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CATEGORY A: ANALYTICAL CHEMISTRY: METHODS DEVELOPMENT AND APPLICATIONS
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W. C. Andersen1 , S. B. Turnipseed1 , C. M. Karbiwnyk1 , L. E. Carr2 , R. H. Lee2 , M. R. Madson2 , K. S. Kreuzer2 , 1Animal Drug Research Center, FDA, Denver, CO 80225, 2Denver District Laboratory, FDA, Denver, CO 80225
Background: The FDA analyzes hundreds of shellfish samples annually to screen for illegal residues of chloramphenicol (CAP). Current methodology is based on mass spectrometric determination of sample extracts using a triple quadrupole LC-MS-MS. While the method provides excellent sensitivity and selectivity, shellfish requires a lengthy extraction and cleanup to render samples suitable for analysis. Surface plasmon resonance (SPR) biosensor technology based on an analyte-specific binding reaction has become standard methodology for the analysis of drug residues in food in Europe, but it has not yet been adopted for this use by the FDA.
Methods: Shrimp or crab was mixed with ethyl acetate then centrifuged. The organic phase was collected, evaporated, and redissolved in HBS-EP buffer. The sample was defatted with cyclohexane, and the aqueous phase collected for direct analysis. A Biacore Q Biosensor instrument and commercially available CAP test kit was used to analyze samples.
Results: The Biacore Q was evaluated by analyzing 400 shrimp and crab samples for CAP residues. Samples had previously been analyzed using the current FDA LC-MS-MS testing method. The method provides a limit of detection of 0.073 ppb for shellfish, exceeding the FDA requirements for shellfish testing. Biosensor and LC-MS-MS methods were compared with respect to residue detection, instrument performance, extraction efficiency, analysis time, and cost for sample preparation and analysis.
Conclusions: The biosensor method was found to be well-suited for routine shellfish testing providing lower CAP detection limits, higher sample throughput, and lower instrumentation cost than the current LC-MS-MS method.
D. L. Anderson, W. C. Cunningham, CFSAN, FDA, College Park
Background: Instrumental neutron activation analysis (INAA) is a non-destructive technique used by CFSAN for multi-element analysis of foods. High levels of NaCl in foods lead to high limits of detection (LODs) for other elements because of spectral background interferences. To improve INAA's capabilities, a procedure involving background suppression was developed.
Methods: Compton suppression INAA electronics were implemented and procedures developed and used to analyze foods for 16 elements with short half-life irradiation products. Irradiation and gamma-ray counting conditions were optimized for iodine detection to provide quality assurance analyses for FDA's Total Diet Study.
Results: Iodine mass fractions (0.075 to 2.03 mg/kg) were measured in 19 of 42 foods analyzed. LODs ranged from 0.03 to 1.4 mg iodine/kg for foods containing 0.007 to 3.6% NaCl. Al, Br, Ca, Cl, Cu, K, Mg, Mn, Na, Sr, Ti, V, and Zn mass fractions were determined for some or all foods. Control analyses of certified reference materials yielded very good agreement with known values.
Conclusions: Compton suppression spectrometry significantly improved INAA results. Compared with previous procedures, iodine LODs were lowered by about a factor of 4. Similar improvements were found for Al, As, Ba, Br, Ca, Cl, Cu, K, Mg, Mn, Na, Sr, Ti, V, and Zn. Improvements are due to the combined factors of Compton suppression, greater detection efficiency, count timing and times, and choice of irradiation location.
T. H. Begley, W. Hsu, G. W. Diachenko, CFSAN, FDA, College Park, MD
Perfluorochemicals because of their stability and chemical resistance are widely used in the manufacturing and processing of a vast array of consumer goods, including electrical wiring, medical devices, clothing, household, and automotive products. Furthermore, relatively small quantities of perfluorochemicals are also used in the manufacturing of food contact substances (FCS) which represent potential sources of oral exposure to these chemicals. The most recognizable products to consumers are the uses of perfluorochemicals in non-stick coatings (polytetrafluoroethylene (PTFE)) for cookware and also their use in paper coatings for oil and moisture resistance in microwave popcorn bags. Recent epidemiology studies have demonstrated the presence of two particular perfluorochemicals, perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in human serum at very low part per billion (ppb) levels. These perfluorochemicals are biopersistent and are the subject of numerous studies investigating the many possible sources of human exposure. Because of this potential for biopersistence, FDA is evaluating the migration characteristics of perfluorochemicals from food contact paper.
In this paper, the types of perfluoro chemicals used in food contact paper will be illustrated, along with methods for their determination. Additionally, research will be presented on the migration of these chemicals into foods or food simulating liquids. Results from migration tests show that fluorotelomers from the paper additives/coatings do transfer to food.
H. W. Yang1 , H. T. Mai1 , A. Weisz2 , 1Office of Cosmetics and Colors, Color Certification Branch, FDA College Park, MD, 20740, 2Office of Cosmetics and Colors, Color Technology Branch, FDA College Park, MD 20740
Background: D&C Red No. 21 (R21, Colour Index No. 45380:2, mainly 2',4',5',7'-tetrabromofluorescein), its disodium salt, D&C Red No. 22 (R22, Colour Index No. 45380, Eosin Y), and their lakes are color additives listed in the U.S. Code of Federal Regulations (CFR) for use in drugs and cosmetics (Only material which has been batch certified by the FDA in accordance with CFR specifications is appropriately identified with the D&C designation.) R21 and R22 are manufactured by condensing phthalic anhydride (or acid) with two equivalents of resorcinol, partially purifying, and brominating the resulting fluorescein to yield the main component of R21. R21 is hydrolyzed with sodium hydroxide to produce the main component of R22. During manufacture, various impurities may occur. CFR specifications include certain side-reaction products: brominated resorcinol, not more than 0.4%, and 2-(3,5-dibromo-2,4-dihydroxybenzoyl)benzoic acid, not more than 0.5%; and the intermediate phthalic acid, not more than 1%. Currently, the Color Certification Program determines these impurities using gravity-column chromatography followed by UV-Vis spectrophotometry. A more efficient, automated method was needed for their determination.
Methods: Brominated resorcinol was purified for use as a reference standard. Appropriate standards were obtained for the other two analytes. A high-performance liquid chromatographic (HPLC) method was developed to determine the three specified impurities in R21 and R22.
Results: Five-point calibration curves were prepared by analyzing samples of R21 spiked with varying amounts of the three analytes. Twenty previously certified samples of R21, R21 lakes, and R22, representing eight manufacturers, were analyzed by the new method. The results compared well with those for the gravity-column chromatography method.
Conclusions: The present study reports the development of an automated, efficient, and reliable high-performance liquid chromatographic (HPLC) method for the determination of two side-reaction products and an intermediate that often contaminate batches of R21 and R22.
I. Vertsman, Y. T. Cain, Wyeth Research, Wyeth
Background: Sample preparation is the most time consuming part of the analytical testing. To be a front runner in today's competitive market place, dramatically increase in efficiency and productivity is crucial. Wyeth has recognized that utilizing new technology and automation is the answer. One of the techniques to automate sample preparation is using Tablet Processing Workstation (TPW).
