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PubMed1990

Bicarbonate dependence of cAMP accumulation induced by phorbol esters in hamster spermatozoa.

Phorbol esters stimulate cyclic adenosine 3',5'-monophosphate (cAMP) accumulation in hamster spermatozoa under conditions for in vitro capacitation. The 20-50-fold elevation of cAMP levels induced by 1 microM phorbol 12-myristate 13-acetate (PMA) in spermatozoa depends on the presence of sodium bicarbonate in the medium (ED50: 15 mM) and it is independent of extracellular pH. Sodium bicarbonate stimulates adenylate cyclase activity in membrane preparations by 4-fold (ED50: 40 mM). After solubilization, the bicarbonate-sensitive moiety elutes as a single peak of 55 kDa in a gel filtration column. Blockers of bicarbonate chloride antiporters diisothiocyanate stilbene 2,2'-disulfonic acid (DIDS) or acetamido 4'-isothiocyanate stilbene 2,2'-disulfonic acid (SITS) inhibit the bicarbonate dependent PMA effect on cAMP in living spermatozoa (ED50: 100 microM). Maximal (85%) inhibition in cAMP accumulation is observed at 1 mM. Motility is inhibited only at high concentrations of the blockers. Pretreatment of living cells with 1 mM DIDS does not affect membrane adenylate cyclase activity which remains responsive to bicarbonate. These results suggest that controlled transport of bicarbonate through the sperm plasma membrane could be associated to the regulation of cAMP synthesis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Expression of arylhydrocarbon hydroxylase, epoxide hydrolases, glutathione S-transferase and UDP-glucuronosyltransferases in H5-6 hepatoma cells.

1. The presence of arylhydrocarbon hydroxylase (cytochrome P-450 IA1 dependent), glutathione S-transferase, two distinct forms of epoxide hydrolases and UDP-glucuronosyltransferases was detected in H5-6 hepatoma cell homogenates using model substrates, selective inhibitors and specific antibodies. 2. The activity of arylhydrocarbon hydroxylase decreased strongly at the first days after plating and remained at a minimal value (1.5 pmol/min per mg) after 5 days of culture. 3. The hydratation of trans-stilbene oxide catalyzed by the soluble form of epoxide hydrolase was very low (11.0 pmol/min per mg), whereas the hepatoma cells contained appreciable amounts of the membrane-bound epoxide hydrolase and glutathione S-transferase measured with cis-stilbene oxide as substrate (maximal specific activity: 1.46 and 2.73 nmol/min per mg, respectively). 4. These cells also glucuronidated 1-naphthol efficiently (6 nmol/min per mg) and, at a lower extent, bilirubin (12 pmol/min per mg). 5. Addition of fenofibrate (70 microM) into the culture medium for 1-3 days failed to significantly stimulate the activity of cytosolic epoxide hydrolase. Only bilirubin glucuronidation increased 2-fold after 2 days of presence of the drug.

Animals↗
PubMed1987

Antagonists of GABA responses, studied using internally perfused frog dorsal root ganglion neurons.

Responses of frog dorsal root ganglion neurons to GABA were studied under conditions of internal perfusion. Conductances to Na, Ca and K were pharmacologically blocked, C1 concentrations were maintained equal on both sides of the membrane and a small holding potential was used. Under these conditions GABA-induced C1 currents could be studied in isolation without shifts in EC1 occurring after GABA application. GABA currents were blocked by a variety of agents. The blockade by bicuculline and Zn was competitive, while that to penicillin was competitive at low concentrations (6 x 10(-5) M) and non-competitive at high concentrations (3 x 10(-4) M). Picrotoxin was non-competitive at all concentrations studied. The time course of the GABA-induced currents was changed in the presence of antagonists, including those that were competitive. These actions appear to be due to a change in the rates of receptor desensitization rather than shifts in EC1. Pretreatment with antagonists increased the degree of inhibition only for picrotoxin as compared to simultaneous application of GABA plus antagonist. The voltage dependence of the GABA response was altered by penicillin but not by other antagonists. GABA responses on frog dorsal root ganglion cell were also depressed by a variety of other metal ions (Cd, Ni, Cu, Co, Mn) and other drugs (strychnine, curare, 4-acetamide, 4'-isothiocyano-stilbene-2,2'-dilsulfonic acid disodium salt, 4,4'-diisothiocyano-stilbene-2,2'-dilsulfonic acid disodium salt trihydrate, bemegride and folic acid). In this preparation bicuculline and the heavy metal ions appear to block at or very near to the agonist binding site, while penicillin probably blocks the ion channel. The non-competitive action of picrotoxin appears not to be channel blockade, but to be due to a slowly equilibrating action at a site different from either the agonist binding site or the channel.

