Percutaneous transhepatic cholangioscopy for benign and malignant biliary disease using a novel short single-operator cholangioscope.

BACKGROUND: The length of conventional single-use cholangioscopes poses a challenge for percutaneous or laparoscopic approaches for direct visualization of the biliary tract. The aim of this retrospective observational clinical study was to assess the use of a dedicated percutaneous short single-operator cholangioscope (PSSOC) for diagnosis and treatment of benign or malignant biliary diseases. METHODS: Retrospective analysis of a prospectively maintained database including all consecutive patients undergoing percutaneous transhepatic cholangioscopy with the PSSOC between 06/2021 and 01/2023. RESULTS: Forty patients were included (22F/18 M, age 58.7 ± 16.7 years). The diagnostic and therapeutic management plan was based on procedural findings. Indications were bile duct obstruction associated with complex anatomy (n = 13), choledocholithiasis (n = 11), suspected malignant stenosis of the biliary tract (n = 11), biliary stent placement (n = 2) and removal (n = 1), and failed endoscopic retrograde cholangiopancreatography (n = 2). The cholangioscopies were diagnostic (n = 5), therapeutic (n = 20) or both simultaneously (n = 15). The most frequent procedures were electrohydraulic lithotripsy (n = 25) and biopsy sampling (n = 12). Complications occurred in 7 cases (17.5%), including cholangitis (n = 4, B2), pleural perforation (n = 1, B2), portal bleeding (n = 1, B3), and Tako-Tsubo syndrome (n = 1, B3), classified according to the Society of Interventional Radiology classification. Intraprocedural visual diagnosis was confirmed by the histopathologic result in 11/12 patients in which biopsies were performed (91.7%). PSSOC was relevant to avoid surgery in 2 patients (5%) with indeterminate strictures, allowing to rule out malignancy and treat the lithiasis. CONCLUSIONS: Direct visualization of the biliary tract enabled targeted biopsies for histopathological diagnosis. The visual and histopathological diagnoses were concordant in all but one case. Percutaneous cholangioscopy with a dedicated PSSOC allows to optimize identification and treatment of complex biliary disease including biliary lithiasis while assessing bile duct patency. The clinical use of the novel PSSOC system was safe and effective and could prevent surgical exploration in select patients.

Metabolism of norethisterone in the greyhound.

RATIONALE: Norethisterone has been used as a successful oral contraceptive in humans for many years. It was recently permitted for use as an oestrus suppressant in racing greyhounds. To monitor the use of norethisterone as part of a routine drug surveillance programme, knowledge of its metabolism was required to enable detection. METHODS: Gas chromatography/mass spectrometry and selective derivatisation techniques have been used to identify urinary metabolites of norethisterone following oral administration to the greyhound. Metabolites were extracted using solid-phase and liquid-liquid extraction techniques. RESULTS: Several metabolites were identified, including reduced, mono-, di- and trihydroxylated steroids. The major metabolites observed were 17α-ethynyl-5ß-estrane-3α,17ß-diol, 17α-ethynyl-5α-estrane-3ß,17ß-diol, three 17α-ethynylestranetriol stereoisomers and two 17α-ethynylestranetetrol stereoisomers. The major metabolites were predominantly excreted as glucuronic acid conjugates and detection of the administration of norethisterone was possible for up to 8 days post-dose using the methods described. The nandrolone metabolites, 19-norepiandrosterone, estranediol and 19-noretiocholanolone, were also identified in the post-administration samples collected up to 8 h after dosing the treated animals. CONCLUSIONS: The urinary metabolites identified in this study have further increased the knowledge of steroid metabolism in the greyhound, providing information to support routine drug testing programmes for greyhound racing.

Application of a novel material in the inguinal region using a totally percutaneous approach in an animal model: a new potential technique?