Methods: Automated sample preparation methods using TPW were developed for the content uniformity testing in supporting of a several late stage development projects. TPW system weighs each tablet, adds assigned amount of solvents, performs extraction, filters sample, dilutes and transfers in the vials for the further analysis. Analyst time required for the set up of the instrument is about 2 hrs per 50 samples.
Results: Excellent equivalency was obtained between manual and automated sample preparation methods. Difference between manual and automated results was in the range of 0% to 1.8%. Reproducibility (Precision and Intermediate Precision) of the automated results was less then 1%. Recovery of the known standard concentration, processed through the TPWII system the same way as samples ranged between 97.9 and 102.1%.
Conclusions: The biggest advantage of automated sample preparation technique was observed for high volume projects, especially process validation testing, when robotic systems can prepare samples 24 hrs a day 7 days a week. Time consuming manual preparation steps can be fully automated. Minimum analyst time is required for the set up of the instruments, which increases efficiency about 4 times.
Y. T. Cain, M. Meyers, Wyeth Research, Wyeth
Background: Ion Mobility Spectrometry (IMS) has been successfully implemented as a rapid analysis technique for cleaning verification/validation.
Methods: IMS is a sensitive and fast analytical technique that has proven advantageous when used in cleaning validation/verification applications. The instrument is easy to operate and has a lower cost of operation in comparison with HPLC. Two examples of technical challenges that have been overcome are discussed. IMS technology has been enhanced using the new High Performance Injection (HPI) system. Improved precision and method simplification via elimination of dilutions have been demonstrated with HPI. Future applications using IMS with HPI are discussed.
Results: Several cleaning methods have been developed and validated using the limit test approach. The technology has been deployed to several commercial manufacturing sites and successful technical transfers have been completed. Sensitivity is a function of the attributes of the analyte, but detection limits at the low pictogram level have been achieved with data acquisition times as short as one minute per injection. HPI technology significantly improves reproducibility both short-term (precision) and long-term (signal drift).
Conclusion: IMS provides rapid, low cost results which dramatically increases cleaning sample throughput and reduces manufacturing equipment down time.
Y. T. Cain, M. Meyers, Wyeth Research, Wyeth
Background: The development of a fiber optic probe coupled to a fluorescence spectrophotometer has made possible the rapid identification of pharmaceutical tablet dosage forms with minimal sample preparation.
Methods: UV light at the excitation wavelength is transmitted from the spectrophotometer source directly to the solid tablet surface, in which the coating has been removed with light sanding, by way of the fiber optic bundle and probe. Fluorescence from the tablet is transmitted back though the bundle to the detector. The active pharmaceutical ingredient (API) is positively identified by its fluorescence spectrum.
Results: Label claims with similar API to excipient ratios are differentiated though volume adjusted concentration differences of the API in the bulk tablet compared with a standard in powder form.
Conclusion: A tablet can be identified in a matter of seconds with this technique.
Y. T. Cain, J. L. Edgar, Wyeth Research, Wyeth
Background: The future of dissolution is moving toward increasing productivity in laboratories. This can be accomplished is by utilizing dissolution with online fiber optic UV analysis.
Methods: Fiber optics changes the traditional way of sampling for dissolution by bringing the UV spectrometer to the sample solution in-situ.
Results: The use for fiber optics allows real-time determination of drug release levels in-situ and effectively eliminates many common issues with traditional dissolution testing and analysis including carryover, sipper, line or filter malfunction and cost of consumables such as filters, test tubes and HPLC solvents. Since no sample is removed from the dissolution vessels, readings can be taken as often as every few seconds if necessary; thereby setting a new precedent for dissolution profiles. Direct comparison of fiber optic analysis with conventional quartz cell UV analysis shows agreement well within 2%.
Conclusion: The use of fiber optic analysis for dissolution testing saves analyst time, which reduces laboratory operating costs. Product development data is more meaningful since the number of sampling time points is not limited by volume removal. This technology has been successfully implemented at Wyeth research and commercial manufacturing sites.
S. Smith, C. S. Cheely, C. Gieseker, R. Reimschuessel, M. C. Carson, CVM, FDA, Laurel, MD
Background: Prior to approving a drug for food animals, a method for determining residues is needed and a depletion study is conducted using this method. A microbiological (micro) method for erythromycin was used in the salmon depletion study, however a liquid chromatography (LC) assay developed by NCTR gave much lower concentrations in incurred tissue than the micro assay. Using LC-ion trap mass spectrometry, we identified at least two additional compounds in dosed salmon, N-demethylery A and anhydroery A. These may contribute to the difference between the NCTR LC method and the micro method.
Method: Ground salmon is extracted with acetonitrile, the extract defatted with hexane, evaporated, and reconstituted in a solution of Ery C, the internal standard (IS). Extracts are chromatographed on a phenyl column. Detection is by + ESI, with monitoring of ion trap transition products of m/z 734.5 (Ery A), 720.5 (Ery C and N-demethylery A), 718.5 (Ery B), and 716.5 (anhydroery A).
Results: Accuracy ranged from 65% at 50 µg/kg to about 80% at concentrations of 500 µg/kg and above. CVs were 7-20%, reflecting the variable nature of the ion trap. Limits of detection were estimated to be <10 µg/kg. Salmon were dosed with 100 mg/kg Ery A and sacrificed following 1 to 28 days. Ery A and both metabolites were detected at all time points. By 3 days withdrawal, metabolite exceeded parent concentration. Parent Ery A depleted to <100 µg/kg by ~3 weeks, but total residue remained above 1 mg/kg.
Conclusion: Ery A is metabolized in salmon to compounds which deplete much slower than the parent drug.
P. Chu, M. Lopez, CVM, FDA, Laurel, MD
Background: Nitrofurans are antibacterials commonly used in the treatment of aquatic and terrestrial animals to control bacterial and protozoan infections. Although widely used since the 1940s, nitrofurans have been banned for use in food-producing animals in most countries because of their mutagenicity and potential carcinogenicity. Recently, concerns about the illegal use of this class of compounds in food-producing animals have developed. Therefore, methods are needed to monitor nitrofuran residues in food and for research purposes. We describe a method capable of detecting and quantitating total bound and free residues of nitrofurans in milk.
Method: To 2 mL of milk is added 5 mL of 0.125 M HCl followed by 400 µL of 50 mM 2‑nitrobenzaldehyde. The mixture is placed in an incubator at 37°C overnight with gentle shaking. During this step, the side-chains of the bound residues of nitrofurans are released through hydrolysis and simultaneously derivatized with 2-nitrobenzaldehyde. After adjustment of pH to ~7, the sample is cleaned up on a solid-phase extraction column. The derivatives are detected and quantitated using LC-MS/MS in the positive ion mode.
Results and Conclusions: The method has been validated at 1, 2, and 4 ppb using fortified milk and milk obtained from a treated cow. The method accuracy is >60% with coefficients of variation <20%. Among the four nitrofurans studied in milk of the treated animal, nitrofurantoin exhibits the lowest level of residues.