Animals↗
PubMed1994

Depolarization-induced alkalinization of astrocytes in gliotic hippocampal slices.

Depolarization-induced, intracellular alkaline shifts were studied in reactive astrocytes within slices of gliotic hippocampus. Slices were prepared 10-28 days after sterotaxic injection of kainic acid into the hippocampus of anesthetized rats. Astrocytes in gliotic CA3 were impaled with double-barreled pH sensitive microelectrodes and depolarized by iontophoresis of K+ from an adjacent micropipette. Elevation of extracellular K+ produced an intracellular alkalinization that grew with increasing membrane depolarization, ranging from approximately 0.10 to 0.30 pH units. Exposure to Ba2+ depolarized the cells and produced a similar alkalinization. In the presence of Ba2+, the K(+)-induced depolarization and the associated alkaline shift were abolished. The depolarization-induced alkaline shifts were partially inhibited (40 +/- 8.9%) in Na(+)-free media and were enhanced in bicarbonate versus HEPES-buffered saline. The alkalinizations were unaffected by incubation in chloride-free media, or by the stilbene 4,4'-dinitrostilbene-2,2'-disulfonic acid. It is concluded that the depolarization-induced alkaline shift of reactive astrocytes is mediated in part by a Na+ and HCO3(-)-dependent mechanism that is insensitive to stilbenes. These characteristics correspond well with the properties of depolarization-induced acid secretion in the gliotic tissue. In addition, a separate, Na(+)-independent mechanism contributes to the depolarization-induced alkalinization. In view of the absolute Na+ dependence of acid secretion in the gliotic slices, we propose that the latter mechanism does not extrude acid across the plasma membrane.

Alkalies↗
PubMed1986

Effect of aging on testicular epoxide hydrolase and glutathione-S-transferase activities in mice.

The effect of aging on epoxide hydrolase (EH) and glutathion-S-transferase (GST) activities was investigated in testes of C57BL/6 mice 1-30 months of age. Microsomal EH (mEH) activity, as monitored with cis-stilbene oxide (CSO), showed statistically insignificant changes throughout the lifespan of mice. Although cytosolic EH (cEH) was detected in testes by immunoblotting, the enzyme activity towards trans-stilbene oxide (TSO) could not be measured under the experimental conditions used. Gonadal GST monitored with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate displayed an increasing trend until the mice reached senescence, showing a 3.7-fold increase in the enzyme activity in old animals (30 months) when compared with that in young animals (2 months). However, with CSO as substrate, GST showed no change in activity in mice of different ages.

Aging↗
PubMed1985

Age-development and inducibility of hepatic glutathione S-transferase activities in mice, rats, rabbits and guinea-pigs.

Hepatic glutathione S-transferase (GST) activities towards 1-chloro-3,4-dinitrobenzene (DNCB), 3,4-dichloronitrobenzene (DCNB), sulfobromophthalein (BSP), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), trans-4-phenyl-3-buten-2-one (TPBO) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP) were determined in mice, rats, rabbits and guinea-pigs during ageing and after pretreatment with enzyme inducers. Variations were observed in the developmental patterns and in the phenobarbital-, benzo(a)pyrene-, pregnenolone-16 alpha-carbonitrile-, butylated hydroxyanisole-, trans-stilbene oxide-inducibility of hepatic GST activities in the same species towards different substrates. For example, in rats GST activities for EA, DCNB and TPBO increased respectively, 2.3-, 4.8- and 25-fold during age-development, and after treatment with TSO 1.2-, 3.6- and 1.3-fold. Species differences were found in the maturation and in the inducibility of GST activities. For instance, GST activity toward EA at birth is mature in guinea pigs but not in the other species; phenobarbital treatment increased GST activities in mice and rats but not in rabbits and guinea-pigs; treatment with trans-stilbene oxide enhanced GST activity for TPBO 4.5-fold in mice but not at all in rats. It is concluded that hepatic glutathione conjugation exhibits functional heterogeneity which may be due to species dependent variations in the responsiveness of GST isoenzymes to endogenous and exogenous influences.

