Effect of amino acid enriched diets on hemolymph amino acid composition in honey bees.

Amino acids (AAs) are an abundant class of nectar solutes, and they are involved in the nectar attractiveness to flower visitors. Among the various AAs, proline is the most abundant proteogenic AA, and γ-amino butyric acid (GABA) and ß-alanine are the two most abundant non-proteogenic AAs. These three AAs are known to affect insect physiology, being involved in flight metabolism and neurotransmission. The aim of this study was to investigate the effects of artificial diets enriched with either ß-alanine, GABA, or proline on consumption, survival, and hemolymph composition in honey bees belonging to two different ages and with different metabolism (i.e., newly emerged and foragers). Differences in feed intake among diets were not observed, while a diet enriched with ß-alanine improved the survival rate of newly emerged honey bees compared to the control group. Variations in the hemolymph AA concentrations occurred only in newly emerged honey bees, according to the diet and the time of hemolymph sampling. A greater susceptibility of young honey bees to enriched diets than older honey bees was observed. The variations in the concentrations of hemolymph AAs reflect either the accumulation of dietary AAs or the existence of metabolic pathways that may lead to the conversion of dietary AAs into different ones. This investigation could be an initial contribution to studying the complex dynamics that regulate hemolymph AA composition and its effect on honey bee physiology.

Development of a dual-fluorescence reporter system for high-throughput screening of L-aspartate-α-decarboxylase.

ß-Alanine (3-aminopropionic acid) holds great potential in industrial application. It can be obtained through a chemical synthesis route, which is hazardous to the environment. It is well known that l-aspartate-α-decarboxylase (ADC) can convert l-aspartate to ß-alanine in bacteria. However, due to the low activity of ADC, industrial production of ß-alanine through the green biological route remains unclear. Thus, improving the activity of ADC is critical to reduce the cost of ß-alanine production. In this study, we established a dual-fluorescence high-throughput system for efficient ADC screening. By measuring the amount of ß-alanine and the expression level of ADC using two different fluorescence markers, we can rapidly quantify the relative activity of ADC variants. From a mutagenesis library containing 2000 ADC variants, we obtained a mutant with 33% increased activity. Further analysis revealed that mutations of K43R and P103Q in ADC significantly improved the yield of ß-alanine produced by the whole-cell biocatalysis. Compared with the previous single-fluorescence method, our system can not only quantify the amount of ß-alanine but also measure the expression level of ADC with different fluorescence, making it able to effectively screen out ADC variants with improved relative activity. The dual-fluorescence high-throughput system for rapid screening of ADC provides a good strategy for industrial production of ß-alanine via the biological conversion route in the future.

The Role of Amino Acids in Neurotransmission and Fluorescent Tools for Their Detection.

Neurotransmission between neurons, which can occur over the span of a few milliseconds, relies on the controlled release of small molecule neurotransmitters, many of which are amino acids. Fluorescence imaging provides the necessary speed to follow these events and has emerged as a powerful technique for investigating neurotransmission. In this review, we highlight some of the roles of the 20 canonical amino acids, GABA and ß-alanine in neurotransmission. We also discuss available fluorescence-based probes for amino acids that have been shown to be compatible for live cell imaging, namely those based on synthetic dyes, nanostructures (quantum dots and nanotubes), and genetically encoded components. We aim to provide tool developers with information that may guide future engineering efforts and tool users with information regarding existing indicators to facilitate studies of amino acid dynamics.

Space Flight Diet-Induced Deficiency and Response to Gravity-Free Resistive Exercise.

Immune system dysregulation is among the many adverse effects incurred by astronauts during space flights. Omega-3 fatty acids, ß-alanine, and carnosine are among the many nutrients that contribute to immune system health. For space flight, crewmembers are prescribed a diet with a macronutrient composition of 55% carbohydrate, 30% fat, and 15% protein. To quantify omega-3 fatty acid, ß-alanine and carnosine intakes from such a diet, and to examine each nutrient's impact on exercise performance, 21 participants adhered to the aforementioned macronutrient ratio for 14 days which was immediately followed by a workout performed on gravity-independent resistive exercise hardware. Results included daily omega-3 fatty acid intakes below the suggested dietary intake. Daily omega-3 fatty acid, ß-alanine and carnosine intakes each correlated with non-significant amounts of variance from the workout's volume of work. Given the nutritional requirements to maintain immune system function and the demands of in-flight exercise countermeasures for missions of increasingly longer durations current results, in combination with previously published works, imply in-flight supplementation may be a prudent approach to help address the physiological and mental challenges incurred by astronauts on future space flights.

