Identification and determination of propafenone and its principal metabolites in human urine using capillary gas chromatography/mass spectrometry

The capillary gas chromatography/mass spectrometry of trimethylsilyl-trifluoroacetyl, trifluoroacetyl and pentafluoropropionyl (PFP) derivatives of the antiarrhythmic agent propafenone (Rytmonorm), as well as its main metabolites N-despropyl-propafenone and 5-hydroxy-propafenone, have been investigated. Both electron impact and positive isobutane chemical ionization mass spectrometry using the Ion Trap Detector have been evaluated. The presence of propafenone and its co-extracted metabolites in human urine at time intervals after the oral administration of 150 mg Rytmonorm to healthy volunteers was established, and the urinary excretion of propafenone and 5-hydroxy-propafenone was calculated using selective chemical ionization mass spectrometric detection. Only a few per cent of the dose was excreted unchanged in the urine. Large intersubject variabilities had been observed also. The large dynamic range of the Ion Trap Detector and the high correlation coefficients (0.92-0.99) of the calibration curves were striking.     

Determination of human fibroblasts metabolism in vitro by gas chromatography-mass spectrometry of cell-excreted metabolites

In order to develop an approach to the study of cell metabolism in vitro, we undertook the determination of metabolites excreted by human skin diploid fibroblasts into culture medium using high-resolution gas chromatography in combination with mass spectrometry. Chromatographic and mass spectrometric characteristics of 29 metabolites have been obtained, and 11 of the metabolites have been identified. The excreted metabolites reflect the activity of certain metabolic processes in fibroblasts in vitro. A comparison of chromatographic and mass spectrometric characteristics of the cell metabolites and of those excreted from the body in urine showed most of the metabolites excreted by fibroblasts to be different from the urine metabolites. The possibility of secondary conversion of cell metabolites in the organism and the specificity of metabolism in cells of different tissues are discussed.     

Chromatographic fractionation and analysis by mass spectrometry of conjugated metabolites of bis(2-ethylhexyl)phthalate in urine

Mono(2-ethylhexyl)phthalate (MEHP), the primary metabolite of the plasticizer bis(2-ethylhexyl)phthalate (DEHP), was given to guinea pigs and mice and the methods for the isolation, separation and analysis of its metabolites in urine were developed. Following solid-phase extraction with octadecylsilane-bonded silica, individual metabolites were purified and separated using a combination of ion-exchange chromatography on lipophilic gels and reversed-phase high performance liquid chromatography. Analysis of intact conjugates, as well as nonconjugated metabolites, was performed by fast atom bombardment mass spectrometry (FAB-MS) and, after derivation, by gas chromatography-mass spectrometry. Enzymatic methods were used for further characterization. The study confirms glucuronidation as the major conjugation pathway for MEHP in the investigated species. Although less important quantitatively, glucosidation is shown to be an alternative conjugation pathway in mice. The methods developed were applied to a sample of urine from a hyperbilirubinemic newborn infant subjected to DEHP-exposure in conjunction with an exchange transfusion. It was demonstrated that metabolites of DEHP were excreted in amounts which could be analyzed by FAB-MS.     

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.¹ The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 μm wavelength utilize the sudden excitation of water that results in phase explosion.² Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.^3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.¹² This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.     

PAXgene fixation enables comprehensive metabolomic and proteomic analyses of tissue specimens by MALDI MSI

