A.Weisz1, D.Andrzejewski2, 1Office of Cosmetics and Colors, CFSAN, FDA, Chantilly, VA 20151, 2Office of Scientific Analysis and Support, CFSAN, FDA, College Park, MD 20740
The present work describes (a) the identification and characterization of a newly-found contaminant, 2-bromo-3, 4, 5, 6-tetrachloroaniline (2BTCA), in the color additives D&C Red Nos. 27 and 28 (phloxine B) and (b) the determination of the extent and level of 2BTCA contamination in certified lots of these colors. For these purposes, 2BTCA and its positional isomer 4-bromo-2, 3, 5, 6-tetrachloroaniline (4BTCA) were synthetically prepared. 4BTCA was used as the internal standard for the quantification of 2BTCA in the colors. Test portions from 35 certified lots of D&C Red Nos. 27 and 28 were analyzed for 2BTCA using a solid-phase microextraction/GC-MS method. Those lots were submitted for certification by both domestic (seven) and foreign (four) manufacturers during the past four years. Of the test portions analyzed, 22 (62.9%) contained 2BTCA in amounts ranging from 0.15 ppm to 435.7 ppm with an average value of ~131.7 ppm. The remaining 13 (37.1%) test portions contained no 2BTCA or less than 0.01 ppm, which is the limit of quantification of the present method. The analyses revealed substantial differences in the level of 2BTCA across lots from the same manufacturer as well as among different manufacturers. The wide range of 2BTCA levels found in the analyzed lots suggests that the presence of 2BTCA in D&C Red Nos. 27 and 28 may be avoided or significantly reduced during the manufacturing process. A chemical pathway that could explain the presence of 2BTCA in these color additives is also proposed.
C.Beaudry1, P.Eilers2, S.Hall1, 1Office of Seafood, FDA, Laurel, MD, 2Office of Seafood, FDA, Dauphin Island, AL
The Washington Seafood Lab currently uses a modification of the AOAC method for HPLC analysis of domoic acid (amnesic shellfish poison). With the standard method, tryptophan in seafood extracts often coelutes with domoic acid confounding analysis. Triethylamine, a mobile phase modifier, has been found to help resolve tryptophan and domoic acid (Eilers 1996). In order to better understand this effect and optimize conditions, the current study examined the effects of different triethylamine concentrations and pH levels on domoic acid and tryptophan separation and efficiency. Twenty mobile phases were made at five concentrations of triethylamine and four levels of pH. Test mixtures of domoic acid and tryptophan were analyzed. Triethylamine was found to work by reducing retention time and tailing of the tryptophan peak, while having little effect on domoic acid. Increased pH caused a more marked reduction in tryptophan retention time relative to domoic acid but this benefit was offset by lower efficiency. The observations can be explained by competition of triethylamine with the analytes for free silanol sites on the column, and by the relative pKa values of tryptophan and domoic acid. Ideal routine run conditions for the column used were 1 mM triethylamine at pH 2.5.
J.Brower1, J.C.Reepmeyer1, T.Moore1, L.F.Buhse1, M.M.Nasr1, A.S.Hussain2, 1CDER, Division of Pharmaceutical Analysis, St. Louis, MO, 2CDER, Office of Pharmaceutical Science, Rockville, MD
Government stockpiles of drugs needed for bioterrorism include only adult solid dosage forms. For effective pediatric application, the drugs need to be ground and mixed with appropriate food/drinks. Additionally, the dosage form must have good stability and dose uniformity as well as reasonable taste and palatability. To study doxycycline stability and dose uniformity, tablets were ground, visibly divided into halves and quarters and mixed with low fat chocolate milk. Stability of the ground material over several days was also investigated. The following preparations were used to evaluate 24 hour stability (room temperature and refrigerator): water, apple juice with table sugar, low fat milk, low fat chocolate milk, regular chocolate milk, chocolate pudding, grape jelly, strawberry jelly, yogurt with cherry flavor, and simple syrup with FlavoRx sour apple flavor. Doxycycline was found to be stable as a ground powder for one week or for at least 24 hours in the food/drinks evaluated. Dose uniformity for the half tablet portions was good; the quarter tablet portions, exhibited higher variability.
D.Toler1, J.Brower1, M.M.Nasr1, L.F.Buhse1, N.Sadrieh2, 1CDER, Division of Pharmaceutical Analysis, St. Louis, MO, 2CDER, Office of Pharmaceutical Science, Rockville, MD
The government has a large supply of some drugs including Cipro for use in the event of an emergency for inhalation anthrax. For Cipro, the dosage form recommended for children is a suspension, but the government pharmaceutical stockpile does not include a suspension formulation for this drug. In order to deliver effective dosages to children, the tablets must be disintegrated and mixed with appropriate food/drinks. The prepared dosages must have good stability and dose uniformity as well as reasonable taste and palatability. In this study, suspensions of the tablets in water were mixed with various foods/drinks (water, apple juice, Log Cabin Syrup, Similac, strawberry jelly, chocolate milk, and chocolate syrup). Stability of these preparations was studied over 7 days. Dose uniformity using common dispensing cups was determined. Stability data show that Ciprofloxacin HCl suspended in water and mixed with Log Cabin Syrup, apple juice, strawberry jelly, Similac and chocolate milk can be stored for 24 hours in a refrigerator. The suspension mixed with water or chocolate syrup can be stored for 1 week in the refrigerator.
J.Brower1, L.Revelle1, B.J.Westenberger1, M.M.Nasr1, L.F.Buhse1, N.Sadrieh2, 1CDER, Division of Pharmaceutical Analysis, St. Louis, MO, 2CDER, Office of Pharmaceutical Science, Rockville, MD
Lindane is commonly used to treat scabies and lice. Although the label recommends using non-permeable gloves when applying the lotion or shampoo, the extent to which Lindane can cross the glove barrier, or the relative permeability of various types is unknown. Because Lindane is stored in body fat and adrenal glands, observed toxic effects will be cumulative. Four types of gloves were used in this study (vinyl, latex, latex blend with neoprene, and nitrile). Gloves were inverted and approximately 40mL of shampoo or lotion were evenly distributed in the glove. Gloves were suspended in water at 37C, with stirring, and samples of the water were taken at 5, 30, and 60 min. Samples were extracted by solid phase extraction and analyzed by Gas Chromatography with electron capture detection. All gloves were found to be impermeable for short duration exposure (5 minutes). At one hour, nitrile and latex with neoprene gloves showed no Lindane permeability while vinyl and natural latex gloves showed considerably greater permeability. The latex glove with the greatest permeability, allowed <0.1% permeation of the applied Lindane concentration. At long exposures, greater permeability was observed for the shampoo than for the lotion.
U.R.Cieri, FDA
Reserpine , rescinnamine and deserpidine, alkaloids obtained from the roots of Rauwolfia plants are used as hypotensive agents. Commercial products containing reserpine alone or in combination with a diuretic were very common thirty or forty years ago but their use has dropped dramatically in recent years because other more effective antihypertensive agents were developed and marketed. Today there are still a few products containing reserpine alone or in combination. Rescinnamine and deserpidine never enjoyed great popularity but occasionally a product containing one of these two alkaloids appears in the market. Atropine (d-l Hyoscyamine) sulfate and l-Hyoscyamine Sulfate, alkaloids obtained from Atropa belladonna or similar plants , have identical chemical structure but have different stereo configuration. Commercial products containing a belladonna alkaloid are used as anticholinergic, mydriatic or antidiarrheal agents. A review is presented of methods, both official and non official, for the analysis of commercial products containing a Rauwolfia or a Belladonna alkaloid. An evaluation is also made on the specificity and accuracy of each method; the issue of detecting impurities and degradants is also discussed briefly.
K.K.Cook1, D.Fitelson2, P.Delmonte1, K.D.White1, E.Grundel1, M.P.Yurawecz1, J.I.Rader1, 1CFSAN, FDA, College Park, MD, 2JIFSAN, UMD College Park, MD
While sales of herbal supplements have increased significantly in recent years, information about the composition of botanical raw materials has not kept pace. Pyrrolizidine alkaloids (PAs) are common toxins produced by many species of flowering plants. These alkaloids may be carcinogenic, mutagenic, teratogenic and, with chronic use, hepatotoxic. Plants containing these alkaloids, including comfrey, may pose significant health hazards to those consuming them in certain kinds of teas , tinctures, salves or other traditional remedies. We purchased authenticated comfrey (Symphytum x uplandicum) leaves and roots from Botanical Liaisons, Boulder, CO. Retrorsine, a PA standard, was purchased from Sigma-Aldrich, Inc. (St. Louis, MO) Retrorsine was used to predict the specta and chromatographic behavior of other PAs contained in this species of comfrey. Thin layer chromatography (TLC), mass spectrometry (MS) and authenticated standards symphytine, symlandine and echimidine, generously provided by Research Triangle Institute (Research Triangle Park, NC), have been used to identify and target other PAs for collection and further identification. Various extraction procedures (e.g., extraction in methanol: ammonium hydroxide and straight methanol) have been utilized and the alkaloid compositions of the resulting extracts compared using HPLC and TLC.
L.S.de Jager, G.A.Perfetti, G.W.Diachenko, OFAS, CFSAN, FDA, College Park, MD
There is a growing movement in the United States towards using natural therapies and herbal dietary supplements as alternatives to western drugs. Since the passage of the Dietary Supplement Health and Education Act of 1994, marketing of supplements containing herbal ingredients has increased. Over the past few years, several food products containing botanical ingredients have appeared on the market. These “functional foods” occupy a tenuous position between dietary supplement and food. Most dietary supplement ingredients have not been pre-approved as food additives or determined to be Generally Recognized as Safe (GRAS). Although many methods have been developed to detect marker compounds from botanicals, little work has been done for the detection of these compounds in complex food matrices. In this study, analytical methodology has been developed for the detection of characteristic bioactive compounds of St. John’s wort (Hypericum perforatum L.) and kava (Piper methysticum) in functional food and drink products. Using various sample preparation techniques, in conjunction with LC, detection limits in the low ppb range have been obtained. Using these methods, a variety of food and drink products purporting to contain St. John’s wort and/or kava were analyzed. LC-MS analysis was conducted on sample extracts for confirmation.
A.V.Del Grosso, J.C.May, CBER, FDA, Rockville MD 20852
Plant pollen source materials used to manufacture allergenic extracts are often processed using perchloroethylene (PCE). In this process, pollens are separated from soil and other contaminants based on density differences while suspended in PCE. Since PCE is a known carcinogen, a study was planned to measure PCE content in representative samples of pollen source materials. A solvent extraction and GC/MS SIM procedure for the determination of residual PCE in pollen source materials has been developed. 10 mg of pollen is extracted with 1 mL methanol and 1, 1, 2-trichloroethane added as an internal standard. Chromatography is on a 5% phenyl column with dimensions of 0.25mm x 30m and 0.25 micron film thickness, operated isothermally at 70oC. Sample is introduced using a 1:20 split injection and detection is by selected ion monitoring. Total chromatographic analysis time is 4.5 minutes. Recoveries of > 90% were obtained from pollen samples spiked with quantities that corresponded to 10% to .01% w/w PCE/pollen. PCE content was determined in 41 lots of pollen source materials representing 32 different types of pollen. Measured concentrations ranged from 15.2 % w/w to less than the assessed limit of quantitation (0.01% w/w).
