High-Resolution Mass Spectrometry Non-Targeted Detection of Per- and Polyfluoroalkyl Substances in Roe Deer (Capreolus capreolus).

Among wildlife species, roe deer stands out as a valuable indicator of environmental pollution due to its ecological significance and role as a game animal. The assessment of poly- and perfluoro substances (PFASs) bioaccumulation is of the utmost importance, relying on the liver and muscles as the main organs of interest. The study concerned the identification of 60 PFAS through a non-target workflow analysis based on HPLC Q-Exactive Orbitrap High-Resolution Mass Spectrometry in a homogeneous group of 18 female roe deer species. The developed strategy allowed us to individuate the 60 PFAS compounds with different levels of confirmation. Apart from seven PFASs identified via analytical standards, the remaining fifty-three features were identified with CL 2 or 3. Moreover, by applying a differential statistic approach, it was possible to distinguish the bioaccumulation patterns in the liver and muscle, identifying 12 PFAS upregulated in the muscle and 20 in the liver. The analysis reveals that specific PFAS compounds present exclusively in either the muscle or in the liver. The study emphasises the specificity of the liver and muscle as significant bioaccumulation sites for PFAS, raising questions about the underlying mechanisms of this process. In conclusion, the presented non-targeted PFAS analysis workflow evidenced promising and reliable results, successfully demonstrating its feasibility in the field of environmental research.

Influence of Area, Age and Sex on Per- and Polyfluorinated Alkyl Substances Detected in Roe Deer Muscle and Liver from Selected Areas of Northern Italy.

Due to their physicochemical properties, per- and polyfluorinated alkyl substances (PFASs) persist and bioaccumulate in living organisms, causing adverse health effects. Since exposure to xenobiotics is influenced by factors related to both the living organism and the considered compounds, biomonitoring PFASs' presence in the environment is of crucial importance. This study aimed to detect and quantify 15 PFASs in the muscle and liver of 40 roe deer from a specific area in Northern Italy by UPLC-HRMS. In the roe deer, liver PFAS concentrations were higher than those seen in muscle (p < 0.05). Although PFAS content in animals from urbanized areas was higher than those found in deer from rural areas, this difference was not statistically significant. In female roe deer, the concentration was higher than in males (p < 0.05); moreover, older animals showed higher concentrations of PFASs in the liver than younger animals (p < 0.05). In conclusion, the amount of PFASs was higher in tissues from roe deer belonging to urbanized areas, showing that this species might serve as a good bioindicator due to its territorial behavior. PFAS content was significantly higher in female roe deer, although the reason is not fully known. Finally, PFAS concentration was higher in the liver of older animals, probably due to compromised hepatic function.

First Investigation of the Physiological Distribution of Legacy and Emerging Perfluoroalkyl Substances in Raw Bovine Milk According to the Component Fraction.

Bovine milk is a pillar of the human diet and plays a key role in the nutrition of infants. Perfluoroalkyl substances (PFASs) are well-recognized highly stable organic compounds that are able to pollute ecosystems persistently and threaten both human and animal health. The study aimed to analyze the distribution of 14 PFASs within the milk matrix by comparing their content in whole milk, and its skimmed and creamed fractions. Raw milk samples were individually collected from 23 healthy cows (10 primiparous and 13 multiparous) reared on a farm in Northern Italy not surrounded by known point sources of PFASs. Each sample was fractioned in whole, skim, and cream components to undergo PFAS analysis using liquid chromatography-high-resolution mass spectrometry. All samples contained at least one PFAS, with perfluorobutanoic acid (PFBA) being the primary contaminant in all three fractions, followed by perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA). PFOS was shown to be significantly (p < 0.001) more concentrated in cream than in raw and skimmed milk. Multiparous cows showed a higher frequency of positive samples in all analyzed fractions. Further research is necessary to assess the risk of dairy diets and high-fat dairy products and to investigate the toxicological effects of PFASs on cattle, even in environments without known PFAS sources.

Quantification of cortisol and its metabolites in human urine by LC-MSn: applications in clinical diagnosis and anti-doping control.

