G. Pompa

Investigation on possible transformations of cortisol, cortisone and cortisol glucuronide in bovine faecal matter using liquid chromatography―mass spectrometry

Given the close resemblance of the ring A structure of prednisolone and prednisone on the one hand, and of androstadienedione on the other, the transformation of cortisol and cortisone into prednisolone and prednisone in cattle faeces was evaluated. A simple method that does not involve extraction but only the 1:100 dilution of cattle faeces, spiking with 400ng/mL cortisol, cortisone or cortisol glucuronide and incubation of the suspension, was used. The analyses were performed by HPLC-MS(3) to detect the supposed Delta(1) dehydrogenation of the glucocorticoids. The decision limits (CCalpha) and detection capabilities (CCbeta) were 2.0 and 3.0ng/mL for cortisol, cortisone and prednisolone, 3.0 and 4.0ng/mL for cortisol glucuronide and 7.0 and 10.0ng/mL for prednisone, respectively. Intra-day and inter-day coefficients of variation (CV%), were 5.6-6.2 and 5.2-6.6 for cortisol glucuronide, cortisol, cortisone and prednisolone, and 16.0 and 16.2 for prednisone, respectively. The recoveries were in the range 110-143% for all analytes. Regression coefficients (R2) were in the range 0.996-0.999 for all analytes. The results show the hydrolysis of the conjugated form and the dehydrogenation in ring A in diluted faeces. It is therefore predicted that urine contaminated with faeces may be positive for prednisone and prednisolone in the same way as they are positive for boldenone, i.e. as a result of microbiological dehydrogenase activity on cortisol and cortisone.

Dioxin and PCB contamination of fish and shellfish: Assessment of human exposure. Review of the international situation

Abbreviations: ADI, acceptable daily intake; LOAEL, least observable adverse effect level; NOAEL, no observable adverse effect level; PCB, polychlorinated biphenyl; PCDD/F, polychlorinated dibenzodioxins and dibenzofurans; SCF, Scientific Committee on Food; TCDD, tetrachlorodibenzodioxin; TDI, tolerable daily intake; TEF, toxic equivalency factor; TEQ, toxic equivalent of 2,3,7,8-TCDD; TWI, tolerable weekly intake

Investigation of the origin of prednisolone in cow urine

After a two-year period of the frequent detection of prednisolone-positive bovine urine samples in the Italian region of Lombardy, studies were initiated to investigate the source. Because the majority of positive samples were detected at the slaughterhouse, researchers hypothesised that, together with increased cortisol and cortisone, a small quantity of prednisolone could be produced by the cows in stressful situations. In the present study, three dairy cows underwent intramuscular treatments with tetracosactide hexaacetate, a synthetic analogue of adrenocorticotropic hormone, to simulate stress. The animals were slaughtered at the end of the study. The results indicated that prednisolone could be detected occasionally in the non-stressful state, but was consistently found in the urine of stressed cows (concentrations ranged from 1.01 to 4.08 ng/mL). To confirm the stress condition, urinary cortisol and cortisone were also detected at high concentrations in the urine, typically at concentrations of hundreds of nanograms per millilitre. The results of this preliminary study did not reveal the metabolic pathway responsible for prednisolone but suggested that this corticosteroid could be produced endogenously.

Neoformation of boldenone and related steroids in faeces of veal calves

Abstract Conflicting findings regarding the boldenone content of bovine faeces suggest it may be synthesized de novo in emitted faeces. We tested this hypothesis by analysing uncontaminated urine, fresh and various forms of dried faeces from 10 calves (not given boldenone) by liquid chromatography/tandem mass spectrometry for 17α- and 17β-boldenone (α and β BOL); 1,4-androstadiene-3,17-dione (ADD); 4-androstene-3,17-dione (AED), testosterone (T) and epitestosterone (ET). Urine contained no α BOL, β BOL or ADD. The analysed substances were variably present in the rectal faeces, and at generally higher levels in faeces scraped from skin or stall floor. In pooled rectal faeces naturally dried for 13 days, α BOL, ADD, AED and ET levels were extremely high (much higher than accounted for by increases due to drying), and β BOL and T were absent. It is concluded that de novo synthesis of α BOL and metabolites occurs naturally in bovine faeces and only uncontaminated urine should be analysed for illegal boldenone.

Aflatoxin B1 binding to sorbents in bovine ruminal fluid.

A recent approach to the problem of contamination of agricultural products by aflatoxin B1 (AFB1) is to add non-nutritional adsorbents to animal diets in order to sequester ingested aflatoxins. We conducted in vitro experiments to develop a rapid and cheap model using ruminal fluid to assess the ability of sorbent materials to bind AFB1. Seven sorbents (hydrated sodium calcium aluminosilicate; clinoptilolite; zeolite; two types of bentonite; sepiolite; and PHIL 75), commonly added to bovine diets were incubated in water and ruminal fluid in the presence of AFB1. Hydrated sodium calcium aluminosilicate, sepiolite and one of the bentonites bound 100% of the AFB1 in the presence of both ruminal fluid and water; clinoptilolite bound about 80% of AFB1 in both liquids; whereas the affinities for the mycotoxin of zeolite (50%) and the other sample of bentonite (60%) in water seem to be increased by about 40% in ruminal fluid incubations. PHIL 75 had the poorest binding ability: about 30% in water and 45% in ruminal fluid. In view of the differences in toxin binding in water and ruminal fluid, it is preferable to use the ruminal fluid model for the in vitro pre-screening of sorbent materials potentially useful as adjuvants to ruminant feeds.

