Hundreds of Unrecognized Halogenated Contaminants Discovered in Polar Bear Serum.

Exposure of polar bears (Ursus maritimus) to persistent organic pollutants was discovered in the 1970s, but recent evidence suggests the presence of unknown toxic chemicals in their blood. Protein and phospholipid depleted serum was stirred with polyethersulfone capillaries to extract a broad range of analytes, and nontarget mass spectrometry with "fragmentation flagging" was used for detection. Hundreds of analytes were discovered belonging to 13 classes, including novel polychlorinated biphenyl (PCB) metabolites and many fluorinated or chlorinated substances not previously detected. All analytes were detected in the oldest (mid-1980s) archived polar bear serum from Hudson Bay and Beaufort Sea, and all fluorinated classes showed increasing trends. A mouse experiment confirmed the novel PCB metabolites, suggesting that these could be widespread in mammals. Historical exposure and toxic risk has been underestimated, and these halogenated contaminants pose uncertain risks to this threatened species.

Potentiation of ecological factors on the disruption of thyroid hormones by organo-halogenated contaminants in female polar bears (Ursus maritimus) from the Barents Sea.

As apex predators, polar bears (Ursus maritimus) are among the most heavily polluted organisms in the Arctic. In addition to this anthropogenic stressor, climate warming has been shown to negatively affect their body condition, reproductive output and survival. Among potential underlying physiological mechanisms, thyroid hormones (THs), which control thermoregulation, metabolism and reproduction, can be affected by a variety of both natural and anthropogenic factors. While THs have been extensively used as proxies for pollution exposure in mammals, including polar bears, there is a lack of knowledge of their natural variations. In this context, we examined seasonal variations in body condition and circulating TH concentrations in free-ranging female polar bears. Females with variable reproductive status (i.e., solitary, with cubs of the year or with yearlings) were sampled from locations with contrasted sea ice conditions. Furthermore, we studied THs in relation to levels of organo-halogenated contaminants. As predicted, solitary females were in better condition than females caring for offspring, especially in spring. In addition, TH levels were lower in autumn compared to spring, although this seasonal effect was mainly observed in solitary females. Finally, the negative relationships between organochlorine and perfluoroalkyl substances and some THs suggest a possible alteration of homeostasis of THs. Since the latter relationships were only observed during spring, we emphasize the importance of considering the ecological factors when using THs as proxies for pollution exposure. Yet, the combined effects of natural and anthropogenic stressors on THs might impair the ability of polar bears to adapt to ongoing climate changes.

Influence of maternal and sociodemographic characteristics on the accumulation of organohalogen compounds in Argentinian women. The EMASAR study.

The occurrence of organohalogen compounds in venous serum from post-partum mothers from two Argentinian cities, Salta and Ushuaia, has been investigated (n = 698). 4,4'-DDE was the most abundant compound in these cities, with geometric means of 33 and 67ng/g lipid weight, respectively. City of residence, age and parity were the main determinants of the accumulation of these compounds. Hexachlorobenzene (HCB) was the second most abundant pollutant in Ushuaia, 8.7ng/g lipid, and ß-hexachlorocyclohexane (ß-HCH) in Salta, 7.8ng/g lipid. Decabromodiphenyl ether was higher in Ushuaia than Salta, 8.2 and 4.1ng/g lipid, respectively. The predominance of ß-HCH, 4,4'-DDE and 4,4'-DDT in Salta was related with higher use of pesticides for agricultural applications. The observed higher concentrations of 4,4'-DDE and 4,4'-DDT in the mothers from rural+semi-urban sites than in urban areas were consistent with this agricultural origin. In addition, the most volatile organochlorine compounds included in this study, HCB and α-HCH, were mainly found in Ushuaia. The concentrations of the studied organohalogen pollutants in Argentina were lower than those found in other similar studies which is consistent with the location of these cities in the southern hemisphere. Age, mainly for 4,4'-DDE and polychlorobiphenyl (PCB) congeners 138, 153 and 180, and parity, mainly for HCB, ß-HCH, 4,4'-DDT and PCB congener 118, were the second main determinants of the concentrations of these compounds. Gestational weight gain also influenced on the maternal levels of HCB, ß-HCH, 4,4'-DDT and PCB congeners 118, 138 and 153. Higher weight accumulation during pregnancy involved dilution of these persistent pollutants. Body mass index (BMI) was a statistically significant determinant for 4,4'-DDT, α-HCH and PCB congeners 153 and 180. The observed direct correspondence between higher BMI and 4,4'-DDT concentrations was in agreement with the above reported inputs related with agricultural applications. The reverse correspondence of BMI with α-HCH and the PCB congeners indicated higher dilution at higher weight increase.