D. Dai, S. Wang, NRL, FDA, Jamaica, NY
The cephalosporins are a group of antibiotics closely related to the penicillins. The presence of penicillin residues in cephalosporins has the potential to cause health effects to patients allergic to penicillin.
The presence of trace levels of spiked penicillin G in cephalosporins was determined by HPLC with a PDA detector using a Waters Symmetry C18 5µm, 4.6 x 250 mm column; a gradient elution of 0.01M orthophosphoric acid/acetonitrile from 80/20 to 40/60 in 20 min. The UV spectra were collected from 190 to 400 nm and extracted at 220 nm. Identification was confirmed by LC/MS/MS method.
This HPLC method provided excellent separation between penicillin G and 12 cephalosporins and PDA detection provides spectra identity of the peaks. Excellent linear response of penicillin G was found in the range of 0.5 to 100 µg/mL and the estimated MDL was 0.04 µg/mL. A peak very close to the retention time of penicillin G was detected in the screening of selected cephalosporins APIs and finished products; however, the UV and mass spectra data indicated it was not penicillin G. The stability of penicillin G and Cephalosporins in various solvents has been examined. The optimum solvent for dissolving and diluting penicillin G and Cephalosporins was found to be the mixture of acetonitrile and water at a neutral pH. Penicillin G rapidly changed to its isomer in an acidic environment. In the presence of methanol, penicillin G rapidly converted to its methanol adduct.
L. S. de Jager, G. A. Perfetti, G. W. Diachenko, CFSAN, FDA, College Park MD
Vanilla extract is widely used as a flavoring in food. Because authentic vanilla extract is expensive, artificial vanilla flavorings containing synthetic vanillin and ethyl vanillin are often used. Some foreign manufacturers add coumarin to vanilla products to increase the vanilla flavor perception. Coumarin is a phytochemical found in many plant species, with the main source being the tonka bean. It has a sweet herbaceous odor and has been used in food, tobacco, and cosmetics as a flavoring and fragrance material. Coumarin has been shown to be hepatotoxic and has been prohibited from being added to food in the U.S. since 1940 (CFR part 189.30).
Two official AOAC methods have been published for the detection of coumarin in vanilla extract. One involves separation using column chromatography followed by spectophotometric detection and quantification (AOAC Official Method 955.3) and the other is a purely qualitative TLC method (AOAC Official Method 964.11). These methods have limited selectivity and sensitivity. A solid phase microextraction (SPME) GC-MS method for determining coumarin and vanilla extract components has been developed. This method has greater sensitivity than the AOAC methods and provides MS confirmation. Method development, optimization and validation and the results of a survey of vanilla extract products will be presented.
Q. Ge, R. V. Bahulekar, P. M. Amin, M. W. Diamond, F. M. Buevich, F. T. Do, R. A. Ebelle, S. K. Pulapura, A. C. Moses, W. C. McJames, TyRx Pharma, Inc.
Background: Gravimetric analysis is accepted and the standard method for measuring the mass loss of bioresorbable polymers (ASTM Standard F1635-04a). The current method involves drying the sample to a constant weight, prior to weighing. Since resorbable polymers degrade via hydrolysis, there is often an initial increase in mass at earlier assessment time points, which one can assume is the result of bound water molecules. The introduction of the use of polyarylate polymers on implantable medical devices has permitted the development and use of an HPLC method that calculates mass loss by measuring the polymer degradants as they become solubilized in the buffer over time.
Methods: Polyarylate polymer-coated polypropylene mesh was incubated in PBS at 37°C with no agitation. PBS buffer was changed twice each week and the buffer analyzed via HPLC for the presence of tyrosine-related compounds (i.e., monomers and end stage degradants). The mass loss at each time point was calculated by adding the mass of the measured compounds and the mass of equivalent molar amounts of the non-measurable degradants to the mass equation. The experiment was terminated when no degradants were detected at three consecutive time points.
Results: This experiment characterized the in vitro mass loss profile and degradation rate of the polyarylate polymer coating of the polypropylene mesh.
Conclusions: HPLC is a reliable and accurate method to measure the mass loss of resorbable polymers containing optically active compounds. When it is possible to use it, it may be more accurate than gravimetric analysis.
S. M. Etheridge1 , J. R. Deeds1 , S. M. Conrad1 , S. Hall1 , P. DiStefano2 , M. Ellwanger2 , K. Chu3 , F. Pettengill4 , M. Hickey5 , D. Couture6 , 1OS, CFSAN, FDA, Laurel, MD, 2OS, CFSAN, FDA, College Park, MD, 3NOAA Fisheries Service, Gloucester, MA, 4Div. of Marine Fisheries, Gloucester, MA, 5MA Marine Fisheries, Pocasset, MA, 6Dept. of Marine Resources, W. Boothbay Harbor, ME
An extensive Alexandrium bloom occurred off the New England coast from May to July 2005 creating an unprecedented paralytic shellfish poisoning (PSP) event severely impacting the shellfish industry with toxicity exceeding the action level (80 micrograms saxitoxin equivalents per 100 grams tissue). High-resolution coastal sampling by management programs allowed safe shellfish to be marketed, whereas shellfish beds threatened by PSP were closed to protect public health. At the request of FDA, NMFS closed approximately 15,000 square miles of federal waters in the northwestern Atlantic Ocean on 14 June. A time-series of offshore shellfish toxicity was monitored since the beginning of the closure. The receptor binding assay was used as the primary detection method, with the AOAC mouse bioassay providing confirmation for regulatory decisions. Shellfish toxicities varied with species, with the highest toxicity (2045 micrograms saxitoxin equivalents per 100 grams) found in whole scallops sampled in July. Toxicity decreased over time with depuration rates differing between species. Analytical data supported reopening a portion of the closure on 9 September (except for whole and roe-on scallops); although the remaining northern area still remains closed. Due to an effective, long-standing cooperative shellfish program managed by FDA and implemented by states, there were no human illnesses despite remarkably high toxicity in the unmarketed product. Current/future efforts to improve seafood biotoxin risk management include: 1) implementing a dockside testing protocol and 2) conducting a collaborative investigation with academia, government, and industry to establish a comprehensive regional-scale understanding of Alexandrium blooms and associated shellfish toxicity.
R. B. Shah, Y. Yang, M. A. Khan, P. J. Faustino, Division of Product Quality Research, Office of Testing and Research, Office of Pharmaceutical Science, FDA, Silver Spring, MD
Purpose. Development of a validated SEC method to efficiently assess the MW of polysaccharides. Heparin, a mucopolysaccharide whose antithrombic activity depends on MW was investigated.
Methods. Separation was achieved on two gel filtration columns (TSK-GEL-G4000SWXL) with a mobile phase of sodium azide (0.02%, pH, 6.2) delivered isocratically at 0.6 ml/min with refractive index detection. Standard curves were constructed using dextran MW standards (1 to 670 KDa). The method was validated according to ICH. Heparin sodium from porcine intestinal mucosa (Product A) and bovine lung tissue (Product B) in different pH media were analyzed.
