Bulk to Nanometer-Scale Infrared Spectroscopy of Pharmaceutical Dry Powder Aerosols.

Solid state chemical analysis of pharmaceutical inhalation aerosols at the individual particle level has been an analytical challenge. These particles can range from a few nanometers to micrometers and are a complex mixture of drugs and excipients. Conventional analytical techniques cannot resolve the distribution of excipients and drugs at the submicrometer scale. Understanding the nanochemical composition of individual particles can be critical for pharmaceutical scientists to evaluate drug and excipient stability as well as the drug-drug or drug-excipient interactions that affect the aerosol performance of powders. Herein, we show the novel application of a combination of optical photothermal infrared (O-PTIR) spectroscopy and atomic force microscopy infrared (AFM-IR) spectroscopy to probe nanochemical domains of powders containing the inhaled corticosteroid fluticasone propionate and long-acting ß2-agonist salmeterol xinafoate, which are widely used to treat asthma and chronic obstructive pulmonary disease. Three types of powder formulation were analyzed, including the commercial product Seretide, which is a physical mixture of the drugs with crystalline lactose, and two spray-dried powders containing the drugs along with either amorphous or crystalline lactose. We obtained spatially resolved O-PTIR and AFM-IR spectra confirming the presence of peaks related to fluticasone propionate at 1743, 1661, and 1700 cm-1, salmeterol xinafoate at 1580 cm-1, and lactose at 1030 and 1160 cm-1. The location of the drugs and lactose among the particles varied significantly, depending on the formulation type. For the first time, it was possible to map the drug distribution in individual aerosol particles. This is significant as such information has been lacking, and it will open an exciting research direction on how drug distribution affects the aerosol performance of powders and the consistency of dose uniformity. Further, these advanced spectroscopic techniques can be applied to study a wide range of pharmaceutical formulations.

Steroid Profiling and Immunohistochemistry for Subtyping and Outcome Prediction in Primary Aldosteronism-a Review.

PURPOSE OF REVIEW: Steroid profiling and immunohistochemistry are both promising new tools used to improve diagnostic accuracy in the work-up of primary aldosteronism (PA) and to predict treatment outcomes. Herein, we review the recent literature and present an outlook to the future of diagnostics and therapeutic decision-making in patients with PA. RECENT FINDING: PA is the most common endocrine cause of arterial hypertension and unilateral forms of the disease are potentially curable by surgical resection of the overactive adrenal. Recent studies have shown that adrenal steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be helpful for subtyping unilateral and bilateral forms of PA, classifying patients with a unilateral aldosterone-producing adenoma (APA) according to the presence of driver mutations of aldosterone production in APAs, and potentially predicting the outcomes of surgical treatment for unilateral PA. Following adrenalectomy, immunohistochemistry of aldosterone synthase (CYP11B2) in resected adrenals is a new tool to analyze "functional" histopathology and may be an indicator of biochemical outcomes after surgery. Biochemical and clinical outcomes of therapy in PA vary widely among patients. Peripheral venous steroid profiling at baseline could improve diagnostic accuracy and help in surgical decision-making in cases of a suspected APA; results of "functional" histopathology could help determine which patients are likely to need close post-surgical follow-up for persistent aldosteronism.

Adrenal hormones and circulating leukocyte subtypes in stroke patients treated with reperfusion therapy.

Ischemic stroke sets in motion a dialogue between the central nervous and the immune systems that includes the sympathetic/adrenal system. We investigated the course of immune cells and adrenocortical and adrenomedullary effectors in a cohort of 51 patients with acute stroke receiving reperfusion therapy (intravenous alteplase or mechanical thrombectomy) and its correlation with stroke outcomes and infarct growth. Cortisol increased rapidly and fleetingly after stroke, but 39% of patients who had larger infarctions on admission showed a positive delta cortisol at day 1. It was associated with enhanced infarct growth (p = 0.002) and poor outcome [OR (95% CI) 5.30 (1.30-21.69)], and correlated with less lymphocytes and T cells at follow up. Likewise, fewer circulating lymphocytes, T cells, and Tregs were associated with infarct growth. By contrast, metanephrines did not increase at clinical onset, and decreased over time. Higher levels of NMN correlated with more Treg and B cells. Eventually, complete reperfusion at the end of therapy headed the identification of more circulating Tregs at day 1. Then activation of cortical or medullar compartments of the adrenal gland result in specific signatures on leukocyte subpopulations. Manipulation of the adrenal gland hormone levels warrants further investigation.

