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CALL FOR POSTERS!
AOAC PNW Section invites you to submit a Poster Presentation at the 2008 Pacific Northwest Meeting.

[Click here to view Abstracts for submitted posters.]

The poster topic areas are:

  • Botanicals and Dietary Supplements
  • Detection and Measurement of Natural Toxins
  • Emerging Issues in Food Safety and Security
  • Feeds, Fertilizers and Related Agricultural Topics
  • Food Nutrition and Food Allergens
  • Microbiological Reference Methods
  • Pharmaceutical Analysis, Authenticity and Safety
  • General Analytical Methods, Quality Assurance and Accreditation
  • Seafood Toxins
  • Environmental Microbiology/Chemistry
  • Clinical Microbiology/Chemistry
  • Molecular Biology
  • Physical Sciences

Poster Presentations will be held on June 18, Wednesday 11:30 am to 2:00pm.
To submit a Poster Presentation, at least one of the poster authors must register for AOAC PNW Section. To register for the Annual Meeting please visit our website. All poster submitters MUST pre-register by May 31, 2008. If you do not pre-register by May 31, your name will not be printed in the Final Program.

If you are planning to present a poster please let the Poster Committee Carlos Abeyta, Jr. (carlos.abeyta@fda.hhs.gov or 425.483.4890), Mike Grant (mike.grant@fda.hhs.gov or 425.402.3179), Fred Krick (pestchem@msn.com or 206.522.8013) or Don Bark (don.bark@fda.hhs.gov or 425.402.3165) know by May 1, 2008.

Submission of Abstracts: Abstracts will be published at our website. Submit abstracts to Carlos Abeyta, Jr. (see email above). Please include the following:
1. Your name and title.
2. Affiliation and address
3. Title of your paper
4. Abstract body.

For example:
“A Rapid Method for Determining E. coli 0157:H7”
John Smith, Microbiologist, U.S. Food and Drug Administration, 22201 23 rd. Dr. S.E. Bothell Washington 98021-4421
Abstract Body

GUIDELINES FOR Preparation of Posters:
1. Dimension of poster should not exceed 1 meter X 1 meter.
2. Freestanding boards will be provided for presenting posters and pins will be provided.
3. All posters must be written in English. If the author is non-English speaking, consider having the poster reviewed by an English-speaking person before submitting.
4. All posters should include the title and author information, abstract, methods, results and discussion.

 

 

POSTERS:

Quality Assurance - Why & How Should Laboratories Seek Accreditation?

Jerry Hirsch, JH Techright Consulting Services, #1 – 2842 Whatcom Road, Abbotsford, BC Canada V3G 3B8, hirschj@shaw.ca

 Laboratory accreditation has become an important issue in Canada, the USA and internationally for both commercial and government analytical laboratories. Accreditation, while not guaranteeing that a given analytical result is correct, provides some assurance of the technical and professional competence of a laboratory. Laboratories need to address issues such as Quality Management, Quality Assurance and Quality Control as part of the process in developing an effective Quality Management System that generates analytical results meeting client requirements. This paper describes the steps and basic procedures that need to be followed for an analytical laboratory to become accredited ISO 17025.


Disparities between antibiotic resistance genotypes and phenotypes from calf-origin, commensal Escherichia coli

Margaret A. Davis, Thomas E. Besser, Amelia Lanie, Shira Broschat, Lisa Orfe, Daniel New, Douglas R. Call. (Dept. of Veterinary Microbiology & Pathology, Washington State University, Pullman, WA; WSU-Zoonosis Unit, Washington State University, Pullman, WA; School of Electrical Engineering and Computer Science, Washington State University, Pullman, WA)