Relationships between concentrations of selected organohalogen contaminants and thyroid hormones and vitamins A, E and D in Faroese pilot whales.

Pilot whales (Globicephala melas) from the Faroe Islands, North-East Atlantic, have high body concentrations of organohalogenated compounds (OHCs), such as polychlorinated biphenyls (PCBs), organochlorinated pesticides (OCPs) and brominated flame retardants (BFRs). The aim of the present study was to examine if and to what extent blood plasma and liver concentrations of several groups of these OHCs are related to concentrations of relevant nutritional and hormonal biomarkers in pilot whales. Thyroid hormones (THs: total and free thyroxine and total and free triiodothyronine) and vitamin A (retinol), D (25-hydroxyvitamin D3) and E (α-tocopherol) were analysed in plasma (n=27) and vitamin A (total vitamin A, retinol and retinyl palmitate) and E (α- and γ-tocopherol) were analysed in liver (n=37) of Faroe Island pilot whales. Correlative relationships between the biomarkers and OHC concentrations previously analysed in the same tissues in these individuals were studied. The TH concentrations in plasma were significantly higher in juveniles than in adults. Vitamin D concentrations in plasma and α- and γ-tocopherol in liver were higher in adults than in juveniles. Multivariate statistical modelling showed that the age and sex influenced the relationship between biomarkers and OHCs. Some significant positive relationships were found between OHCs and thyroid hormone concentrations in the youngest juveniles (p<0.05). In plasma of juvenile whales α-tocopherol was also positively correlated with all the OHCs (p<0.05). Only few significant correlations were found between single OHCs and retinol and vitamin D in plasma within the age groups. There were significant negative relationships between hepatic PBDE concentrations and retinol (BDE-47) and γ-tocopherol (BDE-49, -47, -100, -99, -153) in liver. The relationships between OHCs and THs or vitamins suggest that in pilot whales OHCs seem to have minor effects on TH and vitamin concentrations.

Residue profiles of organohalogen compounds in human serum from e-waste recycling sites in North Vietnam: Association with thyroid hormone levels.

This study demonstrated the contamination levels of polychlorinated biphenyls (PCBs), hydroxylated PCBs (OH-PCBs), polybrominated diphenyl ethers (PBDEs), methoxylated PBDEs (MeO-PBDEs), hydroxylated PBDEs (OH-PBDEs), and bromophenols (BPhs), and their relationships with thyroid hormones (THs), in the serum of human donors from an e-waste recycling site and a rural site in Hung Yen province, Vietnam. Occupationally related exposure was indicated by significantly higher residue levels of PCBs, OH-PCBs, PBDEs, and BPhs in the serum of donors from the e-waste recycling site (median: 420, 160, 290, and 300pgg(-1) wet wt, respectively) than those in the serum of donors from the rural site (median: 290, 82, 230, and 200pgg(-)(1) wet wt, respectively). On the other hand, levels of OH-/MeO-PBDEs were significantly higher in serum of donors from the reference site (median: 160 and 20pgg(-1) wet wt, respectively) than in those from the e-waste recycling site (median: 43 and 0.52pgg(-1) wet wt, respectively). In addition, we implemented stepwise generalized linear models to assess the association between the levels of TH and PCBs, PBDEs, and their related compounds. In females, we found positive associations of PCBs and OH-PCB concentrations with total thyroxine, free thyroxine, total triiodothyronine, and free triiodothyronine, and a negative association with thyroid-stimulating hormone concentrations.