W.H.Doub1, W.P.Adams2, J.A.Spencer1, L.F.Buhse1, M.M.Nasr1, P.J.Treado3, M.P.Nelson3, D.S.Lester4, A.S.Hussain5, 1DPA, OPS, CDER, FDA, St. Louis, MO, 2OPS, CDER, FDA, Rockville, MD, 3ChemImage, Pittsburgh, PA, 4Pharmacia Corp., 5OPS, CDER, FDA, Rockville, MD
A feasibility study was conducted to examine whether Raman imaging is capable of determining the chemical identity, particle size and PSD of micronized drug substance, principally corticosteroids, in aqueous suspension formulations of nasal sprays. Raman spectra were obtained for the active pharmaceutical ingredient (API) and all excipients for a common formulation of aqueous suspension nasal spray. The formulation was sprayed onto a microscope slide, and brightfield as well as API-specific chemical images were obtained of the dried particles. These images were processed using ChemImage’s software to obtain the PSD of the API particles. Calibration of the imaging system was performed using NIST—traceable microparticles.
API particles were clearly distinguishable from those due to excipients. We also observed API particles adhering to excipient particles. This phenomenon may be an artifact of sample preparation and studies are planned to address this issue. The use of Raman-based chemical imaging enables the qualitative identification of corticosteroid in the presence of microcrystalline cellulose as is found in an aqueous suspension nasal inhalation product. Studies are currently underway to investigate the technique for quantitative particle sizing.
This work was presented at AAPS 2002.
W.H.Doub1, A.M.Wokovich1, J.C.Black1, W.P.Adams2, 1DPA, OPS, CDER, FDA, St. Louis, MO, 2OPS, CDER, FDA, Rockville, MD
Several configurations of the Andersen Cascade Impactor (ACI) were evaluated to assess its utility to provide sufficient precision for the measurement of “fines” in aqueous nasal sprays when used for in vitro bioequivalence (BE) studies. Deposition of nasal inhalation pharmaceuticals were assessed with the full (8–stage), standard (ACI28.3) and 90 lpm (ACI90/28.3) configurations, both at a flowrate of 28.3 lpm. These results were compared to those obtained using the ACI28.3 and ACI90/28.3 configurations employing only three stages (top two and final plus filter). Additionally, three different spherical induction chambers (1L, 2L, and 5L) were examined both with and without the Andersen preseparator.
Early experiments showed the 1L chamber to be too small for use with aqueous nasal sprays and it was eliminated from all subsequent comparisons. Using either remaining chamber, the ACI90/28.3 configuration allows as much as 40% more drug to be collected in the CI, although absolute amounts are very small (0.5-1%) with both configurations. A dramatic increase in the measurement precision is observed when the 2L chamber is used. The use of the 3–stage ACI configurations yields additional improvement in the precision of the measurement of “fines”, particularly with the 2L chamber.
This work was presented at AAPS 2002.
R.J.Mobley, J.C.Archer, P.Barnes, V.Litman, S.Shojaee, M.K.Halbert, J.J.Eckert, FDA/ORA/Arkansas Regional Laboratory, 3900 NCTR Rd., Jefferson, AR 72079
Typical sample preparation for dioxin analysis utilizes an acidified silica gel or acetonitrile cleanup that may be followed by further cleanup using activated carbon. Cleanup of high fat samples with acid silica requires large quantities of reagents. Under these conditions, the recoveries are inconsistent. The traditional cleanup using acetonitrile suffers from low recoveries. An improved method, FDA LIB 4084 uses a silica gel cleanup followed by a reusable carbon column then a micro acid silica/alumina fractionation. Although LIB 4084 generally gives acceptable results for low fat samples, frequent problems have been encountered with high fat matrices. The analysis of samples such as vegetable oils, butter, shortening, fish oil, and Vitamin E frequently result in carryover problems with analyses when reusing carbon columns. Dioxin elution profiles with toluene reveal the elution solvent volume is significantly affected by the matrix. This results in the need to replace the carbon column or use large volumes of toluene to clean the carbon column between analyses. This paper proposes the use of a single-use carbon column with high lipid samples. The benefits of this approach are elimination of re-extractions caused by carryover from previous extractions, lower solvent usage and ability to use an auto-feed technique.
G.Hirsch, M.K.Halbert, R.V.Furth, FDA/ORA/Arkansas Regional Laboratory, 3900 NCTR Rd., Jefferson, AR 72079
Blue fish sample extracts that have previously been prepared and analyzed for dioxin / furan analyses were re-analyzed for three, co-planar, polychlorinated biphenyls (co-PCBs); PCB 77, PCB 126 and PCB 169. These co-PCBs have been given toxic equivalency factors (TEFs) that when added with the dioxin / furan TEF contributions could result in a total toxic equivalent (TEQ) that is of concern. Currently, the United States has no set TEQ tolerance level, however any consumable part of a fish near 1 pg/g usually indicates a need for follow-up studies. TEQ values, from dioxin / furan contributions, ranged from 0.9 to 2.3 pg/g for five blue fish extracts. The addition of these three co-PCBs elevated the TEQ range to 1.6 -11 pg/g.
S.Shojaee, J.C.Archer, R.Vocque, FDA/ORA/Arkansas Regional Laboratory, 3900 NCTR Rd., Jefferson, AR 72079
There are several advantages to using the Accelerated Solvent Extractor (ASE-300) over conventional Soxhlet extraction for animal feeds. These include:
For the work reported in this presentation, five grams of various feed samples were spiked with the internal standard 13C12-labeled dioxin and furan congeners, with Ottawa sand serving as the method blank. The ASE-300 extraction conditions were completed at 125°C under 1500 psi in approximately 20 minutes. The solvents used were 1:1 hexane:dichloromethane. The extracts were subjected to a clean-up process of multi-layered silica gel and alumina columns and then analyzed by gas chromatography/high resolution mass spectrometry (GC/HRMS).
C.Earnheart, L.M.Pence, M.K.Halbert, G.Hirsch, J.C.Archer, FDA/ORA/Arkansas Regional Laboratory, 3900 NCTR Rd., Jefferson, AR 72079
When utilizing and adopting the Chemically Activated LUminescence eXpression (CALUX) bioassay to detect dioxins efficiently, recovery methodologies must provide results without affecting the cells or analyses. Different recovery methods are available for CALUX® analysis. This cellular dependent bioassay system detects compounds that activate the Aryl hydrocarbon Receptor (AhR).
The recovery method recommended by the manufacturer uses a surrogate technique in which one of the duplicate extracts is spiked with a 14C12 dioxin. The recovery is then calculated from a scintillation counter response. Another method calculates percent recoveries from a single extract per sample batch. Using only a batch recovery for calculations assumes no matrix affect and thus is not a true recovery.
Our approach to achieving recoveries is to add 12C12 1, 2, 3, 4-TCDD to each sample and quantitatively determine recoveries by GC/Mass Spectrometry. Advantages to this approach include:
J.C.Archer, L.M.Pence, L.Bluhm, C.Earnheart, J.J.Eckert, FDA/ORA/Arkansas Regional Laboratory, 3900 NCTR Rd., Jefferson, AR 72079
In the Chemically Activated LUminescence eXpression (CALUX®) bioassay, the presence of dioxins and furans in a sample triggers the production of light via a genetically engineered cell line containing the luciferase gene. The CALUX® methodology allows for the simultaneous analysis of a large number of samples, thereby making it a useful screening method for prioritizing sample analyses and reducing the number of samples analyzed by traditional means, namely Gas Chromatography/High Resolution Mass Spectrometry (GC/HRMS). Before the CALUX® method could be used routinely, it was necessary to show that the assay was as sensitive as traditional methods and that there was good agreement between the methods according to established guidelines. Samples representing various types of matrices (feed and feed components; vitamins, minerals, fats, etc.) were analyzed using the CALUX® method. The results from the CALUX® method were then compared to results obtained by traditional methods (extraction, GC/HRMS). Archived samples representing high, moderate and low total Toxic Equivalency (TEQ) values were chosen for the comparison in order to assess the accuracy, precision and sensitivity of the detection method over a wide range.
M.Farahani, CDER, FDA, Rockville MD
The least studied and understood type of DNA damage in cells is the cross-linking between DNA and proteins. Hydroxyl radicals induce cross-linking between phenylanaline (Phe) and 2-deoxyribose (dR) via formation of corresponding free radical intermediates. In this study, aqueous solutions of the mixture of Phe (0.5 x10-3 M, pH 6.3) and dR (0.92 x10-3 M), were saturated with oxygen-free N 2O for 30 min and irradiated in a 60Co gamma source dose range 110-440 Gy, dose rate 110 Gy/min. Samples (10 mg) dried with a rotary evaporator were trimethylsilylated (TMS) in Teflon-capped Hypovials with 0.1 mL each of Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine (1:1) by heating for 30 min at 140°C. The cross-linked products were separated and identified by capillary gas chromatography-mass spectrometry. In the present work, we find that hydroxyl radical induced cross-links between (Phe) and (dR) can take place in model aqueous systems. The newly discovered cross-link between 2-deoxyribose and Phenylalanine may also serve as a model for radiation or free radical induced cross-linking between DNA and proteins and in general between sugar moieties and amino acids.
P.J.Faustino1, A.B.Ciavarella1, E.B.Asafu-Adjaye1, C.R.Brownell1, L.X.Yu2, R.C.Lyon1, 1Division of Product Quality Research, Kensington, MD 20895, 2Office of Generic Drugs, Rockville, MD 20855
A simple, efficient and highly specific reverse phase HPLC method was developed that simultaneously measures plasma concentrations of hydrochlorothiazide (diuretic) and propranolol (B-blocker) in patients given an FDA-approved combination solid oral dosage form tablet. Analytes were extracted from human plasma with silica based (SPE) C-18 cartridges used in combination with unendcapped Cyano SPE cartridges. Hydroflumethiazide and labetalol were added to the plasma samples and standards as internal standards (IS) in the two-phase continuous SPE extraction. Samples were analyzed on an Agilent 1100 series HPLC equipped with a 1046A fluorescent detector and an 1100 series UV detector. Separation was achieved on a 250 X 4.6 mm, 5 micron Phenomenex C-18 (2) Luna column using a 20% ACN/20 mM PO4, pH=3.0 reverse phase isocratic method. Drugs and IS’s were detected simultaneously utilizing fluorescent detection (EX 232 EM 400) for propranolol and labetalol (IS) and ultra-violet detection (272 nm) for hydrochlorothiazide and hydroflumethiazide (IS). The method was validated using FDA’s Guidance for Industry, “Bioanalytical Method Validation”. Hydrochlorothiazide and propranolol met all validation acceptance criteria as defined in the guidance. Hydrochlorothiazide and propranolol had average recoveries ranging from >90-102% and r2 of 0.993 to 0.998 over the analytical range of 2.5-150 ng/mL.
M.L.Gay, R.A.Niemann, S.M.Musser, FDA, College Park, MD
As part of our efforts to develop methods for the quantitation and confirmation of identity of ingredients in dietary supplements, we have extended our previously reported analytical method for the determination of ephedrine-type alkaloids from Ephedra sinica (Ma Huang) in dietary supplements to include synephrine. Synephrine is structurally related to the ephedrine-type alkaloids and occurs naturally in bitter orange (Citrus aurantium). It is often an ingredient in dietary supplements, including those containing Ephedra. We also developed an MS/MS method for the analysis. Results of the analysis of 25 finished products by this method were compared with those obtained by APCI using increased tube lens voltage to achieve adequate fragmentation. A plot of the results obtained by LC/MS/MS vs. those obtained by LC/MS. (i.e. y vs. x) gives excellent agreement, as demonstrated by the slope of 0.986 and the correlation coefficient (R2) of 0.9991. Comparison of the results for synephrine in the eight products found to contain it also demonstrated good agreement.