Perinatal exposure to venlafaxine leads to lower anxiety and depression-like behavior in the adult rat offspring.

Depression during pregnancy and in the post-partum period is a growing health issue. Venlafaxine, a representative of serotonin and noradrenaline reuptake inhibitors, is used to treat a wide spectrum of mood disorders. However, the limited number of prenatal and perinatal studies raises the question about the long-term consequences of venlafaxine therapy. The aim of this study was to investigate the effect of venlafaxine exposure during pregnancy and lactation on anxiety-like and depression-like behaviors, as well as adrenocortical hormone concentrations in the adult rat offspring. For this purpose, rat dams were treated orally with venlafaxine from day 15 of gestation to postnatal day 20 at doses of 7.5, 37.5, and 75 mg/kg. Administration of venlafaxine during gestation and lactation affected anxiety-like and depression-like behaviors in adult rat offspring of both sexes. The animals exposed through their mothers to venlafaxine, particularly at the lowest and middle doses, were less anxious and less depressive in several relevant behavioral tests, which can be considered a deviation from the normal state. At clinically relevant doses, venlafaxine did not alter circulating level of corticosterone and aldosterone in the adult offspring. In general, the consequences of venlafaxine were dose dependent and more apparent in females. Together, these results suggest that prenatal and early postnatal exposure to venlafaxine may interfere with functional development of the brain, though not necessarily in a negative way.

The concentration of fear: mice's behavioural and physiological stress responses to different degrees of predation risk.

Predation is an unavoidable and dangerous fact in the lifetime of prey animals and some sign of the proximity of a predator may be enough to trigger a response in the prey. We investigated whether different degrees of predation risk by red foxes (Vulpes vulpes) evoke behavioural and physiological stress responses in wood mice (Apodemus sylvaticus). We examined the variation in mice responses due to individual factors (sex and reproductive status) and related them to the concentration of the volatile compounds from fox faeces over time. In our experiment, we introduced predation cues into four plots, each subjected to a different concentration treatment (0, 10, 50 and 100% concentration of fresh faeces of red fox), based on the following outline: initial odourless phase 0, phase1 in which predation treatment was renewed daily, and phase 2 in which we renewed the treatment only on the first day. Wood mice were live trapped during all three phases and the physiological response was measured non-invasively by analysing faecal corticosterone metabolites (FCM) in freshly collected faeces. Data were analysed by Generalized Linear Mixed Models. Overall, males were trapped less often than females, and reproductively active individuals from both sexes avoided traps more than non-reproductively active individuals, especially in medium- and high- concentration plots. Variations in FCM concentrations were explained by plot, the interaction between plot and treatment phase, and the interaction between the treatment phase and the reproductive status. During phase 1, we detected a significant rise in FCM levels that increased with predator faecal odour concentration. Additionally, reproductively active individuals showed a strong physiological response during both phases 1 and 2 in all plots, except the control plot. Our results indicated that wood mice are able to discriminate different degrees of predation risk, which allows them to trigger gradual changes in their behavioural and physiological stress responses.

Supplementing electronic health records through sample collection and patient diaries: A study set within a primary care research database.

PURPOSE: To describe a novel observational study that supplemented primary care electronic health record (EHR) data with sample collection and patient diaries. METHODS: The study was set in primary care in England. A list of 3974 potentially eligible patients was compiled using data from the Clinical Practice Research Datalink. Interested general practices opted into the study then confirmed patient suitability and sent out postal invitations. Participants completed a drug-use diary and provided saliva samples to the research team to combine with EHR data. RESULTS: Of 252 practices contacted to participate, 66 (26%) mailed invitations to patients. Of the 3974 potentially eligible patients, 859 (22%) were at participating practices, and 526 (13%) were sent invitations. Of those invited, 117 (22%) consented to participate of whom 86 (74%) completed the study. CONCLUSIONS: We have confirmed the feasibility of supplementing EHR with data collected directly from patients. Although the present study successfully collected essential data from patients, it also underlined the requirement for improved engagement with both patients and general practitioners to support similar studies.

Application of microwave-assisted extraction and ultra-high performance liquid chromatography-tandem mass spectrometry for the analysis of sex hormones and corticosteroids in sewage sludge samples.