Aging↗
PubMedDate not supplied

The disruption of the third extracellular loop of the red cell anion exchanger AE1 does not affect electroneutral Cl-/HCO3- exchange activity.

The red cell anion exchanger (band 3; AE1) is a multispanning membrane protein that traverses the bilayer up to 14 times and mediates the stilbene-disulfonate-sensitive, electroneutral exchange of chloride and bicarbonate. Previous studies showed that the integrity of the third extracellular loop (EC3) of the protein was not essential for stilbene-disulfonate-sensitive chloride uptake. Here we demonstrate that the chloride uptake mediated by assemblies separated at EC3 represents the physiological electroneutral Cl(-)/HCO(3)(-) activity associated with intact AE1 protein. This provides further evidence that the 1:5 and 6:14 regions of the protein form discrete folding domains and confirms that the third extracellular loop does not play a pivotal role in AE1 transport function.

Animals↗
PubMed2005

Determination of fluorescent whitening agents in laundry detergents and surface waters by solid-phase extraction and ion-pair high-performance liquid chromatography.

A simple method was developed to detect four stilbene-type disulfonate and one distyrylbiphenyl-type fluorescent whitening agents (FWAs) in household laundry detergents and surface waters by ion-pair high-performance liquid chromatography. The FWA concentrations in detergents were measured directly. The contents of FWAs in water samples were extracted by solid-phase extraction (C18-SPE) with ion-pairing reagent, and were then determined by an isocratic ion-pair chromatography (IPC) using a C18 column, applying tetrabutylammonium hydrogensulfate (TBA) as the ion-pairing reagent in mobile phase, and equipped with fluorescence detection. Water samples at various pH conditions for SPE were evaluated. Experimental results indicate that the proposed method is precise and sensitive in analyzing FWAs, and enables quantitation of 0.01-0.1 microg/l in 100 ml water samples. The recovery rates of FWAs in spiked water samples were between 73 and 89%, and the precision (RSD) ranged from 2.6 to 8.9%. Over 7200 microg/g of 4,4'-bis(2-sulfostryl)-biphenyl (DSBP) and 2320 microg/g of 4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazine-2-yl)-amino]stilbene-2,2'-disulfonate (DAS1) were detected in household laundry detergents. Trace amounts of DSBP were detected in surface water samples ranging from 0.2 to 3.7 microg/l.

Chromatography, High Pressure Liquid↗
PubMed2006

Hot-water and solid-phase extraction of fluorescent whitening agents in paper materials and infant clothes followed by unequivocal determination with ion-pair chromatography-tandem mass spectrometry.

A comprehensive method for the determination of four stilbene-type disulfonate and one distyrylbiphenyl-type fluorescent whitening agents (FWAs) in paper materials (napkin and paper tissue) and infant clothes was developed. FWAs were extracted from paper material and cloth samples using a hot-water extraction, and the aqueous extracts were then preconcentrated with the newly developed Oasis WAX (mixed-mode of weak anion exchange and reversed-phase sorbent) solid-phase extraction cartridge. The analytes were unequivocal determined by ion pair chromatography coupled with negative electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS-MS), applying a di-n-hexyl-ammonium acetate (DHAA) as the ion-pairing reagent in mobile phase. Limits of quantitation (LOQ) were established between 0.2 and 0.9 ng/g in 2 g of samples. Recovery of five FWAs in spiked commercial samples was between 42 and 95% and RSD (n = 3) ranging from 2 to 11%. The method was finally applied to commercial samples, showing that two stilbene-type disulfonates were predominant FWAs detected in napkin and infant cloth samples.

Chromatography, High Pressure Liquid↗
PubMed2006

Analysis of phenolic compounds in rhubarbs using liquid chromatography coupled with electrospray ionization mass spectrometry.