George.M.Hanna, NERL, FDA, Jamaica NY
1H and 13C NMR methodologies are described for the determination and the characterization of melatonin (N-acetyl-5-methoxytryptamine), a hormone used as supplementary drug in the alleviation of jet-lag and other sleep disorders, utilizing a 400 MHz spectrometer without the need of pure reference standards. The developed methodologies are able to differentiate between melatonin and its precursor neurotransmitter serotonin, assess chemical structure of these compounds, and determine the quantity in the dietary supplement formulation. The NMR methodology was found suitable to monitoring the photo-stability, with the ability to detect degradation products. The quantitative analysis was based on the integrals for the methyl proton of 2-methyl-2-propanol served as an internal standard at 1.24 ppm and the methoxy protons at 3.86 ppm. The precision was established by analyzing synthetic mixtures of the analyte and internal standard. Excellent agreement was verified between the assay results and the quantities of analyte in the mixture.
C.B.Nochetto, D.N.Heller, FDA, Laurel, MD
CVM's Office of Research is developing a novel approach to the higher throughput analysis of food tissues from animals for presence of drug residues. This approach is based liquid chromatography-electrospray tandem mass spectrometry (LC/MS/MS), which has the ability to detect a wide variety of drug classes. Ionization conditions were used that were compatible with a generic, wide-range LC gradient. Acquisition parameters were tailored for compounds that eluted at various times during the gradient. The second multi-class extraction method to be combined with this approach uses solid phase extraction (SPE) of eggs with a silica phase. This method was capable of extracting multiple compounds from two major drug classes: polyether ionophores (e.g., salinomycin, monensin) and macrolides (e.g. erythromycin, tylosin). Sensitivity and selectivity by LC/MS/MS was very good: residues could be detected below10 ng/g (ppb). Residue-incurred eggs, i.e., eggs from hens dosed with these drugs, were analyzed during method development. The method is designed for use in surveillance for potential misuse of drugs in eggs.
P.J.Kijak1, J.D.von Bredow1, C.B.Nochetto1, R.J.Condon2, W.T.Hammack3, A.Brown3, 1CVM, FDA, Laurel, MD, 2RJC Associates, Inc. Myersville, MD, 3Florida Department of Agriculture and Consumer Services, Tallahassee, FL
The US, EU, Canada, and many other countries prohibit the use of Chloramphenicol (CAP) in food producing animals because it has been associated with aplastic anemia in humans. Early in 2002, sub ppb amounts of CAP were discovered by the EU in shrimp and honey imported from China. In order to provide Federal and state laboratories with validated screening methods for CAP, we evaluated two commercial screening tests for CAP, the Charm II test and the RIDASCREEN test in shrimp and honey. A statistically designed protocol was developed to evaluate the selectivity and sensitivity of the screening tests for CAP. Both tests were capable of detecting CAP at 0.3 ppb in shrimp; however, both tests exhibited a positive bias for negative samples. If the tests were used as labeled, this bias would lead to an unacceptably high rate of false positive results. For both kits, we were able to develop alternative protocols for data interpretation that would give a significantly lower percentage of false positive results while maintaining the ability to detect samples containing 0.3 ppb CAP in shrimp. Both test kits were also evaluated for the detection of CAP in honey.
Board A-29 Validation of Methods to Confirm Chloramphenicol at 0.1 ppb in Shrimp, Crabmeat, and Honey: Collaboration between FDA CVM, FDA Office of Regulatory Affairs, Florida Dept. of Agriculture and Consumer Affairs, and the Canadian Food Inspection Agency
M.C.Carson, C.B.Nochetto, D.N.Heller, K.Ferbos, P.J.Kijak, Division of Residue Chemistry, Office of Research, CVM, Laurel MD 20708
Chloramphenicol (CAP) is a potent and cheap antibiotic that is associated with aplastic anemia and other toxic effects in humans. It is banned from use in food-producing animals in the US. Low levels of CAP were detected by analysts in Europe, Canada, and some US states in imported shrimp, honey, and other commodities. Existing FDA methods could only detect 1-2 parts per billion CAP. In July 2002 the Commissioner committed the FDA to begin analyzing imported foods with methods capable of confirming CAP at 0.3 ppb, consistent with enforcement levels used in other countries, and also consistent with the claimed detection limits of marketed screening assays. FDA needed to quickly validate methods to confirm CAP at sub-part per billion concentrations. The Florida Department of Agriculture and Consumer Services and the Canadian Food Inspection Agency had recently developed appropriate LC-MS-MS methods for shrimp and honey, respectively, which they shared with the FDA. We validated Florida’s method to confirm 0.1 ppb CAP in shrimp and crabmeat, and we validated Canada’s method to confirm 0.1 ppb CAP in honey. We adapted FDA’s existing shrimp method (LIB 4284) for analysis on a triple quadrupole, lowering its limit of confirmation from 1 ppb to 0.1 ppb, and validated the modified method. Of three locally purchased imported canned crabmeat samples, two contained CAP. One locally purchased honey sample contained CAP.
L.P.Lue1, A.Vancura2, 1NRL, ORA, FDA, Jamaica, NY 11433, 2St. John's University, Jamaica, NY 11439
Cephalosporins (CEF) are b-lactam antibiotics with cephalosporinic nucleus, produced together with penicillins (PNC) by a fungus Cephalosporium acremonium. Chemical synthesis involves conversion of penicillin nucleus into a cephalosporinic nucleus. Therefore, it is very common to find the contaminant of penicillin in active pharmaceutical ingredient (API) and dosage forms of cephalosporins. However, this contamination phenomenon of penicillins in cephalosporins is not acceptable in medicinal practice. The HPLC on C18 reversed phase columns with UV or fluorescence(FD) detection has been utilized to detect penicillin in API’s of cefadroxil and cefoperazone. FD enhanced the detection level ten times. The low levels of penicillins in cephalosporine (ppb) after derivatization with HgCl2 or 4-fluoro-7-nitro-benzofuran were detected by UV detector or FD , respectively.
T.P.McNeal1, C.Olivo2, T.H.Begley1, 1OFAS, FDA, College Park, MD, 2Virginia Tech, Blacksburg, VA
Vinyl chloride is the building block for polyvinyl chloride (PVC) polymers. Polyvinyl chloride production is second only to polyethylene in the US. Polymers of PVC are used in building construction, housewares, water pipes and food packaging. Vinyl Chloride monomer (VCM) is a human carcinogen. Thus the levels of residual VCM in food packaging are of interest to the Food and Drug Administration (FDA). In 1986, FDA determined that thick walled PVC food packaging, i.e., bottles and blister packages, were safe provided the polymer contained < 10 parts-per-billion (ppb) residual VCM. Since then, no data are available that show residual VCM levels in these food packages are < 10 ppb. To determine whether the residual VCM levels in PVC containing food packages in current use are < 10 ppb, a survey and analysis of PVC containing food packages was conducted.
The analytical procedure employed for these determinations utilizes automated static headspace sampling and capillary GC with a PLOT capillary with mass selective detection. Polyvinyl chloride and Saran™ films, PVC bottles and blister packages were analyzed for residual VCM. Residual VCM levels found in the packages ranged from none detected (< 1 ppb) to ca. 275 ppb. The package containing 275 ppb residual VCM was a not a food contact material. The Repeatability for VCM in PVC polymers at the 5 parts-per-billion level had a coefficient of variation of < 1%. Recovery of VCM at the 5 ppb level was essentially 100%. Data on residual VDCl levels in Sarans ™ also will be presented.
M.Miyahara1, T.Mashimizu2, Y.Kobayashi3, T.Maitani1, 1National Institute of Health Sciences (NIHS), Setagaya, Tokyo, 2Japan Electron Optics Laboratory (JEOL), Akishima, Tokyo, 3Japan Atomic Energy Research Institute (JAERI), Takasaki, Gunma
After extensive studies on irradiated food for years, food irradiation technology has been utilized for food safety recently. Detection methods for irradiated foods are still required by leveling regulation. In this study we discuss detection method for irradiated foods using ESR. Bones and shells consist of hydroxyapatite Ca10(PO4)6OH2 or of calcium carbonate. Those compounds retain radicals in the crystal lattices, which are induced by irradiation. ESR is a radical monitor. Samples were prepared by modified procedures suggested by EN. Bone and shell samples were washed to remove muscles and dried over phosphorus oxide (V) in vacuum. ESR can detect those radicals in bones (fish, frog legs, wing sticks) and shells of mussels and shrimps. Spectra of irradiated bones of fish, frog legs, wing sticks, and shells of mussels were same as these of hydroxyapatite. The signals were detectable at 0.5kGy about for one year. Those results were comparable with the results of EN method. Irradiated shells of shrimps and prawns were different from those of bones. Thus preliminary results showed new scope of the method.
P.J.Nyman, G.W.Diachenko, G.A.Perfetti, CFSAN/OFAS, FDA, College Park, MD
A survey of soy sauces and related products available in the U.S. was conducted to determine the levels of 3-monochloropropane-1, 2-diol (3-MCPD) and 1, 3-dichloro-2-propanol (1, 3-DCP) in these products. Fifty-five retail samples were purchased and analyzed for 3-MCPD. 3-MCPD determinations were made according to a gas chromatography/mass spectrometry method that was validated by a collaborative trial. Eighty-five percent of the samples analyzed were found to contain greater than the detection limit of 0.005 ppm (g/g) for 3-MCPD. Thirty-three percent were found to contain greater than 1 ppm; the highest level was 876 ppm 3-MCPD. Thirty-nine of the samples analyzed for 3-MCPD also were analyzed for 1, 3-DCP by using a modified method developed and validated in-house. Fifty-six percent of the samples analyzed for 1, 3-DCP were found to contain greater than the detection limit of 0.055 ppb (ng/g) for 1, 3-DCP; the highest level was 9.8 ppm 1, 3-DCP. Products manufactured in Asia contained the highest chloropropanol levels.
P.J.Nyman, G.W.Diachenko, G.A.Perfetti, CFSAN/OFAS, FDA, College Park, MD
A GC/MS method for 3-MCPD in foods and food ingredients was modified for the determination of 1, 3-DCP in soy and related sauces. The method was validated by using a blank soy sauce. The detection limit, quantitation limit, and recoveries were determined and identities were confirmed by MS on the basis of analyses of test portions spiked with 1, 3-DCP at 10, 25, 50, and 100 ng/g. The spiked test portions were quantitated by using an internal standard calibration curve. For the spiked test portions, the mean internal standard-corrected recovery for 1, 3-DCP was 100 percent with a relative standard deviation of 1.32 percent. The limits of detection and quantitation were determined to be 0.055 and 0.185 ng/g, respectively. The method also was compared with a headspace GC/MS method that was recently developed by the U.K.’s Central Science Laboratory. Results from the method comparison showed that the recoveries for the spiked test portions, as well as the amounts of 1, 3-DCP found in the retail products, were comparable.
J.C.Reepmeyer, CDER, FDA, St. Louis, MO 63101
Fraudulent over-the-counter topical products, sold for the treatment of psoriasis, have been found to contain undeclared designer corticosteroids, which possess modified ester substituents. Corticosteroids, extracted from topical products, were analyzed by HPLC on a Waters Symmetry C-18 column with a mobile phase gradient consisting of either acetonitrile-water or methanol-water using diode-array detection and positive ion atmospheric pressure chemical ionization liquid chromatography mass spectrometry (APCI LC-MS). Betamethasone-17-propionate-21-butyrate (BMPB), -21-isobutyrate (BMPI), and -21-stearate (BMPS), potential designer corticosteroid candidates, were synthesized as standards. Known corticosteroid pharmaceuticals, such as betamethasone-17, 21-dipropionate (BMD), were analyzed for comparison. All corticosteroids studied gave prominent molecular ions [MH]+ by positive ion APCI LC-MS. BMD gave fragments corresponding to [MH - HF]+, [MH - CH3CH2COOH]+, [MH - HF - CH3CH2COOH]+, and [MH - HF - CH3CH2COOH - H2O]+. Interestingly, BMPB gave fragments with the same losses as BMD, and thus gave atomic mass units that were 14 units above those for BMD. Fragments due to loss of butyrate were absent or negligible, which demonstrated selective fragmentation of the 17-propionate ester over the 21-butyrate ester. A similar phenomenon was observed with BMPI and BMPS. BMPB and BMPS were identified as compounds present in fraudulent topical products.