Hormonal compounds are a concern to the international community because they can affect the aquatic biota and are therefore considered to be endocrine-disrupting compounds. These compounds have lipophilic properties, so they tend to accumulate in solid matrices, such as sewage sludge. This work presents the optimization of a microwave-assisted extraction process combined with ultra-high performance liquid chromatography-tandem mass spectrometry for the determination of 15 hormonal compounds in sludge samples. The proposed method has relative standard deviations below 23 %, good recoveries (over 71 %) for all compounds, detection limits that ranged from 1.1 to 7.9 ng g(-1) and quantification limits which ranged from 3.7 to 26.3 ng g(-1). The method was used to analyse sludge samples from four different wastewater treatment plants of Gran Canaria (Spain) with different wastewater treatments. 17ß-estradiol, 17α-ethynylestradiol, norgestrel and cortisone were detected in sludge samples at concentrations that ranged from 17.3 to 1.44 × 10(3) ng g(-1). The developed method permits the use of small quantities of sample and organic solvents, presents short extractions times and is the first one based on microwave-assisted extraction for the analysis of both sex hormones and corticosteroids.

Analysis of salicylic acid, arbutin and corticosteroids in skin whitening creams available in Pakistan using chromatographic techniques.

BACKGROUND: A simple, new and efficient reversed-phase high-performance liquid chromatography method was developed and validated for the separation of most popular ingredients in skin whitening creams. METHODS: For RP-HPLC analysis, a Hibar(®) C18 250 mm × 4.6 mm, 5 µm column (Merck Millipore, Carolina, USA) as stationary phase with a mobile phase consisting a mixture of acetonitrile, methanol and water 40 : 40 : 20 (pH 7.0), respectively, at flow rate of 0.8 mL min(-1) (total run time 10 min) at room temperature was used. Detection was performed at 254 and 280 nm using photodiode array detector. The method was validated in accordance with ICH guidelines with respect to linearity, accuracy, precision, specificity, limit of detection and quantification. RESULTS: The method results in excellent separation of skin whitening agents in cosmetic creams. The method is specific for salicylic acid, arbutin, cortisone, hydrocortisone, betamethasone valerate and betamethasone dipropionate. The calibration curve of skin whitening agents was linear with the regression analysis showed r(2) ≥ 0.999. %RSD for inter- and intraday precision were determined as 0.461 and 0.329 for salicylic acid, 0.427 and 0.317 for arbutin, 0.360 and 0.346 for cortisone, 0.336 and 0.350 for hydrocortisone, 0.463 and 0.339 for betamethasone valerate and 0.385 and 0.372 for betamethasone dipropionate, respectively. LOD and LOQ were calculated as 0.48 and 1.20 µg mL(-1) for salicylic acid, 0.09 and 0.22 µg mL(-1) for arbutin, 0.07 and 0.18 µg mL(-1) for cortisone, 0.06 and 0.24 µg mL(-1) for hydrocortisone, 0.07 and 0.20 µg mL(-1) for betamethasone valerate and 0.02 and 0.06 µg mL(-1) for betamethasone dipropionate. The recovery of skin whitening agents were 97.18% for salicylic acid, 97.99% for arbutin, 98.30% cortisone, 97.63% for hydrocortisone, 98.65% for betamethasone valerate and 98.18% for betamethasone dipropionate, respectively. According to this study, salicylic acid is present in 87.88% skin whitening creams, arbutin in 96.97%, cortisone in 60.60%, hydrocortisone in 48.48%, betamethasone valerate in 15.15% and betamethasone dipropionate present in 12.12% cosmetic creams available in Pakistan.

Liquid chromatography/tandem mass spectrometry for simultaneous determination of undeclared corticosteroids in cosmetic creams.