Rhubarb is an important herbal medicine for the treatment of constipation, inflammation, and cancer. In this study, a facile method based on liquid chromatography coupled with electrospray ionization tandem mass spectrometry has been established for the analysis of bioactive phenolic compounds in rhubarbs. From six rhubarb species, official (Rheum officinale, R. palmatum, and R. tanguticum) and unofficial (R. franzenbachii, R. hotaoense, and R. emodi), a total of 107 phenolic compounds were identified or tentatively characterized based on their mass spectra. These compounds include sennosides, anthraquinones, stilbenes, glucose gallates, naphthalenes, and catechins. Ion chromatograms for the identified compounds of different rhubarbs were then compared. Consistent with previous reports, sennosides and rhein were only detected in official rhubarbs. Unexpectedly, we found that R. officinale contained very different phenolic compounds from the other two official species. Sennoside A, which has been considered as the major purgative component of rhubarb, was only detected in R. officinale, while its close isomers were observed in R. palmatum and R. tanguticum. In addition, the predominant anthraquinone glycosides in R. officinale were found to be rhein 8-O-glucoside and emodin 1-O-glucoside, whereas those in R. palmatum and R. tanguticum were rhein 1-O-glucoside and emodin 8-O-glucoside. Stilbenes, which are the major constituents of unofficial rhubarbs, were also different among the species. Our results clarify the chemical composition of rhubarbs comprehensively for the first time. Due to the significant differences in chemical components of rhubarbs, we suggest that different Rheum species be used separately in clinical practice.

Anthraquinones↗
PubMed2006

An evaluation of four commercial HPLC chiral detectors: a comparison of three polarimeters and a circular dichroism detector.

With increasing frequency, new drug candidates being introduced into pharmaceutical drug pipelines are chiral. Often only one enantiomer exhibits the desired biological activity and the other enantiomer may exhibit undesired side effects, thereby making chiral purity an important parameter. The introduction of chiral analysis adds additional complications in drug development. The pharmaceutical industry is constantly striving to streamline processes and improve efficiencies in an effort to move molecules to market quickly. In order to simplify the process of chiral method development, chiral screening can be set up, however a successful chiral screen depends on optimizing two factors: the column and the detector. The following work investigated the second factor and evaluated two types of commercially available chiral detectors for their possible use in chiral method development and screening: polarimeters and circular dichroism (CD) detectors. Linearity, precision, and the limit of detection (LD) of six compounds (trans-stilbene oxide, ethyl chrysanthemate, propranolol, 1-methyl-2-tetralone, naproxen, methyl methionine) on four commercial detectors (three polarimeters and one CD detector) were determined experimentally and the limit of quantitation (LQ) calculated from the experimental LD. Trans-stilbene oxide worked well across all the detectors, showing good linearity, precision and low detection limits. However, the other five compounds proved to be more discriminating and showed that the circular dichroism detector performed better as a detector for chiral screens, over the polarimeters.

Algorithms↗
PubMed2006

18F-labeled styrylpyridines as PET agents for amyloid plaque imaging.

Positron emission tomography (PET) imaging of beta-amyloid (Abeta) plaques in the brain is a potentially valuable tool for studying the pathophysiology of Alzheimer's disease (AD). It may also be applicable for measuring the effectiveness of therapeutic drugs aimed at lowering Abeta plaques in the brain. We have successfully reported a series of 18F-labeled fluoropegylated stilbenes for PET imaging studies. Encouraging results clearly demonstrated the usefulness of 18F-labeled stilbenes as potential Abeta plaque-imaging agents. In the present study, we applied a similar approach to a styrylpyridine backbone structure. Among all derivatives examined, (E)-2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-5-(4-dimethylaminostyryl)-pyridine (2) displayed high binding affinity in postmortem AD brain homogenates (Ki=2.5+/-0.4 nM, with [125I]IMPY as radioligand). No-carrier-added [18F]2 was successfully prepared by [18F]fluoride displacement of the corresponding tosylate precursor with a high labeling yield (30-40%) and a high radiochemical purity (>99%). Specific activity at the end of synthesis was determined to be 1500-2000 Ci/mmol. The tracer [18F]2 showed adequate lipophilicity (log P=3.22). In vivo biodistribution of [18F]2 in normal mice exhibited excellent initial brain penetration and rapid washout (7.77% and 1.03% dose/g in the brain at 2 and 30 min after intravenous injection, respectively)--properties that are highly desirable for Abeta-plaque-specific brain imaging agents. Autoradiography of AD brain sections and homogenate binding with postmortem AD brain tissues confirmed the high binding signal of [18F]2 due to the presence of Abeta plaques. These preliminary results suggest that novel PET tracers may be potentially useful for the imaging of Abeta plaques in the living human brain.