Bacterial genomes undergo a low rate of genetic mutation whereby deleterious mutations are purged from populations and advantageous mutations increase in frequency. Many enteric bacteria harbor antibiotic resistance genes that are irrelevant to conventional antibiotic use. For example, in the United States, streptomycin and chloramphenicol have very limited uses in agricultural animal husbandry. Consequently, we expect these rarely selected genes will undergo mutations that are selectively neutral even if these mutations eliminate the ability of the gene to produce a functional protein. In an equilibrium state, dysfunctional genes will be purged from the population, but before equilibrium we should observe cases where antibiotic resistance genes are present in strains of enteric bacteria, but no corresponding phenotypes. To test this hypothesis we compared the resistance genotypes and phenotypes of 52 commensal E. coli isolates from calves. Enteric E. coli populations in young animals are dynamic, and likely do not reach equilibrium before adulthood of the host. Thirty isolates were from a dairy calf-raising operation and 22 were from a beef cow-calf operation; the former represents an environment with intense antimicrobial selection pressure while the latter is more representative of an environment with little to no antibiotic selection pressure. We determined resistance phenotypes using a standard disk-diffusion assay with a panel of 12 antibiotics. To assay resistance genotypes, we hybridized bacterial genomic DNA with an oligonucleotide microarray developed for the project and containing 203 resistance and virulence gene probes. Microarray hybridizations identified 13/52 isolates (26%) with genotypes that did not exhibit resistance phenotypes. There was no significant difference between the two types of cattle operations (6/30 compared to 7/22, Chi sq P=0.33). Although our hypothesis that resistance genes would be more likely to degrade in the absence of antimicrobial selection pressure was not supported by these data, the overall high proportion of isolates with non-functioning resistance genes was unexpected. To characterize the genetic events underlying this resistance gene degradation, we examined the DNA sequences for unexpressed resistance genes and found evidence for nonsynonymous point mutations and potential inversions that may explain the failure of these genes to express correctly. We also observed eight cases whereby resistance phenotypes were present in the absence of corresponding genotypes. These cases probably represent instances where our microarray probes do not correspond to a known gene, or cases of novel genes that warrant further investigation.


A Multiplex Assay for Rapid Detection and Differentiation of Campylobacter jejuni and Campylobacter coli Using Real-Time PCR
Don H. Bark, Carlos Abeyta Jr., Heidi Marks (US FDA - Bothell, WA), Jinxin Hu (Washington State Health Laboratory - Shoreline, WA)
The thermophilic Campylobacter species, particularly C. jejuni and C. coli are among the most frequently isolated cause of bacterial. Reports have shown that Campylobacter jejuni causes more disease than Shigella spp. and Salmonella spp. combined. The rapid detection of bacterial pathogens from food has become increasingly important, both for consumer protection, and for trace back investigations of outbreaks. This work describes a rapid identification method for C. jejuni and C. coli. The technique is a real-time multiplex PCR method that provides genus and species identification of these two bacteria in 2 hours from isolated colonies. The method is also amenable to identification directly from enrichment. The target for the primers and probe are sections of the ceuE that codes for a membrane protein responsible for transfer of enterochelin-iron complexes across the cell wall. Polymorphic sections of this gene (C. jejuni accession X82427, C. coli accession X88849) were selected to design specific primers/probe sets to differentiae C. jejuni and C. coli. The amplicons generated by this RT-PCR method are 61 and 81 BP, respectively. Routine PCR analysis was conducted on the SMART Cycler. Results have shown that this method is sensitive (~1 target detected in monoplex mode) and specific. The method did not recognize any of the non-Campylobacter bacteria from a challenge panel that included Salmonella gaminara, Listeria monocytogenes, E. coli, Klebsiella pneumonia, Morganella morganii, Hafnia alvei, Vibrio cholerae, and Shigella youngae, Staphylococcus aureus, Proteus mirabilis, Enterococcus faecalis. and Pseudomonas aeruginosa.