Sources of overestimation in the analysis of acrylamide-in coffee by liquid chromatography mass spectrometry.

Analysis of acrylamide in coffee by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is prone to interferences. According to our study, unknown background ions can entail an overestimation by up to 40% in case of coelution with acrylamide. In order to develop a precise and accurate quantification method for acrylamide, identification and removal of these interfering ions is requested. We thus studied potential isobaric impurities of acrylamide using liquid chromatography-high resolution mass spectrometry (LC-HRMS). An in-source fragment of N-Acetyl-ß-alanine, a substance not yet reported in coffee, was identified as the main interfering ion. The characterization of the interference further triggered modification of the mobile phase-pH to alter the retention of N-Acetyl-ß-alanine and achieve an interference free acrylamide determination. Two other compounds closely related to acrylamide namely 3-aminopropanamide and lactamide were also susceptible to in-source fragmentation, highlighting the pivotal role of chromatographic conditions to ensure a reliable quantification of acrylamide.

Reduction of ß-ODAP and IP6 contents in Lathyrus sativus L. seed by high hydrostatic pressure.

Grass pea (Lathyrus sativus L.) seeds contain an endogenous neurotoxic non-proteinogenic amino acid, ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), a major limiting factor-for their human consumption. Furthermore, phytate (IP6), a well-known antinutrient is present in concentration capable of hindering bioavailability of iron (Fe), zinc (Zn), calcium (Ca), phosphorus (P) and other micronutrients from the seeds. Due to the reported capability of high hydrostatic pressure (HHP) to reduce the content of certain antinutritional/toxic agents in seeds and grains, the impact of HHP on the reduction of ß-ODAP and IP6 were investigated. The contents of ß-ODAP of accessions from different regions in Ethiopia were found to be in the range of 51.94 to 806.52 mg/100 g. Accession (GF1- Alemu, AK) exhibiting the highest ß-ODAP content was selected for HHP treatment in soaked and batter forms using Central Composite Face Centered Design of experiments. The best HHP conditions in respect to ß-ODAP reduction were also applied to the accession (GP-240038) with the lowest ß-ODAP-content, a genetically improved variety (Wassie) and a variety from Germany (GR). The HHP treatment at 600 MPa for 25 min of seeds soaked for 6 h and 12 h exhibited the maximum reduction of ß-ODAP (232.11 mg/100 g) and IP6 (21.11 mg/100 g) respectively. The combined incremental effect of pressure and soaking time resulted in a more significant (p ≤ .001) reduction in both compounds than the interaction of pressure with holding time (p ≤ .05). A reduction of ß-ODAP from 36.00 to 71.22% by soaked-HHP treatment was observed. ß-ODAP reductions were always higher for soaked compared to batter grass pea seeds. IP6 contents after HHP treatment ranged from 33.65 mg/100 g to nill. It can be concluded that pressure, soaking and holding time as well as the grass pea seed accession/variety had great impact on molecular structure changes, enhancement of enzyme activity and reduction in ß-ODAP and IP6 content.

Genome-wide association studies identified variants for taurine concentration in Japanese Black beef.

We performed genome-wide association studies (GWAS) using the BovineSNP50 array to detect significant single nucleotide polymorphisms (SNPs) that may affect the concentration of 22 free amino acids and three peptides in Japanese Black beef cattle. A total of 574 Japanese Black cattle and 40,657 SNPs from the array were used for this study. Genome-wide significant SNPs were detected for ß-alanine (three SNPs on chromosomes 22 and 29) and taurine (26 SNPs on chromosome 22). Importantly, the top two SNPs for taurine were highly significant (p = 6.2 × 10-21 ), and the frequency of the increase-concentration allele (Q) for taurine was found to be 0.73. The Q allele frequency of this population was similar to that of the other unrelated Japanese Black cattle, but different from that of the other breeds. In addition, the significant SNPs were not associated with carcass traits or fatty acid compositions. Interestingly, the top three of the four most significant SNPs for taurine were located near solute carrier family 6, member 6 (SLC6A6), which is a membrane transporter for taurine. We also found two associated variants in the 5'-upstream region of SLC6A6; however, they were less significantly associated than the SNPs from the BovineSNP50 array.

