Quantitation of tetramethylene disulfotetramine in human urine using isotope dilution gas chromatography mass spectrometry (GC/MS and GC/MS/MS)

Tetramethylene disulfotetramine (tetramine) is a rodenticide associated with numerous poisonings was extracted and quantified in human urine using both gas chromatography/mass spectrometry (GC/MS) and GC/tandem mass spectrometry (MS/MS). 1200 μL samples were prepared using a ¹³C₄-labeled internal standard, a 96-well format, and a polydivinyl-benzene solid phase extraction sorbent bed. Relative extraction recovery was greater than 80% at 100 ng/mL. Following extraction, samples were preconcentrated by evaporation at 60 °C, and reconstituted in 50 μL acetonitrile. One-microliter was injected in a splitless mode on both instruments similarly equipped with 30 m × 0.25 mm × 25 μm, 5% phenyl-methylpolysiloxane gas chromatography columns. A quantification ion and a confirmation ion (GC/MS) or analogous selected reaction monitoring transitions (GC/MS/MS) were integrated for all reported results. The method was characterized for precision (5.92–13.4%) and accuracy (96.4–111%) using tetramine-enriched human urine pools between 5 and 250 ng/mL. The method limit of detection was calculated to be 2.34 and 3.87 ng/mL for GC/MS and GC/MS/MS, respectively. A reference range of 100 unexposed human urine samples was analyzed for potential endogenous interferences on both instruments—none were detected. Based on previous literature values for tetramine poisonings, this urinary method should be suitable for measuring low, moderate, and severe tetramine exposures.     

Improvement and validation the method using dispersive liquid-liquid microextraction with in situ derivatization followed by gas chromatography-mass spectrometry for determination of tricyclic antidepressants in human urine samples

A simple, rapid and sensitive method termed dispersive liquid-liquid microextraction (DLLME) combined with gas chromatography-mass spectrometry (GC/MS) was developed for the determination of tricyclic antidepressants (TCAs) in human urine sample. An appropriate mixture of methanol (disperser solvent), carbon tetrachloride (extraction solvent), and acetic anhydride (derivatization reagent) was injected rapidly into human urine sample. After extraction, the sedimented phase was analyzed by GC/MS. The calibration curves obtained with human urine were linear with a correlation coefficient of over 0.99 in the range of 2.0/5.0-100 ng mL(-1). Under the optimum conditions (carbon tetrachloride: 10 μL, methanol: 150 μL), the detection limits and the quantification limits of the tricyclic antidepressants were 0.5-2.0 ng mL(-1) and 2.0-5.0 ng mL(-1), respectively. The average recoveries of TCAs were 88.2-104.3%. Moreover, the inter- and intra-day precision and accuracy was acceptable at all concentrations. The results showed that DLLME is applicable to the determination of trace amounts of TCAs in human urine sample.     

Gas chromatography–ion trap mass spectrometry method for the simultaneous measurement of MDMA (ecstasy) and its metabolites,MDA, HMA,and HMMA in plasma and urine

The investigation of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) abuse requires very robust methods with high sensitivity and wide linearity ranges for the quantification of this drug of abuse and its main metabolites in body fluids. An optimized gas chromatography–ion trap mass spectrometry (GC–IT/MS) methodology with electron impact ionization addressing these issues is presented. The sample preparation involves an enzymatic hydrolysis of urine and plasma for conjugate cleavage, a SPE extraction, and a derivatization process. The method was fully validated in rat plasma and urine. Linearity for a wide concentration range was achieved for MDMA, and the metabolites 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA). Limits of quantification were 2 ng/mL in plasma and 3.5 ng/mL in urine using a Selected Ion Monitoring detection mode. Selectivity, accuracy, precision, and recovery met the required criteria for the method validation. This GC–IT/MS method provides high sensitivity and adequate performance characteristics for the simultaneous quantification of MDMA, MDA, HMA and HMMA in the studied matrices.     