PURPOSE: To evaluate the feasibility and safety of a new percutaneous image-guided surgery technique to simulate a hernia repair using hydrogel. MATERIALS AND METHODS: A comparative prospective study was conducted in animals, with survival. Five pigs without any hernias were used. A hydrogel was injected at a site corresponding to the preperitoneal inguinal region. This procedure was performed bilaterally. An image-guided needle (ultrasound and computed tomography) was used, through which the material was injected. After survival, the local and systemic inflammatory reaction generated by the new material, was studied. RESULTS: All animals survived the procedure. No hemorrhagic or infectious complications were reported. The solidification of the material occurred as expected. In eight out of ten cases, the material was found in the planned site. No systemic inflammatory reaction secondary to the administration of hydrogel was reported. The adhesion of the material to surrounding tissues was satisfactory. CONCLUSION: The introduction of a liquid material which solidifies after injection in a short time (hydrogel) using a needle is feasible. The combined CT-scan and US image guidance allows for the percutaneous placement of the needle in the required location. The introduced hydrogel remains in this space, corresponding to the inguinal region, without moving. The placed hydrogel compresses the posterior wall composed of the transversalis fascia, supporting the potential use of hydrogel for hernia defects.

Application of stable carbon isotope analysis to the detection of 17beta-estradiol administration to cattle.

The use of anabolic agents in food producing animals is prohibited within the EU since 1988 (96/22/EC directive). The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as far as no definitive method and non-ambiguous analytical criteria are available. The ability of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of 17beta-estradiol to bovine has been investigated in this paper. By comparison of 13C/12C isotopic ratio of main urinary estradiol metabolite, i.e. 17alpha-estradiol, with two endogenous reference compounds (ERCs), i.e. dehydroepiandrosterone (DHEA) and 5-androstene-3beta,17alpha-diol, the differentiation of estradiol metabolite origin, either endogenous or exogenous, has been proved to be achievable. After treatment, the delta(13)C(VPDB)-values of 17alpha-estradiol reached -27 per thousand to -29 per thousand, whereas delta13CVPDB-values of DHEA remained between -13 per thousand and -20 per thousand depending on the diet, maize and grass, respectively. A significant difference of delta13CVPDB between ERCs and 17alpha-estradiol was measurable over a period of 2 weeks after estradiol ester administration to the animal.

Steroids in equine testes: the identification of endogenous 19-hydroxy and 19-nor neutral steroids by gas chromatography--mass spectrometry.

After homogenization of testicular tissue from stallions aged 1, 2 and 5 years, the unconjugated and conjugated steroids were isolated by a combined solvent-solid extraction procedure. The conjugates were further separated into glucuronides and sulphates by chromatography using Sephadex LH-20. After enzyme hydrolysis and solvolysis of the respective conjugate classes, the three extracts, unconjugated steroids, aglycones and solvolysed sulphates, were purified by chromatography using Kieselgel 60H columns. Five fractions were resolved from each extract; an aliquot of each fraction was derivatized to form the methoxime-trimethylsilyl ethers and the steroids were identified by combined gas chromatography-mass spectrometry. The results have shown that in stallion testes (1) steroidogenesis proceeds by both the 4-ene and the 5-ene pathways, (2) age-linked changes occur in both unconjugated and sulphoconjugated steroid fractions and (3) 19-hydroxy androgens and the 19-nor (C18) neutral steroids (19-norandrostenedione and 19-nortestosterone) are detected only in the unconjugated fraction whereas oestrene, the isomers of oestradiol and of 5(10)-oestrene-3,17-diol are the only steroids detected in the sulphoconjugate fraction. It is suggested that the unconjugated 19-oxygenated androgens present in stallion testes are converted to 19-nor neutral steroids by a reverse aldol reaction and a mechanism showing the putative intermediates in their formation is illustrated.

3-Hydroxy- and 3-keto-3-phenylpropionic acids: novel metabolites of benzoic acid in horse urine.