Biomarkers of food intake and nutrient status are associated with glucose tolerance status and development of type 2 diabetes in older Swedish women.

Background: Diet is frequently associated with both the development and prevention of type 2 diabetes (T2D), but there is a lack of objective tools for assessing the relation between diet and T2D. Biomarkers of dietary intake are unconfounded by recall and reporting bias, and using multiple dietary biomarkers could help strengthen the link between a healthy diet and the prevention of T2D.Objective: The objective of this study was to explore how diet is related to glucose tolerance status (GTS) and to future development of T2D irrespective of common T2D and cardiovascular disease risk factors by using multiple dietary biomarkers.Design: Dietary biomarkers were measured in plasma from 64-y-old Swedish women with different GTS [normal glucose tolerance (NGT; n = 190), impaired glucose tolerance (IGT; n = 209), and diabetes (n = 230)]. The same subjects were followed up after 5 y to determine changes in glucose tolerance (n = 167 for NGT, n = 174 for IGT, and n = 159 for diabetes). ANCOVA and logistic regression were used to explore baseline data for associations between dietary biomarkers, GTS, and new T2D cases at follow-up (n = 69).Results: Of the 10 dietary biomarkers analyzed, ß-alanine (beef) (P-raw < 0.001), alkylresorcinols C17 and C19 (whole-grain wheat and rye) (P-raw = 0.003 and 0.011), eicosapentaenoic acid (fish) (P-raw = 0.041), 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) (fish) (P-raw = 0.002), linoleic acid (P-raw < 0.001), oleic acid (P-raw = 0.003), and α-tocopherol (margarine and vegetable oil) (P-raw < 0.001) were associated with GTS, and CMPF (fish) (OR: 0.72; 95% CI: 0.56, 0.93; P-raw = 0.013) and α-tocopherol (OR: 0.71; 95% CI: 0.51, 0.98; P-raw = 0.041) were inversely associated with future T2D development.Conclusions: Several circulating dietary biomarkers were strongly associated with GTS after correction for known T2D risk factors, underlining the role of diet in the development and prevention of T2D. To our knowledge, this study is the first to use multiple dietary biomarkers to investigate the link between diet and disease risk.

Determination of ß-N-oxalyl-L-α,ß-diaminopropionic acid and homoarginine in Lathyrus sativus and Lathyrus cicera by capillary zone electrophoresis.

BACKGROUND: Lathyrus species as legumes represent an alternative protein source for human and animal nutrition. Heavy consumption of these species can lead to lathyrism, caused by the non-protein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP). Currently, there is no well-defined level below which ß-ODAP is considered non-toxic. In this work, the ß-ODAP content was determined in L. sativus and L. cicera samples to assess their potential toxicity. Homoarginine is another non-protein amino acid found in Lathyrus spp. with interesting implications for human and animal nutrition. RESULTS: The level of ß-ODAP found in these two species ranged from 0.79 to 5.05 mg g(-1). The homoarginine content of the samples ranged from 7.49 to 12.44 mg g(-1). CONCLUSION: This paper describes an accurate, fast and sensitive method of simultaneous detection and quantification of ß-ODAP and homoarginine by capillary zone electrophoresis in L. cicera and L. sativus seeds. Moreover, several methods of extraction were compared to determine the highest performance.

Amino acid metabolic dysfunction revealed in the prefrontal cortex of a rat model of depression.

Major depressive disorder (MDD) is a debilitating mood disorder. However, the molecular mechanism(s) underlying depression remain largely unknown. Here, we applied a GC-MS-based metabonomic approach in the chronic unpredictable mild stress (CUMS) model, a well-established rodent model of depression, to investigate significant metabolic changes in the rat prefrontal cortex (PFC). Multivariate statistical analysis - including principal component analysis, partial least squares-discriminate analysis, and pair-wise orthogonal projections to latent structures discriminant - was applied to identify differential PFC metabolites between CUMS rats and healthy controls. As compared to healthy control rats, CUMS rats were characterized by lower levels of isoleucine and glycerol in combination with higher levels of N-acetylaspartate and ß-alanine. These findings should provide insight into the pathophysiological mechanism(s) underlying MDD and preliminary leads relevant to diagnostic biomarker discovery for depression.