Quantification of six herbicide metabolites in human urine

We developed a sensitive, selective and precise method for measuring herbicide metabolites in human urine. Our method uses automated liquid delivery of internal standards and acetate buffer and a mixed polarity polymeric phase solid phase extraction of a 2 mL urine sample. The concentrated eluate is analyzed using high-performance liquid chromatography-tandem mass spectrometry. Isotope dilution calibration is used for quantification of all analytes. The limits of detection of our method range from 0.036 to 0.075 ng/mL. The within- and between-day variation in pooled quality control samples range from 2.5 to 9.0% and from 3.2 to 16%, respectively, for all analytes at concentrations ranging from 0.6 to 12 ng/mL. Precision was similar with samples fortified with 0.1 and 0.25 ng/mL that were analyzed in each run. We validated our selective method against a less selective method used previously in our laboratory by analyzing human specimens using both methods. The methods produced results that were in agreement, with no significant bias observed.     

Validation of a gas chromatography-mass spectrometry method for the analysis of sterol oxidation products in serum

A validated gas chromatography-mass spectrometry (GC-MS) detection method for the quantitative analysis of sterol oxidation products (SOPs) in serum is described. After a lipid extraction procedure with chloroform-methanol, a cold saponification and purification by solid phase extraction, oxysterols were derivatized to form trimethyl-sylil-ethers which were subjected to GC-MS analysis. Calibration curves for cholesterol oxidation products showed determination coefficient (R(2)) of 1.0, with low values for the coefficient of variation of the response factors (< 1%). Detection and quantification limits were below 5 ng/mL and 10 ng/mL, respectively. Recovery data were between 77.65% and 110.29% (CV < 10% for all compounds). Good results were obtained for within- and between-day repeatability, with values below 10%. In conclusion, the method performed is suitable for the determination and quantification of SOPs in serum.     

Simultaneous determination of urinary cortisol, cortisone and corticosterone in parachutists, depressed patients and healthy controls in view of biomedical and pharmacokinetic studies

A rapid and sensitive reversed-phase liquid chromatographic method (RP-LC) with UV detection has been developed for the determination of free cortisol, cortisone and corticosterone in human urine. The assay was performed after a solid-phase extraction procedure (SPE) with dexamethasone as the internal standard. Chromatographic separation was carried out on a Nucleosil 100 C(18) analytical column using a mixture of acetonitrile and water (30 : 70, v/v) as a mobile phase at a flow-rate of 1 mL min(-1). Spectrophotometric detection was performed at 240 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The absolute recoveries of glucocorticoids were above 94.6%. The limits of detection (LOD) and quantification (LOQ) were 0.5 and 2 ng mL(-1), respectively, for all analytes. Linearity was confirmed in the range of 2-300 ng mL(-1) with a correlation coefficient greater than 0.9997 for all steroid hormones. The proposed method was sensitive, robust and specific allowing reliable quantification of steroid hormones. This method was successfully applied for determination of three endogenous glucocorticoid levels in human urine. The studies were performed on 20 sedentary healthy volunteers in comparison to two socially diversified groups, namely 10 parachutists before and after jump and 10 patients with depression. Pharmacokinetic studies performed on these groups indicated that urinary free cortisol and cortisol-to-cortisone ratios can be treated as biomarkers of stress and depressive disorders.     

Development of a liquid chromatography-tandem mass spectrometry method for the determination of 23 endogenous steroids in small quantities of primate urine

A quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed for the simultaneous determination of 23 endogenous steroids in primate urine. The introduced method includes estrone, pregnandiol, cortisol, testosterone and several human urinary glucocorticoid and androgen metabolites. As the method is intended for the analysis of steroid hormones in behavioral studies on wild-living primates, it was adapted for a sample volume of 200microL urine. The sample preparation consisted of an enzymatic hydrolysis of steroid glucuronides using beta-glucuronidase from E. coli followed by a solvolytic cleavage of steroid sulfates employing sulfuric acid/ethyl acetate. The extraction of steroids from urine was optimized with respect to pH during extraction, type of ether and the amount of enzyme necessary for complete hydrolysis of glucuronides. The recovery of steroids spiked into urine before hydrolysis was 58.9-103.7% with an intra-day precision of 2.7-14.3% and an inter-day precision of 2.9-14.8%. Detection limits ranged from 0.1-0.5ng/mL. The reproducibility of the whole sample preparation process was also demonstrated for unspiked urine (CV 1.2-16.5%). The proportion of steroid hormone excreted as sulfate was determined for 21 steroids in chimpanzee urine. The solvolysis proved to be essential for all investigated steroids except for pregnandiol, tetrahydrocortisol and tetrahydrocortisone, which were found to be less then 10% in the solvolysis fraction.     