Genetic Control of Flowering Time in the Allopolyploid Arabidopsis suecica
Natalie Henkhaus, University of Puget Sound, Department of Biology, Tacoma, WA 98416
The species Arabidopsis suecica was derived from allopolyploidization (hybridization concomitant with genome doubling) of the two species A. thaliana and A. arenosa, some 20,000 years ago in a single hybridization event. Allopolyploidization has been implicated in widespread genomic instability (genomic shock), epigenetic changes, gene activation and gene silencing, genomic rearrangements and transposon activation. We hypothesized that the molecular consequences of genomic shock in allopolyploids may lead to greater phenotypic variability in the allopolyploid offspring than in siblings of the original diploid parents. Using thirteen different geographic accessions of the natural allopolyploid A. suecica, we observed phenotypic variation in germination rate, flowering time, flower morphology, seed yield, and pollen viability. Many genes are known to influence flowering in Arabidopsis through genetic and epigenetic mechanisms. These genes are regulated internal (i.e. increase in sugar production) and external (i.e. changes in temperature) factors. Here, we analyze the activity of thirteen genes in pre-bolting leaf tissue to determine if differential expression of genes relating to flowering-time is contributing to the observed difference in phenotype.


Determination of Fluoroquinolone Residues in Food Products Using LC/MS/MS

Cathy S. Hetrick, Restek Corporation, Bellefonte, PA 16823
Fluoroquinolones are a family of broad-spectrum antibiotics that show great efficacy toward both gram-positive and gram-negative bacteria. In the United States, they are not approved for use in farm-raised aquatic animals. Environmental exposure from these food contaminants is suspected to increase antibiotic resistance to this important class of antibiotics. Studies from the Center for Disease Control (CDC) have shown that there may be a linkage between the use of fluoroquinolones in food animals and the spread of drug-resistant strains of Campylobacter in humans.1, 2 The Food and Drug Administration (FDA) recently announced import controls on seafood from China, where the use of fluoroquinolones in food animals is permitted. All incoming shipments will be detained at the border until proven to be free of drug residues. These current events have created an urgent need for the fast and accurate analysis of fluoroquinolones in food stuffs.




Enhancing Retention and Selectivity of Unsaturated Compounds Using a Unique Biphenyl Stationary Phase

Cathy S. Hetrick, Restek Corporation, Bellefonte, PA 16823
Phenyl stationary phases are commonly used for analyses of neutral or aromatic compounds in reversed phase applications, in which they exhibit distinct selectivity differences, relative to butyl (C4) through octadecyl (C18 / ODS) alkyl chain stationary phases. However, a downside to phenyl phases is that they typically show only moderate retention when compared to ODS phases. As a result, advancements have been made in phenyl bonding chemistries to increase their overall retention capacity. One such advancement is the development of a Biphenyl stationary phase. A Biphenyl phase offers a more concentrated phenyl arrangement, and a sterically favorable positioning of the phenyl groups, by presenting a surface chemistry that consists of two phenyl groups bonded end-to-end.


Shopping Smart: Eating Sustainably by Planning Ahead
Sara Montone, University of Puget Sound, Environmental Studies Department, Tacoma, WA 98416

 The average American household discards 10 to 15% of the food they purchase. This makes up the largest component of our residential waste. Shop Smart, a resource-efficient meal-planning system, was the brainchild of a marketing class in fall of 2006. In spring of 2007 their program was tested by the Environment and Society class, taught by Dan Sherman. The class tested three families and several student groups to determine if weekly meal planning with nested ingredients reduced food and packaging waste. As a continuation of that pilot test the aim of this summer 2007 research is to gather a background foundation of supporting research. This includes a Literature Review, surveys conducted at local Proctor and Tacoma Farmer's Markets, and a testing of a variety of methods for pre-planning meals to determine waste/cost per portion to see it the initial claim made by the marketing class was best. The aim of this research is to better prepare the next class of Environment and Society students to conduct a possible second pilot test by providing them with a background of the information already in existence and recommendations for focus.