The objective of the current research was to develop a liquid chromatography-MSn (LC-MSn) methodology for the determination of free cortisol and its 15 endogenous metabolites (6ß-hydroxycortisol, 20α-dihydrocortisol, 20α-dihydrocortisone, 20-ß-dihydrocortisol, 20ß-dihydrocortisone, prednisolone, cortisone, α-cortolone, ß-cortolone, allotetrahydrocortisol, 5α-dihydrocortisol, tetrahydrocortisol, allotetrahydrocortisone, 5ß-dihydrocortisol, tetrahydrocortisone) in human urine. Due to its optimal performance, a linear ion trap operating in ESI negative ion mode was chosen for the spectrometric analysis, performing MS3 and MS4 experiments. The method was validated for limit of detection (LOD) and limit of quantification (LOQ) (0.01 ng mL-1 and 0.05 ng mL-1, for all compounds, respectively), intra- and inter-day precision (CV = 1.4-9.2% and CV = 3.6-10.4%, respectively), intra- and inter-day accuracy (95-110%), extraction recovery (65-95%), linearity (R2 > 0.995), and matrix effect that was absent for all molecules. Additionally, for each compound, the percentage of glucuronated conjugates was estimated. The method was successfully applied to the urine (2 mL) of 50 healthy subjects (25 males, 25 females). It was also successfully employed on urine samples of two patients with Cushing syndrome and one with Addison's disease. This analytical approach could be more appropriate than commonly used determination of urinary free cortisol collected in 24-h urine. The possibility of considering the differences and relationship between cortisol and its metabolites allows analytical problems related to quantitative analysis of cortisol alone to be overcome. Furthermore, the developed method has been demonstrated as efficient for antidoping control regarding the potential abuse of corticosteroids, which could interfere with the cortisol metabolism, due to negative feedback on the hypothalamus-hypophysis-adrenal axis. Lastly, this method was found to be suitable for the follow-up of prednisolone that was particularly important considering its pseudo-endogenous origin and correlation with cortisol metabolism.

The presence of prednisolone in complementary feedstuffs for bovine husbandry.

BACKGROUND: According to European Union legislation, prednisolone, a steroid that belongs to the glucocorticosteroid group, is banned as a growth promoter in cattle husbandry and therefore should not be present in bovine feedstuffs. As our preliminary investigations detected prednisolone in this matrix, we performed a study on different commercially available complementary feedstuffs, stored at the farm and/or in the laboratory, in order to verify whether its presence was due to neo-formation during storage. RESULTS: Prednisolone was detected in almost all (95%) feedstuffs collected at the farm.When the feedstuffs were stored at the laboratory, the frequency (31%) and the concentration of prednisolone-positives were lower. This difference, which is likely due to different environmental conditions, implies the possibility of its neo-formation. CONCLUSION: Our data indicate that the neo-formation of prednisolone can occur in feedstuff, and that the frequency and concentration could be related to the storage conditions. The individuation of an objective parameter that is useful for the identification of the compliance of feed is therefore essential.

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/MS(n). 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.

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.

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.

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.

Evaluation of boldenone formation and related steroids transformations in veal faeces by liquid chromatography/tandem mass spectrometry.

It is established that bovine urine can result positive for boldenone and androstadienedione in consequence of faecal contamination. The simple transfer of steroids to urine is one minor aspect of faecal contamination. A high de novo production of steroids in faeces after deposition and in faeces-contaminated urine is almost certainly due to microbial activity, although the precursor compounds and transformations leading to the presence of these illegal steroids are unclear. We developed a simple in vitro method - incubation of faecal matter suspended in 0.9% saline - to induce steroid transformations in faeces, and analyzed the products by liquid chromatography/tandem mass spectrometry, without the need for prior extraction. Norethandrolone was the internal standard. The linearity (R(2): 0.987-0.999), sensitivity (LODs: 0.3 to 1.0 ng/mL; LOQs: 1.0 to 3.0 ng/mL), precision (intra-day CVs: 2.6-8.2; inter-day CVs: 4.5-11.5) and accuracy (percentage recovery: 89-120%) were calculated for the studied steroids. Androstenedione, androstadienedione, alpha- and beta-boldenone, testosterone and epitestosterone transformations were investigated. Mutual interconversion of steroids was observed, although 17beta-hydroxy steroids had low stability compared with 17alpha-hydroxy and 17-keto steroids. The results suggest that this simple in vitro system may be an effective way of studying hormone transformations in faeces and, after analogue studies, in faeces-contaminated urine.

A case of fatal intoxication from metformin.

A case of fatal intoxication from metformin is presented. The decedent was an obese 58-year-old-woman with type II diabetes, in whom severe lactic acidosis secondary to metformin accumulation was precipitated by acute renal failure. She had been on metformin 500 mg twice a day. Postmortem blood was deproteinated with acetonitrile, washed with dichloromethane, and the resulting supernatant injected into high-performance liquid chromatography system. Separation was performed on a analytical 125 x 4 mm i.d. RP-8 column. The wavelength was set at 235 nm. The mobile phase was acetonitrile (40%), sodium lauryl sulfate, and sodium dihydrogen phosphate adjusted to pH 5.1 (60%) at a flow rate of 1.0 mL/min. The concentration of metformin in postmortem blood was 77.3 microg/mL. The qualitative result was also confirmed by LC/APCI/MS/MS analysis.

Rapid test by liquid chromatography/tandem mass spectrometry to evaluate equine urine reactivity towards 17beta-OH steroids.

Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, demonstrating the stereoselectivity of the reaction. A rapid test was performed by spiking one of the four 17beta-OH steroids in samples of diluted equine urine. The steroids were transformed into their respective ketones in the presence of bacterial activity. The test allows direct injection of diluted samples into the LC/MS system, without the need for prior extraction. Results show that the best method of storage is freezing at -18 degrees C. Urine specimens should be analyzed as soon as possible after thawing. This allows bacterial degradation of equine urine to be arrested temporarily, so that the urine can be used for qualitative or quantitative analysis of 17beta-OH steroids.