Variations in thyroid hormone levels in endangered St. Lawrence Estuary belugas: Potential linkage with stress and organohalogen contaminant exposure.

The St. Lawrence Estuary (SLE) beluga (Delphinapterus leucas) population is highly exposed to an array of contaminants that were identified as one of the causes to the non-recovery of this endangered and declining population. In the last decade, an increasing number of parturition-associated complications and calf mortality has been reported in this population. It was suggested that elevated exposure to organohalogens (e.g., the halogenated flame retardants polybrominated diphenyl ethers [PBDEs]) and stress could play a role in this phenomenon by perturbing thyroid hormones. The objective of this study was to investigate the impact of concentrations of organohalogen contaminants and stress (cortisol levels) on thyroid hormone variations in adult male and female SLE belugas. Because plasma could not be collected in SLE belugas for ethical reasons, skin biopsy (n = 40) was used as a less-invasive alternative matrix to determine organohalogens (PBDEs and other halogenated flame retardants, polychlorinated biphenyls, and organochlorine pesticides), cortisol, and thyroid hormones (triiodothyronine [T3] and thyroxine [T4]), and their metabolites reverse T3 and 3,5-diiodothyronine [3,5-T2]). Cortisol and thyroid hormones were analyzed by ultra-performance liquid chromatography-multiple reactions monitoring mass spectrometry (UPLC-MRM/MS). This method was compared using skin and plasma samples obtained from Arctic belugas. Comparisons of linear models showed that cortisol was a weak predictor for T4, rT3 and 3,5-T2. Specifically, there was a weak significant negative association between T4 and cortisol levels. Moreover, in male SLE belugas, a weak significant positive association was found between T3 and Σ34PBDE concentrations in skin. Our findings suggest that stress (i.e., elevated skin cortisol levels) along with organohalogen exposure (mainly PBDEs) may be associated with thyroid hormone level perturbations in skin of cetaceans.

Reactive oxygen species alteration of immune cells in local residents at an electronic waste recycling site in northern China.

The health effects of exposure to pollutants from electronic waste (e-waste) pose an important issue. In this study, we explored the association between oxidative stress and blood levels of e-waste-related pollutants. Blood samples were collected from individuals living in the proximity of an e-waste recycling site located in northern China, and pollutants, as well as reactive oxygen species (ROS), were measured in comparison to a reference population. The geometric mean concentrations of PCBs, dechlorane plus, and 2,2',4,4',5,5'-hexabromobiphenyl in plasma from the exposure group were 60.4, 9.0, and 0.55 ng g(-1) lipid, respectively, which were 2.2, 3.2, and 2.2 times higher than the corresponding measurement in the reference group. Correspondingly, ROS levels in white blood cells, including in neutrophil granulocytes, from the exposure group were significantly higher than in those from the reference group, suggesting potential ROS related health effects for residents at the e-waste site. In contrast, fewer ROS were generated in the respiratory burst of neutrophil granulocytes for the exposure group, indicating a depressed innate immune function for the individuals living at the e-waste site. These findings suggest a potential linkage between exposure to pollutants from e-waste recycling and both elevated oxidative stress and altered immune function.

Comparative study of organohalogen contamination between two populations of Eastern Atlantic loggerhead sea turtles (Caretta caretta).