The development of an ultra performance liquid chromatography-coupled atmospheric pressure chemical ionization mass spectrometry assay for seven adrenal steroids

An ultra performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (UPLC-APCI-MS) method was developed for the separation and quantification of adrenal steroid metabolites from heterologous expression media. Steroids were extracted by liquid-liquid extraction, separated on a Waters UPLC BEH C18 column, ionized by APCI, and detected using a triple quadrupole mass spectrometer in APCI positive mode with single ion monitoring. The incorporation of UPLC enabled the detection of seven structurally closely related steroids at between 5 and 40 ng/ml using run times of 11 min. The adrenal steroidogenic enzyme cytochrome P450 17-hydroxylase/17,20-lyase (CYP17) was expressed in the yeast Pichia pastoris and in nonsteroidogenic COS-1 cells, and used as a model system to evaluate the detection and quantification of adrenal steroid metabolites by UPLC-APCI-MS.     

Simultaneous measurement of urinary polyols using gas chromatography/mass spectrometry

In the present study, we simultaneously measured several polyols, such as adonitol, arabitol, dulcitol, glucose, myo-inositol, mannitol, sorbitol, and xylitol, in urine by gas chromatography/mass spectrometry-positive chemical ionization. We also examined possible relationship between the levels of these metabolites and age in normal individuals. In order to proceed to its quantification by GC/MS, 200 microL of a urine sample were diluted with 3 ml of distilled water, lyophilized, acetylated, and then analyzed them. Using this method, we were able to quantify as little as 0.5-1.0 ng/microL, and we made the calibration curves to be linear from 0.25 to 250 ng/microL (r(2)>0.991). Analytical recoveries were over 89.4%, and the inter-day and intra-day variability for accuracy and reproducibility was less than 20%. In the normal urine sample, the levels of polyols were gender-differentiated and age-related. This simple GC/MS method is sensitive and allows the measurement of wide ranges of polyols using small amounts of urine. We conclude that the quantitation of urinary polyols using GC/MS appears to be a clinically useful method for assessing polyol-pathway activity.     

Determination of cyanide and volatile alkylnitriles in whole blood by headspace solid-phase microextraction and gas chromatography with nitrogen phosphorus detection

Simultaneous determination of cyanide and volatile alkylnitriles such as acetonitrile, cis- and trans-crotononitrile, allylnitrile and butyronitrile at low ppb concentration on whole blood (rat and mice) by headspace solid-phase microextraction (HS-SPME) followed by gas chromatography (GC) with nitrogen phosphorus detection has been achieved for the first time. SPME extraction time and temperature were optimized using a star experimental design. Optimum conditions for cyanide extraction were chosen to analyze unspiked blood samples containing alkylnitriles as that analyte occurs at the lowest concentrations. For all analytes, the developed methodology yielded good quality parameters. In all cases, good reproducibility (relative standard deviation < or =12%), detection limits (<3ng mL(-1)) and quantification limits (<4 ng mL(-1)) were recorded.     

Development and validation of a gas chromatography/ion trap-mass spectrometry method for simultaneous quantification of cocaine and its metabolites benzoylecgonine and norcocaine: application to the study of cocaine metabolism in human primary cultured renal cells

Acute renal failure is a common finding in cocaine abusers. While cocaine metabolism may contribute to its nephrotoxic mechanisms, its pharmacokinetics in kidney cells is hitherto to be clarified. Primary cultures of human proximal tubular cells (HPTCs) provide a well-characterized in vitro model, phenotypically representative of HPTCs in vivo. Thus, the present work describes the first sensitive gas chromatography/ion trap-mass spectrometry (GC/IT-MS) method for measurement of cocaine and its metabolites benzoylecgonine (BE) and norcocaine (NCOC) using a primary culture of HPTCs as cellular matrix, following solid phase extraction (SPE) and derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). The application of this methodology also enables the identification of two other cocaine metabolites: ecgonine methyl ester (EME) and anhydroecgonine methyl ester (AEME). The validation of the method was performed through the evaluation of selectivity, linearity, precision and accuracy, limit of detection (LOD), and limit of quantification (LOQ). Its applicability was demonstrated through the quantification of cocaine, BE and NCOC in primary cultured HPTCs after incubation, at physiological conditions, with 1 mM cocaine for 72 h. The developed GC/IT-MS method was found to be linear (r2 > 0.99). The intra-day precision varied between 3.6% and 13.5% and the values of accuracy between 92.7% and 111.9%. The LOD values for cocaine, BE and NCOC were 0.97±0.09, 0.40±0.04 and 20.89±1.81 ng/mL, respectively, and 3.24±0.30, 1.34±0.14 and 69.62±6.05 ng/mL as LOQ values.     