J.C.Reepmeyer, CDER, FDA, St. Louis, MO 63101
Ivermectin and clorsulon are antiparasitic agents used in veterinary medicine, sometimes used in combination. The purpose of this work was to develop a method for the simultaneous analysis of ivermectin and clorsulon in injection solutions by high-performance liquid chromatography (HPLC), to differentiate between two ivermectin analogs, and to confirm structures by ultraviolet (UV) spectroscopy and mass spectrometry. Clorsulon (CSL), ivermectin methyl analog (IVM-B1b), and ivermectin ethyl analog (IVM-B1a) were separated by reversed-phase HPLC on a short (4.6 mm x 50 mm) C-18 column using a mobile phase gradient consisting of acetonitrile-methanol-water, and detected by UV at 244 nm and by negative ion atmospheric pressure chemical ionization liquid chromatography mass spectrometry (APCI LC-MS). UV area measurements were found to be linear for all three compounds. Repeatability measurements (n = 6) for CSL, IVM-B1b, and IVM-B1a gave 0.13, 0.29, and 0.08% relative standard deviations, respectively. The two ivermectin analogs gave molecular ions [M-H]- as their base peaks by negative ion APCI LC-MS. Clorsulon also gave a molecular ion with prominent isotope peaks due primarily to the presence of three chlorine and two sulfur atoms, but the base peak was due to loss of hydrogen chloride [M-H-HCl] -.
F.J.Schenck1, J.E.Hobbs1, S.J.Lehotay2, 1ORA, FDA, Atlanta, GA, 2ARS, USDA, Wyndmoor, PA
A fast and inexpensive method for the analysis of pesticide residues in fruits and vegetables (produce), named QuEChERS, developed at a USDA-ARS Laboratory, was evaluated. The procedure entails vortexing 10 g of produce sample with 10 mL of acetonitrile, and effecting a phase separation by salting out with MgSO4 (4 g) and NaCl (1 g). Cleanup of the acetonitrile extract was performed using dispersive SPE, which entails vortexing the extract with a small quantity of SPE sorbent and MgSO4. Gas chromatography or liquid chromatography was then utilized to perform quantitative analysis of the pesticides. Produce samples containing various incurred organochlorine, organophosphorus and N-methyl carbamate pesticide residues were analyzed using the QuEChERS method, a published acetonitrile extraction method used by the Canadian Pest Management Regulatory Agency and the acetone extraction (Luke) method used by the FDA. Similar results were obtained using all three methods.
L.V.Podhorniak1, F.J.Schenck2, A.J.Krynitsky3, F.Griffith1, 1OPP, EPA, Fort Meade, MD, 2ORA, FDA, Atlanta, GA, 3CFSAN, DPIC, College Park, MD
A reverse phase liquid chromatographic method with both fluorescence and mass spectrometric detection is presented for the analysis of 13 parent N-methyl carbamate pesticides and their metabolites, as well as piperonyl butoxide, for a total of 24 compounds found in selected fruits and vegetables. The commodities chosen were of special concern to EPA as these commodities had the least amount of monitoring data for dietary exposure estimates used in risk analysis. The method uses an acetone extraction, followed by an aminopropyl SPE cleanup. Determination of residues is by reverse phase liquid chromatographic column fitted with either fluorescence or a mass spectrometric detector. A set of six samples can be prepared in one working day. Recovery data are presented from selected commodities for some of EPA's fruit and vegetable crop groupings.
V.A.Vega, F.J.Schenck, ORA, FDA, Atlanta, GA
An extraction technique is described for aflatoxins B1, B2, G1, and G2 in peanut butter using matrix solid phase dispersion (MSPD) in conjunction with immunoaffinity column (IAC) clean up. 2.0 grams of peanut butter sample is blended with 3 grams of C-18 SPE sorbent. A column prepared with the C-18-peanut butter matrix is washed with 5 mL water, and then the aflatoxins are eluted with 15 mL methanol. The eluate is diluted with water and subjected to an IAC clean up. The aflatoxins are then quantitated by reversed phase liquid chromatography with fluorescence detection. Recoveries will be determined using a negative control sample which will be fortified from 0.5 ppb to 20.0 ppb. The method provides a rapid, specific and easily controlled assay for the analysis of aflatoxins in peanut butter, with minimal solvent usage. The MSPD method reduces organic solvent consumption by 85% and hazardous waste by 80% compared with the AOAC method.
S.L.Snellings1, S.C.Harris2, M.A.Jackson2, D.W.Milller1, 1NCTR, FDA, Jefferson AR 72079, 2Northrop Grumman, NCTR, FDA, Jefferson AR 72079
The Scientific Imaging and Analysis System (SIAS) was developed at the National Center for Toxicological Research (NCTR) in the Division of Chemistry as a way to perform colorimetry and pseudo-spectrophotometry in a cost-effective, rapid manner. The potential benefits to the FDA are numerous, including the documentation of gels, animals, tumors, and microorganisms. Other applications include explosive detection, food analysis, and organic chemistry. SIAS uses a digital camera to image objects in color or grayscale. Images can be collected individually or as a time series, which allows real-time color analysis. Each image is automatically corrected for lighting and camera effects using custom color standards. Using SIAS custom software, specific pixels in an image can be analyzed, and calibration curves, concentration, diffusion kinetics, thickness, and spectral data can be calculated. Data analysis can be performed on a single image or a template can be applied to a series of images using auto-analysis. SIAS can be a benchtop or handheld unit, which is perfect for field analysis. One use of SIAS is the colorimetric analysis of tuna fillets that may have undergone carbon monoxide treatment. SIAS quantitates the level of carbon monoxide in the tuna based exclusively on the color of the meat.
J.A.Spencer1, L.F.Buhse1, M.M.Nasr1, N.J.Westenberger1, J.P.Jasper2, 1Div. Pharmaceutical Analysis, 2Molecular Isotope Technologies
We wished to learn whether Isotope Ratio Mass Spectrometry (IRMS) could distinguish between API materials made by different manufacturers and between different lots of material by the same manufacturer. Four sample sets, each representing a particular drug substance, were created from API materials. Each sample set for IRMS consisted of five identical vials to be analyzed. Analysts were given the name of each of the four pharmaceutical samples for safety reasons but no other details. The sample sets were distinguished as follows:
Bivariate plots (d15N vs. d13C; dD vs. d18O) showed a high degree of clustering for Set A and less clustering for Set B. Set C showed tight clustering for each of the two manufacturers, but strong differentiation between the two sources. Set D showed widely separated points in all the plots. For the API samples examined, distinctions were readily made between different manufacturers of the same drug substance. IRMS instrumental precision was high enough to discern different lots of a pure active pharmaceutical made by a single manufacturer.
J.A.Spencer1, L.F.Buhse1, M.M.Nasr1, N.J.Westenberger1, A.S.Hussain2, E.H.Jefferson2, 1Div. Product Quality Research
‘Process Signature’ denotes measurable variances in chemical and physical properties of a material that are process dependent. This was a study to learn whether NIR reflectance spectra of powdered APIs can establish sameness of samples from the same lot and, conversely, can distinguish between APIs from different lots or sources. Samples consisted of five individual vials of four different API materials as follows: Set A — five samples, same manufacturer, same lot; Set B — same manufacturer, five lots; Set C — two manufacturers, one lot from each; and Set D — five manufacturers. NIR reflectance scans were made of each of the API samples through the bottom of 1ml glass vials. The direct NIR reflectance spectra for Set A show a very low standard deviation as a function of wavelength, whereas the five spectra for Set D differed substantially in slope and offset. Set B exhibited about four times and Set D ~ 20 times the standard deviation of Set A. For these API samples, NIR reflectance spectral variations demonstrate sameness of multiple samples from a single lot. Meaningful spectral differences can be observed between the lots of API and even larger variations could be seen between different manufacturers.
J.A.Spencer1, L.F.Buhse1, M.M.Nasr1, A.S.Hussain2, 1Div. Pharmaceutical Quality Research
In 2001 the FDA initiated a program to stimulate the development of Process Analytical Technology (PAT) in the manufacture of pharmaceuticals. This calls for substantial changes in manufacturing technology, the philosophy of product quality control, the drug approval process and, ultimately, the regulatory atmosphere. There are a variety of fast, non-destructive instruments and sensors such as Near Infrared, Raman and particle sizing. These are now commonplace in non-regulated industries and will play an important role in PAT-based pharmaceutical manufacture. Measurements from such combinations of devices will inevitably be joined into multidimensional process control models. Central to the implementation of PAT is the use of chemometric tools such as PCA and PLS. In pharmaceutical manufacturing these will need to be implemented with detailed validation protocols that can be properly managed by the manufacturer and understood by the FDA.
Two popular techniques, NIR and Raman, were used to measure spectra of a series of Active Pharmaceutical Ingredients (APIs). The sample sets came from different manufacturers and lots. We show that data preprocessing such as derivativization or MSC which is typically done for chemical analysis, can dramatically reduce the amount of process dependent information (process signature) contained in the raw NIR spectra.
P.R.Sundaresan, K.D.White, J.I.Rader, CFSAN, FDA, College Park, MD
Teucrium species are rich in neoclerodane diterpenoids. Teucrium species known as germander have been used in traditional folk medicine for their stimulant, diuretic, antipyretic and antiseptic properties. However, the furano-neoclerodane diterpenoids present in germander have been implicated in the in vivo hepatotoxicity of this botanical. The lack of commercially available reference standards prompted us to develop in-house standards for Teucrin A and other diterpenoids. Authenticated germander was used as the source material. Acetone extracts of powdered plant material were prepared. They were analyzed by HPLC using Synergi Max-RP columns with monitoring at 220 nm. Limited amounts of Teucrin A and other diterpenoid standards were analyzed on a Synergi Max-RP column to determine their retention times and correlation coefficients. The same standards of Teucrin A and other diterpenoids were subjected to concurrent mass spectral analysis. Teucrin A and diterpenoids such as dihydroteugin, teuflin, teucrin H1 and teucvidin were identified by LC/MS. Isolation and characterization of germander standards on a preparative scale are under way. The chemical identities of these potential reference standards will be confirmed by NMR spectroscopy.
S.Wang, Lab E, FBC, Northeast Regional Laboratory, ORA, FDA, Jamaica, NY 11433
The ion trap has an important limitation: its maximum ion storage capacity is small, approximately 107ions. When the number of ions exceeds 105, space-charge repulsion occurs, thus, its mass resolution deteriorates. To overcome the limitation, the AGC is applied. The AGC is implemented by automatic control of the ionization time for EI or CI, or the injection time for ESI or APCI/AAPI. The AGC software sets up to maintain the optimum quantity of ions into the ion trap according to a linear equation: the number of ions stored in the ion trap is proportional to the ionization time or ion injection time. The linear equation is based on a linear approximation of first order kinetics. The linear approximation is based on the assumption that only a small proportion of ions initially present (less than 20%) is consumed, but the assumption is not true very often. The linear approximation of first order kinetics applied by AGC only is a special case of a more general and accurate kinetics. In order to better quantitatively understand the MS data calculated by the AGC, the more accurate and general kinetics must be developed for ion trap MS. Accurate and general kinetics are presented.