Total Synthesis and Evaluation of Two N-Acetylmuramic Acid Derivatives
Kelsey Pobanz, University of Puget Sound, Department of Chemistry, Tacoma, WA 98416
T4 lysozyme, an enzyme produced by T4 bacteriophage, hydrolyzes the _(1-4) glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in polysaccharides chains of the bacterial cell wall. The T4 lysozyme also requires that NAM be substituted with a peptide side chain in order for hydrolysis to occur. The only available substrate is actual cell wall, which is heterogeneous, preventing detailed, accurate kinetic analysis. Synthesis of a smaller, well-defined and convenient substrate, and the ability to modify the peptide side chain in that substrate, would allow us to study the kinetics and mechanism of T4 lysozyme. O-nitrophenyl and p-nitrophenyl-b-glycosides of N-acetylmuramic acid linked to Ala-D-Glu(Lys) peptides were synthesized and purified by HPLC and characterized by NMR. Synthesis of the substrates of the T4 lysozyme will be discussed.

A paleomagnetic study of the Early Jurassic Talkeetna Formation, Peninsular Terrane, Alaska.
Erika E. Kercher and Mike Valentine, Geology Department, University of Puget Sound, Tacoma, WA

 Southern Alaska consists of an accretionary complex with over 50 terranes accreted over the past 200 million years. A paleomagnetic study was conducted in the Talkeetna Mountains to better define the poorly understood past movements of the Peninsular terrane and the sequence of accretion between the Peninsular and Wrangellia terranes. Early Jurassic basalts from the Talkeetna Formation were sampled from ten different sites. 8-14 cores were drilled from each site. Thermal and alternating field demagnetization studies were preformed to try to determine primary thermal remanent paleomagnetic directions from each site. Units sampled did not yield consistent inclinations and therefore did not indicate a consistent early Jurassic paleolatitude for the Peninsular terrane. Within-site scatter of paleomagnetic directions and inconsistent demagnetization behavior suggest hydrothermal alteration of magnetic minerals in the samples. A study of the magnetic mineralogy through reflected light microscopy will determine the extent of alteration and help constrain the alteration history of the early Jurassic Talkeetna Formation in the Talkeetna Mountains.


Optimizing Dispersive Solid Phase Extraction and Splitless Injection-Gas Chromatography for QuEChERS Fruit and Vegetable Extracts
Cathy Hetrick, Restek Corporation, 110 Benner Circle, Bellefonte, PA

 QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) is a widely-adopted batch sample preparation method for analysis of pesticides in fruits, vegetables, and other food. Dispersive solid phase extraction (dSPE), adding sorbent, e.g. primary secondary amine, directly to the QuEChERS extract, is used to provide a quick cleanup for samples prior to their analysis with gas and/or liquid chromatography. The effects on recoveries from using different dSPE sorbents on strawberry extracts containing pesticides with a variety of chemical functionalities are shown. Preliminary results are illustrated from splitless injection optimization for a liner containing a CarboFritTM.


Molecular Dynamics Simulations of Cirrus-Like ice Crystal Growth and Sublimation

 Erin K. Nugent, University of Puget Sound, Tacoma, WA
We present classical dynamics simulations of cirrus-like ice crystals on a time scale of tens of nanoseconds and a spatial scale of 20 x20 angstroms. The crystal is represented as a slab, 4-5 molecules thick, with cubic periodic boundary conditions. We report residence times of water molecules striking the slab, as a function of temperature and surface structure. We assess the robustness of these results with respect to simulation time and modeled slab thickness.


Synthesis of a Phosphonate Nucleotide Analog with a 3’-Carbon-Phosphorous Bond
Steven Melhorn, University of Puget Sound, Dept. of Chemistry, Tacoma, WA
The enzyme BamH1 cleaves the backbone of DNA at a specific sequence between two guanine bases. In order to study the mechanism by which BamH1 cleaves DNA, a DNA analog is being synthesized. This analog is identical to an actual segment of DNA except the 3'-oxygen-phosphorus bond in the backbone will be replaced by a 3'-carbon-phosphorus bond, allowing the enzyme to be able to bind with, but not cleave, the DNA. The 3'-oxygen in a DNA segment cannot simply be exchanged for a 3'-carbon, therefore, it is necessary to first prepare a phosphonate nucleotide analog and then incorporate it into the desired DNA sequence. The first seven reactions in this multi-step synthesis have been established, and the last steps are being developed. This research found that the deoxygenation of the 2'-hydroxy group were particularly problematic steps. The hydroxy group was most successfully removed by thionization with phenyl chlorothionoformate in a 1:1 solution of pyridine:chloroform, and subsequently deacylated with N-tetrabutylammonium peroxydisulfate. The final steps involve the attachment of the dimethoxytrityl group and the hydrolysis of the phosphonate diester.