Results. Log-linear function was used for MW standard curve (12 to 670 KDa). A second order polynomial fit was applied for the MWs below 5 KDa. Accuracy ranged from 90-103% and precision was <2.7%. Product A showed three MW fractions of 580, 30, and 1.5 KDa at pH 3, 4, and 5. However at pH 6.2, only two fractions were obtained (590 and 2 KDa). At pH 7.4, the two significant fractions were 23 and 1 KDa. Product B showed two fractions of different MW from pH 3-7.4. The major fraction at pH 3, 4, and 5 was 30 KDa. However at pH 7.4, a fraction of 12 KDa was achieved.
Conclusions. A highly specific, selective, and efficient SEC method was developed for the accurate MW determination of polysaccharides. Two heparin products exhibited different MW fractions depending upon isolation source and media pH. The method can be used to assess the quality of therapeutic polysaccharides.
S. R. Gratz, R. A. Flurer, M. R. Witkowski, C. L. Flurer, Forensic Chemistry Center, FDA, Cincinnati, OH
For several years, synthetic phosphodiesterase type 5 (PDE-5) inhibitors have been identified routinely in "all natural" herbal remedies and dietary supplements, as well as in counterfeit and unapproved pharmaceutical products. A liquid chromatography-electrospray mass spectrometry (LC-MS) method was developed and implemented at the Forensic Chemistry Center to screen for sildenafil (Viagra®), tadalafil (Cialis®) and vardenafil (Levitra®) in both herbal and pharmaceutical matrices. More recently, there has been a trend toward the development of designer drugs, or analogs, based on sildenafil, tadalafil and vardenafil. As a result, methods that only screen for the FDA-approved compounds are inadequate. Among the growing list of such compounds are homosildenafil, hydroxyhomosildenafil, acetildenafil, hydroxyacetildenafil, aminotadalafil and piperadino-vardenafil. Based on the structural similarities of these drugs to their FDA-approved counterparts, it is reasonable to expect that they would exhibit similar biological activity.
The general approach used for identification of these analogs will be shown. The method of choice for initial identification of this class of compounds is LC-MS, while Fourier-Transform infrared spectrometry also provides valuable information. Elucidation of analog structures is typically accomplished through MSn experiments, and by comparing spectra to those of sildenafil, tadalafil and vardenafil. Fourier-Transform ion cyclotron resonance mass spectrometry is used for accurate mass determinations, to further characterize analog structures.
Z. Gao, T. Moore, A. Smith, W. H. Doub, B. J. Westenberger, L. F. Buhse, FDA
Background: In-vitro dissolution tests on finished dosage forms have been performed for many years. Currently, the repeatability and reproducibility of dissolution tests are of concern to the pharmaceutical industry. The large variability observed in dissolution test results may be a result of uneven distribution of hydrodynamic forces within the dissolution vessel. Random positioning of tablets/capsules, even in well calibrated USP apparatus 2 dissolution vessels, can lead to high variability in dissolution results. Method: The gauge repeatability and reproducibility (Gauge R&R) method was used to analyze variability in a dissolution testing system. Additionally, perturbation studies were performed to study the variability caused by changes in the sample position. Results: Evaluation of dissolution testing results at 30 minutes using an internal DPA NCDA #2 tablet indicate that the main contribution to total variance (~70% of total variance) is due to the sample tablets, ~25% of the total variance arises from the vessels (apparatus) and ~5% of the total variance is due to the operators. In addition, the sample position (centered and off-centered positions) can have a serious effect on the dissolution results. Conclusions: Gauge R&R analysis is a useful tool for determining the sources of variability in a dissolution measurement system. Although there are sample position effects, the impact of variability from sample position depends on the drug product.
P. E. Nwakama, S. H. Haidar, Y. S. Yang, B. M. Davit, D. P. Conner, L. X. Yu, CDER, FDA, Rockville, MD
Background: Bioequivalence (BE) studies are conducted "...to ensure therapeutic equivalence between a pharmaceutically equivalent test (T) drug product and a reference (R) listed drug." The current confidence interval limits of 80 -125% for Cmax and AUC ratios were established based on physicians' thinking that as much as a 20% difference in dose would not be clinically significant for most drugs. This study was to evaluate performance of the BE criteria, by examining differences in extent (AUC) and rate (Cmax) of bioavailability between reference listed drugs (RLD) and their generic equivalents.
Methods: With few exceptions, data used in this study were from single-dose, fasting BE studies of approved oral drug products submitted to the Office of Generic Drugs from 1996 to 2005. Statistical analysis was performed on differences in extent and rate of absorption between the RLD and its generic equivalent using point estimates for AUC and Cmax, respectively.
Results: A total of 1636 fasting BE studies were examined; the sample size of each study ranged from 12 to 127 subjects. The mean (±S.D.) of the absolute value of the difference in point estimates |T - R| for AUC0-T was 3.19% (±2.72) and 3.12% (±2.66) for AUC0-inf. Mean difference for Cmax was 4.50% (±3.57). All were well within the 90% CI criteria of 80 - 125%.
Conclusion: The results show that generic products have averaged less than 4% difference in extent of absorption relative to the RLD. This illustrates the effectiveness of the BE acceptance method and criteria used in the approval of generic drugs in the U.S.
M. K. Halbert, J. C. Archer, ARL, FDA
Background: According to the guidelines of the International Olive Oil Council, olive oil may by labeled as "extra virgin" if it has no more than 0.8 percent acidity and has not undergone any treatment which would lead to alterations in the oil. Oils not labeled as "virgin" have undergone some refinement to alter the acidity, taste and appearance, and may be a mixture of refined and virgin oils. Since virgin olive oils have undergone no refinement, they may contain higher concentrations of contaminants, such as dioxins and furans.
Methods: Thirty-eight samples of olive oil were collected from store shelves during the period 2003-2005, and were analyzed for dioxins and furans using EPA Method 1613, including HRMS analysis. These oils were described variously as "extra light tasting," "100% pure," "extra virgin," or simply "olive oil."
Results: The oils were divided into two categories for the sake of comparison —"extra virgin olive oil" and "non-virgin olive oil." The extra-virgin oils exhibited an average toxic equivalence (TEQ) overall of 0.035 pg/g, with a range of 0.0014-0.10 pg/g. The average TEQ for the non-virgin olive oils was lower, at 0.010 pg/g, with a range of (non-detect)-0.041 pg/g. The greatest contributors to TEQ for both groups were 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF, with these congeners reduced by 73% and 77%, respectively, in the non-virgin olive oils. 1,2,3,7,8-PeCDD was reduced to a non-detect level in the non-virgin olive oils from an average of 0.0058 pg/g in the extra-virgin oils.
Conclusions: As expected, refinement of the olive oils apparently reduces the levels of dioxins and furans, although the levels in all the samples were acceptably low.