RATIONALE: Undeclared corticosteroids in creams intended for frequent use might cause serious side-effects, especially in children. In order to prevent this or find the cause, it was essential to develop a method for quick detection and quantification of low levels of corticosteroids. METHODS: Eleven corticosteroids were used in this study: prednisolone, methylprednisolone, prednisolone-21-acetate, fluocinolone acetonide, fluocinolone acetonide-21-acetate, hydrocortisone-21-acetate, dexamethasone, betamethasone, betamethasone dipropionate, clobetasol propionate and triamcinolone. Separation was achieved via liquid chromatography (LC), and mass spectrometric analysis was conducted by electrospray ionization triple-quadrupole mass spectrometry (MS/MS) in the multiple reaction monitoring mode using corticosterone as internal standard. RESULTS: Good separation by using a gradient-elution LC/MS/MS method with run time of 25 min enabled the use of a segmented detection method and consecutive decrease in detection limits. The proposed method has been validated in the linearity range of 10-1000 ng/mL with coefficients of determination higher than 0.990. The method has shown to have very low limits of quantification (0.75-3 ng/mL) with satisfactory precision and accuracy for each of the corticosteroids. CONCLUSIONS: An LC/MS/MS method for the rapid and simultaneous determination of low levels of eleven topical corticosteroids in creams was developed, optimized and validated. The proposed method can be used for testing of different products indicated for the treatment of atopic dermatitis, including "natural products", and "herbal creams" with "miraculous effects".

Corticosteroid adulteration in proprietary Chinese medicines: a recurring problem.

OBJECTIVES: To investigate adulteration of proprietary Chinese medicines with corticosteroids in Hong Kong. DESIGN: Case series with cross-sectional analysis. SETTING: A tertiary clinical toxicology laboratory in Hong Kong. PATIENTS: All patients using proprietary Chinese medicines adulterated with corticosteroids and referred to the authors' centre from 1 January 2008 to 31 December 2012. MAIN OUTCOME MEASURES: Patients' demographic data, clinical presentation, medical history, drug history, laboratory investigations, and analytical findings of the proprietary Chinese medicines were analysed. RESULTS: The records of 61 patients who consumed corticosteroid-adulterated proprietary Chinese medicines were reviewed. The most common corticosteroid implicated was dexamethasone. Co-adulterants such as non-steroidal anti-inflammatory drugs and histamine H1-receptor antagonists were detected in the proprietary Chinese medicine specimens. Among the patients, seven (11.5%) required intensive care, two (3.3%) died within 30 days of presentation, and 38 (62.3%) had one or more complications that were potentially attributable to exogenous corticosteroids. Of 22 (36.1%) patients who had provocative adrenal function testing performed, 17 (77.3% of those tested) had adrenal insufficiency. CONCLUSION: The present case series is the largest series of patients taking proprietary Chinese medicines adulterated with corticosteroids. Patients taking these illicit products are at risk of severe adverse effects, including potentially fatal complications. Adrenal insufficiency was very common in this series of patients. Assessment of adrenal function in these patients, however, has been inadequate and routine rather than discretionary testing of adrenal function is indicated in this group of patients. The continuing emergence of proprietary Chinese medicines adulterated with western medication indicates a persistent threat to public health.

Confirmation of protein biomarkers of corticosteroids treatment in veal calves sampled under field conditions.

In veal calf production, growth promoters are still illicitly used. Surveillance of misuse of such molecules is necessary to preserve human health. Methods currently adopted for their analysis are based on liquid chromatography-tandem mass spectrometry, but their efficacy can be affected by undetectable residual concentrations in biological matrices due to treatments at low-dosage or based on unknown anabolic compounds. The development of screening methods to identify the indirect biological effects of administration of growth promoters can improve the efficiency of drug residue monitoring. To this purpose, an integrated approach has been used to further validate the set of protein biomarkers defined in a previous controlled study to detect the use of corticosteroids through the changes caused in muscle protein expression. The thymus morphology of 48 samples collected under field conditions was evaluated to assess the presence of potential corticosteroids treatment. Animals were divided on the basis of their thymus characteristics in negative or suspected for illegal corticosteroids treatment. Drug residue analyses were performed on the liver, giving a satisfactory correlation with the histological examination (∼85%). Finally, the proteomics analysis of muscle protein extracts was carried out by 2D differential in gel electrophoresis, and proteins that were differentially expressed between the two animal groups (p value <0.01) were selected for MALDI-MS/MS analysis. This approach allowed us to identify 29 different proteins, and our findings indicate that the altered protein expression pattern can be used as an indirect method for the detection of illicit corticosteroids administration. A subset of the identified proteins was already reported in a previous controlled study, proving that these biomarkers can be used to develop a screening assay to improve the tools currently available for the detection of corticosteroids abuse in bovine meat production.

Amphiphilic polymeric micelle as pseudostationary phase in electrokinetic chromatography for analysis of eight corticosteroids in cosmetics.