We evaluated the presence of 37 organohalogen contaminants in plasma samples from 162 juvenile and 197 adult loggerhead turtles (Caretta caretta) from the archipelagos of the Canary Islands and Cape Verde, respectively, and compared the contamination profiles found. We detected five organochlorine pesticides (OCP) and 16 polychlorinated biphenyls (PCBs). The concentrations of the two groups of contaminants were higher in turtles from the Canary Islands (OCPs, 1.04 vs. 0.37 ng/mL; PCBs, 1.92 vs. 0.08 ng/mL). We also observed a different profile of PCB contamination between the two populations. In addition, there was a negative correlation between body size and the total concentration of PCBs in the Canary Islands turtles, but not in turtles from Cape Verde. The present study presents the first data on the organochlorine contaminants (OCs) of live turtles from Canary Islands. In addition, we perform a comparison of the levels and profiles of OCs between these two different groups of loggerhead sea turtles from the Eastern Atlantic.

Influence of prenatal organohalogen levels on infant male sexual development: sex hormone levels, testes volume and penile length.

BACKGROUND: Prenatal exposure to endocrine disruptors, like organohalogen compounds (OHCs), might be responsible for the increased aberrations in human male sexual development (hypospadias, cryptorchidism, testicular cancer and fall in sperm count) observed over the past decades. This development is established during fetal life, and reflected in sex hormone levels, testes volume and penile length post-partum. The present study investigates the correlation between prenatal OHC levels and male sexual development outcomes. METHODS AND RESULTS: Levels of eight neutral [2,2'-bis-(4-chlorophenyl)-1,1'-dichloroethene (4,4'-DDE), 2,2',4,4',5,5'-hexachlorobiphenyl, 2,2',4,4'-tetrabromodiphenyl ether (BDE)-47, -99, -100, -153, -154 and 1,2,5,6,9,10-hexabromocyclododecane, HBCDD] and four phenolic [(pentachlorophenol (PCP), 4OH-CB-107 (4-hydroxy-2,3,3',4',5-pentachlorobiphenyl), -146 and -187)] OHCs were determined in 55 maternal serum samples taken at 35 weeks of pregnancy. Eight sex development-related hormones [testosterone, free testosterone, sex hormone-binding globulin (SHBG); LH, FSH, estradiol (E(2)), free E(2) (FE(2)) and inhibin B (InhB)] were determined in their sons at 3 months of age, and testes volume and penile length at 3 and 18 months of age. The following prenatal OHC levels correlated significantly with sex hormone levels: PCP with SHBG and InhB (ρ = 0.30 and -0.43, respectively), 4OH-CB-107 with testosterone (ρ = 0.31) and BDE-154 with FE(2), E(2) and InhB (ρ = 0.49, 0.54 and 0.34, respectively). BDE-154 levels correlated positively with testes volume at 18 months of age (ρ = 0.34). CONCLUSIONS: Prenatal OHC exposure is correlated with aspects of sexual development outcome in boys up to 18 months of age.

Differential exposure biomarker levels among cigarette smokers and smokeless tobacco consumers in the National Health and Nutrition Examination Survey 1999-2008.

Assessment of biomarkers is an appropriate way to estimate exposure to cigarette mainstream smoke and smokeless tobacco (SLT) constituents in tobacco consumers. Using the US National Health and Nutrition Examination Survey (NHANES, 1999-2008), biomarkers of volatile organic compounds, halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), acrylamide, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and metals were evaluated. In general, biomarker levels in SLT consumers were significantly lower than in smokers (excluding NNK and some HAHs) and were not significantly different compared with nonconsumers (excluding NNK and some PAHs). These results provide useful information for science-based risk assessment and regulation of tobacco products.

Assessment of prenatal exposure to persistent organohalogen compounds from cord blood serum analysis in two Mediterranean populations (Valencia and Menorca).