An alcohol-based non-crosslinking tissue fixative, PAXgene Tissue System, has been proposed as alternative fixation method to formalin, providing superior and morphological preservation. To date, metabolites have not been assessed in PAXgene-fixed tissues. The study focuses on a comparison between PAXgene and standard formalin fixation for metabolomic analysis by MALDI mass spectrometry imaging. Therefore, fifty-six samples from seven mice organs were fixed with PAXgene (PFPE) or formalin (FFPE), embedded in paraffin, and processed to a tissue microarray. PAXgene was able to spatially preserve metabolites in organs achieving an overlap of common metabolites ranging from 34 to 78% with FFPE. Highly similar signal intensities and visualization of molecules demonstrated negligible differences for metabolite imaging on PFPE compared to FFPE tissues. In addition, we performed proteomic analysis of intact proteins and peptides derived from enzymatic digestion. An overlap of 33 to 58% was found between FFPE and PFPE tissue samples in peptide analysis with a higher number of PFPE-specific peaks. Analysis of intact proteins achieved an overlap in the range of 0 to 28% owing to the poor detectability of cross-linked proteins in formalin-fixed tissues. Furthermore, metabolite and peptide profiles obtained from PFPE tissues were able to correctly classify organs independent of the fixation method, whereas a distinction of organs by protein profiles was only achieved by PAXgene fixation. Finally, we applied MALDI MSI to human biopsies by sequentially analyzing metabolites and peptides within the same tissue section. Concerning prospective studies, PAXgene can be used as an alternative fixative for multi-omic tissue analysis.     

The recording of human fibroblast metabolism in vitro by the chromatographic-mass spectrometric determination of the metabolites excreted

Metabolites excreted into culture medium by human skin fibroblasts have been studied by high resolution gas chromatography and mass spectrometry. Parameters for 29 metabolites have been obtained and 11 of them have been identified. Excreted metabolites reflect activity of certain metabolic processes in fibroblasts. Comparison of chromatographic and mass spectrometric parameters of cellular metabolites with the metabolites excreted with urine revealed that most metabolites excreted from fibroblasts differ from urine metabolites. The possibility for secondary transformation of cell metabolites in organism and specificity of metabolism in different tissues has been discussed.     

Measurement of the cortisol production rate in two sisters with 17 alpha-hydroxylase deficiency using [1,2,3,4-13C]cortisol and isotope dilution mass spectrometry

[1,2,3,4-13C]cortisol was i.v. administered to two sisters aged 11 yr (patient I) and 3 yr (patient II) who suffer from 17 alpha-hydroxylase deficiency. This is the first time that the cortisol production rate (CPR) in patients with 17 alpha-hydroxylase deficiency has been measured with a stable labelled tracer using the urinary method. The urine was collected for 3 days. High-performance liquid chromatography (HPLC) of approximately 100 ml urine extracts was carried out to isolate the small amount of cortisol metabolites excreted. The cortisol metabolites were oxidized to 11-oxo-aetiocholanolone. The isotope dilution in the methyl oxime tert-butyldimethylsilyl ether derivatives was measured by selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The CPR calculated from tetrahydrocortisone (THE) and the cortolones was 765 and 536 nmol/day, respectively in patient I. The CPR in patient II was only calculated from THE and was 62 nmol/day. If radioactive labelled cortisol had been used, much larger quantities of urine would have been needed for isolation of sufficient mass of metabolites, even then purification may have been difficult. Steroid profiling of 1 ml urine samples by GC and identification by GC/MS revealed high concentrations of pregnenolone, progesterone, 11 beta-hydroxy progesterone and corticosterone metabolites. Tetrahydrocorticosterone and 5 alpha-tetrahydrocorticosterone were found in urine at elevated excretions of 2.5 and 5.7, 0.9 and 2.0 mumols/24 h, in patients I and II respectively. No cortisol metabolites were detected by routine GC or GC/MS as the low amounts excreted co-eluted with the relatively abundant corticosterone metabolites.     

Evaluation of isotope ratio (IR) mass spectrometry for the study of drug metabolism

Isotope ratio (IR) mass spectrometry was evaluated for the study of drug metabolism and balance using 13C, 15N2-labelled antipyrine (AP) as a test drug. Rats were given 40 mg kg-1 (13C,15N2)AP intraperitoneally. Breath, urine, faeces and blood were collected. Except for breath, samples were combusted in sealed quartz tubes. The resulting CO2 and N2 were analysed for excess 13C and 15N, relative to pre-dose samples, by IR mass spectrometry. In addition, blood levels of AP and cumulative excretion of urinary AP metabolites were determined by gas chromatography/mass spectrometry/selected ion monitoring (GC/MS/SIM) and high-performance liquid chromatography (HPLC) respectively. Excess 13C and 15N levels in blood were comparable with observed levels of AP, and urinary recoveries of 13C (42%) were in good agreement with those calculated from HPLC data (45%). N-Demethylation, one of the important pathways of AP metabolism, was most rapidly determined by excess 13CO2 excretion in breath (8%). The IR mass spectral analysis complemented gas chromatographic/mass spectrum and HPLC analyses, and was less complex.     