The metabolism of benzoic acid has been examined in the horse, using 14C- and deuterium-labelled compounds. Chromatographic analysis of the urine showed the presence of hippuric acid, benzoyl glucuronide and benzoic acid and a discrete band which accounted for 2% of the dose administered. This material was isolated by solvent extraction and HPLC and, following treatment with diazomethane, examined by GC/MS. The major component of this fraction was 3-hydroxy-3-phenylpropionic acid methyl ester, which was accompanied by very much smaller amounts of cinnamic acid methyl ester and acetophenone. The two latter minor components have been shown to be artefacts produced during workup and analysis. Cinnamic acid methyl ester arises by the thermal decomposition of 3-hydroxy-3-phenylpropionic acid methyl ester on the GC column. It is proposed that acetophenone has formed, during workup, by decarboxylation of 3-keto-3-phenylpropionic acid. It is suggested that 3-hydroxy and 3-keto-3-phenylpropionic acids, which are also endogenous in horse urine, have arisen by an addition of a 2 carbon fragment to benzoyl CoA, in a sequence analogous to the reactions of fatty acid biosynthesis. Some implications of the metabolic interrelationships between xenobiotic acids and fatty acids are discussed.

Investigations into the biosynthetic pathways for classical and ring B-unsaturated oestrogens in equine placental preparations and allantochorionic tissues.

In on-going studies of 'classical' and ring B-unsaturated oestrogens in equine pregnancy, the products of metabolism of [2,2,4,6,6-2H(5)]-testosterone and [16,16,17-2H(3)]-5,7-androstadiene-3 beta,17 beta-diol with equine placental subcellular preparations and allantochorionic villi have been identified. Using mixtures of unlabelled and [2H]-labelled steroid substrates has allowed the unequivocal identification of metabolites by twin-ion monitoring in gas chromatography-mass spectrometry (GC-MS). Two types of incubation were used: (i) static in vitro and (ii) dynamic in vitro. The latter involved the use of the Oxycell cartridge (Integra Bioscience Systems, St Albans, UK) whereby the tissue preparation was continuously supplied with supporting medium plus appropriate cofactors in the presence of uniform oxygenation. [2H(5)]-Testosterone was converted into [2H(4)]-oestradiol-17 beta, [2H(4)]-oestrone and [2H(3)]-6-dehydro-oestradiol-17 alpha in both placental and chorionic villi preparations, but to a greater extent in the latter, confirming the importance of the chorionic villi in oestrogen production in the horse. On the basis of GC-MS characteristics (M(+) m/z 477/482 (as O-methyl oxime-trimethyl silyl ether), evidence for 19-hydroxylation of testosterone was found in static incubations, while the presence of a 6-hydroxy-oestradiol-17 alpha was recorded in dynamic incubations (twin peaks in the mass spectrum at m/z 504/507, the molecular ion M(+)). It was not possible to determine the configuration at C-6. The formation of small, but significant, quantities of [2H(4)]-17 beta-dihydroequilin was also shown, and a biosynthetic pathway is proposed. In static incubations of placental microsomal fractions, the 17 beta-dihydro forms of both equilin and equilenin were shown to be major metabolites of [2H(3)]-5,7-androstadiene-3,17-diol. Using static incubations of chorionic villi, the deuterated substrate was converted into the 17 beta-dihydro forms of both equilin and equilenin, together with an unidentified metabolite (base peak, m/z 504/506). The isomeric 17-dihydroequilins were also obtained using the dynamic in vitro incubation of equine chorionic villi, together with the 17 beta-isomer of dihydroequilenin. Confirmation of the identity of 17 beta-dihydroequilin and 17 beta-dihydroequilenin was obtained by co-injection of the authentic unlabelled steroids with the phenolic fraction obtained from various incubations. Increases in the peak areas for the non-deuterated steroids (ions at m/z 414 (17 beta-dihydroequilin) and 412 (17 beta-dihydroequilenin) (both as bis-trimethyl silyl ether derivatives) were observed. Biosynthetic pathways for formation of the ring B-unsaturated oestrogens from 5,7-androstadiene-3 beta,17 beta-diol are proposed.