Urinary steroids in men with male-pattern alopecia

Enzyme hydrolysis, solid phase extraction, methoxym-silyl derivatization and capillary gas chromatographic analysis were used to examine the changes in urinary steroid metabolites in men with androgenic alopecia. A total of 23 men with androgenic alopecia and 7 age-matched control healthy men collected 24-h urine. Significantly increased values were found in the metabolites of testosterone (T): androsterone (A) (p<0.02), and etiocholanolone (E) (p<0.05) in patients with androgenic alopecia, compared to the control values. Elevated levels of 16-hydroxy-dehydroepiandrosterone (16-OHD) (p<0.03) and cortisol (F) (P<0.05) were found, but the levels of cortisol metabolites were unchanged. Calculating the ratio of total 5 alpha/5 beta metabolites provided information on the activity of 5 alpha-reductase. The ratio of total 5 alpha/5 beta metabolites was increased in the patients showing the increased 5 alpha-reductase activity. The elevated 16-OHD level could be indicative of patients who had mild hyperadrenal activity.     

Gas chromatographic and mass spectrometric analysis of conjugated steroids in urine

This study was carried out qualitatively and quantitatively to investigate the presence and the concentrations of anabolic steroids in urine collected from orally administered humans. Microanalysis of conjugated steroids by gas chromatography and mass spectrometry (GC/MS) has been carried out. Following oral administration three major metabolites of anabolic steroid drugs have been detected and partially characterized. The six steroids can be analysed at the same time in 17 min. The lower detection limit was 10 ng/ml in 5 ml of urine. The conjugated steroids from urine were centrifuged to 2,430g for 10 min, the supernatant solution passed through Amberlite XAD-2 column and the steroids eluted fraction esterified by using MSTFA and TMSI. The rate of metabolism and urinary excretion seem to be reasonably fast.     

HPLC analysis of PAMAM dendrimer based multifunctional devices

6-OXO, a new nutritional supplement commercially available on the internet, is sold as an aromatase-inhibitor and contains androst-4-ene-3,6,17-trione as active ingredient. This anabolic steroid is a prohibited substance in sports. Androst-4-ene-3,6,17-trione is metabolised to androst-4-ene-6alpha-ol-3,17-dione and androst-4-ene-6alpha,17beta-diol-3-one. A fast, sensitive and accurate LC/MS method was developed and validated for the quantification of androst-4-ene-3,6,17-trione and its metabolites in urine. The method is capable of determining the stereochemical position of the hydroxy-group at C-6 of the metabolites and consists of a liquid-liquid extraction step with diethylether after enzymatic hydrolysis, followed by separation on a reversed phase column. Ionisation of the analytes is carried out using atmospheric pressure chemical ionisation. The limit of quantification of the method was 5 ng/mL for all compounds. The accuracy ranged from 14.8 to 1.3% for androst-4-ene-3,6,17-trione, 9.4 to 1.6% for androst-4-ene-6alpha-ol-3,17-dione and 4.1 to 3.2% for androst-4-ene-6alpha,17beta-diol-3-one in the range of 5-1000 ng/mL. Using this method androst-4-ene-6alpha-ol-3,17-dione was identified as a major urinary metabolite, whereas androst-4-ene-6alpha,17beta-diol-3-one as a minor metabolite. While the parent compound is predominantly excreted in conjugated form, both metabolites are solely excreted as conjugates.     