Amphiphilic polymeric micelle, as a novel pseudostationary phase in EKC was used to determine eight kinds of corticosteroids namely hydrocortisone, prednisolone, hydrocortisone acetate, prednisone, cortisone acetate, prednisolone acetate, dexamethasone, and triamcinolone acetonide in cosmetics. Amphiphilic random copolymer poly(methyl methacrylate-co-methacrylic acid) (P(MMA-co-MAA)) was micellizated via neutralization in alkaline aqueous solution. The influences of the molar ratio of monomer MMA to MAA, the concentration of polymer and pH on the polymeric micelle microstructure and EKC performances were investigated. As molar ratio of MMA to MAA in P(MMA-co-MAA) increased, both CMC and environmental polarity of the inner core in polymeric micelle decreased dramatically. With increasing monomer ratio, the size of polymeric micelles increased firstly, and then decreased, finally increased again. ζ potential of the micelle had a slight decline trend. As increment of polymer concentration, the size of the polymeric micelle increased steadily. By optimizing the monomer ratio, the polymer concentration, and pH of the running buffer, as well as operation conditions such as separation voltage and temperature, the eight analytes could be separated within 16.5 min using 7.5 mg/mL polymer with the monomer ratio of 7:3 dissolved in pH 9.2 borax buffer as the running buffer. The method has been used for analysis of corticosteroids in cosmetic samples with simple extraction; the recoveries for eight analytes were between 85.9 and 106%. This method was of accuracy, repeatability, pretreatment simplicity, and could be applied to the quality control of cosmetics.

LC-MS/MS determination of potential endocrine disruptors of cortico signalling in rivers and wastewaters.

A targeted analytical method was established to determine a large number of chemicals known to interfere with the gluco- and mineralocorticoid signalling pathway. The analytes comprise 30 glucocorticoids and 9 mineralocorticoids. Ten out of these corticosteroids were primary metabolites. Additionally, 14 nonsteroids were included. These analytes represent a broader range of possible adverse modes of action than previously reported. For the simultaneous determination of these structurally diverse compounds, a single-step multimode solid-phase extraction and pre-concentration was applied. Extracts were separated by a short linear HPLC gradient (20 min) on a core shell RP column (2.7 µm particle size) and compounds identified and quantified by LC-MS/MS. The method provided excellent retention time reproducibility and detection limits in the low nanograms per litre range. Untreated hospital wastewater, wastewater treatment plant influent, treated effluent and river waters were analysed to demonstrate the applicability of the method. The results show that not all compounds were sufficiently eliminated by the wastewater treatment, resulting in the presence of several steroids (∼20 ng/L) and nonsteroids in the final effluent, some of them at high concentrations up to 200 ng/L. Most of the detected mono-hydroxylated steroidal transformation products were found at significantly higher concentrations than their parent compounds. We therefore recommend to include these potentially bioactive metabolites in environmental toxicity assessment.

Microstructure of microemulsion modified with ionic liquids in microemulsion electrokinetic chromatography and analysis of seven corticosteroids.

In this work, the influences of ionic liquid (IL) as a modifier on microemulsion microstructure and separation performance in MEEKC were investigated. Experimental results showed that synergetic effect between IL 1-butyl-3-methylimidazolium tetrafluoro-borate (BmimBF4 ) and surfactant SDS gave a decreased CMC. With increment of IL in microemulsion, negative ζ potential of the microdroplets reduced gradually. The influence of IL on the dimensions of microdroplet was complicated. At BmimBF4 less than 8 mM, IL made microemulsion droplet smaller in size. While at BmimBF4 more than 10 mM, the size increased and reached to a maximum value at 12 mM, where the microdroplets were larger than that without IL. After that, the micreodroplet size decreased again. Relative fluorescence intensity of the first vibration band of pyrene to the third one (I1 /I3 ) enhanced as IL was added to microemulsion, which indicated that this addition increased environmental polarity in the inner core of microdroplets. Prednisone, hydrocortisone, prednisolone, hydrocortisone acetate, cortisone acetate, prednisolone acetate, and triamcinolone acetonide were analyzed with MEEKC modified with IL to evaluate the separation performance. Cortisone acetate and prednisolone acetate could not be separated at all in typical microemulsion. The seven analytes could be separated by the addition of 10 mM BmimBF4 into the microemulsion system. The method has been used for analysis of corticosteroids in cosmetic samples with simple extraction; the recoveries for seven analytes were between 86 and 114%. This method provides accuracy, reproducibility, pretreatment simplicity, and could be applied to the quality control of cosmetics.