Isolation and characterisation of a C(18) neutral steroid, oestra-5(10),7-diene-3,17-diol, from pregnant mare urine and allantoic fluid. Facile oxidation to yield oestra-5(10),6,8-triene-3, 17-diol (diol of Heard's ketone).

Oestradiene-3,17-diol and oestratriene-3,17-diol (or the diol of Heard's ketone (3-hydroxy-5(10),6,8-oestratriene-17-one) have been extracted on a large scale from pooled urines and allantoic fluid obtained from pregnant mares. Initial purification was achieved using column chromatography, and further purification by high performance liquid chromatography or silver nitrate (argentation) thin layer chromatography. The steroids were characterised using gas chromatography-mass spectrometry. Positions of the double bonds in ring B of oestradienediol were deduced on the basis of results of ultraviolet (UV) and nuclear magnetic resonance (NMR) spectroscopy, hydrogenation, and incubation studies with the enzyme 5-ene-3beta-hydroxysteroid dehydrogenase/steroid-4,5-isomerase. The reference steroid, 5,7-cholestadien-3beta-ol (7-dehydrocholesterol), with its conjugated double bond system, behaved entirely differently to oestradienediol, consistent with the latter having no conjugated system. These data, together with detailed results of NMR studies, have led us to designate the positions of the double bonds in oestradienediol as 5(10),7-. The instability of the dienediol became apparent when the steroid was converted to its bis-trimethylsilyl (TMS) ether. The phenomenon was exacerbated when derivatisation was performed at elevated temperatures or when the fraction containing the dienediol was stored at 4 degrees C prior to being derivatised. The facile oxidation product was shown to be 5(10),6, 8-oestratriene-3,17-diol, implying that the two steroids are related and, furthermore, that all the sites of unsaturation are in the B ring. Because of the facile oxidation of oestradienediol to oestratrienediol (the diol of Heard's ketone), we propose, that this, and by implication, Heard's ketone itself, are artefacts of the isolation procedures which were utilised in the original studies. A possible mechanism is proposed for the biosynthesis of 5, 7-oestradienediol from a ring-B unsaturated C(19) compound, involving C(19) demethylation without aromatisation.

Cannulation in situ of equine umbilicus. Identification by gas chromatography-mass spectrometry (GC-MS) of differences in steroid content between arterial and venous supplies to and from the placental surface.

Equine umbilicus was cannulated in utero and a series of cord plasma samples removed for analysis. After steroid extraction and derivatisation, gas chromatographic-mass spectrometric (GC-MS) analysis demonstrated large differences in steroid content between the plasma samples obtained from the umbilical artery and vein, the blood supplies leading to and from the placental surface, respectively. 3Beta-hydroxy-5,7-androstadien-17-one, dehydroepiandrosterone, pregnenolone, 3beta-hydroxy-5alpha-pregnan-20-one, 5-pregnene-3beta,20beta-diol and 5beta-pregnane-3beta,20beta-diol were identified as major constituents in extracts from umbilical arterial plasma samples, mostly as unconjugated steroids. Together with 5alpha-pregnane-3,20-dione, these steroids were identified in extracts from umbilical venous plasma samples but at significantly reduced levels to those determined in arterial plasma samples. Oestradiol-17alpha, dihydroequilin-17alpha and dihydroequilenin-17alpha were identified in extracts (mostly sulphate-conjugated) from both umbilical arterial and venous plasma samples, much larger amounts being detected in the plasma sampled from, rather than to, the placental surface. Equilin, equilenin, oestrone, oestradiol-17beta, dihydroequilin-17beta and dihydroequilenin-17beta were not detected in the present studies. Isomers of 5(10)-oestrene-3,17beta-diol together with 5(10),7-oestradiene-3,17beta-diol and its possible oxidative artifact, 5(10),7,9-oestratriene-3,17beta-diol, were tentatively identified only in sulphate-conjugated extracts from umbilical venous plasma samples. No glucuronic acid-conjugated steroids could be detected. The implications of this work in the elucidation of the biosynthetic pathways leading to both the formation of oestrogens and C18 neutral steroids at the placental surface are discussed.