Quantitation of 4-hydroxycyclophosphamide/aldophosphamide in whole blood

There is considerable interest in determining 4-hydroxycylcophosphamide/aldophosphamide (4-HO-CP/AP) blood levels in patients receiving the prodrug, cyclophosphamide (CP). Phosphoramide mustard (PM), the alkylating metabolite of CP, is relatively impermeable to cell membranes and it is generally believed that circulating intermediary metabolites, including aldophosphamide, the immediate precursor of PM, is transported by circulating blood to tumor tissue. Therefore, circulating 4-HO-CP/AP blood levels should more closely reflect the oncostatic and cytotoxic effects of CP than the parent drug. We have developed a gas chromatographic electron-impact mass spectrometric (GC-EIMS) method suitable for routine monitoring of 4-HO-CP/AP levels in whole blood over the range 0.085 μM (25 ng/ml) to 34 μM (10 μ/ml). The unstable metabolites were derivatized with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine-HCl to form a stable aldophosphamide oxime derivative (PBOX). [²H₄]PBOX was used as an internal standard. For clinical samples, tubes were prepared prior to blood drawing, which contained the derivatizing reagent solution and the internal standard. These solutions were stable for up to 3 months when stored at room temperature. Following addition of blood to the reaction tubes, PBOX formation was rapid and the resulting derivative was stable under these conditions for up to 8 days at room temperature. Application of the method was demonstrated by quantitating 4-HO-CP/AP blood levels in patients receiving 4 g/m² intravenous infusion of CP over a period of 90 min.     

Distribution and breakdown of labeled coenzyme Q10 in rat

Radioactive coenzyme Q(10) ([(3)H]CoQ) was synthesized in a way that the metabolites produced retained the radioactivity. Administration of the lipid to rats intraperitoneally resulted in an efficient uptake into the circulation, with high concentrations found in spleen, liver, and white blood cells; lower concentrations in adrenals, ovaries, thymus, and heart; and practically no uptake in kidney, muscle, and brain. In liver homogenate most [(3)H]CoQ appeared in the organelles, but it was also present in the cytosol and transport vesicles. Mitochondria, purified on a metrizamide gradient, had a very low concentration of [(3)H]CoQ, which was mainly present in the lysosomes. All organs that took up the labeled lipid also contained water-soluble metabolites. The majority of metabolites excreted through the kidney and appeared in the urine. Some metabolites were also present in the feces, which further contained nonmetabolized [(3)H]CoQ, excreted through the bile. The major metabolites were purified from the urine, and the mass spectrometric fragmentation showed that these compounds, containing the ring with a short side chain, are phosphorylated. Thus, the results demonstrate that CoQ is metabolized in all tissues, the metabolites are phosphorylated in the cells, transported in the blood to the kidney, and excreted into the urine.     

Metabolism of the Fungicide S-n-Butyl S′-p-tert-Butylbenzyl N-3-Pyridyldithiocarbonimidate (S-1358) in Rats

S-1358 was rapidly absorbed, metabolized and readily excreted via urine and feces from orally dosed rats. Excretion of radioactivity was almost complete within 4 days. The radioactivity was distributed mainly in stomach, intestines, liver and kidneys. It seems that S-1358 and its metabolites do not persist in organs and tissues following a single oral dosing.

Major urinary metabolites of the benzyl-labeled S-1358 were p-(1,1-dimethyl-2-hydroxyethyl)benzyl methyl sulfide [B], p-(1,1-dimethyl-2-hydroxyethyl)benzyl methyl sulfone [A], p-(1-methyl-1-carboxylethyl)benzyl methyl sulfide [D], p-(1-methyl-1-carboxylethyl)benzyl methyl sulfone [C] and their glucuronide conjugates. Fecal metabolites were S-n-butyl S′-(1, 1-dimethyl-2-hydroxyethyl)benzyl N-3-pyridyldithiocarbonimidate [MR], A, B, C and D. These metabolites were also found in the bile. The pyridine-labeled S-1358 gave rise to 2-(3′-pyridylimino)-4-carboxylthiazolidine [HM] and 3-aminopyridine [AP] in the urine, and MR and AP in the feces. Intact S-1358 was a major component of the fecal radioactivity.     