Evaluation of the fecal steroid concentrations in Alouatta belzebul (Primates, Atelidae) in the National Forest of Tapirape-Aquiri in Pará, Brazil.

BACKGROUND: The studies on fecal steroid metabolites published with free-living primates are limited mainly by the difficulty in obtaining samples. METHODS: A radioimmunoassay was used to measure the fecal steroid concentrations in Alouatta belzebul in the National Forest of Tapirape-Aquiri in Brazil. RESULTS AND CONCLUSIONS: Androgens were significantly higher for the adult males from the Area of Influence (AI-I group) when compared to those from the Control Area (CA group) (P < 0.05). Progestin and estrogen concentrations were higher in the females from the CA group than in those from the AI-I for both the adult females and females with offspring; however, P < 0.05 was only observed in the concentrations of fecal progestins from the adult females. The physiological differences between the AI-I and CA groups suggest that the cause was a sum of factors, such as an exposure to sound waves, feeding habits, daily activity patterns, and the habituation of the animals.

In-house validation and factorial effect analysis of a liquid chromatography-tandem mass spectrometry method for the determination of corticosteroids in bovine and porcine muscle tissue.

A sensitive and robust liquid chromatography-tandem mass spectrometry method allowing the rapid screening and confirmation of ten synthetic corticosteroids in bovine and porcine muscle tissue was developed and validated. The validation was conducted according to Commission Decision 2002/657/EC, Sect. 3.1.3 ("Validation according to alternative models"), by applying a matrix-comprehensive in-house validation concept. The decision limit, detection capability, recovery, repeatability, within-laboratory-reproducibility and measurement uncertainty were calculated. Furthermore, a factorial effect analysis was conducted to identify factors that have a significant influence on the method. To this end, factors considered to be relevant for the method in routine analysis (e.g. operator, duration of storage of the extracts before measurement, different lots of the cartridges and different species) were systematically varied on two levels during the validation study. Subsequently, the extent to which these factors influence the measurement results of the individual analytes was examined.

Hormonal and behavioral variation in pied tamarins housed in different management conditions.

Pied tamarins are an endangered Amazonian primate that has limited breeding success in zoos. Unfortunately, little is known about their reproductive biology and adrenocortical activity. Objectives were: (1) determine if fecal hormones could be utilized to monitor gonadal and adrenocortical activity; (2) characterize male and female gonadal and adrenocortical hormones; and (3) determine if there were differences between adrenocortical activity and behavior in a nonbreeding, on-exhibit (NB-ON) pair compared to a breeding, off-exhibit (B-OFF) pair. Fecal samples were collected from four (two males; two females) individuals. Hormones were analyzed for fecal progesterone (FPM), androgen (FAM), and glucocorticoid (FGM) metabolites by enzyme immunoassay. Behavioral observations were conducted for 6 months. Data were collected on instantaneous behavior, location, and all occurrences of intraspecific behaviors. Fecal progesterone metabolites were validated by pregnancy (mean ± SE, pregnant: 28.47 ± 1.60 µg/g; nonpregnant: 8.63 ± 0.89 µg/g). Fecal androgen metabolites were higher (T = 31,971, P < 0.05) in the B-OFF male (863.66 ± 46.30 µg/g) than the NB-ON male (838.63 ± 60.70 µg/g). Fecal glucocorticoid metabolites were validated by response to veterinary procedure with elevated values (7.31 ± 1.48 µg/g) seven times the baseline (0.37 ± 0.04 µg/g) at 24-hr postphysical. Females had higher baseline FGM than the males (P < 0.05). Baseline FGM were higher (P < 0.05) in the NB-ON female (0.93 ± 0.03 µg/g) compared to the B-OFF female (0.38 ± 0.02 µg/g). Similarly, the NB-ON male's FGM baseline (0.71 ± 0.03 µg/g) were higher (P < 0.05) than the B-OFF male (0.21 ± 0.01 µg/g). Behavioral data revealed stereotypical behaviors in the NB-ON pair but no stereotypical behaviors in the B-OFF pair. Fecal hormone monitoring and behavioral analysis may provide insight on the limited breeding success of pied tamarins in zoos.

Quantitation of bioactive components in infant formulas: Milk oligosaccharides, sialic acids and corticosteroids.