Low-level Mercury Determination in Wastewater Effluent Using CVAFS
Maggie Day, Mercury Product Specialist, CETAC Technologies, 14306 Industrial Rd. Omaha Nebraska 68144.

Both local and regional regulations may require lower mercury detection limits for wastewater effluent, prompting more sensitive analytical techniques. Current regulatory standards do not require detection at the low-level; therefore, low-level reporting data is labeled ‘non-detect’. This reporting could potentially result in biased data and does not give a true representation of the total mercury in the effluent. The current determinative technique, cold vapor atomic absorption spectrometry (CVAAS), does not allow for sub-ppt mercury quantitation. This paper will present low-level mercury determination of wastewater effluent using cold vapor atomic fluorescence spectrometry (CVAFS). This technique allows for analysis over a large dynamic range to obtain accurate, quantitative data, with minimal analysis time and detection limits ranging from 0.05ppt to 100 ppb. Calibration, quality controls and digested wastewater effluent samples will be analyzed to compare and contrast single amalgamation and non-amalgamation with CVAFS using both EPA Method 245.7 and EPA Method 1631, Revision E.


The NIH/ODS Analytical Methods and Reference Materials Program for Dietary Supplements: Five-Year Accomplishments and Future Directions.

Joseph M. Betz, Leila G. Saldanha, Kenneth D. Fisher, Paul M. Coates (Office of Dietary Supplements, National Institutes of Health, Bethesda, MD, 20892 USA), Agnes Nguyen Pho (U.S. Food and Drug Administration, Silver Spring, MD 20993 USA)
Quality of botanical products is one of the greatest uncertainties that consumers, clinicians, regulators, and researchers face. Definitions of quality abound, and include specifications for sanitation, adventitious agents (pesticides, metals, weeds), and content of natural chemicals. Validated analytical methods and reference materials to ensure the identity, purity, quality, and strength of constituents in natural health products and dietary supplements are essential. Researchers need these materials and methods to characterize test articles used in research and ensure that the materials are of sufficient quality that studies can be reproduced. Regulators and industry need them in dealing with regulatory, safety, labeling, quality control, and manufacturing issues. Because these products and their ingredients are often complex mixtures, they pose analytical challenges and methods validation may be difficult. In response to widespread concerns about product quality and the need for validated and publicly available methods for DS analysis, in 2002 the U.S. Congress directed the Office of Dietary Supplements (ODS) at the National Institutes of Health (NIH) to accelerate an ongoing methods validation process, and the Dietary Supplements Methods and Reference Materials Program was created. The program was constructed from stakeholder input and incorporates several U.S. federal procurement and granting mechanisms in a coordinated and interlocking framework. The framework facilitates validation of analytical methods, analytical standards, and reference materials. The major accomplishments of the first five years of the Dietary Supplements Methods and Reference Materials program are collaborative efforts with FDA, AOAC, and NIST. The ODS/FDA/AOAC project has resulted in 18 collaborative studies of methods for dietary supplement constituents. Seven of the studied methods have been approved as First Action Official Methods of Analysis (OMA) and 3 additional methods have been approved as Final Action OMA. An additional 6 collaborative study reports were being reviewed by the AOAC Official Methods Board as of 3/31/08. The ODS/FDA/NIST project has resulted in the production of 5 suites of dietary supplement Standard Reference Materials, with an additional 12 suites in various stages of completion. NIST has also created a pilot Dietary Supplement Laboratory Quality Assurance Program that will assist participating laboratories to become proficient at dietary supplement analysis. A more detailed account of these accomplishments and an outline of the future scope and direction of the program will be presented.