Studies into aromatase activity associated with fetal allantochorionic and maternal endometrial tissues of equine placenta. Identification of metabolites by gas chromatography mass spectrometry.

Maternal endometrial and fetal allantochorionic tissues were separated manually from the placentae of seven healthy thoroughbred and three pony mares, ranging in gestational age from 100 to 318 days. The homogeneity of subcellular fractions prepared from these tissues was assessed initially using the marker enzymes, succinate dehydrogenase, NADPH cytochrome C reductase and lactate dehydrogenase for the mitochondrial, microsomal and cytosolic fractions, respectively. Light microscopy and histochemical analysis demonstrated that the separated fetal allantochorionic membrane, which is made up of allantoic and chorionic epithelia, contained no significant contamination of maternal tissues. The maternal endometrium, however, was found to contain appreciable amounts of fetal chorion torn off during the separation process. Tissue homogenates and subcellular fractions were incubated with testosterone together with [4-(14)C] and [(2)H5 or (2)H3] labelled analogues in either an NADPH (1 mM) or a NADPH-regenerating environment; control experiments (without additional cofactor) were also performed. After extraction of the tissue homogenates, neutral and phenolic (oestrogen) unconjugated steroids were separated by column chromatography. Radiolabelled studies revealed that in allantochorionic tissue incubations 67-77% of testosterone was converted to oestrogenic material, subcellular fractionation indicating that oestrogen production was largely confined to the microsomal fraction and time-course studies showing that the rate of formation appeared to be linear up to 90 min. In contrast, only 5-25% conversion occurred using maternal endometrial tissues, which could be accounted for by the contaminating presence of fetal chorion. No oestrogen production was detected in control incubations. These radiolabelled studies demonstrate that aromatase activity is located on the fetal allantochorionic surface and, together with the histochemical data, further delineate this activity to the chorion in mature equine placenta. Gas chromatographic-mass spectrometric (GC-MS) analysis of the phenolic extracts from allantochorionic tissue homogenate incubations indicated the presence of substrate-derived oestradiol-17beta (E2), 6-oxo-oestradiol-17beta (6-oxo-E2) and 6beta-hydroxyoestradiol-17beta (6beta-OH-E2). Whereas all three oestrogens were identified as metabolites from testosterone in incubations performed using allantochorionic tissue homogenates and post-mitochondrial suspensions (PMS), only E2 was identified from incubations performed using microsomal fractions prepared from this tissue. We conclude that both the microsomal and cytosol fractions are required for the conversion of E2 to the 6-oxygenated species in vitro. Using stable isotope-labelled substrates and GC-MS analysis the mechanism of formation of these metabolites from these in vitro incubation studies may be inferred. GC-MS analysis of the neutral extracts from allantochorionic tissue homogenate incubations confirmed the presence of small quantities of substrate-derived 5(10)-oestrenediols. No substrate-derived 5(10)-oestrene-3,17-diols were detected in extracts from microsomal preparations incubated in the absence of cytosol. These data suggest that demethylation of C19 steroids to produce C18 neutral steroids may require the synergistic action of enzymic activities that appear to reside both in the microsomal and cytosolic fractions of equine allantochorionic tissues.

Electrospray collision-induced dissociation of testosterone and testosterone hydroxy analogs.