G.M.Ware, FDA
Hypoglycin A (HG-A) is a water-soluble natural toxin found in ackee fruit. It is similar to leucine and isoleucine in chemical structure, chromatographic and photometric properties. This paper describes a liquid chromatographic (LC) method for the resolution of HG-A, leucine and isoleucine. Chromatographic stationary phases C18, C8 and C2 showed little selectivity for the separation of HG-A from leucine and isoleucine. Resolution was obtained on phenyl, diol and polar group end capped C18 stationary phases using a mobile phase of acetonitrile/ methanol/water. Leucine, isoleucine and HG-A were detected at 210 nm. Method validation included the determination of the selectivity, (a), chromatographic resolution, (Rs) detection and quantitation limits for leucine, isoleucine and HG-A. The purity of HG-A standard containing 1% leucine and isoleucine was characterized. Low milligrams of pure HG-A were isolated by analytical column. Chromatographic conditions used for the analytical separation were scaled up to a semi-preparative method capable of isolating 25 to 50 mg HG-A per injection. UV and IR spectra for pure HG-A were used to confirm purity. Hypoglycin A standard was further characterized by GC/MS.
L.Xu, T.Heinze, A.Pogge, W.Slikker, Jr., L.C.Schmued, NCTR, FDA, Jefferson AR
Fluoro-Jade B contains a number of structural analogues of Fluoro-Jade, a well-known and useful dye for detection of degenerating neurons in tissue sections. It exhibited a higher affinity for degenerating neurons than Fluoro-Jade. The present research focused on isolation, purification and identification of Fluoro-Jade B components and will ultimately distinguish the most biologically active compound(s). The reaction mechanism suggests about nine possible reaction products corresponding to a 1:1, 2:1, 3:1 and 4:1 ratio of the starting material resorcinol and benzenetetracarboxylic dianhydride. The molecular weights of the predicted structures based on these ratios could be 346 (two isomers), 438 (two isomers), 420, 512 (two isomers) and 586 (two isomers), respectively. The analytical HPLC method consisted of a reversed phase C18 column and a mobile phase with a pH gradient. The LC/MS data supported the theoretical predictions. The quantitative separations were performed by silica gel column chromatography, TLC and preparative reversed phase HPLC. For each fraction collected, the negative-ion electrospray mass spectra showed the expected [M-H-] ions and reasonable fragmentation with in-source collision-induced dissociation (CID).
A.X.Yang, B.Bhattacharya, J.Mejido, J.Han, R.K.Puri, Microarray Program, Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, CBER, FDA, Bethesda, MD 20892
CBER has joined forces with the National Cancer Institute and developed a state-of-the-art microarray program. In this program, not only do we provide hands-on-training to our researchers/reviewers, we have been optimizing the production of high quality arrays for use in scientific experiments. Recently, we purchased a human oligo set consisting of 16, 659 70-mer oligos. Each oligo was selected from the 3’ end of the gene and optimized using BLAST search. The gene array list (GAL) file was generated in collaboration with John Powell of Center for Information Technology (CIT), National Institutes of Health (NIH). We created the human oligo nucleotide ‘chip’ by using GeneMachine spot arrayer. Oligos were deposited on the surfaces of poly-L-lysine coated glass slides and arranged in 32 subarrays with 23 x 23 spots. After printing, oligo chips were cross-linked and slides were blocked with succinic anhydride to reduce background hybridization with fluorochrome dyes. The quality controls of these chips included scanning of autofluorescence of SSC, and hybridization with 9-mer Cy3-labeled oligo nucleotide probe. Chips were only acceptable if >95% spots were uniformly printed. The quality of these chips was further confirmed by hybridization with total standard RNA obtained from human brain tumor cells. GenePix 4000B and ScanArray Express HT were used for image acquisition and data collection. Data were further analyzed using different softwares, primarily the mAdb database, CIT, NIH. Current efforts are focused on quality control and standards development. These activities are expected to enhance knowledge base and ability to evaluate results of microarray experiments in product development or clinical trial design.
G..C.Ziobro, CFSAN, FDA, College Park, MD 20740
Concerned about the presence of filth contaminants such as rat, mouse, and other animal hairs and excreta; whole insects, insect parts, and excreta; and other extraneous materials which, because of their repulsiveness, would not knowingly be eaten or used, Dr. Wiley hired B.J. Howard to develop microscopic methods to isolate, identify, and quantify these contaminants in foods, drugs, and cosmetics. Over the past 100 years Howard and his successors have analyzed over 48,000 regulatory samples and have published several hundred papers on the detection of these materials. The most recent series of papers detailed the regulatory action criteria for filth and other extraneous materials that are the scientific basis of Compliance Policy Guide Sec. 555.600. This poster will outline the history of filth method development how it influenced America's health.
F.A.Bencsath, L.Quinonez, H.Valentin-Perez, J.D.Barnett
Diesel oil is one of the largest volume petroleum products spilled at sea by ship accidents. The spills may contaminate seafood and result in the closure of the affected fisheries. Taint-free seafood is required for reopening. We used instrumental analyses to quantify contamination as a complement to the qualitative assessment of the human sensory panels. We used an electronic nose instrument and dynamic headspace gas chromatography-mass spectrometry (GCMS). Oysters were tainted by exposing them for 48hours to seawater contaminated with 10ppm diesel oil, then were depurated in clean seawater for six weeks. After the exposure, the oysters were found tainted by sensory assessment. GCMS analysis identified C10-C17 alkanes and 60 aromatic compounds: benzene, indane, tetralin and naphthalene homologues. The total hydrocarbon concentration decreased exponentially during depuration, from the initial 26ppm to 0.5ppm (the threshold of human olfaction) in 24 days. The electronic nose, calibrated by a mixture of six representative aromatic hydrocarbon compounds, provided very similar quantitative data for the oyster samples with total hydrocarbon concentration of 2ppm and above.
F.J.Schenck1, D.J.Donoghue2, J.E.Hobbs1, 1ORA, FDA, Atlanta GA, 2Univ. of Arkansas, Fayetteville AR
A method for the analysis of N-methyl carbamate pesticide residues in eggs is presented. The purpose of the study is to provide residue data for exposure estimates used in risk analysis. The method uses an acetonitrile extraction, a graphitized carbon and aminopropyl SPE column cleanup and determination by reverse phase liquid chromatography with fluorescence detection. The average recoveries of 11 fortified (1.0 — 10.0 ppb) carbamates from eggs ranged from 80-100%. Single Comb White Leghorn hens were treated with carbaryl. Hens dusted once with a 10% carbaryl powder produced eggs containing 76, 54, and 42 ppb carbaryl and 69, 33 and 34 ppb of the carbaryl metabolite 1-napthol on days 1, 2 and 6 post-dosing, respectively. Hens sprayed with a 1% carbaryl solution produced eggs containing 14, 15, 23, 14, and 19 ppb carbaryl and 5, 6, 6, 6 and 15 ppb 1-napthol on days 2, 3, 4, 5 and 7 post-dosing, respectively.
T.Liu, J.Muller, Z.Ye, CBER/FDA/Rockville MD
The matrix protein (M1) of influenza virus plays a central role in viral replication. In relation to viral growth and morphology, we studied the RNP-binding activity of M1s from high-growth strain A/Puerto Rico/8/34 (A/PR8/34) and the relatively low-growth wild-type strain A/Nanchang/933/95. The RNP-binding strength of M1 was studied by disruption of M1 from M1/RNP complexes with salt and acidic condition. Our results indicated that binding of M1 of high-growth A/PR8/34 was more difficult to break than the binding of M1 of low-growth A/Nanchang/933/95. Consistent with the presence of M1 in A/PR8/34, binding of M1 of Resvir-9, a reassortant containing P, M and NS genes from A/PR8/34 and the rest of genes from A/Nanchang/933/95 and retaining relative high-growth characteristic, was relatively difficult to break than the binding of M1 of A/Nanchang/933/95. Physical properties of morphological features of these viruses were studied by velocity sucrose gradient centrifugation and transmission electron microscopy of purified viral particles, and by immunofluorescence staining of hemagglutinin expressed on the surface of infected cells. The results demonstrated that high-growth strains, A/PR8/34 and a relative high-growth reassortant, Resvir-9, had characteristics associated predominantly with spherical particles, while the low-growth strain, A/Nanchang/933/95, had characteristics of filamentous particles. These studies indicate that the binding between M1 and RNP complex might determine viral growth and morphology.
Board B-01 Microarray Detection of Antibiotic Drug Resistant Strains of Mycobacterium tuberculosis (MTB)
X.Tang1, S.L.Morris2, J.J.Langone1, S.A.Khan3, P.M.Regan4, L.E.Bockstahler1, 1CDRH, FDA, Rockville, MD 20857, 2CBER, FDA, Rockville, MD 20857, 3NCTR, FDA, Jefferson, AR 72079, 4WEAC, ORA, FDA, Winchester, MA 01890
Global public health is threatened through the emergence of potentially dangerous antibiotic drug-resistant strains of MTB. We are developing a model MTB system to establish a protocol utilizing microarray technology for rapid and accurate detection of clinically relevant point mutations in MTB genes associated with drug resistance. DNA templates of MTB rpoB and katG genes containing clinically relevant point mutations were generated by recombinant PCR techniques. Template sequences were confirmed by DNA sequencing. We prepared Cy5 dye-labeled single-stranded DNA target fragments from the templates by a modified asymmetric PCR technique. Wild type and mutant MTB gene-specific oligonucleotide probes (15-25 bases) were spotted onto glass slides. Following hybridization of the DNA target fragments with the array bound oligo probes, the results were determined using a laser scanner. We have used this technique successfully to detect and identify (<24 hrs) on a single microarray slide a number of wild type and clinically relevant mutant rpoB and katG gene target sequences. We plan to expand our microarray system to the detection of mutations in the other MTB genes associated with drug resistance. Validation and further refinement of our microarray protocol will be required for its application to clinical diagnoses.
W.H.Doub1, A.M.Wokovich1, G.J.Singh2, W.P.Adams3, 1DPA, OPS, CDER, FDA, St. Louis, MO, 2DPE, OPS, CDER, FDA, Rockville, MD, 3OPS, CDER, FDA, Rockville, MD
Nasal spray drug products are characterized in part via measurement of plume geometry and spray pattern as suggested in the U.S. Food and Drug Administration (FDA) June 1999 Draft Guidance for Industry, Bioavailability and Bioequivalence Studies for Nasal Aerosols and Nasal Sprays for Local Action. At the present time, plume geometry is typically measured using high-speed photography often with manual actuation. Spray pattern is assessed by collecting the spray on a glass or plastic sheet followed by manual measurement.
We have evaluated the SprayVIEW NSP System (Image Therm Engineering, Sudbury, MA) with respect to its ability to characterize both plume geometry and spray pattern. The SprayVIEW NSPis a nonimpaction system using a sheet laser and high speed digital camera to image the plume geometry and spray pattern. This system provides intensity profiles that increase objectivity for the determination of the spray pattern perimeter and shape.
We also see areas where improvements might be made. We find a need for the user to assess the homogeneity of the laser beam as this factor has a profound effect on the image of the spray plume. Additionally, there is still much subjectivity involved in measuring parameters which describe plume geometry.
T.J.Flynn, P.W.Wiesenfeld, S.C.Sahu, CFSAN/OSCI/OARSA, FDA, Laurel, MD
Hepatotoxicity is the leading cause of post-market withdrawal of drugs and of warning notices for dietary supplements. The present study evaluated the effects of model compounds, some with known hepatotoxicity, on a human (HepG2/C3A) hepatocyte cell line. Cells were cultured on 96-well plates and exposed to 9 concentration levels of test agent for 48 hr. Specific endpoint assays, which could all be conducted in a plate reading fluorometer and which reflect known mechanisms of hepatotoxicity, included: generation of reactive oxygen species; depolarization of mitochondria; steatosis; and induction or inhibition of cytochrome P450 activities. All parameters were normalized for total DNA content per well as a measure of number of viable cells. Each model compound generated a unique response profile based on which endpoints showed a concentration-related increase, decrease, or no change. Only 3 of 12 compounds tested showed cytotoxicity as measured by a significant decrease in total DNA. For some model compounds that are human drugs and have known hepatotoxicity (e.g., valproic acid), some endpoints responded at concentrations comparable to therapeutic blood levels. These findings suggest that this test system may serve as a high throughput screening assay for hepatotoxicity of food- or dietary supplement-related compounds.