Evidence for false-positive results for Boldenone testing of veal urine due to faecal cross-contamination during sampling

European Directive 96/22/EC, which controls veterinary residues in animals, does not permit the presence of synthetic growth promoters in products of animal origin or in livestock. Boldenone is categorized in class A3 (growth promoters — steroids) and is thus a banned substance. Testing of veal urine for banned substances is part of the European Union statutory programme for animals going into the food chain. In relation to this monitoring, three studies were conducted to investigate the apparent presence of the banned growth promoter boldenone in veal urine, which was suspected as being caused by interference from faecal contamination of the sample. In the first study, urine samples were collected at different times (time 0 and after 30 min) using (1) a conventional zoonotechnical apron and (2) a technique designed specifically to avoid faecal contamination (‘kettle’). This resulted in samples that were, respectively, positive and negative for the presence of α-boldenone (α-BOL). In a second study, urine samples negative to α-BOL were collected from eight veal calves, but became positive after deliberate faecal contamination. In a third study, data obtained from the Italian RNP (Residual National Program) indicated that 18.1% of 3295 urine samples collected using the zootechnical apron were positive for α-BOL and 2.1% for β-boldenone (β-BOL), whilst of 902 samples collected using the kettle, β-BOL was not detected in any samples and only 0.2% were positive to α-BOL, in concentrations lower than 2 ng ml−1. These results further support the supposition that faecal contamination of the urine during sample collection can lead to false-positive results during boldenone analysis.

Investigation on the origin of prednisolone in urine and adrenal glands of cows

Prednisolone is a steroid belonging to the corticosteroid group. The results obtained in the application of the 2008 and 2009 Italian Residue Control Plans show the frequent detection of prednisolone traces in cow’s urine. Since most of the positive samples were detected at the slaughterhouse, the researchers hypothesised that, together with an increase of cortisol concentration, traces of prednisolone could be produced endogenously during stressful situations due to transport and handling before slaughter. In the present trial, 52 lactating cows housed in seven different farms in Lombardy, Italy, were studied. Urine samples were collected at the farm (after urethral catheterisation) and immediately after slaughter (from urinary bladder) together with 40 adrenal gland samples belonging to the same animals. All the samples were analysed for the determination of prednisolone and cortisol by LC/MSn. The results demonstrated that prednisolone can be endogenously produced in dairy cows and, furthermore, its endogenous presence in bovine urine seems to be strongly related to a state of stress in the animals (at the farm and at the slaughterhouse). The data from adrenal glands do not, however, clarify if the endogenous production occurs, partially or totally, in this organ.

Investigation of the presence of endogenous prednisolone in equine urine by high-performance liquid chromatography mass spectrometry and high-resolution mass spectrometry

RATIONALE After the detection of low concentrations of prednisolone in racehorse urine samples collected at Italian racetracks, a study was initiated to investigate the accuracy of the analytical protocol used and the possible endogenous origin of detected prednisolone. METHODS Multiple reaction monitoring (MRM) MS(2) acquisition with a triple quadrupole (n = 780) and full scan MS(2) and MS(3) (n = 180) acquisition with a linear ion trap were checked. As a further confirmation, ten urine samples were analysed by high-resolution mass spectrometry (HRMS). RESULTS The study showed the difficulty of identifying prednisolone, probably due to interfering compounds with the same molecular weight (360 Da) present in the matrix. The characteristic transitions for prednisolone were identified, both in MS(2) and MS(3), as the ions 187 and 280; the ion 295 was also used for identification. The concentrations detected with the triple quadrupole and the linear ion trap were not statistically different. The exact mass of prednisolone formiate (the adduct acting as a molecular ion) was identified by HRMS. CONCLUSIONS The very high frequency of prednisolone detection in the samples (78.5%), the low concentration of this steroid and, importantly, the narrow range of the 95% confidence limits (0.97-1.05 in MS(2) mode and 0.88-1.04 in MS(3) mode), could represent evidence that its presence is endogenous. In the light of these results, this hypothesis seems the most probable, even if further studies are required to confirm it. Furthermore, a microbiological origin (i.e. fermentation of cortisol after sample collection) could not be disregarded.

Presence of endogenous prednisolone in human urine

The possibility of an endogenous presence of the glucocorticoid prednisolone has already been demonstrated in bovine and horse urine, with the aim of clarifying its origin in this matrix, which is used by official agencies for the control of illicit treatments. From this point of view, the endogenous nature of prednisolone could be a major topic in doping control of both amateur and professional human athletes. A study was therefore made on 34 human volunteers (13 males and 21 females; aged 22-62) to detect the presence of prednisolone in their urine by HPLC-MS(3). One of the volunteers underwent vernal allergy treatment with betamethasone for two subsequent years. An investigation was carried out with the aim of verifying if the suppression, and the circadian rhythm, of cortisol urinary levels could also apply to prednisolone. The results of the study show that prednisolone was present in the urine of all 34 volunteers, with a concentration very close to 100-times lower that of cortisol, with no dependence on gender. The same ratio (1/100) was observed in the prednisolone and cortisol levels detected during the 24h together with the suppression of prednisolone by betamethasone treatment. These data demonstrate the endogenous nature of low concentrations of prednisolone in human urine, and motivate further studies about the biosynthetic pathways of this corticosteroid and its relationship with stress in humans, as already described in cows.

Membrane lipid composition of subcellular fractions from lungs of rats treated with paraquat.

Summary Paraquat (125 mg/kg/os) affects body weight, ceruloplasmin plasma levels but not lungs weights of rats within a 192 h observation period. The analyses of cholesterol and fatty acid composition of the subcellular fractions from lungs of the treated rats reveal a significant, early and transient increase of cholesterol in mitochondrial membrane and marked, late reductions of palmitic, stearic, oleic and linoleic acids in cell debris and nuclei as well as in microsomal membranes. The results obtained indicate a probable weakening of the lipid structure of the membranes of these subcellular components.