Complications with the gas chromatographic analysis of steroids prompted the use of alternative techniques for their identification. High-performance liquid chromatography/mass spectrometry with atmospheric pressure ionization allowed the collection of data for structural identification of these compounds. The objective of this study was to investigate the up-front collision-induced dissociation (UFCID) electrospray ionization (ESI) mass spectra of testosterone and monohydroxylated testosterones. The positive ion UFCID ESI mass spectrum of testosterone showed three significant ions at m/z 97, 109 and 123. The relative abundance of these ions in the UFCID ESI mass spectra of monohydroxylated testosterones varied with the position of the hydroxy group. Statistical data allowed the prediction of hydroxy group position on testosterone by evaluation of the relative abundance of the m/z 97, 109, 121 and 123 ions. Data from the ESI mass spectral analysis of testosterone in a deuterated solvent and from the analysis of cholestenone and 4-androstene-3 beta, 17 beta-diol indicated that the initial ionization of testosterone occurred at the 3-one position. CID parent ion monitoring analyses of the m/z 97, 109 and 123 ions indicated that each resulted from different fragmentation mechanisms and originated directly from the [M + H]+ parent ion. The elemental composition of these fragment ions is proposed based on evidence gathered from the CID analysis of the pseudo-molecular ions of [1,2-2H2]-, [2,2,4,6,6-2H5]-, [6,7-2H2]-, [7-2H]-, [19,19,19-2H3]- and [3,4-13C2]testosterone. The structure and a possible mechanism of formation of the m/z 109 and 123 ions is presented. The results of this study advance the understanding of the mechanisms of collision-induced fragmentation of ions.

Immunoaffinity chromatography combined on-line with high-performance liquid chromatography-mass spectrometry for the determination of corticosteroids.

On-line coupled immunoaffinity chromatography-reversed-phase high-performance liquid chromatography (IAC-HPLC) with detection by quadrupole ion trap mass spectrometry using a particle beam interface has been developed for the determination of the steroids, dexamethasone and flumethasone. HEMA (polyhydroxyethylmethacrylate) was evaluated as a support material for the anti-dexamethasone antibodies used in IAC. Antibody cross-reactivity and non-specific binding have been investigated for the HEMA bound anti-dexamethasone IAC column. The on-line IAC-HPLC-MS determination of dexamethasone and flumethasone in post-administration equine urine samples showed precisions (R.S.D.) of 8.0 and 7.1%, respectively, with limits of detection in the range 3-4 ng/ml.

The use of stable isotopes and gas chromatography/mass spectrometry in the identification of steroid metabolites in the equine.

Stable isotope gas chromatography/mass spectrometry has been used successfully in the elucidation of structures of urinary steroid metabolites in the horse and in the identification of metabolites isolated from in vivo perfusion and in vitro incubation studies using equine tissue preparations. Deuterium-labeled steroids, testosterone, dehydroepiandrosterone, and 5-androstene-3 beta,17 beta-diol have been synthesized by base-catalyzed isotope exchange methods and the products characterized by gas chromatography/mass spectrometry. [16,16(-2)H2]Dehydroepiandrosterone (plus radiolabeled dehydroepiandrosterone) was perfused into a testicular artery of a pony stallion and was shown to be metabolized into 2H2-labeled testosterone, 4-androstenedione, isomers of 5-androstene-3,17-diol, 19-hydroxytestosterone, and 19-hydroxy-4-androstenedione. In further studies, equine testicular minces have been incubated with 2H2-labeled and radiolabeled dehydroepiandrosterone and 5-androstene-3 beta, 17 beta-diol. The metabolites, whose identity was confirmed by stable isotope gas chromatography/mass spectrometry, proved the interconversion of the two substrates, as well as formation of testosterone and 4-androstenedione. The aromatization of dehydroepiandrosterone was also confirmed, together with the formation of an isomer of 5(10)-estrene-3,17-diol from both substrates showing 19-demethylation without concomitant aromatization. In studies of the feto-placental unit, the allantochorion was shown to aromatize [2H5]testosterone to [2H4]estradiol, the loss of one 2H from the substrate being consistent with aromatization of the A ring. The formation of 6-hydroxyestradiol was also confirmed in this study. The same technique has been valuable in determining the structure of two metabolites of nandrolone isolated from horse urine.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection and identification of carprofen and its in vivo metabolites in greyhound urine by capillary gas chromatography-mass spectrometry.