Comportamentos e práticas sexuais de homens que fazem sexo com homens / Sexual behaviors and practices of men who have sex with men / Conductas y prácticas sexuales de los hombres que tienen sexo con hombres

Objetivou-se identificar comportamentos e práticas sexuais de homens que fazem sexo com homens no contexto da vulnerabilidade ao HIV/AIDS. Estudo transversal, exploratório descritivo. Foi realizado em um local de sociabilidade gay de Fortaleza, no Estado do Ceará, entre novembro de 2010 e março de 2011, por meio de entrevista com 189 homens que fazem sexo com homens. Encontrou-se uma amostra composta, majoritariamente, por jovens, solteiros e com alto nível educacional. A história sexual demonstrou o início precoce da vida sexual, com prevalência elevada de relação sexual com parceira do sexo oposto. Houve alta frequência de testagem para o HIV. As práticas sexuais revelaram prevalência superior da realização de sexo oral e anal, bem como altos níveis de proteção no sexo anal, apesar de baixa no sexo oral. Constatou-se uma maior incorporação das práticas de prevenção em relação ao panorama nacional do início da epidemia.

The objective was to identify behaviors and sexual practices of men who have sexual relations with other men in the context of vulnerability to HIV/AIDS. This was a cross-sectional, exploratory and descriptive study. It was carried out in a gay sociability place in Fortaleza, Ceará, Brazil, between November 2010 and March 2011, through interviews with 189 men who have sex with men. The ethical aspects were respected. We found a sample consisting mostly by young, single, and highly educated men. The sexual history demonstrated the early onset of sexual activity, with a high prevalence of sexual intercourse with a partner of the opposite sex. There was also a high prevalence of HIV testing. Sexual practices revealed high prevalence of performing oral and anal sex, as well as high levels of protection in anal sex, despite the low protection in oral sex. A greater incorporation of prevention practices was found compared to the national scene in the beginning of the disease outbreak.

El objetivo fue identificar los comportamientos y las prácticas sexuales de los hombres que tienen sexo con hombres en el contexto de la vulnerabilidad al VIH/SIDA. Fue un estudio transversal, descriptivo y exploratorio. Se celebró en una sociabilidad local gay de Fortaleza, Ceará, Brasil, entre noviembre de 2010 y marzo de 2011, a través de entrevistas con 189 hombres que tienen sexo con hombres. Se encontró una muestra compuesta en su mayoría por jóvenes, solteros y con alto nivel de educación. La historia sexual demostró el inicio temprano de la actividad sexual, la alta prevalencia de relaciones sexuales con una pareja del sexo opuesto. Hubo alta prevalencia de la prueba del VIH. Las prácticas sexuales revelaron una alta prevalencia de realizar sexo oral y anal, así como altos niveles de protección en el sexo anal, a pesar de la baja protección en el sexo oral. Se encontró una mayor incorporación de las prácticas de prevención en relación con la escena nacional en el inicio de la epidemia.

ß-alanine biosynthesis in Methanocaldococcus jannaschii.

One efficient approach to assigning function to unannotated genes is to establish the enzymes that are missing in known biosynthetic pathways. One group of such pathways is those involved in coenzyme biosynthesis. In the case of the methanogenic archaeon Methanocaldococcus jannaschii as well as most methanogens, none of the expected enzymes for the biosynthesis of the ß-alanine and pantoic acid moieties required for coenzyme A are annotated. To identify the gene(s) for ß-alanine biosynthesis, we have established the pathway for the formation of ß-alanine in this organism after experimentally eliminating other known and proposed pathways to ß-alanine from malonate semialdehyde, l-alanine, spermine, dihydrouracil, and acryloyl-coenzyme A (CoA). Our data showed that the decarboxylation of aspartate was the only source of ß-alanine in cell extracts of M. jannaschii. Unlike other prokaryotes where the enzyme producing ß-alanine from l-aspartate is a pyruvoyl-containing l-aspartate decarboxylase (PanD), the enzyme in M. jannaschii is a pyridoxal phosphate (PLP)-dependent l-aspartate decarboxylase encoded by MJ0050, the same enzyme that was found to decarboxylate tyrosine for methanofuran biosynthesis. A Km of ∼0.80 mM for l-aspartate with a specific activity of 0.09 µmol min(-1) mg(-1) at 70°C for the decarboxylation of l-aspartate was measured for the recombinant enzyme. The MJ0050 gene was also demonstrated to complement the Escherichia coli panD deletion mutant cells, in which panD encoding aspartate decarboxylase in E. coli had been knocked out, thus confirming the function of this gene in vivo.