LC-MS analysis of trimethoxyamphetamine designer drugs (TMA series) from urine samples

A sensitive liquid chromatography-mass spectrometric (LC-MS) method for quantification of an active psychedelic hallucinogenic drugs (trimethoxyamphetamines) in human urine after solid-phase extraction (SPE) with C(18) cartridge was developed and validated. Chromatographic separation was achieved on reversed-phase Phenomenex 3.0 microm Polar Plus column (150 mm x 2.1 mm) with acetonitrile -0.2% acetic acid as mobile-phase and the step gradient elution resulted in a total run time of about 20 min. The analytes were detected by using an electrospray positive ionization mass spectrometry in selected ion monitoring (SIM) mode. In the evaluated concentration range (10-200 ng/mL) (R(2) > or = 0.998) a good linear relationship was obtained. The lower limits of detection (LLODs) and quantification (LLOQs) ranged from 4.26 to 9.12 ng/mL and from 13.18 to 29.22 ng/mL, respectively. Average recoveries ranged from 68.52 to 97.90% in urine at the concentrations of 25, 50 and 100 ng/mL. Intra- and inter-day relative standard deviations were 3.70-10.77% and 7.63-12.94%, respectively. This LC-MS method proved to be robust and reliable, and suitable for the use as a confirmation method in clinical urine drug testing.     

Quantification of phenolic metabolites of environmental chemicals in human urine using gas chromatography-tandem mass spectrometry and isotope dilution quantification

We have developed a method to measure 12 urinary phenolic metabolites of pesticides or related chemicals. The target chemicals for our method are 2-isopropoxyphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; carbofuranphenol; 2,4,5-trichlorophenol; 2,4,6-trichlorophenol; 3,5,6-trichloro-2-pyridinol; para-nitrophenol, ortho-phenylphenol, pentachlorophenol, 1-naphthol and 2-naphthol. The sample preparation involves enzyme hydrolysis, isolation of the target chemicals using solid phase extraction cartridges, a phase-transfer catalyzed derivatization, cleanup using sorbent-immobilized liquid/liquid extraction cartridges, and concentration of the sample. Derivatized samples are analyzed by capillary gas chromatography-tandem mass spectroscopy using isotope dilution calibration for quantification. The limits of detection are in the mid ng/L range and the average coefficient of variation was below 15% for most of the analytes. Using our method, we measured concentrations of the target chemicals in urine samples from the general population.     

Capillary gas chromatography as a tool for characterization of urinary steroid excretion in patients with congenital adrenal hyperplasia

Urinary steroid excretion was studied by capillary gas chromatography in 23 patients with congenital adrenal hyperplasia. In 5 patients the estimated excretion rates of pregnanetriol were in or below the normal range and 7 patients presented supranormal excretion rates of tetrahydro-cortisone and/or other glucocorticoid metabolites. Deficiency of 21-hydroxylase was nevertheless demonstrated in each patient by an increased ratio of excreted precursors vs products of 21-hydroxylase, e.g. of pregnanetriol/tetrahydro-cortisone. Due to this relative deficiency of glucocorticoids the patients' steroid excretion was further characterized by a predominance of 5 alpha-hydrogenated C19O3 metabolites (11-keto-androsterone, 11-hydroxy-androsterone) over their 5 beta-hydrogenated homologues (11-keto-etiocholanolone, 11-hydroxy-etiocholanolone). An apparent preponderance in the excretion of pregnenetriol over that of pregnanetriol was found in 4 patients, but the presence of pregnenetriol was not confirmed by mass spectrometry following prepurification of the urine samples by thin-layer chromatography indicating interference of an unidentified steroid metabolite with the initial gas chromatographic analysis. The simultaneous determination of steroids serving as precursors or products of 21-hydroxylase by capillary gas chromatography helps to establish the diagnosis of 21-hydroxylase deficiency and to characterize the pattern of steroid excretion in this syndrome even in patients where the estimation of single urinary steroids may lead to erroneous conclusions.     

Simultaneous quantification of alprazolam, 4- and alpha-hydroxyalprazolam in plasma samples using liquid chromatography mass spectrometry

A sensitive and specific method was developed for quantification of alprazolam and its two metabolites 4-hydroxyalprazolam and alpha-hydroxyalprazolam in plasma. The work up procedure was solid phase extraction. Liquid chromatography-mass spectrometry (LC-MS) was used for separation, detection and quantification of the analytes. The limit of quantitation (LOQ) was 0.05 ng/mL for alprazolam and the two metabolites. The extraction recovery was more than 82% for alprazolam and its metabolites. The within- and between-assay coefficients of variation were in the range of 1.9-17.9%. The method was used for determination of the pharmacokinetics parameters of alprazolam and its two metabolites in healthy Caucasian subjects who ingested 1mg of alprazolam.     