Rimadyl (carprofen) was administered orally to the racing greyhound at a dose of 2.2 mg kg(-1). Following both alkaline and enzymatic hydrolysis, postadministration urine samples were extracted by mixed mode solid-phase extraction (SPE) cartridges to identify target analyte(s) for forensic screening and confirmatory analysis methods. The acidic isolates were derivatised as trimethylsilyl ethers (TMS) and analysed by gas chromatography-mass spectrometry (GC-MS). Carprofen and five phase I metabolites were identified. Positive ion electron ionisation (EI(+)) mass spectra of the TMS derivatives of carprofen and its metabolites show a diagnostic base peak at M(+)*. -117 corresponding to the loss of COO-Si-(CH(3))(3) group as a radical. GC-MS with positive ion ammonia chemical ionisation (CI(+)) of the compounds provided both derivatised molecular mass and some structural information. Deutromethylation-TMS derivatisation was used to distinguish between aromatic and aliphatic oxidations of carprofen. The drug is rapidly absorbed, extensively metabolised and excreted as phase II conjugates in urine. Carprofen, three aromatic hydroxy and a minor N-hydroxy metabolite were detected for up to 48 h. For samples collected between 2 and 8 h after administration, the concentration of total carprofen ranged between 200 and 490 ng ml(-1). The major metabolite, alpha-hydroxycarprofen was detected for over 72 h and therefore can also be used as a marker for the forensic screening of carprofen in greyhound urine.

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.

A retrospective study of increased plasma progestagen concentrations in compromised neonatal foals.

Plasma progestagen concentrations were measured daily by radioimmunoassay (RIA) in 35 sick foals for the duration of their illness. The foals were divided into three groups on the basis of time to stand after birth. Foals were given intensive care treatment according to the severity of their illness. Plasma and urine concentrations of pregnenolone (P5) and pregnenediol (P5 beta beta) were measured by gas chromatography--mass spectrometry; plasma cortisol concentrations were measured by RIA and the foals' renal and respiratory status were assessed by creatinine clearance ratios and arterial oxygen concentrations respectively. Five patterns of plasma progestagen concentrations were identified; in general, values increased when the foal's clinical condition deteriorated and decreased as the foal improved. Median progestagen concentrations decreased over the first three days post partum in Group 1 foals but remained elevated in foals from Groups 2 and 3. Similar changes were observed in plasma P5 and P5 beta beta concentrations. Plasma cortisol concentrations were highest in foals from Groups 2 and 3 (P < 0.01) compared with foals from Group 1. Regardless of foal group, mean cortisol concentrations were highest (P < 0.001) in those foals treated with adrenocorticotrophic hormone compared with those treated with dexamethasone or with neither drug. There was no relationship (r2 = 0.21) between plasma cortisol and progestagen concentrations. Results from renal clearance, steroid conjugation and respiratory status suggest that these factors did not play a significant role in elevating progestagen concentrations in sick foals. It is hypothesized that there may be a relationship between adrenal stimulation and an enzyme block resulting in overproduction of P5 and P5 beta beta in the sick neonatal foal.

ELISA screening with GC-MS confirmation of the tranquilizer chlorprothixene administered in subtherapeutic doses to horses.

A commercially available generic promazine ELISA kit is available which shows cross-reactivity for the tranquilizer chlorprothixene (CPT). The ELISA test readily detects the presence of CPT or its metabolites in equine urine for up to 24 h after the i.v. and i.m. administration of sub-therapeutic doses (4.5 mg) to three horses. Maximum concentrations (CPT equivalents) are obtained 2 h after i.v. dosing. No distinct concentration peak values are observed after i.m. administration. Following solid-phase extraction, confirmation of CPT and its metabolites by electron impact mass spectrometry after sub-therapeutic administration is not successful. The use of chemical ionization mass spectrometry however revealed the presence of at least four metabolites including; chlorprothixene sulphoxide, hydroxylated chlorprothixene and hydroxylated chlorprothixene sulphoxide.

Effects of maternally administered depot ACTH(1-24) on fetal maturation and the timing of parturition in the mare.