L.Zhang1, E.F.Lizzio1, E.Gubina1, T.Chen1, H.Mostowski2, S.Kozlowski1, 1DMA OTRR, 2OCGT
There are two distinct phenotypes of T-cell cytokine responses that lead to different effector functions and to different outcomes in disease processes. Although evidence suggests a possible role of the local microenvironment in the differentiation or localization of T-cells with these phenotypes, there are no examples of divergent T-cell cytokine phenotypes with the same antigen specificity concurrently existing in different tissue compartments. Utilizing a CD8+ T-cell adoptive transfer model for GVHD, we demonstrate a potent type 2 cytokine response develops in the spleen while a potent type 1 cytokine response simultaneously develops in the testis. These experiments demonstrate for the first time that cytokine production can be oppositely polarized in different organs of the same individual. This may have important implications for organ specific pathology in infection or autoimmunity: infections or autoimmune diseases that affect multiple organs may have heterogeneity in tissue cytokine responses that are not revealed in systemic lymphocyte cytokine responses. Therefore, attempts to modulate the immune response phenotype may ameliorate pathology in one organ while exacerbating pathology in another.
D.B.Lyle, J.A.Barrett, J.C.Shallcross, S.C.Wood, J.J.Langone, CDRH, FDA, Rockville MD
Damage to endothelial cells, which line the interior of blood vessels, can occur in balloon angioplasty and/or the placement of stents within blood vessels. A key aspect of minimizing injury and subsequent restenosis of the vessel is the proliferation of endothelial cells which re-line and cover the denuded, exposed extra-cellular matrix. We have adapted a fluorescent plate reader methodology which uses the DNA-binding dye Hoechst 33342, for easily and quickly quantifying proliferation of the porcine aorta endothelial cell line PECSV.46 in 96-well culture plates during and following exposure to a variety of potential proliferation-altering drugs or biomaterials. PECSV.46 was optimally labeled with 50 mg/ml Hoechst 33342 for 1 hr. Binding of the dye to DNA resulted in a dose-dependent increase in fluorescence intensity within the wells. A linear response was observed for cell numbers between 2, 000 to 20, 000 cells/well, corresponding to 10% and 100% confluency. Detection did not require washing the dye from the wells, thus minimizing possible disturbance or loss of cells. This model system will facilitate the study of contrast agents, drugs used on drug-eluting stents, or device biomaterials for their potential to cause or modulate vascular toxicity.
M.E.Bishop1, S.Dertinger2, J.McNamee3, M.M.Moore1, A.Aidoo1, S.Harper4, R.Frobish5, J.L.Weaver6, W.Witt1, W.Slikker, Jr.1, C.Hotchkiss1, M.Hayashi7, J.T.MacGregor8, 1NCTR, FDA, Jefferson, AR, 2Litron Laboratories, Rochester, NY, 3Health Canada, Ottawa, Ontario, 4CFSAN, FDA, Laurel, MD, 5CVM, FDA, Laurel, MD, 6CDER, FDA, Laurel, MD, 7NIHS, Tokyo, Japan, 8NCTR, FDA, Rockville, MD
Pre-market testing of regulated products includes assessment of chromosomal damage in vivo. The traditional assay is assessment of the induction of micronuclei (cellular DNA fragments) in reticulocytes from mouse bone marrow using microscopic scoring. We are evaluating a flow cytometric assay that may permit routine monitoring of micronucleus frequency using peripheral blood from the rat, dog, non-human primate, and human, using mechanistically characterized agents known to induce micronuclei. The use of peripheral blood in these species has previously been limited by splenic activity that selects against micronucleated cells. The proposed method, which uses anti-CD71 labeling to identify young reticulocytes and a malaria standard that allows cross-laboratory standardization, may overcome this splenic selection problem. We have compared the new method with established methods in rats, and have identified suitable antibodies for flow cytometric assay in the beagle dog. Flow cytometric scoring gave superior counting statistics and lower variability than manual scoring. With appropriate validation, it may be possible to integrate the flow cytometric assessment into routine testing and eliminate the need for a separate mouse assay.
O.A.Maximova, R.E.Tafts, K.L.Pomeroy, D.McMahon, D.M.Asher, Laboratory of Bacterial, Parasitic & Unconventional Agents, DETTD, OBRR, CBER, FDA, Rockville MD 20852
Abnormal prion protein (PrP) associated with transmissible spongiform encephalopathies (TSEs), often called “scrapie-type PrP” (PrPSc), can be detected in tissues using immunohistochemistry (IHC). Evaluation of PrPScby IHC is often subjective–based on recognition by an experienced evaluator of specific PrPSc staining in tissue sections, sometimes reported semi-quantitatively using a four-point scale (0 to 4+). Evaluations are often unambiguous but may be inconsistent between evaluators or by the same evaluator on different days. FDA has sometimes had to base decisions regarding safety of products on information that includes results of PrPSc IHC to diagnose TSE in donors of blood or tissue or in patients who underwent invasive procedures with reusable FDA-regulated instruments. Quantitative IHC criteria would aid FDA in making such decisions. We sought to develop a system facilitating objective and consistent decisions about PrPSc IHC using computer image analysis to reduce variability and inconsistency in evaluations of tissue specimens. Using commercially available computer software (MetaMorph® Universal Imaging), we optimized capture and analysis of tissue images from sections of scrapie-infected hamster brain to quantify positive-signal intensity and percentage of selected tissue areas occupied by deposits of PrPSc. We believe that this approach may be useful for TSE research and for improved immunodiagnostics.
O.A.Maximova1, R.E.Taffs1, K.L.Pomeroy1, J.Ridge1, D.McMahon1, V.Ogryzko2, G.Amexis3, E.M.Dragunsky3, K.M.Chumakov3, D.M.Asher1, 1Laboratory of Bacterial, Parasitic & Unconventional Agents, DETTD, OBRR, CBER, FDA, Rockville MD 20852, 2NICHD/NIH, Bethesda MD 20895, 3Laboratory of Methods Development, DVP, OVRR, CBER, FDA, Rockville MD 20852
Detection of abnormal “scrapie-type” prion protein (PrPSc) in tissue is the gold standard for histopathological diagnosis of transmissible spongiform encephalopathies (TSEs). Immunohistochemistry (IHC) most often uses formalin-fixed paraffin-embedded (FFPE) tissue to identify PrPSc accumulations and characterize distribution patterns. Considerable disparity in IHC protocols can cause substantial variation in results. To reduce variation, we developed and optimized IHC for two types of specimens–necropsy tissues and cell cultures. We found a strong correlation between results of infectivity assays and PrPSc immunoreactivity in sections of scrapie-infected hamster brain. We developed a novel technique using “touch” prints of freshly cut brain tissue directly onto glass slides, facilitating rapid IHC detection of PrPSc. We modified the basic IHC protocol to detect the normal “cellular” prion protein (PrPC) and mutant forms of PrPC in fixed transfected SY5Y human neuroblastoma cell cultures expressing multiple copies of mutant PrP-encoding (PRNP) genes associated with familial Creutzfeldt-Jakob disease and similar TSEs. PrPC was detected in transfected cultured SY5Y cells but not in normal progenitor cells. The methods may help to improve early diagnosis of TSEs in potential donors of human tissues and in animals used as sources of raw materials and in-process reagents in FDA-regulated biological products.
S.L.Snellings1, L.A.Groves1, T.C.Schmitt1, D.W.Miller1, W.B.Parsons2, 1National Center for Toxicological Research, 2Arkansas Regional Laboratory, Jefferson, AR 72079
Ammonia, dimethylamine, and trimethylamine are volatile bases produced during seafood decomposition. Human sensory analysis is the method most commonly used to characterize seafood in terms of freshness or quality. An ion chromatography analytical method with conductance detection has been described for measuring the formation of these constituents as decomposition products in shrimp. The salient elements of the method begin with the extraction of the volatile bases with brine containing 0.02M sulfuric acid. Next, the acid salts are rapidly transformed to their respective free volatile bases by the addition of 40% sodium hydroxide. Lastly, the basic solution is purged with argon and the volatile gases are trapped in 5 mM methanesulfonic acid solution. Subsequent analysis with ion chromatography allows the quantification of ammonia, dimethylamine, and trimethylamine in the 5 mM methanesulfonic acid solution. We will describe a colorimetric method for determining total volatile bases that is based on the sample preparation for the IC method. The IC, colorimetric and human sensory method will be scrutinized for consistency.
V.J.Tomazic-Jezic, A.D.Lucas, B.A.Sanchez
Adverse reactions to NRL proteins are caused by topical, parenteral or respiratory exposures. NRL proteins bound to glove powder can cause serious reactions in NRL glove users. We evaluated protein extracts from medical gloves with and without accompanying powder. The ratios did not correlate with either the total amount of protein or powder on the glove, or glove weight, indicating that other factors may affect the protein binding. To investigate this possibility, we exposed clean powder to NRL antigens under various conditions and evaluated protein levels on starches and respective protein solutions. We found that protein binding was dose-dependent; with the saturation point at the concentrations above 200 µg/ml. Marked differences were observed among the individual starch preparations. Two cross-linked corn starch powders showed a strong affinity to attach proteins, while the protein binding to cooking corn starch and oat starch was much lower. Those differences were stronger with proteins in water than in PBS. The data indicate that physico-chemical properties of glove powder and some procedural factors play a role in the protein binding affinity.
V.J.Tomazic-Jezic, B.A.Sanchez
Allergenicity of NRL glove powders is well known, but the quantitative relationship to the allergenicity of the entire glove is not established. This study evaluated binding of individual NRL allergens to several glove powders. Two cross-linked corn starch powders, cooking corn starch and oat starch were exposed to ammoniated (AL) or non-ammoniated (NAL) raw NRL proteins. The amounts of starch-bound allergens were determined using the FIT Biotech allergen kit. The ratios of four individual allergens were similar for all starches, although the total level was different. However, the allergen ratio on starches was significantly different from the ratios in either AL or NAL extracts. While Hev b 6.02 comprised about 60% of total allergen in NAL extract, only 12-30% Hev b 6.02 was bound to starches. In contrast, there was only 3-7% of Hev b 1 allergen in NAL extract, but on powders, 35-45% of total allergen was Hey b 1. All samples demonstrated significantly higher binding of Hev b 1, and lower propensity for Hev b 6.02 allergen. These indicate that allergenic properties of NRL glove and glove powder may be different.
P.W. Wiesenfeld, T.J. Flynn and S.C Sahu, CFSAN/OSCI/OARSA, FDA, Laurel, MD
Hepatotoxicity is one of the most frequently reported adverse findings in safety assessment of food additives, drugs and dietary supplements. It is important, therefore, to develop in vitro screening assays and end-points that can quickly and accurately identify potential liver toxicants. Human and rodent hepatocyte cell lines (HepG2/C3A, WRL-68, CL9) were used to evaluate endpoints for apoptosis and cytotoxicity. Among the endpoints used were: cell viability, cytochrome P450 activity, programmed cell death (apoptosis), liver enzyme activity in the medium, and an indicator of steatosis. Caspase 3 activity was compared with classical markers of cytotoxicity such as, release of lactate dehydrogenase (LDH) into the medium. Caspase 3 and cytotoxicity end-points were dose, time, and cell-line dependent. C3A cells exposed to progesterone for 4-hours, did not produce any change in DNA concentration or Caspase 3 activity, but increased LDH release by 25%. Caspase 3 activity decreased with increasing dose and time (2, 4 and 6 hr) when C3A cells were exposed to androstendione (1.5 - 200 µg/ml). Under these conditions, there was no change in LDH activity and only a small decrease in total DNA content. In CL9 cells, 48-hour exposure to different concentrations of galactosamine increased release of LDH, decreased DNA content and Caspase 3 activity. Multiple cell lines, and end-points are needed to predict hepatotoxicty of model hepatotoxic compounds.