Analysis of Aflatoxins in a Dietary Ingredient
Michael R. Zahn, Research Associate, Unigen Inc., Lacey, WA 98516

 A method for the analysis of aflatoxins B1, B2, G1, and G2 in a dietary ingredient (Product A) consisting of Scutellaria baicalensis and Acacia catechu extracts has been developed at Unigen. Sample cleanup was challenging due to the product’s unique and complex matrix. Current official and industrial methods, such as HPTLC, ELISA test strips, and immuno-affinity columns were tested but found to be unsuccessful due to the presence of interferences. The method developed, modified from AOAC 990.33, utilizes partition in conjunction with a silica gel column clean up and then pre-column derivatization before RP-HPLC analysis at 365nm and 440nm as the excitation and emission wavelengths, respectively. Results showed that the average recovery for total aflatoxins was approximately 70% and none of the samples tested contained detectable aflatoxins.

Brinkmann Titrator Delivery Analysis
Teresa Wenda, Dairy Scientist and Cole Monnahan, Agricultural Statistician, USDA Federal Milk Market Administrator, Bothell, WA
The Brinkmann Titrator was suspected of causing a pattern of errors in analysis through inaccurate delivery volumes. Delivery accuracy was tested by “titrating” a particular volume of HCl into a narrow mouthed Erlenmeyer flask on an analytical balance (0.0001g precision). The mass, delivered volume, and temperature of certified 0.1000N HCl were recorded. A calculated volume was obtained using the mass and density of HCl and compared to the delivered volume. Differences in density due to HCl temperature variations were observed, and data was “corrected” for these variations. Multiple operators with unique techniques, all performed analyses on different dates throughout a range of volumes and pump speeds. Our particular analysis involves raw milk protein content as determined by the Kjeldahl method. The theory was that at higher delivery volumes the error in delivery was greater, causing larger protein differences. In testing the Brinkmann, it was discovered that the HCl temperature has a noticeable effect on the final results; however upon further analysis, this effect was found to be small in comparison to the inherent variation of the Kjeldahl method. Nevertheless, the most significant outcome of this study was that the results when tabulated at working temperatures made the Brinkmann look as if it failed; yet, when tabulations were corrected for temperature differences from the reference of 20°C, the Brinkmann proved to work with guaranteed precision.


Pervasive Crustal Melting on a Regional Scale: Sr-Nd Isotopic Evidence from Eocene Intrusions in NE Washington
Matthew Loewen, University of Puget Sound Geology Department, Tacoma, WA

During the Eocene the Pacific Northwest was the site of a short-lived but voluminous and geochemically diverse magmatic episode, commonly termed the Challis event. To investigate the origins of this event we have measured whole rock Sr and Nd isotopic compositions of 12 plutonic and hypabyssal samples, ranging from basalt to two-mica granite, collected along a 250 km transect across NE Washington. This transect crosses the 0.706 line (Armstrong 1977), the boundary between dominantly Mesozoic crust to the west and older crust to the east. The results reveal a wide spread in isotopic compositions (87Sr/86Srm = 0.7041 - 0.7262; ?Ndm = +3.8 to -18.5) with no systematic relationship between isotopic composition and bulk composition (e.g., MgO). This decoupling of isotopic composition and bulk chemistry suggests mixing between mantle and crustal melts was of minimal importance, and that these rocks are dominantly of crustal origin. The range in ?Ndm also indicates melting of crustal sources that varied considerably in age. Samples with ?Ndm > +2 range from basalt (13 wt. % MgO) to two-mica granite (0.3 wt.% MgO). Such juvenile ?Ndm in a two-mica granite precludes significant involvement of ancient metasedimentary material and implies rapid intracrustal differentiation of a mantle-derived source, which may have been deep arc crust of Mesozoic age. At the other end of the spectrum, samples with ?Ndm < -15 come from Springdale (87Sr/86Srm = 0.7071; ?Ndm = -18.5) and Medical Lake (87Sr/86Srm = 0.7143; ?Ndm = -15.2) at the eastern side of the study area. Data from these sites, both east of the 0.706 line, are similar to values reported for the nearby Silver Point Quartz Monzonite and attributed to melting of late Archean to Early Proterozoic crust (Whitehouse et al. 1992). Other samples analyzed in this study are broadly similar in isotopic composition ?Ndm = +1 to -8; 87Sr/86Srm = 0.706-0.709) to rocks of the Colville Igneous Province and probably formed by melting of Proterozoic arc crust (Morris 2000). Geographic variability in Sr-Nd data indicates that isotopically distinct crustal domains are juxtaposed laterally and/or vertically, in some cases on a small scale. The sample with the highest 87Sr/86Srm (0.7262; ?Ndm = -13.3) is a dacite porphyry well west of the 0.706 line, while at Porcupine Bay adjacent plutons differ by almost 10 ?Nd units (-7.4 and 2.2). Ongoing work is designed to further characterize the crustal sources and better understand the nature of the thermotectonic event that drove such widespread crustal melting.