The aims of this study were to ascertain 1) whether fetal maturation could be induced precociously by maternal administration with adrenocorticotrophic hormone (ACTH) and 2) whether maturation could be achieved without significant risk to mare or fetus. Twenty-two mares received either 1 mg (low dose, LD, n = 6) or 4 or 5 mg (higher dose, HD, n = 16) synthetic Depot ACTH(1-24) at 300, 301 and 302 days gestation. Because, during the course of the study, ACTH appeared to have a greater influence on mares mated during the later part of the breeding season, the HD group were divided retrospectively into those mated before (HDE, n = 6), or after (HDL, n = 10), 1st July. All LD mares were mated before 1st July. Control injections were not performed but gestational data were compared retrospectively with 64 untreated, spontaneously foaling pony mares mated between May and October. Plasma progestagen and cortisol concentrations increased significantly (P<0.05) following ACTH administration in all groups, but progestagens were higher and cortisol elevated for longer in HD mares. ACTH stimulated mammary development and milk electrolyte changes in HD mares. Mean +/- s.e. gestation period (days) was significantly (P<0.01) shorter in HDL mares (318 +/- 1.8) compared with LD (335 +/- 3.7), HDE (340 +/- 4.3) and untreated mares mated after 1st July (327 +/- 1.3). All foals were mature except 2 HDL foals which were stillborn. HDL foals had a higher MCV and lower mean bodyweight, indicating they were delivered before full term. In conclusion, maternal ACTH administration appears to accelerate fetal maturation and delivery in pony mares given high doses and mated late in the breeding season. Further work is required to establish the optimal gestational age and dosage for maternal ACTH administration before clinical recommendations can be given for this therapy.

Increase in plasma progestagen concentrations in the mare after foetal injection with CRH, ACTH or betamethasone in late gestation.

Maternal plasma progestagen concentrations increase about 20 days before parturition. The major contributors to the increase are reduced metabolites (ie 5 alpha-pregnanes). Precocious increases (ie less than 310 days of gestation) in these metabolites may occur in abnormal pregnancies. The effects of CRH, ACTH or betamethasone administered to the foetus at gestational ages ranging from about 250 to 320 days were examined. Sixteen healthy pony mares were used for foetal injection employing aseptic techniques. Water or normal saline were used as controls. Maternal plasma progestagen concentrations were measured using a commercial radioimmunoassay (RIA) progesterone kit and results were confirmed using gas chromatography-mass spectrometry (GC-MS). Results demonstrated clearly that an increase in maternal plasma progestagen concentrations occurred after injection of ACTH, CRH or betamethasone to the foetus, irrespective of gestational age. A comparable increase was not observed in the control animals. Of the 16 mares in which the foetus was injected, 13 produced viable foals at gestational ages ranging from 307 to 339 days whereas 3 mares delivered non-viable foals at 284 to 306 days gestation. The results support the hypothesis that the pre-parturient rise in progestagens occurring in the mare is the result of foetal adrenocortical activity.

Cortisol concentrations in post competition horse urine: a French and British survey.

The purpose of the present report was to estimate the population parameters of cortisol concentrations in urine, an endogenous hormone used as a 'doping' agent and for which an international threshold (1.0 micrograms/ml) has been proposed. Two data bases (French and UK) corresponding to 112 and 142 samples, respectively were considered. Urine was collected under specific post competition conditions. Cortisol concentrations were obtained by validated methods (HPLC for the French samples, and GC-MS for UK samples). No difference was observed between the 2 data sets and statistical analyses were carried out on the two merged files. The overall geometric mean cortisol concentration was 48 ng/ml. Distribution was not Gaussian. A log-normal distribution was not rejected (for P > 0.05). Using the log-normal distribution, it was calculated that the probability of exceeding a cortisol concentration in urine of 1.0 micrograms/ml was 1.1 x 10(-4). It was concluded that the actual international threshold is specific i.e. robust with regard to the risk of erroneously declaring an unmedicated horse as positive.