Animals↗
PubMed2006

Prooxidant DNA breakage induced by caffeic acid in human peripheral lymphocytes: involvement of endogenous copper and a putative mechanism for anticancer properties.

Plant-derived dietary material contains several classes of polyphenols such as flavonoids, curcuminoids, stilbenes and hydroxycinnamic acids. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing cellular DNA degradation in the presence of transition metal ions such as copper. Earlier we have shown that the stilbene resveratrol is able to mobilize endogenous copper ions leading to oxidative breakage of cellular DNA. In this paper, we show that caffeic acid (a hydroxycinnamic acid), which is a major constituent of coffee, is also capable of DNA breakage in human peripheral lymphocytes. Incubation of lymphocytes with neocuproine inhibited the DNA degradation confirming that Cu(I) is an intermediate in the DNA cleavage reaction. Further, we have also shown that caffeic acid generates oxidative stress in lymphocytes, which is inhibited by scavengers of reactive oxygen species and neocuproine. These results are in further support of our hypothesis that anticancer mechanism of plant polyphenols involves mobilization of endogenous copper, possibly chromatin bound copper, and the consequent prooxidant action.

Antineoplastic Agents↗
PubMed2004

Thrombin stimulation of Cl-/HCO3- exchange in human platelets.

The presence of one acidifying Cl-/HCO3- exchange mechanism in human platelets has not been previously reported. This paper demonstrates that this mechanism does function and that it increases its activity after stimulation with thrombin. On resuspension of BCECF-loaded platelets in a chloride-free medium (gluconate replaced) that contains bicarbonate, cytosolic pH (pHi) increased and stabilized after 10 min at an alkaline value. After addition of 50 mM NaCl, pHi fell rapidly reaching steady state in the succeeding 5 min. The stilbene derivative 4-acetamido-4'-isothiocyanato stilbene-2,2' disulfonic acid (SITS) inhibited both, the alkalization in chloride-poor solution and the recovery from the alkaline load after chloride enrichment. The decline in pHi was observed whether chloride was delivered to the solution in the form of LiCl or NaCl, or when the later was applied after blockage of the Na+/H+ exchanger. The recovery in chloride-containing solution was in contrast to the effect of a similar change in osmolarity by addition of 50 mM sodium gluconate that did not produced a significant variation of pHi. Posterior addition of NaCl after 5 min in high gluconate reproduced the pHi fall of the control experiment. Alkali loads produced by 25 mM trimethylamine hydrochloride (TMA) were also counteracted by HCO(3-)-equivalent efflux via Cl-/HCO3- exchange. One of the major observations of the present study is that HCO3- equivalent efflux was twice as high when the platelets were previously stimulated with 0.1 IU of thrombin, but thrombin did not produce significant changes of the pHi recovery rate in a bicarbonate-free solution. The increase of the decline in pHi elicited by preexposure to thrombin was still observed in the presence of an inhibitor of the Na+/H+ exchange or in sodium-free solutions. It is concluded that a Na-independent Cl-/HCO3- exchange mechanism mediates the recovery of pHi from alkalosis in platelets and that thrombin activates this exchanger by a direct regulatory pathway.

Alkalosis↗
PubMed2001

Involvement of anion exchange in the hypoxia/reoxygenation-induced changes in pH(i) and.

The involvement of Cl(-)/HCO(3)(-) exchange in hypoxia/reoxygenation-induced changes in pH(i) and Ca(2+) concentration ([Ca(2+)](i)) was examined in rat ventricular myocytes. During 10-min hypoxia, the initial pH(i) (7.21+/-0.04) fell to below 6.8. Subsequent reperfusion with reoxygenated buffer returned this acidic pH(i) to the neutral range with increases in [Ca(2+)](i). These responses were reduced by the removal of Cl(-) or HCO(3)(-) and by the addition of anion exchange inhibitors, SITS (4-acetamido-4'isothiocyanato-stilbene-2,2'disulfonic acid) and DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid), while inhibitors for the Cl(-) channel and Na(+)/K(+)/2Cl(-) cotransport were without effects. The hypoxia-induced acidification was attenuated by protein kinase C inhibitors, calphostin C and chelerythrine, but not by a protein kinase A inhibitor, KT5720. Under normoxic condition, protein kinase C activation induced a SITS-sensitive acidification. Furthermore, in electrically driven rat papillary muscle, SITS and DIDS improved the recovery of developed tension during the reoxygenation. These results suggest that the hypoxia-induced decrease in pH(i) is mediated at least in part by anion exchange stimulation through protein kinase C activation, and this exchange takes part in the reoxygenation-induced Ca(2+) overload as well as contractile dysfunction.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2003

Does Cl-/HCO3- exchange play an important role in reperfusion arrhythmias in rats?