Urine volume dependency of specific dehydroepiandrosterone (DHEA) and cortisol metabolites in healthy children

Urine volume should be considered as a confounder when using urinary free cortisol (UFF) and cortisone (UFE) to assess glucocorticoid (GC) status. We aimed to examine whether adrenal androgen (AA) metabolites may be also affected by urine volume in healthy children. To compare the flow dependence of GC and AA metabolites, specific GC metabolites were examined. In 24-h urine samples of 120 (60 boys) healthy children (4-10 yr), steroid profiles were determined by GC-MS analysis, UFF and UFE by radioimmunoassay. To assess daily AA and GC secretion rates, 7 quantitatively most important AA (∑C19) and GC (∑C21) metabolites were summed. Sum of DHEA and its 16α-hydroxylated metabolites were denoted as DHEA&M. Association of urine volume with AA (∑C19, DHEA&M, DHEA, 16α-hydroxy-DHEA, 3β,16α,17β-androstenetriol) and GC (∑C21, UFF, UFE, 6β-hydroxycortisol, 20α-dihydrocortisol) were examined in linear regression models. Among the examined AA metabolites, 16α-hydroxy-DHEA (β=0.56, p<0.0001) and DHEA (β=0.43, p=0.05) showed relatively strong association with urine volume. A trend was seen for ∑C19 (β=0.23, p=0.08), but not for DHEA&M (p>0.1). Regarding GC metabolites, urine volume showed a stronger association with cortisol's direct metabolites, i.e., cortisone, 6β-hydroxycortisol and 20α-dihydrocortisol (β=0.4-0.6, p<0.01) than with cortisol itself (β=0.28, p<0.05). ∑C21 was not associated with urine volume. In conclusion, like UFF and UFE, renal excretion of DHEA, 16α-hydroxy-DHEA, 6β-hydroxycortisol, and 20α-dihydrocortisol may also depend on urine volume. The intrarenal production of the latter three and cortisone might explain their relative strong water-flow-dependency. Total AA or GC secretion marker appears not to be relevantly confounded by urine volume.     

Excretion of primary prostanoids and their metabolites during acute volume expansion

Simultaneous determination of urinary excretion rates of primary unmetabolized prostanoids and their enzymatic metabolites were performed by gas chromatography-mass spectrometry (GC/MS) or tandem mass spectrometry (GC/MS/MS). Changes in kidney function were induced by acute (4 h) volume expansion. Despite marked changes in urine flow, GFR, urinary pH, osmolality, sodium and potassium excretion, only a insignificant or transient rise in the enzymatic prostanoid metabolites (2,3-dinor-6-keto-PGF1 alpha, PGE-M, 2,3-dinor-TxB2 and 11-dehydro-TxB2) was observed. The excretion rates of the primary prostanoids were elevated in parallel with the rise in urine flow: PGE2 rose (p less than 0.05) from 14.2 +/- 4.0 to 86.2 +/- 20.7, PGF2 alpha from 60.0 +/- 4.9 to 119.8 +/- 24.0, 6-keto-PGF2 alpha from 7.2 +/- 1.3 to 51.5 +/- 17.0, and TxB2 from 11.2 +/- 3.3 to 13.6 +/- 3.6 ng/h/1.73 m2 (means +/- SEM) at the maximal urine flow. Except for 6-keto-PGF1 alpha and TxB2, this rise in urinary prostanoid levels was only transient despite a sustained fourfold elevated urine flow. We conclude that urine flow rate acutely affect urine prostanoid excretion rates, however, over a prolonged period of time these effects are not maintained. The present data support the concept that urinary levels of primary prostanoids mainly reflect renal concentrations whereas those of enzymatic metabolites reflect systemic prostanoid activity. From the excretion pattern of TxB2 one can assume that this prostanoid represents renal as well as systemic TxA2 activity.