Metabolic fingerprinting of rat urine by LC/MS Part 1. Analysis by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry

Complex biological samples, such as urine, contain a very large number of endogenous metabolites reflecting the metabolic state of an organism. Metabolite patterns can provide a comprehensive signature of the physiological state of an organism as well as insights into specific biochemical processes. Although the metabolites excreted in urine are commonly highly polar, the samples are generally analyzed using reversed-phase liquid chromatography mass spectrometry (RP-LC/MS). In Part 1 of this work, a method for detecting highly polar metabolites by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry (HILIC/ESI-MS) is described as a complement to RP-LC/ESI-MS. In addition, in an accompanying paper (Part 2), different multivariate approaches to extracting information from the resulting complex data are described to enable metabolic fingerprints to be obtained. The coverage of the method for the screening of as many metabolites as possible is highly improved by analyzing the urine samples using both a C(18) column and a ZIC-HILIC column. The latter was found to be a good alternative when analyzing highly polar compounds, e.g., hydroxyproline and creatinine, to columns typically used for reversed-phase liquid chromatography.     

Apolar conjugates of ecdysteroids are not used as a storage form of molting hormone in the argasid tick Ornithodoros moubata

Fifth (last) instar nymphs of the tick Ornithodoros moubata convert ingested 20-hydroxyecdysone (20E) to apolar conjugates AP2, which are then converted to the more polar conjugates AP1. Only small quantities of free hormone were transferred to the hemolymph and the carcass within the first 2 days after the blood meal. The proportion of radiolabel in these two compartments was highest at the time of the endogenous ecdysteroid peak; however, no traces of free [³H]20E were detected. The conversion probably occurs principally in the intestinal cells. Eleven days after ingestion, 84% of the radiolabel is located in the digestive tract, mainly in the form of AP1 conjugates. AP1 obtained in second instar nymphs fed with [³H]ecdysone ([³H]E) remain stable throughout the following nymphal instars. The ecdysteroid moiety of AP1 remained unchanged. The hydrolysis, although not complete, always yielded a peak comigrating with the reference E but never 20E or any other clearly distinct peaks that may have corresponded to metabolites of 20E. Less label per individual was present in adults, but its nature remained the same, viz., AP1 mainly located in the digestive tract. In females, 2.5% of the label was transferred to the progeny during the first ovipositional cycle. Apolar products (mainly AP2) that accumulated in eggs of females injected with [³H]E or [³H]20E during vitellogenesis remained unchanged during the whole embryonic development. During the molting cycle of larvae, there was only a slight conversion of AP2 to AP1, but esterase hydrolysis of these products released the same percentages of E and 20E as in the freshly laid eggs. We conclude that in this tick species apolar conjugates of ecdysteroids are inactivation metabolites that are not reutilized during the development of the animal. These metabolites are mainly retained in the tick, probably because of its peculiar blocked midgut. Several studies have shown that in other arthropod species (ticks, spiders, and insects), these apolar metabolites are excreted in the feces.     