S. Hall, S. M. Etheridge, J. R. Deeds, S. M. Conrad, WSL, OS, CFSAN, FDA, Laurel, MD
Marine biotoxins, produced by unicellular algae and accumulated in seafood, can cause illness and death in human consumers. Purified toxins are needed for determination of structure, for chemical and toxicological characterization, for the development and validation of detection methods, and for the field deployment of those methods. In most laboratories, work with these toxins is constrained by their availability since they tend not to be commercially available and are difficult to obtain or produce. Due to the facilities established and maintained by the Washington Seafood Laboratory for the production and purification of marine biotoxins, the FDA needs for the saxitoxins and domoic acid can be well supported and many other needs can be met as they become priorities. Current priorities include the purification and certification of reference standard saxitoxin to replace the historical standard now in use, purification and characterization of decarbamoylsaxitoxin as an alternative to saxitoxin and as a starting material for producing other needed derivatives, and purification of other saxitoxin congeners for the evaluation of new detection methods.
D. G. Hayward1 , T. S. Pisano2 , 1CFSAN, FDA, College Park, MD, 2JIFSAN, University of Maryland, College Park, MD
Background: Polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBDEs) are known to persistent in the environment. Foods are the major route of exposure to nonoccupationally exposed persons. FDA routinely monitors PCDD/Fs in 1000s of foods of many types annually. PCBs are monitored through the total study program, but no isomer specific data is generated. PBDEs have never been measured routinely by FDA in foods.
Methods: These three compound classes are often determined by separate methods (e.g. EPA 1613b/8290, 1668 and 1614). A study was conducted to evaluated four separate approaches to determining all target analytes by a single method that might be easily automated using four criteria to include or exclude elements in each approach. The method selected must be 1) automated, 2) produce reliable recovery and chromatography, 3) be applicable to any food or feed, 4) use a sample size suitable for detecting congeners with toxic equivalency factors (TEFs).
Results: An automated procedure using accelerated solvent extraction with integrated clean up followed by "express" gel permeation chromatography with on-line SPE clean up made the final selection. Eight fish oils, a corn oil, 3 fish fillets, carrots and butter were successfully measured isomer specifically for 50 target analytes from the three compound classes. Recoveries were assessed using fortified corn oil and native incurred levels in fish fillets measured by a LIB 4084 and the new method. Recoveries fell within the acceptable range (50-100%).
Conclusions: A single automated approach was identified that met the four criteria.
T. S. Pisano1 , D. G. Hayward2 , 1JIFSAN, University of Maryland, College Park, MD, 2CFSAN, FDA, College Park, MD
Background: Persistent organic pollutants (POPs) such as polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), polychlorinated biphenyls (PCBs), and polybrominated biphenyls (PBDEs) are associated with lipid compartment in foods. FDA plans to report milk POPs levels on a lipid weight basis and will need to determine the lipid content of all milk samples as part the PCDD/F analysis. Traditional liquid/liquid extraction (LLE) methods such as AOAC 970.52H, AOAC methods vol.2, p278, require large amounts of solvent and tedious manual manipulations. Pressurized liquid extraction (PLE) uses less solvent and is automated.
Methods: A study was performed to find the optimal conditions for extracting fat from freeze dried cow's milk using PLE then following with purification of the fat for POPs analysis1. Freeze-dried milk was extracted with several solvent systems by PLE. PLE was performed by an ASE300™ (Dionex, Sunnyvale, CA). Using butter as a test matrix, automated fat removal with sulfuric acid silica gel as fat retainer was tested with PLE.
Results: The optimal solvent system was determined to be methanol/ dichloromethane/ hexane (1/2/2). The PLE gravimetric fat results were not significantly different from those by LLE at 95 % confidence level. A 99% fat removal from 4g of butter was achieved with 60g of 44% sulfuric acid on silica gel fat retainer in the ASE with the following conditions: 100% petroleum ether in three 5min extractions at 1500psi, 100ºC and a 5% flush.
Conclusions: PLE based extraction method produces comparable results using less solvent and man-hours for the milk fat determination.
1 Hayward, D.G., Pisano, T.S., 2006 FDA science form, Automation for isomer specific determination of polychlorinated dibenzo-p-dioxins/furans, polychlorinated biphenyls and polybrominated diphenyl ethers in foods and feeds.
N.M. Hepp, Office of Cosmetics and Colors, CFSAN, FDA, College Park, MD
Background: Bone Black is proposed to be listed in the Code of Federal Regulations as the color additive D&C Black No. 3, subject to FDA batch certification. Methods are needed to enforce specifications of <10 ppm for lead (Pb) and <3 ppm for arsenic (As). X-ray fluorescence spectrometry (XRF) standards were prepared and validated by atomic absorption spectrometry. The calibration curves generated from the standards were evaluated for suitability in the routine analysis of Bone Black samples for Pb and As.
Methods: Standards were prepared from Bone Black fortified with varying amounts of Pb and As. The fortified standards were used to develop appropriate grinding and pellet-pressing procedures and to obtain XRF calibration curves. Portions of each standard were analyzed by atomic absorption spectrometry to verify fortification levels. Electrothermal atomization atomic absorption spectrometry was used to determine Pb, and flow injection/hydride atomic absorption spectrometry was used to determine As.
Results: Very good XRF calibration data were obtained for both Pb and As in fortified Bone Black samples using secondary XRF lines to deconvolute overlap of the primary lines for the two analytes. Detection limits for Pb (1.0 ppm) and As (0.3 ppm) indicate that XRF is appropriate for routine batch certification analysis of these elements in Bone Black.
Conclusion: XRF can be used for FDA batch certification to efficiently analyze multiple samples of Bone Black (D&C Black No. 3) at the proposed specification levels for Pb and As.S. C. Hight, J. Cheng, FDA
A method was developed for determination of methylmercury and estimation of total mercury (Hg) in seafood. Hg compounds were extracted from 0.5 g edible seafood or 0.2 g lyophilized reference material by adding 50 ml aqueous 1% w/v L‑cysteine•HCl•H2O and heating 120 min at 60°C in glass vials. Hg compounds in 50 µl of filtered extract were separated by reversed‑phase HPLC using a C‑18 column and aqueous 0.1% w/v L‑cysteine•HCl•H2O+0.1% w/v L‑cysteine mobile phase at room temperature and were detected by ICP-MS at mass‑to‑charge ratio 202. Total Hg was calculated as the mathematical sum of methyl‑ and inorganic Hg determined in extracts. Precision of analyses was ≤5% relative standard deviation (RSD) for methylmercury and ≤9% RSD for inorganic Hg. Recovery of added analyte was 94% for methylmercury and 98% for inorganic Hg. Methyl‑ and total Hg results for reference materials agreed with certified values. Limits of quantitation were 0.007 mg/kg methylmercury and 0.005 mg/kg inorganic Hg in edible seafood and 0.017 mg/kg methylmercury and 0.012 mg/kg inorganic Hg in lyophilized reference materials. Evaluation of analyte stability demonstrated that L‑cysteine both stabilized and de‑alkylated methylmercury, depending on holding time and cysteine concentration. Polypropylene adversely affected methylmercury stability. Total Hg results determined by this method were equivalent to results determined independently by cold vapor‑atomic absorption spectrometry. Methylmercury was the predominant form of Hg in finfish. Ratios of methylmercury/total Hg determined by this method were 93‑98% for finfish and 38‑48% for mollusks.