Prenatal levels of organohalogen compounds (OHCs), including polybromodiphenyl ethers (PBDEs), polychlorobiphenyls (PCBs), hexachlorobenzene (HCB), hexachlorocyclohexanes and DDTs, have been investigated in two different cohorts from the Spanish Mediterranean area, Valencia and Menorca, by analysis of cord blood serum. Comparison of the observed median concentrations to other cohorts shows low PBDE levels (2.8-6.5 ng g(-1) lipid) but high values of HCB (0.68 ng ml(-1), 260 ng g(-1) lipid) and PCBs (0.65 ng ml(-1), 240 ng g(-1) lipid) in the cohort of Menorca indicating that Mediterranean areas, and not only high latitude regions, may contain population groups highly exposed to some of these pollutants. Significant differences in the concentrations of these compounds have been found between the two cohorts. Differences in maternal population such as age or body mass index cannot explain this variation. One possible cause of variability is the seven year time period elapsed between the two cohort recruitments but the strong differences observed do not allow exclusion of local diet differences. Thus, the different average PCB congener distributions between the two cohorts suggest an influence from materials containing diverse PCB mixtures. The congener mixtures found in Menorca could reflect a diet contribution from some western Mediterranean fish species. These results provide baseline information on prenatal OHC background levels in the Mediterranean area.

Integrated analysis of halogenated organic pollutants in sub-millilitre volumes of venous and umbilical cord blood sera.

A rapid, robust and economical method for the analysis of persistent halogenated organic compounds in small volumes of human serum and umbilical cord blood is described. The pollutants studied cover a broad range of molecules of contemporary epidemiological and legislative concern, including polychlorobiphenyls (PCBs), polychlorobenzenes (CBs), hexachlorocyclohexanes (HCHs), DDTs, polychlorostyrenes (PCSs) and polybromodiphenyl ethers (PBDEs). Extraction and clean-up with n-hexane and concentrated sulphuric acid was followed with analysis by gas chromatography coupled to electron capture (GC-ECD) and GC coupled to negative ion chemical ionisation mass spectrometry (GC-NICI-MS). The advantages of this method rest in the broad range of analytes and its simplicity and robustness, while the use of concentrated sulphuric acid extraction/clean-up destroys viruses that may be present in the samples. Small volumes of reference serum between 50 and 1000 microL were extracted and the limits of detection/quantification and repeatability were determined. Recoveries of spiked compounds for the extraction of small volumes (> or = 300 microL) of the spiked reference serum were between 90% and 120%. The coefficients of variation of repeatability ranged from 0.1-14%, depending on the compound. Samples of 4-year-old serum and umbilical cord blood (n = 73 and 40, respectively) from a population inhabiting a village near a chloro-alkali plant were screened for the above-mentioned halogenated pollutants using this method and the results are briefly described.

A kinetic model for human blood concentrations of gaseous halocarbon fire-extinguishing agents.

A simple kinetic model for calculating the blood concentration history of humans exposed to time-varying concentrations of gaseous, halocarbon fire-extinguishing agents is described. The kinetic model was developed to extend experimental physiologically based pharmacokinetic (PBPK) models for arterial blood concentration of halocarbons, obtained from constant concentration exposure of dogs to time-varying exposure conditions for humans. In the present work, the simplified kinetic model was calibrated using published PBPK-derived arterial concentration histories for constant concentration exposure to several common fire-extinguishing agents. The calibrated kinetic model was then used to predict the blood concentration histories of humans exposed to time-varying concentrations of these fire-extinguishing agents in ventilated compartments and the results were compared with PBPK-derived data for the agents. It was found that the properly calibrated kinetic model predicts human arterial blood concentration histories for time-varying exposures as well as the PBPK models. Consequently, the kinetic model represents an economical methodology for calculating safe human exposure limits for time-varying concentrations of gaseous halocarbon fire-extinguishing agents when only PBPK-derived human arterial blood concentration histories for constant exposure conditions are available.

Dechlorination of DDT in frozen blood.

Dechilorination of a commercial mixture of o, p'-DDT and p, ppDDT to p, p'-DDD and p, p'-DDE occiurs in avian blood stored at-20 degrees C, indicatinig nonenzymalic degradation. The subsequent disappearance of these two metabolites suggests conversion to other metabolites which cannot be detected by gas chromatography with an electron-captutre detector.

DDT tissue retention: sudden rise induced by the addition of aldrin to a fixed DDT intake.