H.Golding1, L.R.King2, R.E.Taffs3, M.Merchlinsky2, N.Eller4, D.E.Scott4, J.Manischewitz2, 1DVP, CBER, FDA, Rockville, MD, 2DVP, CBER, FDA, 3DETTD, CBER, FDA, 4DH, CBER, FDA
In anticipation of large-scale smallpox vaccination, clinical trials of new vaccine candidates with improved safety profiles, and new vaccinia immune globulin (VIG) products, are under way.
There is an immediate need to develop new assays to measure vaccinia-specific immune responses. The classical assay to measure vaccinia neutralization, the Plaque Reduction Neutralization Test (PRNT), is slow, labor intensive, and difficult to validate and transfer. Here we describe the development of a novel vaccinia neutralization assay based on the expression of a reporter gene, b-galactosidase. Using a previously constructed vaccinia-b-galactosidase recombinant virus vSC56, we developed a neutralization assay, which is rapid, sensitive and reproducible. The read-out is automated. We show that the neutralizing titers (50% inhibitory dose) for several vaccinia immune globulin products (VIG) measured in our assay were very similar to those obtained in classical PRNT assays. A new FDA VIG standard was established for distribution to other laboratories. The new assay is currently used in several collaborative studies in primates and mice, and to evaluate clinical samples from human vaccine trials. The new assay will serve as an important tool for pre-clinical and clinical trials of new smallpox vaccines, and evaluation of therapeutic agents to treat vaccine associated adverse reactions.
D.N.Busick, S.A.Brown, K.Merritt, Office of Science and Technology, CDRH, FDA, Rockville, MD
To prevent patient-to-patient transmission of Creutzfeldt-Jakob Disease, the World Health Organization (WHO) recommends destroying instruments used on tissues possibly contaminated with CJD. Because it is impractical and costly to destroy instruments that are designed to be reusable, an alternative is to thoroughly decontaminate before reusing instruments that have contacted high-infectivity tissues (e.g., brain, spinal cord) of patients with confirmed or suspected CJD. The WHO and the US Centers for Disease Control and Prevention (CDC) have suggested that one of the following CJD-decontamination methods be used prior to routine cleaning and sterilization of surgical instruments:
- Autoclave in 1 N sodium hydroxide at 121°C for 30 minutes; OR
- Soak in either 1 N sodium hydroxide or 5% bleach for 60 minutes, then autoclave (dry or in water) for 60 minutes.
We subjected a variety of surgical instruments to the WHO-recommended decontamination methods to determine the effects on the instruments. Some instruments tolerated the decontamination procedures well; others did not (for example, gold-plated forceps handles corroded after soaking in bleach). We also subjected identical instruments to two standard methods for testing corrosion resistance, to determine whether the methods could predict the instruments’ response to decontamination.
M.Farahani, CDER, FDA, Rockville MD
Dental clinics are faced with increasingly stringent requirements for the treatment of both inorganic and organic wastes. Electron beam was used to reduce heavy metal ions such as lead and mercury with high efficiency. Radiolytically produced e-aq and H react with the heavy metal ions to reduce them to lower oxidation states, leading to formation of the atomic metal. Radiolysis was performed using an electron accelerator (7 MeV) with a dose rate of 0.4-o.6 kGy/3s pulse. Lead chloride (1x10-3M), mercury chloride (1x10-3M) were prepared for irradiation. All solutions were saturated with either air or N2. Sand was added to the solutions to nucleate precipitation. Following irradiation all solutions were filtered; syringe filters were used in the absence of oxygen for lead-containing samples, and Whatman 42 filter paper for mercury-containing samples. The soluble lead ions after the radiation treatment were measured with Atomic Absorption Spectrometer. The absorbed dose to achieve 99.9% removal of Hg ion from an aqueous solution is as low as 3 kGy. These results demonstrate that that electron beam technique can be used to convert water-soluble heavy metal ions to insoluble particles.
I.K.Ilev1, R.W.Waynant1, K.R.Byrnes2, E.Gorman1, J.J.Anders2, 1CDRH, FDA, Rockville, MD 20857, 2USUHS, Bethesda, MD 20814
A fundamental in vivo/ex vivo study of optical properties and light transmission characteristics of single and multiple tissue layers and blood in a Sprague Dawley rat model is presented. In our experiments, we utilize either coherent laser sources with various energy and spectral characteristics, or incoherent light sources in a broadband spectral range covering the visible and near-infrared. The measurement techniques are based on a minimally invasive fiber-optic approach that includes the use of dual-fiber sensor probes with input fibers for delivery of the illuminating light emission and output fibers for collection and delivery of the detected optical signals. The sensor probes are placed into different tissue layers (skin, sub-cutaneous connective and deep connective tissue, back muscle, bone and spinal cord) or blood, and broadband spectral transmission characteristics of these media are measured in vivo and ex vivo. The transmission spectra are analyzed in order to determine the specificity of interaction of different tissues with light. The main goal is to determine the most effective coherent or incoherent light sources to be used for specific minimally invasive photobiomodulation therapeutic and optical diagnostics techniques.
L.N.Kerr, L.D.Cash, M.P.Chaput, L.G.O'Malley, E.M.Sarhrani, J.C.Teixeira, W.S.Boivin, S.A.Mailhot, WEAC, FDA, Winchester, MA 01890
Current test methods evaluate the presence of defects in finished product, not the dynamics of glove use. A method to assess the durability of medical examination gloves made of various materials was evaluated. For each glove type, three sets of 100 gloves were tested. The first set of gloves was “challenged” using the durability method. The second set was manually donned and conditioned through a series of tasks simulating clinical use. The third set was unstressed control gloves. The gloves from each set were water-leak tested and the results recorded. The durability method created defects in all areas of the gloves. The durability method produced defects at similar rates to the simulated clinical method for all the glove types tested. Defect rates correlated well with target defect rates established from the literature. Durability varied among glove types with powder-free vinyl being the least durable (42% defect rate) and powder-free, textured chloroprene the most durable (3% defect rate). The durability method was reproducible with a coefficient of variation of 2.4%. This method can be used to establish the durability of medical examination gloves of a variety of materials.
W.Limm1, T.H.Begley2, T.Lickly3, S.G.Hentges4, 1OFAS, FDA, College Park. MD, 2OFAS, FDA, College Park, MD, 3Dow Chemical, Midland, MI, 4American Plastics Council, Arlington, VA
Diffusion coefficients of limonene in various linear low density polyethylene (LLDPE) and low density polyethylene (LDPE) resins have been determined from sorption data using a thermogravimetric method. From these data, one can determine whether polymer synthesis parameters such as choice of catalytic process or comonomer result in substantial differences in polymer barrier properties.
For example, LLDPE is currently manufactured using either one of two distinct catalytic processes: Ziegler-Natta (ZN) and metallocene, a single site catalyst. ZN catalysis is a heterogeneous process that has proven itself over the last half-century. It involves a transition metal compound containing a metal-carbon bond that can handle repeated insertion of olefin units. In contrast, metallocene catalysis has fewer than 20 years of history, but has generated much interest due to its ability to produce highly stereospecific polymers in very high yield. In addition to high stereospecificity, metallocene-catalyzed polymers are significantly lower in polydispersity than traditional ZN counterparts.
Absorption and desorption testing of heat-pressed films made from LLDPE and LDPE resins of varying processing parameters indicates that diffusion coefficients of limonene in these resins do not change substantially enough to warrant a change in the way FDA accesses LLDPE barrier properties.
T.O.Woods1, S.A.Brown1, S.G.McNamee1, K.Merritt2, A.D.Lucas2, V.M.Hitchins2, 1Division of Mechanics & Materials Science, CDRH, FDA, Rockville, MD 20850, 2Division of Life Sciences, CDRH, FDA, Rockville, MD 20850
This study addresses two immediate concerns for CDRH reviewers: the effects of reprocessing on OBNU devices and the effects of loading on degradation of absorbable materials. OBNU sutures are commonly reprocessed for reuse, though they are labeled for single use. In addition, the use of absorbable materials in loaded applications in tissue engineering and fracture repair continues to expand, and it is important to establish methods to determine the effects of static and cyclic loading on resorbtion rate. We previously showed that repeated ethylene oxide sterilization (reEtO) can affect the strength of absorbable sutures out of package. Here we continue our investigations to examine the effects of loading on the retained strength after immersion in saline of new and 5x reEtO suture composed of a variety of absorbable materials. A test apparatus was constructed which, in 37ºC saline, simultaneously subjects 5 suture loops to static or cyclic loads without constraining the specimen length. Material degradation was assessed by measuring tensile strength after static or cyclic loading and after immersion with no load. Results indicate that both load and resterilization decrease suture strength after immersion and that resterilization has an increasing effect on degradation with longer immersion times.
L.A.ZarembaCDRH, Rockville MD
MRI examinations of patients with implanted medical devices can produce significant heating of these devices, which is a potential hazard to patients. However, the MRI itself can be used to measure the temperature increase of the implant. The proton resonant frequency changes by 0.01 ppm/0C. This can be used to estimate the temperature distribution using a technique called phase mapping in which a map of the phase of the image signal, rather than its magnitude, is obtained. Phase mapping tests have been conducted in a phantom on the 2 tesla MRI at MOD I. Maps were acquired using a gradient echo sequence with an echo time of 12 msec, producing a phase change of about .064 rad/0C at 2 tesla. The technique was used to examine the temperature distribution surrounding wire configurations simulating implanted medical devices. Results were verified with a Luxtron fiberoptic temperature probe. Phase mapping has the advantage that it produces an image of the temperature distribution, while current methods using probes measure temperature at only a few points. The technique could be used as an alternative to temperature probes in the ASTM standard test method for measuring the heating of implanted medical devices during MRI.
S.Lute1, L.Norling2, J.Brown3, S.Herzer4, M.Voisard4, Y.Xu2, K.Brorson1, 1DMA/OTRR Bethesda MD, 2Genentech, Inc, 3ONDC/CDER Rockville MD, 4Amersham, Inc.
A potential safety concern in biotechnology purification schemes that employ re-use of column media is loss of the virus removal capacity of the chromatographic purification operation. One approach to assure safe reuse of resins is to define chromatography performance attributes that predict retrovirus clearance during extended re-use. In previous small-scale experiments, protein A columns were cycled 150 to 460 times using concentrates of murine hybridoma harvests, standard low pH elution buffers and different cleaning solutions. These studies identified Ab step yield and breakthrough as performance attributes that decay prior to retrovirus log10 reduction value (LRV) when protein A media is multiple-cycled. In these follow-up studies, we are evaluating viral clearance in multiple-cycled anion exchange media. Weak and strong anion exchange media from multiple vendors are being subjected to 250 or more cycles using a variety of cleaning buffers (NaOH, NaCl, and HCl-based). The resins are being challenged with virus-spiked model proteins in both flow-through and bind & elute mode. A variety of performance attributes are being measured including retrovirus clearance using two independent Q-PCR based assays, DNA clearance, and step yield. Our preliminary results emphasize the importance of proper cleaning of resins for chromatographic performance, including viral clearance.