Resistance of heparinase-derived heparin fragments to biotransformation

The biotransformation of heparinase-derived heparin fragments was examined via a combined approach using 35S-labeled heparin fragments as well as unlabeled chemically defined heparin fragments. Rats dosed with either [35S]di-, tetra-, hexa-, or octasaccharide fragments (2 mg/kg body weight, intravenously) excreted 63-69% of the injected radioactivity into the urine within 24 h with two-thirds being excreted during the first 6 h. Gel permeation chromatography of the urinary material shows that the tetra- and octasaccharides have undergone minor (approximately 5%) depolymerization whereas no change was observed for the di- and hexasaccharides. No N-desulfation was demonstrated for any of the substances. The hexa- and octasaccharide metabolites present in the urine 24 h after dosing exhibited the same antifactor Xa activity as that of the injected material. A chemically defined trisulfated disaccharide and a hexasulfated tetrasaccharide were prepared and dosed in a similar manner. Only one metabolite was recovered from animals dosed with disaccharide. This compound was characterized by anion exchange chromatography, proton nuclear magnetic resonance spectroscopy, Fourier transform infrared spectrometry, and mass spectrometry and shown to be identical to the injected disaccharide. Five metabolites were isolated from the urine of rats dosed with the hexasulfated tetrasaccharide. The major metabolite, consisting of at least 65% of the total, was characterized as described for the disaccharide and shown to be identical to the injected compound. The remaining material appeared to be disaccharides and, possibly, a tetrasaccharide conjugate. Taken together, our results show that the heparinase-derived heparin fragments are very resistant to biotransformation compared with heparin and endogenous heparin fragments. These fragments may therefore be useful in defining structure activity relationships in vivo.     

Quantitative metabolome analysis using capillary electrophoresis mass spectrometry

A new approach for the comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS) is proposed. Metabolites are first separated by CE based on charge and size and then selectively detected using MS by monitoring over a large range of m/z values. This method enabled the determination of 352 metabolic standards and its utility was demonstrated in the analysis of 1692 metabolites from Bacillus subtilis extracts, revealing significant changes in metabolites during B. subtilis sporulation.     

Human urine: Epicatechin metabolites and antioxidant activity after cocoa beverage intake

Associations between cocoa consumption in humans, excreted metabolites and total antioxidant capacity (TAC) have been scarcely investigated. The aims of the study were to investigate the epicatechin (( ? )-Ec) metabolites excreted in urine samples after an intake of 40 g of cocoa powder along with the TAC of these urine samples and the relation between both the analyses. Each of the 21 volunteers received two interventions, one with a polyphenol-rich food (PRF) and one with a polyphenol-free food (PFF) in a randomized cross-over study. Urine samples were taken before and during 24 h at 0–6, 6–12 and 12–24 h periods after test intake. The excreted ( ? )-Ec metabolites and the TAC were determined in urine samples by LC-MS/MS and TEAC assay, respectively. The maximum excretion of ( ? )-Ec metabolites and the maximum TAC value were observed in urine samples excreted between 6 and 12 h after PRF consumption. Significance of TAC increase was found in urine samples excreted during 0–6 and 6–12 h (66.6 and 72.67%, respectively, with respect to the 0 h).     

Cortisol metabolism in the domestic cat and implications for non-invasive monitoring of adrenocortical function in endangered felids

Three domestic cats were given i.m. injections of ³H-cortisol to determine the time course and relative proportion of excreted ³H-cortisol metabolites into urine and feces. Most urinary radioactivity was detected in the first sample collected at 3.9 ± 2.5 hr postinjection and accounted for 13.9 ± 2.1% of the total radioactivity recovered. High performance liquid chromatography (HPLC) detected four urinary metabolites, one of which (13.7% urinary radioactivity) eluted with the ³H-cortisol reference tracer and was quantifiable using a commercial cortisol radioimmunoassay (RIA). The majority of cortisol metabolites in feces (85.9 ± 2.1%) was excreted at 22.3 ± 6.2 hr. HPLC analysis detected several fecal metabolites consisting primarily of nonhydolyzable water-soluble forms, none of which eluted with ³H-cortisol or ³H-corticosterone reference tracers. No immunoreactivity was detected in HPLC-separated fecal eluates using the cortisol RIA; however, two of the more polar metabolites were quantifiable using a commerical cortisosterone RIA. The physiological relevance of the immunoreactive fecal metabolites was determined in four domestic cats given an adrenocorticotropin (ACTH) challenge. Increased serum cortisol concentrations were detected within 30 min of ACTH injection, which was maintained for at least 6 hr. A corresponding increase in fecal cortisol metabolite concentrations (ranging from 238% to 826% over individual baseline values) was observed 24–48 hr later. These data indicate that adrenocortical activity can be monitored nonivasively in the cat by measuring cortisol metabolites excreted in feces. This procedure is a potentially valuable tool for endangered felid management to help evaluate responses to physiological and psychological stressors associated with environmental conditions and husbandry practices. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Sex dependence of clearance rates of aldosterone and its metabolites from plasma of intact rats.