The oral administration of aldrin to male and female beagles, whose diet already included a fixed, regular oral dosage of DDT, resulted in a dramatic rise in the concentrations of DDT, DDE, and DDD in blood and fat.

Drivers of maternal accumulation of organohalogen pollutants in Arctic areas (Chukotka, Russia) and 4,4'-DDT effects on the newborns.

BACKGROUND: One of the most worrying consequence of the production and use of persistent organohalogen pollutants (POPs) is the high accumulation in Arctic populations because of long-range transport. Study of the effects in these populations may illustrate human impacts that are difficult to assess in other locations with lower exposure to these compounds and more diverse pollutant influences. OBJECTIVE: We aimed to identify the main maternal characteristics influencing on the accumulation of these compounds and the effects on the newborns in a highly exposed Arctic population (Chukotka, Russia). METHODS: Organochlorine and organobromine compounds were analysed in maternal venous serum (n = 250). The study included data on residence, educational level, age, parity and body mass index (BMI) from self-reported questionnaires and measured anthropometric characteristics of newborns. RESULTS: Concentrations of ß-hexachlorocyclohexanes, hexachlorobenzene, 4,4'-DDT and polychlorobiphenyls were high when compared with those generally found in adult populations later than year 2000. The polybromodiphenyl ethers were negligible. These POP concentrations were higher than in Alaska and Arctic Norway and similar to those in Canada. The Chukotka mothers living in inland areas showed significant lower concentrations than those living in the coast (p < 0.001) except for 4,4'-DDT. The population from the Chukotsky District, a specific coastal area, showed the highest concentrations. Residence was therefore a main concentration determinant (p < 0.001) followed by maternal age, and in some cases parity and BMI (p < 0.05). 4,4'-DDT showed an association with the anthropometric characteristics of the newborns (p < 0.05). Mothers with higher 4,4'-DDT concentrations had longer gestational ages and gave birth to infants with higher weight and length. CONCLUSIONS: The maternal accumulation patterns of POPs were mainly related with residence. Most of these compounds were found in higher concentration in women living at coastal areas except 4,4'-DDE and 4,4'-DDT which were of inland origin. This last pesticide was the pollutant showing positive associations with gestational age and newborn's weight and length. To the best of our knowledge, this is the first study reporting statistically significant associations between maternal 4,4'-DDT exposure and anthropometric characteristics of the newborns.

Organohalogen concentrations in blood and adipose tissue of Southern Beaufort Sea polar bears.

We analyzed 151 organohalogen chemicals (OHCs) in whole blood and subcutaneous fat of 57 polar bears sampled along the Alaskan Beaufort Sea coast in spring, 2003. All major organochlorine pesticides, PCBs, PBDEs and their congeners were assessed. Concentrations of most OHCs continue to be lower among Southern Beaufort Sea polar bears than reported for other populations. Additionally, toxaphenes and related compounds were assessed in adipose tissue, and 8 perflourinated compounds (PFCs) were examined in blood. Perfluorooctane sulfonate (PFOS) concentrations exceeded those of any other contaminant measured in blood. SigmaChlordane concentrations were higher in females, and both SigmaPCBs and SigmaChlordane concentrations in adipose tissue decreased significantly with age. The rank order of OHC mean concentrations; SigmaPCB>Sigma10PCB>PCB153>SigmaChlordane>Oxychlordane>PCB180>SigmaHCH>beta-HCH>SigmaDDT>p,p-DDE>SigmaPBDE>HCB>Toxaphene was similar for compounds above detection limits in both fat and blood. Although correlation between OHC concentrations in blood and adipose tissue was examined, the predictability of concentrations in one matrix for the other was limited.

Prenatal exposure to persistent organic pollutants and methylation of LINE-1 and imprinted genes in placenta: A CHECK cohort study.