J. C. Hubinger, D. C. Havery, Office of Cosmetics and Colors, CFSAN, FDA, College Park, MD
Background: Retinol and retinyl palmitate are frequently used in cosmetic skin care products but may be irritating to the skin at higher concentrations.
Methods: A simple, rapid and sensitive reversed‑phase HPLC method with UV detection was developed for the quantitation of retinol, retinyl palmitate and retinoic acid in cosmetic preparations. The analytes were extracted from a cosmetic/Celite mixture using a solvent system composed of equal amounts of hexane, isopropanol, and ethyl acetate, and the extract was injected directly into an HPLC with a C18 column and UV detector set at 330 nm. Chromatographic separation was achieved by gradient elution with a mobile phase starting with aqueous ammonium acetate buffer/methanol that was gradually changed to methanol/dichloromethane.
Results: The method was validated, and average recoveries of retinol, retinyl palmitate and retinoic acid were 95% or higher. In a survey of 29 consumer cosmetic skin care products, most products were found to contain either retinol or retinyl palmitate at concentrations up to 2.0%, while a few products contained both ingredients. Several cis isomers of retinol and retinoic acid were also isolated from cosmetic products with the method and could be quantitatively distinguished from the all-trans compounds.
Conclusions: The method can be used to accurately quantitate levels of several retinoids and their isomers in cosmetic products.
J. N. Jean-Giles, G. L. George, J. E. LeClerc, T. A. Cebula, Mod-1
Background: In order to evaluate tools for the differentiation of strains of foodborne pathogens, we have applied the multilocus variable-number tandem repeat analysis (MLVA) to a reference collection of diverse isolates of Escherichia coli O157:H7. The method differentiates strains by analysis of the expansion and contraction of short tandem repeats (TRs) in the genome. Since these elements vary in number at several loci, the method discriminates among strains of the same species.
Methods: MLVA analysis using a multiplex of seven targeted loci of TRs (TR1-TR7) was used to compare isolates in a collection of 120 strains of E. coli O157:H7. The MLVA pattern of each strain was determined based on the size (bp) of TRs of each locus to determine the number of repeats. The patterns were categorized using a letter coding system.
Results: From 120 strains of E. coli O157:H7 examined, 89 different TR patterns were determined. The most variable locus analyzed, a tandem repeat of six bp (TR2), showed 30 alleles and as many as 69 repeat elements.
Conclusion: Application of the MLVA analysis to a large set of diverse isolates of E. coli O157:H7 showed the duplication of MLVA patterns in independent strains. While the variability in MLVA patterns showed the genetic diversity present among many of the strains examined, the results demonstrated that additional molecular markers would be required for conclusive strain differentiation. Further evaluation of rapidly evolving loci, such as TR2, is also needed for proper interpretation of MLVA results.
A. N. Joshi, J. Nickelsen, G. Gavini, FDA
Abstract: There is a continuous demand on ORA labs to develop improved qualitative analytical methods for the enforcement of FDA regulations. The method described is for identification of color additives in imported and domestic food and cosmetic products, using FT-IR Spectroscopy. Two parallel procedures are run. One the standard separation and identification procedure and the other using the FTIR identification. The results so obtained are compared for cost, quality, time and reproducibility. If a color is observed and FTIR confirms absence of a certifiable color then it is an indication of non permitted color additives. The results of samples analyzed and spectra generated are reported.
P. A. Jupp, D. Trew, C. H. Turner, Wyeth Research
The measurement of pKa by NMR gives a specific approach to both the determination and the assignment of pKa values to ionisable centres in a molecule. The traditional approach for the measurement of pKa by potentiometric titrations can give the value for the ionisable centre but cannot show where the protons actually resides. Also at low concentrations the influence of the water as a media can distort the values as well as causing determination difficulties if the compound has low solubility. NMR, however can precisely determine the pKa in aqueous as well as mixed solvent systems. Work has been carried out to assess the impact on measuring pKa in organic/aqueous mixtures and clearly demonstrates that protonation is dependant of the nature of the media in which the measurement is made.C. M. Karbiwnyk1 , K. C. Faul2 , S. B. Turnipseed1 , W. C. Andersen1 , K. E. Miller3 , 1ORA, Animal Drugs Research Center, FDA, Denver, CO, 2ORA, FDA, Denver, CO, 3Univ. of Denver, Denver, CO
Background: The drug most commonly used to induce labor in the U.S., oxytocin, is a peptide hormone composed of nine amino acids. Oxytocin is often reconstituted in IV saline solutions at less than 0.05 Units/mL to be delivered at 1 - 4 drops/minute. Existing LC-UV methods for oxytocin do not have sufficient detection limits to quantitate and/or confirm oxytocin in IV solutions. LC-MSn was utilized as a rapid and sensitive method for the determination of oxytocin in a saline solution at less than 0.02 Units/mL.
Method: A determinative and confirmatory method for oxytocin was developed using an LC-MSn ion trap instrument with an electrospray ionization (ESI) interface in positive ion mode. Separation was achieved on a C-18 column with 250 µL/min isocratic flow of 36% acetonitrile:water (1:1) and 64% dilute acetic acid (0.05%) mobile phase.
Results: Calibration standards, prepared in de-ionized water from 0.006 - 0.046 Units/mL, were linear with an R2 value of 0.9983 using results from a SIM scan. A method detection limit (MDL) was tested at 0.003 Units/mL (7.5 ng/mL) and met all criteria for quantitation and confirmation. This LC-MSn method was used to determine the amount of oxytocin in a 0.04 Unit/mL clinical sample that was prepared in 0.9% sodium chloride IV solution. The sensitivity of the method allowed the sample to be diluted 1:1 with de-ionized water to reduce the salt content.
Conclusions: This study demonstrates the application of LC-MSn for low concentration (ng/mL) determination and confirmation of oxytocin in saline solution.
E. Karnaukhova, B. Golding, Y. Ophir, Division of Hematology, CBER, FDA, Bethesda, MD
Human alpha-1-proteinase inhibitor (a1-PI) is the most abundant serine protease inhibitor in plasma. A major function of α1-PI is the inhibition of neutrophil elastase in lungs. Three plasma-derived (pd-) a1-PI products are licensed in the US for augmentation therapy of deficient patients. The recombinant versions (r-α1-PI) provide as an alternative to pd-a1-PI products and have been under intensive investigation. For accurate determination of α1-PI from different sources and in various forms, there is clearly a need for reliable standardized assays. As a part of our multi-step research focused on α1-PI structure-function investigation, we have established an ELISA with high specificity and a low limits of detection (1.10 ng/mL) and quantification (3.34 ng/mL), which allows determination of pd-α1-PI as well as r-α1-PI in complex matrices. A validation of ELISA was performed with the working range of the assay from 3.1 to 50 ng/mL with an average correlation coefficient of 0.995 established by analyses of 108 calibration standard curves.