Investigation of biomolecules trapped in fluid inclusions inside halite crystals by Raman spectroscopy.

Raman spectroscopy was tested for the identification of biomolecules (glycine, L-alanine, ß-alanine, L-serine, and γ-aminobutyric acid) trapped in fluid inclusions inside halite model crystals. The investigated biomolecules represent important targets for future astrobiological missions. We know from terrestrial conditions that organic molecules and microorganisms can be sealed within fluid inclusions and can survive intact even for hundreds of millions of years. Raman spectroscopy is currently being miniaturized for future extraterrestrial planetary exploration (ExoMars 2018). Raman spectroscopy has shown the ability to detect investigated aminoacids nondestructively without any sample preparation, in short measurement times, and in relatively low concentrations. The number of registered Raman bands of investigated aminoacids and their intensity clearly correlate with the given concentration of biomolecules within fluid inclusions.

The achievement of mass balance by simultaneous quantification of floxuridine prodrug, floxuridine, 5-fluorouracil, 5-dihydrouracil, α-fluoro-ß-ureidopropionate, α-fluoro-ß-alanine using LC-MS.

5-Fluoro-2'-deoxyuridine (floxuridine, 5-FdUrd) and 5-fluorouracil (5-FU) are widely used for the treatment of colorectal cancers. The mechanisms of action of 5-FdUrd and 5-FU, as well as the biochemical pathway responsible for their metabolism, are well understood. Identification of every metabolite and achieving mass balance by conventional UV absorption-based HPLC analysis are not feasible because the metabolites beyond 5-FU in the 5-FdUrd metabolic pathway are undetectable by UV light. We therefore established a mass spectrometry method, designed for fast and convenient analysis, for simultaneously measuring 5-FdUrd, 5-FU, and their metabolites. Linearity, precision and accuracy were validated in the concentration ranges studied for each compound. Hydrolysis studies of 5-FdUrd and amino acid mono ester prodrugs of 5-FdUrd in Capan-2 cell homogenates were carried out and the achievement of mass balance was established with this method (recovery of 5'-O-l-leucyl-FdUrd was 96.6-108.2% and that of 5-FdUrd was 79.4-117.4%). This simple LC-MS method achieves reliable quantitation and mass balance of 5-FdUrd, 5-FU, and their metabolites and can be effectively utilized for further kinetic studies.

Vegetarianism, female gender and increasing age, but not CNDP1 genotype, are associated with reduced muscle carnosine levels in humans.

Carnosine is found in high concentrations in skeletal muscles, where it is involved in several physiological functions. The muscle carnosine content measured within a population can vary by a factor 4. The aim of this study was to further characterize suggested determinants of the muscle carnosine content (diet, gender and age) and to identify new determinants (plasma carnosinase activity and testosterone). We investigated a group of 149 healthy subjects, which consisted of 94 men (12 vegetarians) and 55 women. Muscle carnosine was quantified in M. soleus, gastrocnemius and tibialis anterior using magnetic resonance proton spectroscopy and blood samples were collected to determine CNDP1 genotype, plasma carnosinase activity and testosterone concentrations. Compared to women, men have 36, 28 and 82% higher carnosine concentrations in M. soleus, gastrocnemius and tibialis anterior muscle, respectively, whereas circulating testosterone concentrations were unrelated to muscle carnosine levels in healthy men. The carnosine content of the M. soleus is negatively related to the subjects' age. Vegetarians have a lower carnosine content of 26% in gastrocnemius compared to omnivores. In contrast, there is no difference in muscle carnosine content between omnivores with a high or low ingestion of ß-alanine. Muscle carnosine levels are not related to the polymorphism of the CNDP1 gene or to the enzymatic activity of the plasma carnosinase. In conclusion, neither CNDP1 genotype nor the normal variation in circulating testosterone levels affects the muscular carnosine content, whereas vegetarianism, female gender and increasing age are the factors associated with reduced muscle carnosine stores.

Validation of an isocratic HPLC method to detect 2-fluoro-beta-alanine for the analysis of dihydropyrimidine dehydrogenase activity.