Background Challenges During LC/MS Ion Trap Analysis of Marine Toxins

Heidi Marks, Applied Technology Center, Pacific Regional Laboratory Northwest, Food and Drug Administration, Bothell, WA
A new Thermo LTQ-XL ion trap MS coupled with an Agilent 1200 HPLC was used to determine low ppm levels of the common American coastal marine toxins, domoic acid and brevetoxin-3. However, along with the analytes were found a number of extraneous peaks and ions to challenge the chromatography and mass spectral interpretation. A brief picture is presented here to stimulate discussion among new mass and liquid chromatographers. Attention to background ions can reveal: Less than ideal mass resolution of ion trap can lead to confusion with closely eluting peaks of similar mass, or when elucidating unknown ion origin or fragmentation; that adduct ions are common in ion trap – one should be mindful of unexpected combinations or clusters; and that sample preparation materials can contribute greatly to background, notably during residue analysis.


Influence of Female-Specific Ornament on Male Response in Striped Plateau Lizards, (Sceloporus virgatus)
Brianna J. Bean, biology undergraduate, University of Puget Sound, Tacoma, WA 98465

 Male ornamentation has been extensively researched, but few studies have focused on female-specific ornaments. There are essentially two hypotheses for the existence of female-specific ornaments. The first suggests that these ornaments are genetic correlations to male ornaments, while the second proposes direct sexually selection. Striped plateau lizards (Sceloporus virgatus) exhibit a female-specific orange throat ornament, most evident during reproductive receptivity. Previous research has found that this ornament indicates phenotypic quality of the female, and males choose mates based on its color intensity. However, the size of the ornament may better indicate quality and more significantly impact male choice of females. This study aimed to manipulate the ornament size using a paint treatment on S. virgatus in Arizona. In the laboratory, we assessed male behavioral response to different ornament treatments of female lizard, including small, large, asymmetric, and no orange patches. It was found that the size of the throat patch did not influence male courtship behavior, although there was a strong negative trend between male courtship and asymmetric color patches. In addition to the paint manipulation, we examined male-male aggression behavior to determine if a correlation existed between male courtship and male aggression. None was determined, although again trends for a correlation were seen, which suggested that additional trials might aid in determining if any do exist.