Following I.V. injection of 3H-aldosterone, the rates of clearance of plasma 3H-radioactivity was demonstrated to be sex-dependent in intact rats. Even though the percentages of CH2Cl2-extractable plasma radioactivity are greater in female than in male rats, the quantities of CH2Cl2-extractable label are similar until 60 min post-injection. However, the quantities of non-extractable, polar metabolites of aldosterone (NEPD) are markedly greater in the plasma of males and rapidly reach peak levels 10 min post-injection of aldosterone. In females, these polar metabolites (NEPD) are rapidly cleared from the blood. After bile-duct cannulation, the rate of excretion of aldosterone radiometabolites was demonstrated to be rapid and sex-dependent. Within 1 hr., female rats excreted via the bile 82% of the injected dose of 3H-aldosterone, compared to 49% in male rats. In both sexes, greater than 95% of the total radioactivity excreted in the bile are non-extractable polar metabolites of aldosterone (NEPD). The sex hormones appear to influence not only the nature of metabolism of aldosterone in the liver, but also the rates of clearance of aldosterone and its metabolites from the plasma into the bile.     

Behavioural and physiological responses of birds to environmentally relevant concentrations of an antidepressant

Many wildlife species forage on sewage-contaminated food, for example, at wastewater treatment plants and on fields fertilized with sewage sludge. The resultant exposure to human pharmaceuticals remains poorly studied for terrestrial species. On the basis of predicted exposure levels in the wild, we administered the common antidepressant fluoxetine (FLUOX) or control treatment via prey to wild-caught starlings (Sturnus vulgaris) for 22 weeks over winter. To investigate responses to fluoxetine, birds were moved from their group aviaries into individual cages for 2 days. Boldness, exploration and activity levels showed no treatment effects but controls and FLUOX birds habituated differently to isolation in terms of the concentration of corticosterone (CORT) metabolites in faeces. The controls that excreted higher concentrations of CORT metabolites on day 1 lost more body mass by day 2 of isolation than those which excreted lower levels of CORT metabolites. CORT metabolites and mass loss were unrelated in FLUOX birds. When we investigated the movements of birds in their group aviaries, we found the controls made a higher frequency of visits to food trays than FLUOX birds around the important foraging periods of sunrise and sunset, as is optimal for wintering birds. Although individual variability makes interpreting the sub-lethal endpoints measured challenging, our data suggest that fluoxetine at environmentally relevant concentrations can significantly alter behaviour and physiology.     

The metabonomics of aging and development in the rat: an investigation into the effect of age on the profile of endogenous metabolites in the urine of male rats using 1H NMR and HPLC-TOF MS

The effect of aging and development in male Wistar-derived rats on the profile of endogenous metabolites excreted in the urine was investigated using both (1)H NMR spectroscopy and HPLC-TOF MS using electrospray ionisation (ESI). The endogenous metabolites were profiled in samples collected from male rats every two weeks from just after weaning at 4 weeks up to 20 weeks of age. Multivariate data analysis enabled clusters to be visualised within the data according to age, with urine collected at 4 and 6 weeks showing the greatest differences by both analytical techniques. Markers detected by (1)H NMR spectroscopy included creatinine, taurine, hippurate and resonances associated with amino acids/fatty acids, which increased with age, whilst citrate and resonances resulting from glucose/myoinositol declined. A number of ions were detected by HPLC-MS that were only present in urine samples at 4 weeks of age in both positive and negative ESI, with a range of ions, including e.g. carnitine, increasing with age. Age predictions by PLS-regression modelling demonstrated an age-related trend within these data, between 4 and 12 weeks for HPLC-MS and 4-16 weeks for NMR. The possible utility of these techniques for metabonomic investigations of age-related changes in the rat is discussed and the importance of employing suitable control animals in pharmacological and toxicological studies is highlighted.