The analytical performance of the a1-PI ELISA has been demonstrated for: (a) quantification of r-α1-PI in various fermentation mixtures (E. coli and A. niger), (b) investigation of α1-PI enzymatically digested in the conditions of harsh fungal proteolysis, (c) evaluation of α1-PI thermally polymerized, (d) quantification of α1-PI in human serum, as well as (e) for comparative quantification of α1-PI in commercially available products.
E. Karnaukhova, Division of Hematology, CBER, FDA, Bethesda, MD 20895
Human α1-PI derived from plasma is an US FDA licensed product recommended for intravenous treatment of patients with hereditary α1-PI deficiency in order to slow down the progression of emphysema. Retinoic acid (RA) is another FDA-approved (orphan) drug recognized for its activity against acute promyelocytic leukemia. In regards to lung diseases, RA plays a key role in activation of genes involved in lung development and alveolar regeneration.
This study is focused on obtaining complexes of α1-PI with RA, which may have a dual function and thus may possess an enhanced efficacy in regards to current treatment.
The results of our UV/Vis, circular dichroism and fluorescence study show the following: (1) RA does bind to α1-PI in a non-covalent fashion, (2) There are 2 hydrophobic sites in the α1-PI interior available for RA, (3) Due to a complex formation RA, which solubility in water is extremely low (0.2 µM), behaves as water-soluble with concentration range consistent with α1-PI content and stoichiometry of binding (up to 90 µM); (4) Stability of RA in complex with α1-PI is significantly increased.
To the best of our knowledge, this is the first report of a binding of lipophylic ligand to α1-PI. Our study reveals a new structural feature, which may have a significant impact on α1-PI research and development, including clinical application.
E. Karnaukhova, Division of Hematology, CBER, FDA, Bethesda, MD 20895
Human serum albumin (HSA) is an US FDA licensed drug recommended at high doses for various therapeutic indications, including surgery or trauma, cardiopulmonary bypass, hypovolemia, shock, hemodialysis, burns, and acute respiratory distress syndrome.
HSA is the most abundant protein in plasma. As a major carrier protein, HSA is capable of binding a great variety of metabolites and drugs. Binding of new chemical entities to plasma proteins is an important concern confronting the development of new therapeutics.
The scope of this work includes investigation of the interactions between the major plasma proteins and small drugs and metabolites. Retinoic acid and retinaldehyde have been selected as sensitive probes to study these interactions. Titration of HSA with RA over the range of ligand-to-protein ratio from 0.05 to 6 has been monitored by UV/Vis, circular dichroism and fluorescence. The results indicate that retinoic acid specifically binds HSA at least at 2 binding sites of different affinity. Non-covalent binding of retinaldehyde is not specific, but proceeds at higher extent (6 and above). Not only does this research allow characterization of a type of interactions between HSA and different retinoids, but it provides a valuable methodological approach for investigation of protein complexes with small exogenous ligands by exploring phenomena of protein-induced chirality and fluorescence quenching.
We suggest that this approach can be more extensively used for the investigation of the interactions between native plasma proteins, protein therapeutics, and small chemical drugs and/or native metabolites of plasma.
I. P. Leader, P. A. Jupp, P. Weatherhead, V. Huynh, D. Trew, C. H. Turner, T. Day, D. Wheatley, WYETH RESEARCH
Background - Finding an unexpected degradant in a developmental drug formulation is a critical occurrence that requires swift action to either identify or eliminate. Tocopherol succinate polyethylene glycol (TPGS) was used as a wetting surfactant in a developmental drug formulation to aid bioavailability of the active. Analysis of an aged product by HPLC found that at least one unexpected degradant was being formed.
Methods - A combination of reverse-phase HPLC, LC-MS and NMR were utilised to identify the unknown entity. Preparative HPLC was used to collect fractions of the entity; mass spectrometry and NMR were used to elucidate the structure of the entity. Once identified, an HPLC method was developed to quantify levels of both the precursor and degradant species. Further testing and literature searches as to the stability of TPGS showed that the degradation process was actually catalysed by the presence of the drug substance.
Results - The unknown degradant was conclusively identified as tocopherol succinate (TS). With the use of a reference material and a stability indicating method the levels of TPGS and TS were tracked over the stability programme. The most likely pathway of formation seems to be related to a carboxylate functionality present in the drug substance.
Conclusions - A combination of HPLC, MS and NMR have been used to identify and quantify an unknown degradant in a developmental drug formulation. The work also enabled an understanding of how to prevent the formation of the degradant via storage of the product at <5oC.
H. Li, P. J. Kijak, J. von Bredow, A. Chiesa, M. L. Smith, CVM, FDA, Laurel, MD
Background: The misuse of sulfadimethoxine (SDM) in cattle is considered to be one of the major causes of illegal drug residues in slaughtered animals. FDA's official tolerance of SDM in edible cattle tissues is 100 ppb.
Methods: We developed a quantitative method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assay sulfadimethoxine residue and its major metabolite, 4N-acetyl-sulfamethoxine (N-AcSDM), in bovine kidney, liver, plasma, and urine. It includes simple lab procedures to extract the target drug and metabolite residue from these matrices. An additional clean-up step is only necessary for kidney and liver samples. For these tissues, analysis can be performed on a very small sample size (e.g., about 100 mg kidney homogenate or cortex), which makes it possible to analyze laparoscopic samples from live animals.
Results: With appropriate choice of internal standard, the overall method is able to analyze SDM and N-AcSDM in a range of 4,000 to 10,000 fold with lower limit of quantitation (LLOQ) down to low ppb level. Validation of the method is being conducted.
Conclusions: This analytical method will provide quantitative data of SDM and N-AcSDM residue in bovine tissues with high sensitivity, specificity, and wide dynamic range. It will contribute to CVM's tissue-fluid drug residue correlation program. In addition, it will be used to evaluate the FAST test kit (Fast Antibiotic Screen Test) for detecting SDM in bovine kidney.B. S. Liao, T. T. Cain, PRL/SW, FDA, Irvine, CA
Background:
A method has been developed to separate and quantify putrescine, cadaverine, and histamine simultaneously for scombroid fish samples and fishery products by cation ion chromatography. This method is an alternative to the two official methods currently used to analyze for these three biogenic amines. Putrescine and cadaverine are analyzed by Gas Chromatography (AOAC Method 996.07) and histamine is analyzed by Fluorescence Spectroscopy (AOAC Method 977.13). This method eliminates the need for chemical derivatization and the sample preparation is quick and simple.
Methods:
Aqueous 5mM sulfuric acid mobile phase is used with a cation exchange column under isocratic condition at a flow rate 0.8 mL/min and a run time of 30 minutes. Fish samples are homogenized with 75% methanol/water, incubated at 60ºC for 15 minutes and filtered before injection.
Results:
The three biogenic amines are baseline separated regardless of scombroid fish matrix in the order of putrescine, cadaverine, and histamine, without the interferences traditionally seen for tuna with cation ion chromatography. Five different scombroid fish samples, tuna, mackerel, anchovy, mahi mahi and sardines, were spiked in duplicate with standard solutions of the three amines with recoveries within 80% - 120%. The correlation coefficients (r2) of the standard calibration curves for putrescine, cadaverine and histamine are all higher than 0.999. The lowest standard concentrations which were spiked into tuna fish matrix are 0.055 ppm for putrescine, 0.05 ppm for cadaverine, and 1.0 ppm for histamine with RSDs of 1.5%, 5.4%, and 4.7%, respectively.