Preliminary characterization of Multiple Cya-like Genes of the Predatory Bacterium Bdellovibrio bacteriovorus
Jillian Waters, UPS Graduate, Coeur d’Alene, ID 83815

Inspection of the published genome of the predatory Gram-negative ?-proteobacter Bdellovibrio bacteriovorus reveals five cya-like genes. These genes appear related to adenylate cyclase (AC), which synthesizes cAMP. cAMP is a global secondary messenger involved in gene regulation for a number of organisms. Currently, there is no known obvious role for cAMP in Bdellovibrio and genome annotation has not revealed any cAMP receptor protein-like gene, making the cAMP-CRP paradigm of many Gram-negative bacteria problematic. The object of this study was to better characterize the five cya-like genes of Bdellovibrio and determine if they encode functional ACs. Additionally, we are interested in determining if any of the putative ACs are differentially expressed during the biphasic life-cycle of Bdellovibrio. Bioinformatic analysis shows significant differences in sequence and domain structure among the five cya-like genes. Thus far, four of the five cya-like genes have been successfully amplified using PCR with Bdellovibrio specific primers. For each of the cya genes the amplicon was cloned and transformed into E. coli strain PD200 (?cya, crp+) (Dunlap, 1989). Differential plating on MacConkey-maltose medium has demonstrated that two of the four genes, cyaA and cyaK, produce cAMP in E. coli. RT-PCR analysis indicates one of these genes, cyaA, is upregulated during the intraperiplasmic growth phase rather than while the bacterium is seeking new prey cells. Conversely, cyaK appears to be inversely differentially regulated. CyaK is shown to be upregulated during attack phase rather than within the periplasm of the host cell. Thus, we hope to begin to understand the role of cAMP within Bdellovibrio and any possible relationship to the lifecycle of the organism. As both cyaA and cyaK are active in Bdellovibrio (determined functionally and via RT-PCR), the differential gene expression uncovered may shed light on regulatory networks in this predatory bacterium.


Cyanotoxin ELISA Testing in Delaware: Process Development
Edythe M. Humphries, State Harmful Algal Bloom Program Coordinator; Katherine Painter; and Ben Pressly. State of Delaware, DNREC, Environmental Laboratory, Dover, DE 19901
Development of the ELISA test process, using commercial ELISA test kits, included establishing (1) protocols for sample collection, transport, and storage; (2) protocols for sample preparation including lysing cells, (3) quality control acceptance criteria for Laboratory Blanks, standard curve goodness-of-fit, and control sample accuracy/precision; (4) criteria for evaluating sample result precision using duplicate absorbance measurements; (5) acceptance criteria for the value of sample B/Bo% ratio relative to the range of this ratio in the calibration standards; (6) criteria for evaluating temporal stability of manufacturer-supplied calibration standards and controls; (7) a detailed laboratory standard operating procedure including preparation of environmental sample dilutions and spiking solutions, and operation of a Bio-Tek FL600 Fluorescence/Absorbance Plate Reader in absorbance mode; (8) a minimum quantitative reporting level for environmental samples; and (9) data management protocols.


Determination of Hydrastine and Berberine in Goldenseal Raw Materials, Extracts and Dietary Supplements by HPLC-UV: Collaborative Study
Paula N. Brown1, Lori A. Paley1, and Mark C. Roman2. 1NHP Research Group, Technology Centre & School of Health Sciences, BC Institute of Technology, Burnaby, BC. 2 Tampa Bay Analytical Research, Inc., Largo, FL 33777
A multi-laboratory collaborative study was conducted on a High Performance Liquid Chromatography (HPLC) method utilizing Ultraviolet (UV) detection, previously validated using AOAC Single Lab Validation guidelines for the determination of hydrastine and berberine in Goldenseal (Hydrastis canadensis L.) raw materials, extracts, and dietary supplements at levels ranging from 0.4% - 6% w/w. Nine collaborating laboratories determined the hydrastine and berberine content in eight blinded samples. Sample materials included powdered botanical raw materials, whole root material, and four finished product dietary supplements containing either goldenseal powdered root material or extract. Overall laboratory reproducibility was evaluated through the calculation of reproducibility standard deviations, reproducibility relative standard deviations and HorRat values as per AOAC guidelines inter-collaborative validation studies. Results from the laboratories were acceptable and the method of analysis was recommended for Official First Action for determination of hydrastine and berberine in goldenseal raw materials and dietary supplement finished products containing powdered goldenseal and goldenseal extract.