Prenatal exposure to persistent organic pollutants (POPs) has been linked to numerous adverse birth outcomes among newborn infants in many epidemiological studies. Although epigenetic modifications have been suggested as possible explanations for those associations, studies have rarely reported a relationship between POP exposure during pregnancy and DNA methylation in the placenta. In the present study, we investigated the association between prenatal exposure to several POPs, including organochlorine pesticides (OCPs), polybrominated diphenyl ethers (PBDEs), and polychlorinated biphenyls (PCBs), and methylation levels of long interspersed element 1 (LINE-1), as well as imprinted genes in placental DNAs among Korean mother-child pairs (N = 109). We assessed the association of DNA methylation not only with each target POP (single-POP models) but also with multiple POPs applying principal component analysis (multiple-POP models). Potential associations between placental DNA methylation and birth outcomes of newborn infants were also estimated. In single-POP models, significant associations were detected between OCP measurements and placental DNA methylation. Elevated concentrations of ß-hexachlorhexane (ß-HCH) in maternal serum collected during delivery were significantly associated with a decrease in methylation of LINE-1 in the placenta. Higher levels of p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT) in maternal serum were associated with hypermethylation of insulin-like growth factor 2 (IGF2). In multiple-POP models, a significant and positive association between DDTs and IGF2 methylation was also observed. Placental LINE-1 methylation was inversely associated with birth length. Our observations indicate that prenatal exposure to several POPs including DDTs is associated with the changes in methylation of genes, including major imprinted genes in the placenta. The consequences of these epigenetic alterations in placenta during development deserve further investigation.

First in vivo evaluation of a potential SPECT brain radiotracer for the gonadotropin releasing hormone receptor.

OBJECTIVES: In vivo evaluations of a gonadotropin releasing hormone-receptor single photon emission computed tomography radiotracer for non-invasive detection of gonadotropin releasing homone-receptors in brain. RESULTS: We have used a simple, robust and high-yielding procedure to radiolabel an alpha-halogenated bioactive compound with high radiochemical yield. Literature findings showed similar alpha-halogenated compounds suitable for in vivo evaluations. The compound was found to possess nano molar affinity for the gonadotropin releasing hormone-receptor in a competition dependent inhibition study. Furthermore, liquid chromatography-mass spectrometry analysis in saline, human and rat serum resulted in 46%, 52% and 44% stability after incubation for 1 h respectively. In addition, rat brain single photon emission computed tomography and biodistribution studies gave further insight into the nature of the compound as a radiotracer.

Liquid-liquid extraction of strongly protein bound BMS-299897 from human plasma and cerebrospinal fluid, followed by high-performance liquid chromatography/tandem mass spectrometry.

BMS-299897 is a gamma-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Liquid-liquid extraction (LLE), chromatographic/tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for the quantitation of BMS-299897 in human plasma and cerebrospinal fluid (CSF). Both methods utilized (13)C6-BMS-299897, the stable label isotope analog, as the internal standard. For the human plasma extraction method, two incubation steps were required after the addition of 5 mM ammonium acetate and the internal standard in acetonitrile to release the analyte bound to proteins prior to LLE with toluene. For the human CSF extraction method, after the addition of 0.5 N HCl and the internal standard, CSF samples were extracted with toluene and no incubation was required. The organic layers obtained from both extraction methods were removed and evaporated to dryness. The residues were reconstituted and injected into the LC/MS/MS system. Chromatographic separation was achieved isocratically on a MetaChem C18 Hypersil BDS column (2.0 mm x 50 mm, 3 microm). The mobile phase contained 10 mM ammonium acetate pH 5 and acetonitrile. Detection was by negative ion electrospray tandem mass spectrometry. The standard curves ranged from 1 to 1000 ng/ml for human plasma and 0.25-100 ng/ml for human CSF. Both standard curves were fitted to a 1/x weighted quadratic regression model. For both methods, the intra-assay precision was within 8.2% CV, the inter-assay precision was within 5.4% CV, and assay accuracy was within +/-7.4% of the nominal values. The validation and sample analysis results demonstrated that both methods had acceptable precision and accuracy across the calibration ranges.