C.R.Clavet1, A.B.Margolin2, P.M.Regan1, 1University of New Hamphsire
Monoclonal antibodies H1206 (anti-HSV-2 gG-2) and H1379 (anti-HSV-1 gG-1) bound to tosylactivated paramagnetic dynabeads® (Dynal®) have been used to isolate herpes simplex virus type-specific glycoproteins G-1 and G-2 from solubilized HSV infected cell extracts. Monoclonal antibody H1206 was unreactive to the immunomagnetically purified gG-1 and, conversely, monoclonal H1379 was unreactive to immunopurified gG-2. The immunoaffinity purified native glycoproteins were used as markers in conjunction with full Western blot profiles to HSV-1 and HSV-2 to determine the respective serological reactivity to the viruses. Immunoblotting a number of human and rabbit HSV positive antisera obtained from at the Centers for Disease Control (CDC), Atlanta, GA., demonstrated the effectiveness of this methodology. Sera characterized by Western blot and serological reactivity to the immunopurified gG-1 and gG-2 could be used to test the performance characteristics of HSV-1 and HSV-2 clinical serological immunoassays.
Board D-03 Evaluating the Safety and Effectiveness of "Aberration-Free" Ophthalmic Refractive Surgery
B.Drum, M.Eydelman, G.Hilmantel, CDRH, FDA, Rockville, MD
FDA has well-established methods for evaluating spherical and astigmatic refractive surgery. However, new techniques for measuring the optical performance of the eye are now being used with refractive surgical lasers to treat “higher order” aberrations (e.g., coma and spherical aberration) in addition to defocus and astigmatism. Even though many of the effects of higher order aberrations on visual function are still poorly understood, FDA must evaluate the safety and effectiveness of these new treatments. Some specific difficulties include:
1) the potential visual improvement is small and dependent on type of aberration; 2) to date, virtually all refractive surgical treatments have made higher order aberrations worse; 3) treatment outcomes are influenced by uncontrollable factors such as healing, aging, accommodation, pupil size and ocular biomechanical effects; 4) small errors in laser alignment can produce large errors in outcome; and 5) some aberrations may be necessary for optimal visual performance.
FDA’s need to evaluate aberrometry-guided refractive surgery is fueling major research efforts in the vision, ophthalmic and optometric research communities. The results of this research together with outcomes from FDA-regulated trials contribute to a continuing evolution in the types of data and analyses needed for pre-market approval of refractive surgical devices.
Y.Yang1, P.S.Pine2, H.Davis2, P.J.Faustino1, R.C.Lyon1, L.X.Yu3, J.P.Hanig2, 1Division of Product Quality Research, Kensington, MD 20895, 2Division of Applied Pharmacology Research, Laurel, MD 20708, 3Office of Generic Drugs, Rockville, MD 20855
An isocratic reversed-phase HPLC method was developed and validated according to FDA’s Guidance for Industry, “Bioanalytical Method Validation”, for the determination of isotretinoin in plasma and brain tissue from mice following oral single and multiple doses. Plasma sample preparation included deproteination with acetonitrile-perchloric acid followed by centrifugation. Brain tissue was homogenized at 4oC in a solution containing 0.5 mg/mL each of EDTA and ascorbic acid. Homogenates were immediately extracted with acetonitrile-perchloric acid followed by centrifugation. The supernatants were analyzed by HPLC. Benz[a]anthrancene-7, 12-dione was used as the internal standard. Chromatographic separation was achieved on a C18 column (Phenomenex) using an acetonitrile-aqueous 0.5% acetic acid (85:15, v/v) elution. The average extraction efficiency was >95% for plasma and >82% for brain. The lower limit of quantification was 30 ng/mL for plasma and 3.0 ng/0.1g for brain tissue, respectively. The linear range for plasma was 30 to 600 ng/mL, and for brain was 15 to 80 ng/0.1g. The R2 ranged from 0.9891 to 0.998. The peak concentrations (Cmax) of isotretinoin are 2.36 and 2.99 ug/mL in plasma, 34 and 39 ng/0.1mg in brain following a single and multiple doses, respectively. In both cases, Tmax was 1 hr.
T.K.Lee1, E.L.Koo1, A.C.Chang1, D.Sands2, C.Whitton2, C.Longstaff2, 1CBER, FDA, Rockville, MD 20852, 2NIBSC, S. Mimms, UK
An international collaborative study was organized to identify and calibrate a replacement standard for the current US Standard Thrombin, Lot J, and the 1st International Standard (IS) for alpha-Thrombin (89/588). The study involved 25 laboratories from 15 countries. Laboratories were asked to assay two candidate replacement standards, C and D, relative to both the US Standard and the IS using either fibrinogen clotting or plasma clotting methods, and/or chromogenic assays. Data analysis indicated that candidate D is the more suitable replacement for the US Standard and IS. From clotting assays, the potency of candidate D was 106.4 US units/ampoule against the US Standard, and 114.5 IU/ampoule against the IS. Averaging provided 110 Units/ampoule (both US units and IU). The ratios between the potencies derived from the chromogenic and clotting assays indicated that candidate D had a higher proportion of alpha-Thrombin than candidate C. Initial stability studies after 6 months of storage at elevated temperatures showed that both candidates were equally stable with a projected loss of activity of <0.1% per year at -20 °C. It is proposed that candidate D be adopted as the 2nd International Standard for Thrombin with a potency of 110 IU/ampoule.
J.Mauldin1, K.Carbone1, R.Yolken2, S.Rubin1, 1CBER, FDA, Bethesda, Maryland 20892, 2Johns Hopkins University, Baltimore, Maryland 21218
Currently either plaque reduction neutralization assay (PRN) or hemagluttination inhibition assay (HIA) are acceptable measures of protective serological immune responses to mumps. However, there is debate as to whether rapid and quantitative serological assays (e.g., ELISA) can be used as indicators of mumps serological responses. Thus, we compared serological responses to wild type mumps in a group of 75 human sera using two commercially available Mumps IgG ELISAs, Wampole Laboratories (Cranbury, New Jersey, USA) and IBL (Hamburg, Germany), to those obtained using an in-house plaque reduction neutralization (PRN) assay. Because the induction of a neutralizing antibody response is a reasonable index of immunity against mumps virus, results of the PRN assay were used as the standard against which ELISA results were compared. The results indicate that the specificity of the two ELISAs ranged from 83%-91% and the sensitivity ranged from 72%-76%. These results suggest that assays that detect functional antibody, e.g., virus neutralization assays, are of greater utility than ELISAs for assessing mumps immunity.
M.L.Pombo1, I.G.Berthold2, E.Gingrich2, M.T.Jaramillo3, M.Leef2, J.L.Arciniega2, 1FDA, Bethesda MD. National Institute of Hygiene "Rafael Rangel", Venezuela, 2FDA, Bethesda MD, 3FDA, Bethesda MD. National Public Health Laboratory, Mexico
Potency testing of the only anthrax vaccine licensed for human use in the US involves protection of actively immunized guinea pigs from a lethal challenge with a virulent strain of Bacillus anthracis. The objective of this work was to validate an anti-PA (protective antigen) ELISA to measure antibody response in mice. Validation of the assay according to the guidelines of the International Conference on Harmonization (ICH) rendered the following characteristics: linearity (working range: 40-1, 159 EU/ml; r2 = 0.9983), accuracy (91-105% recovery), precision (3-20%CV, repeatability; 1-9%CV, intermediate precision per day and per analyst), detection limit (4 EU/ml), and quantification limit (40 EU/ml). Findings support the use of this ELISA in an alternative immunogenicity (potency) test of PA-containing vaccines in mice and for the standardization of reference materials.
M.W.Trucksess1, V.A.Brewer1, K.M.Williams1, C.D.Westphal1, J.Heeres2, 1CFSAN, FDA, College Park, MD, 2JIFAN/University of Maryland student
Peanuts are one of the eight most common allergenic foods and a large proportion of peanut-allergic individuals have severe reactions, some to minimal exposure. Specific protein constituents in the peanuts are the cause of the allergic reactions in the sensitized individuals who ingest the peanuts. To avoid accidental ingestion of peanut contaminated food, methods of analysis for the determination of the allergenic proteins in foods are important tools. Such methods could help identify foods inadvertently contaminated with peanuts thereby reducing the incidence of allergic reactions to peanuts. Commercial immunoassay kits are available but need study for method performance which requires reference material for within and between laboratory validation. In this study NIST peanut butter, SRM 2387, was used. A polytron homogenizer was employed to prepare an homogenous aqueous peanut butter suspension for the evaluation of method performance of some of the commercially available immunoassay kits such as Veratox Peanut Allergen Test (Neogen Corp.), Ridascreen Peanut (R-Biopharm GmbH), and Bio-Kit Peanut Protein Assay Kit (Tepnel). Each gram of the aqueous peanut butter suspension contained 643 g peanut, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. The suspension was found to be homogenous, stable, reproducible and applicable for adding into ice cream, cookies, breakfast cereals and chocolate for recovery studies at spiked level ranged from 12 mg/kg to 90 mg/g.
H.Wang, A.V.Del Grosso, J.C.May, OVRR
Formaldehyde is often used in bacterial vaccines either as a stabilizer or inactivating agent. Vaccines against anthrax, diphtheria, hepatitis A, influenza, Japanese encephalitis, and tetanus contain residual amounts of free formaldehyde. Less than 0.02% formaldehyde is permitted in vaccine products by FDA based on four decades of safety research. However, the chemical analysis methods used in determining the formaldehyde content of vaccines rely mainly on cumbersome colorimetric procedures (e.g. Hantzsch reaction, Schiff reaction). The aim of this research is to develop a faster and better HPLC method using pre-column chemical derivatization. The selected derivatization reactions such as 2, 4-Dinitrophenylhydrazine, 4-Nitrobenzylhydroxylamine, 1, 3-Cyclohexanedione and Dansyl Hydrazine were compared based on chemical properties of the derivatization reagents, Conditions required for derivatization reaction, derivative stability and HPLC detection sensitivity. HPLC analytical results for several Anthrax Vaccine Absorbed samples were compared to those obtained by traditional colorimetric methods. The recovery of formaldehyde was tested by spiking the vaccine samples since the yields of the derivatization reactions were found either less than quantitative or immeasurable.
P.H.Duffy1, S.M.Lewis2, M.A.Mayhugh2, R.W.Trotter3, 1NCTR, FDA, Jefferson AR, 2Bionetics Corp., Jefferson AR, 3Charles River Corp., Jefferson AR
Ad libitum-fed (AL) obese rats are typically control groups for biomedical studies. Concomitant to weight gain, age- and obesity-related pathologies develop which are modified by dietary controls. A chronic 110-week study was undertaken to determine the effects of 10, 25 and 40% dietary restriction (DR) in male Sprague-Dawley rats. The survival rate for DR groups exceeded that for the AL group; the largest increase in survival occurred between AL and 10% DR. The incidence and severity of cardiomyopathy and nephropathy was significantly lower in all DR vs AL rats. Compared to AL rats, 10% and 40% DR rats had fewer primary neoplasms; 10% DR rats had fewer malignant lesions than 25% and 40% DR rats, and age at which first benign, primary, and malignant tumors occurred was greater in all DR groups. The largest reduction in tumor number occurred between AL and 10% DR groups. Additionally, the percentage of rats with tumors and number of fatal tumors was significantly lower in all DR vs AL groups. This study proved that low-level DR (10%) is effective in increasing survival rate and in preventing or slowing the progression of neoplastic and non-neoplastic obesity-related diseases.
S.Satchithanandam, C.Oles, M.P.Yurawecz, J.I.Rader, ONPLDS, CFSAN, FDA, College Park, MD
The U. S. Food and Drug Administration is finalizing a regulation to include trans fatty acids in nutrition labeling. The purpose of this study was to determine the trans fat contents of currently available foods. Food products were identified on the basis of market share and were purchased from local super markets. AOAC Official Method 996.01 (Fat Analysis in Cereal Products) was modified as needed for the analysis of trans fat. Food