The efficacy of the chemotherapeutic drug 5'-fluorouracil is reduced by catabolism to 2'-fluoro-beta-alanine (FBAL), a three-step reaction in which dihydropyrimidine dehydrogenase (DPD) catalyzes the rate-limiting step. To study in vitro DPD activity, we developed and validated an isocratic, reverse-phase HPLC method to detect and quantify FBAL without using multiple columns or radiolabeled substrates. Pre-column derivatization of FBAL was performed using o-phthalaldehyde in the presence of two sulfur donors, ethanthiol or beta-mercaptoethanol, and the resulting products assayed. Calibration curves were linear over a range of 10-200 microg/ml and the method was successfully applied to the examination of DPD activity in cultured cells.

The effect of germination on antioxidant and nutritional parameters of protein isolates from grass pea (Lathyrus sativus) seeds.

The aim of this research was to study the antioxidant and nutritional (selected objects) properties of protein isolates obtained from grass pea seedlings as compared with soaked and raw seeds. Two percent extract of isolate from 5-day-old seedlings showed the highest total antioxidant activity (25%) and the ability to chelate Fe²+ (2.35 mg/g d.m.) as compared with other isolates. Protein isolates from grass pea seeds had on average 89% total protein, 87% in vitro protein bioavailability, about 5574 TIU/g (d.m.) (trypsin inhibitors activity) and did not contain ODAP. Germination of seeds for 5 days considerably improved the in vitro bioavailability of isolates, by 12%, and profile of sulfur amino acids by 42%, in comparison with isolates obtained from the raw seeds. Isolates from 5-day-old grass pea seedlings had the best antioxidant properties and improved nutritional parameters (as compared with raw seeds), which makes them worthy of being considered as a potential food additive.

Physiological and chemical analysis of neurotransmitter candidates at a fast excitatory synapse in the jellyfish Cyanea capillata (Cnidaria, Scyphozoa).

Motor nerve net (MNN) neurons in the jellyfish Cyanea capillata communicate with one another by way of fast, bidirectional excitatory chemical synapses. As is the case with almost all identified chemical synapses in cnidarians, the identity of the neurotransmitter at these synapses is unclear. MNN neurons are large enough for stable intracellular recordings. This, together with the fact that they can be exposed, providing unlimited access to them and to their synapses, prompted a study of the action of a variety of neurotransmitter candidates, including those typically associated with fast synapses in higher animals. Only the amino acids taurine and beta-alanine produced physiological responses consistent with those of the normal EPSP in these cells. Moreover, chemical analysis revealed that both taurine and beta-alanine are present in the neurons and released by depolarization. These various findings strongly suggest that either or both of these amino acids, or a closely related compound is the neurotransmitter at the fast chemical synapses between MNN neurons.

Gas chromatography-mass spectrometry screening methods for select UV filters, synthetic musks, alkylphenols, an antimicrobial agent, and an insect repellent in fish.

Two screening methods have been developed for simultaneous determination of ten extensively used personal care products (PCPs) and two alkylphenol surfactants in fish. The methods consisted of extraction, clean-up, derivatization and analysis by gas chromatography-mass spectrometry with selected ion monitoring (GC-SIM-MS) or gas chromatography-tandem mass spectrometry (GC-MS/MS) techniques. Among solvents tested to assess recovery of target compounds from 1-g tissue homogenates, acetone was selected as optimal for extracting compounds with dissimilar physicochemical properties from fish tissue. Initial experiments confirmed that GC-SIM-MS could be applied for analysis of lean fillet tissue (<1% lipid) without gel-permeation chromatography (GPC), and this approach was applied to assess the presence of target analytes in fish fillets collected from a regional effluent-dominated stream in Texas, USA. Benzophenone, galaxolide, tonalide, and triclosan were detected in 11 of 11 environmental samples at concentrations ranging from; 37 to 90, 234 to 970, 26 to 97, and 17 to 31 ng/g, respectively. However, performance of this analytical approach declined appreciably with increasing lipid content of analyzed tissues. Successful analysis of samples with increased lipid content was enabled by adding GPC to the sample preparation protocol and monitoring analytes with tandem mass spectrometry. Both analytical approaches were validated using fortified fillet tissue collected from locations expected to be minimally impacted by anthropogenic influences. Average analyte recoveries ranged from 87% to 114% with RSDs <11% and from 54% to 107% with RSDs <20% for fish tissue containing <1% and 4.9% lipid, respectively. Statistically derived method detection limits (MDLs) for GC-SIM-MS and GC-MS/MS methodologies ranged from 2.4 to 16 ng/g, and 5.1 to 397 ng/g, respectively.