The protective effects of Cl(-)/HCO(3)(-) exchange inhibitors, 4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'isothiocyanato-stilbene-2,2'-disulfonic acid (SITS), against reperfusion-induced arrhythmias were investigated in anesthetized rats. Rats were subjected to 5-min occlusion of the left coronary artery followed by 10-min reperfusion. All drugs were intravenously administered 5 min before the onset of occlusion. DIDS (75 mg/kg) reduced the incidence of ventricular fibrillation and mortality to 0%, whereas SITS (75 mg/kg) only decreased these parameters to 60%. DIDS simultaneously decreased the mean blood pressure and heart rate, and prolonged PQ and QRS intervals, whereas SITS produced a weaker effect on these parameters and no change in QRS interval. Mexiletine (5 mg/kg), which had been demonstrated to suppress the arrhythmias and reduce the heart rate and mean blood pressure in this model, was shown to prolong PQ and QRS intervals. Verapamil (0.5 mg/kg) or diltiazem (0.4 mg/kg) suppressed the arrhythmias, simultaneously decreasing the heart rate and mean blood pressure and prolonging PQ interval. The results indicate that the protective effect of DIDS on reperfusion arrhythmias in the anesthetized rats is unlikely to be attributed to the inhibitory action on Cl(-)/HCO(3)(-) exchange, but possibly mediated by its blocking effects on cardiac ion channels, such as Na(+) or Ca(2+) channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Functional reassembly of the anion transport domain of human red cell band 3 (AE1) from multiple and non-complementary fragments.

We constructed cDNA clones encoding N-terminal, C-terminal and internal polypeptide fragments of the human red cell anion exchanger (band 3; AE1). The internal fragments comprised between one and seven putative transmembrane spans with two or more spans deleted from both termini of the membrane domain of band 3. Sets of three, four or five complementary fragments, which together represented the complete amino acid sequence of the membrane domain, were co-expressed in Xenopus oocytes. Stilbene disulphonate-sensitive chloride uptake assays revealed that all six of the three-fragment combinations and two of the four-fragment combinations reassembled functionally in vivo. Unexpectedly, co-expression of a non-complementary pair of fragments comprising the first five and last seven putative transmembrane spans (i.e. entirely lacking spans six and seven) was also found to be sufficient to generate stilbene disulphonate-sensitive chloride uptake.

Animals↗
PubMed1983

Procedure for the gas chromatographic-mass spectrometric confirmation of some exogenous growth-promoting compounds in the urine of cattle.

As gas chromatography-mass spectrometry is the most conclusive confirmation technique available today for the detection of ppb levels of anabolics in the urine of cattle, the following procedure was used. The urine is hydrolysed with Helix pomatia intestinal juice, extracted, and the extract cleaned by gel-permeation chromatography or with Extrelut. In one fraction are eluted diethylstilboestrol, dienoestrol, hexoestrol, methyltestosterone, ethinyloestradiol, zeranol and trenbolone. This fraction is injected into a two-dimensional high-performance liquid chromatography system. From the first column, the fraction containing the above-mentioned compounds is transferred to the second column, where a separation into two fractions is obtained. The first fraction contains zeranol and trenbolone, the second fraction the stilbenes, methyltestosterone and ethinyloestradiol. In general, the compounds are derivatized with an N,O-bis (trimethylsilyl)trifluoroacetamide-trimethylchlorosilane mixture to a trimethylsilyl derivative. With stilbene, confirmatory derivatization into heptafluorobutyryl derivatives is necessary. In combination with a Finnigan 4000/INCOS system, a CP-Sil-5 CB and a CP-Sil-19 CB capillary are used for final confirmation. Two capillaries with different polarities are necessary to overcome problems with possible interferences from compounds extracted from the urine. Recoveries at the ppb level are better than 80%.

Anabolic Agents↗