Human milk is considered the optimal food for infants with abundant nutrients and bioactive components, which play key roles in infant health and development. Infant formulas represent appropriate substitutes for human milk. There are many brands of infant formula with different ingredient sources and functions on the market. The present study aims to quantify important bioactive components, i.e., milk oligosaccharides (MOS), sialic acids (Sia) and corticosteroids, in different infant formulas and compare these to human milk. In total, 12 different infant formulas available on the Dutch market were analyzed in this study. The concentrations of MOS and Sia were characterized by UHPLC-FLD and LC-MS, while corticosteroids were determined using established UHPLC-MS/MS methods. Among infant formulas, 15 structures of oligosaccharides were identified, of which 2'-Fucosyllactose (2'FL), 3'-Galactosyllactose (3'GL) and 6'-Galactosyllactose (6́'GL) were found in all infant formulas. The oligosaccharide concentrations differed between milk source and brands and were 3-5 times lower than in human milk. All infant formulas contained Sia, N-acetylneuraminic acid (Neu5Ac) was dominant in bovine milk-based formulas, while N-glycolylneuraminic acid (Neu5Gc) was major in goat milk-based formula. All infant formulas contained corticosteroids, yet, at lower concentrations than human milk. Insight in concentrations of bioactive components in infant formula compared to human milk may give direction to dietary advices and/or novel formula design.

Adrenal activity in maned wolves is higher on farmlands and park boundaries than within protected areas.

In this study we measured excreted fecal corticoid metabolites (FCM) in maned wolves (Chrysocyon brachyurus) living within a protected reserve, on farmlands or in a boundary zone between the two habitats, and determined the impacts of season and reproductive status on adrenal activity. Feces were collected within a national park (n=191 samples), a park boundary zone (n=39) and on nearby farmlands (n=27), processed and analyzed by enzyme immunoassay. FCM amounts from samples collected on farmlands were higher (P<0.05) than in those collected inside the reserve and from the boundary zone. In relation to seasonality, FCM were elevated (P<0.05) in spring (September-November) when wolf pairs were raising young. We then divided the samples collected during breeding season (March-August) into cycling females and male/non-cycling females based on fecal progesterone: fecal testosterone ratio. FCM concentrations of the former collected inside the park were higher than (P<0.05) than the latter group. However, there were no differences in FCM levels between the two groups for samples collected in the boundary zone and on farmlands. Furthermore, FCM concentrations of male/non-cycling females samples collected on farmlands were 2- to 5-fold higher (P<0.05) than in counterparts collected inside the park. The consistently high FCM concentrations in samples collected on farmlands indicate that, in addition to seasonality, gender and reproductive status, anthropogenic pressures also contribute to elevating adrenal steroid for individuals living in altered habitat.

Noninvasive monitoring of adrenocortical function in captive jaguars (Panthera onca).

Jaguars are threatened with extinction throughout their range. A sustainable captive population can serve as a hedge against extinction, but only if they are healthy and reproduce. Understanding how jaguars respond to stressors may help improve the captive environment and enhance their wellbeing. Thus, our objectives were to: (1) conduct an adrenocorticotrophic hormone (ACTH) challenge to validate a cortisol radioimmunoassay (RIA) for noninvasive monitoring of adrenocortical function in jaguars; (2) investigate the relationship between fecal corticoid (FCM) and androgen metabolite (FAM) concentrations in males during the ACTH challenge; and (3) establish a range of physiological concentrations of FCMs for the proposed protocol. Seven jaguars (3 M, 4 F) received 500 IU/animal of ACTH. Pre- and post-ACTH fecal samples were assayed for corticoid (M and F) and androgen metabolites (M) by RIA. Concentrations of FCMs increased (P80.01) after ACTH injection (pre-ACTH: 0.90 ± 0.12 µg/g dry feces; post-ACTH: 2.55 ± 0.25 µg/g). Considering pre- and post-ACTH samples, FCM concentrations were higher (P80.01) in males (2.15 ± 0.20 µg/g) than in females (1.30 ± 0.20 µg/g), but the magnitude of the response to ACTH was comparable (P>0.05) between genders. After ACTH injection, FAMs increased in two (of 3) males; in one male, FCMs and FAMs were positively correlated (0.60; P80.01). Excretion of FCMs was assessed in 16 jaguars (7 M, 9 F) and found to be highly variable (range, 80.11-1.56 µg/g). In conclusion, this study presents a cortisol RIA for monitoring adrenocortical function in jaguars noninvasively.