Molecular dynamics simulation studies for DNA sequence recognition by reactive metabolites of anticancer compounds

The discovery of novel anticancer molecules 5F‐203 (NSC703786) and 5‐aminoflavone (5‐AMF, NSC686288) has addressed the issues of toxicity and reduced efficacy by targeting over expressed Cytochrome P450 1A1 (CYP1A1) in cancer cells. CYP1A1 metabolizes these compounds into their reactive metabolites, which are proven to mediate their anticancer effect through DNA adduct formation. However, the drug metabolite–DNA binding has not been explored so far. Hence, understanding the binding characteristics and molecular recognition for drug metabolites with DNA is of practical and fundamental interest. The present study is aimed to model binding preference shown by reactive metabolites of 5F‐203 and 5‐AMF with DNA in forming DNA adducts. To perform this, three different DNA crystal structures covering sequence diversity were selected, and 12 DNA‐reactive metabolite complexes were generated. Molecular dynamics simulations for all complexes were performed using AMBER 11 software after development of protocol for DNA‐reactive metabolite system. Furthermore, the MM‐PBSA/GBSA energy calculation, per‐nucleotide energy decomposition, and Molecular Electrostatic Surface Potential analysis were performed. The results obtained from present study clearly indicate that minor groove in DNA is preferable for binding of reactive metabolites of anticancer compounds. The binding preferences shown by reactive metabolites were also governed by specific nucleotide sequence and distribution of electrostatic charges in major and minor groove of DNA structure. Overall, our study provides useful insights into the initial step of mechanism of reactive metabolite binding to the DNA and the guidelines for designing of sequence specific DNA interacting anticancer agents. Copyright © 2014 John Wiley & Sons, Ltd.     

Assessment of urine and fecal testosterone metabolite excretion in Chinchilla lanigera males

Endemic chinchilla (Chinchilla spp.) populations are nearly extinct in the wild (South America). In captive animals (Chinchilla lanigera and C. brevicaudata), reproduction is characterized by poor fertility and limited by seasonal breeding patterns. Techniques applied for studying male reproductive physiology in these species are often invasive and stressful (i.e. repeated blood sampling for sexual steroids analysis). To evaluate endocrine testicular function, the present experiments were designed to (a) determine the main route of testosterone excretion (14C-testosterone infusion in four males); (b) validate urine and fecal testosterone metabolite measurements (HPLC was used to separate metabolites and immunoreactivity was assessed in all metabolites using a commercial testosterone radioimmunoassay, and parallelism, accuracy and precision tests were conducted to validate the immunoassay); and (c) investigate the biological relevance of the techniques applied (quantification of testosterone metabolite excretion into urine and feces from five males injected with hCG and comparison between 10 males and 10 females). Radiolabelled metabolites of 14C-testosterone were excreted, 84.7+/-4.2 % in urine and 15.2+/-3.9 % in feces. A total of 82.7+/-4.2% of urinary and 45.7+/-13.6% of fecal radioactivity was excreted over the first 24 h period post-infusion (metabolite concentration peaked at 8.2+/-2.5 h and 22.0+/-7.0 h, respectively). Several urinary and fecal androgen metabolites were separated by HPLC but only fecal metabolites were associated with native testosterone; however, there was immunoreactivity in more than one metabolite derived from 14C-testosterone. After hCG administration, an increase in androgen metabolite excretion was observed (p<0.05). Males excreted greater amounts daily of urinary androgen metabolites as compared with females (p<0.05); this difference was not evident in feces. Results of the present study indicate that the procedure used is a reliable and non-invasive method to repeatedly monitor variations in testicular endocrine activity in this species. It can be a useful tool that would help ensure the survival of the wild populations as well as to provide the basis for a more efficient use by the fur industry.