Conclusion:
This method is simple, direct, effective, and is also environmentally friendly because no chemical derivatization or time-consuming sample preparation is necessary.
M. Lopez1 , M. Feldlaufer2 , P. Chu1 , 1CVM, FDA, 2ARS, USDA
Background: Many countries, including the United States, have banned the use of nitrofurans in food-producing animals due to the carcinogenicity and mutagenicity of these drugs and their metabolites. Furazolidone, furaltadone, nitrofurantoin, and nitrofurazone are among the most commonly misused nitrofurans. We present a quantitative and confirmatory LC/MS/MS method for the determination of these four nitrofurans as their metabolites in honey at a target level of 1.0 ppb.
Method: The assay begins with a solid-phase-extraction cleanup of the honey sample, followed by overnight acid hydrolysis and derivatization of the nitrofuran metabolites with 2‑nitrobenzaldehyde. After pH adjustment and liquid-liquid extraction, the extract is assayed by LC/MS/MS using electrospray ionization in the positive mode.
Results: The method has been validated using incurred honey and fortified honey at 0.5, 1.0, and 2.0 ppb. For all four metabolites, the reproducibility errors are < 8% and the accuracies > 95%. The details of the quantitative and confirmatory performance of the method for each of the individual nitrofuran metabolites are presented.
Conclusion: The method complies with CVM performance criteria for the analysis of veterinary drug residues in animal products and can, therefore, be used to monitor the presence of nitrofuran metabolites in honey which may result from the misuse of nitrofurans in bee colonies.
J. C. May1 , N. M. Etz1 , H. Wang1 , L. Rey2 , 1CBER, FDA, Rockville, MD, 2Conseiller Scientifique, Lausanne, Switzerland
Residual moisture specifications which are set by the US Food and Drug Administration must be met to ensure potency and stability of freeze-dried biological product throughout the product's licensed shelf life. Thermogravimetry, thermogravimetry/mass spectrometry, and Karl Fischer methodology and vapor pressure moisture measurements are used to provide moisture information for freeze-dried biological products. Residual moisture measurements are compared for a Pertussis Vaccine and other freeze-dried biological products. Residual moisture data for the freeze-dried cake and vapor pressure moisture determinations in the space above the freeze-dried cake in the final container are discussed in regard to stability. Residual moisture stability data are presented and results for biological products in glass vials with elastomeric container closures are studied and compared to freeze-dried products in flame-sealed glass ampoules. Near-infrared spectroscopy measurements in the OH combination bands region (near 1920 nm) for Pertussis Vaccine incubated at 22, 37 and 50o C indicate moisture migration within the flame-sealed glass ampoule.
S. E. McMullen, V. A. Vega, F. J. Schenck, SRL, Atlanta, GA
Aquaculture is rapidly becoming a prominent source of seafood especially for finfish such as salmon, catfish, basa, and tilapia. Currently, there are only 5 veterinary drugs approved in the USA for use in aquaculture. Unfortunately, these drugs do not address all the pests/diseases encountered in aquaculture. Unapproved drug use is widespread especially in third world countries where misinformation on US tolerances is common. Recently, many import fish samples were found to have residues of unapproved fluoroquinolones, specifically enrofloxacin and ciprofloxacin. This poster presents an improved extraction method that facilitates rapid determination (Liquid Chromatography/Fluorescence Detection) and confirmation (Liquid Chromatography/Tandem Mass Spectrometry) of four fluoroquinolone residues including enrofloxacin and ciprofloxacin. This methodology presents several improvements over existing methodology including simplified extraction, less solvent consumption, and a reduction in analysis time.J. C. Moore1 , J. J. Yin2 , L. Yu1 , 1Dept. of Nutrition and Food Science, University of Maryland, College Park, MD, 2FDA, CFSAN, College Park, MD
Growing evidence suggests the role of antioxidants in preventing chronic diseases by scavenging reactive oxygen species such as the highly reactive hydroxyl radical. This has created a need for simple, reliable, high-throughput, in-vitro methods to investigate the free radical scavenging capacities of dietary antioxidants. The objective of this study was to develop an optimized hydroxyl radical scavenging capacity (HOSC) assay to meet the needs of antioxidant research and development. A novel method was developed for micro-plate reader high-throughput analysis using fluorescein as a fluorescent probe and the Fe(III)/H2O2 system to create a steady flux of pure hydroxyl radicals at pH 7.4 verified by electron spin resonance (ESR) spin trapping studies. The method was tested for solvent system compatibility and evaluated for performance characteristics and found to have compatibility with aqueous and 50% acetone solvents, a linear range with trolox of 20-100 µM with a linear correlation of R2=0.99, sensitivity (slope) of 0.4, and a 4.06% relative standard deviation for reproducibility. The method also correlated well with the popular oxygen radical absorbance capacity (ORAC) assay (r = 0.954, P = 0.05). Three 50% acetone extracts from soft wheat grain, hard wheat bran, and cinnamon were tested with the method and found to have values of 38.78, 74.91, and 3009.85 trolox equivalents per gram respectively. This method may provide researchers in the food and human health fields a simple protocol to rapidly evaluate free radical scavenging capacity of pure antioxidative compounds and natural extracts in vitro against the very reactive hydroxyl radical.
H. F. Yancy, D. E. Farrell, J. D. Washington, C. M. Deaver, R. A. Frobish, CVM, FDA, Laurel, MD
The performance characteristics of two lateral-flow test kits, Neogen's Reveal for Ruminant in Feed™ test and Strategic Diagnostic's Feedchek™ test, designed to detect ruminant or terrestrial animal proteins in feeds, respectively and two ELISA test kits, ELISA Technologies MELISA-Tek™ test and Tepnel's Biokit for (Cooked) Species Identification™ test, designed to detect bovine proteins in animal feed, were evaluated. All kits were evaluated using acceptance criteria developed for selectivity, sensitivity, ruggedness, and specificity. A minimum detection level of 0.1% bovine meat and bone meal (BMBM) was required, consistent with the sensitivity of the analytical methods used by FDA The Reveal test met its' label claim of a 1% sensitivity rate but failed to detect feed fortified with 0.5% to 0.1% BMBM. The Feedchek™ test passed the sensitivity assessment, but failed the selectivity assessment. The sensitivity of the Reveal test was affected by the variations in trace mineral concentrations typically present in feed. The Feedchek™ test was not affected by trace mineral levels. The MELISA-Tek™ test failed the selectivity assessment using the acceptance criteria established by the manufacturer, but passed when a set point criteria was applied to those results. It also failed the sensitivity assessment, detecting samples fortified at the 2% level only. The Biokit™ test failed to detect a single BMBM fortified sample. These results indicate that none of the tests are adequate for routine regulatory use for detection of feeds with 0.1% ruminant proteins and/or identification of samples free of ruminant proteins.C. B. Nochetto, C. S. Cheely, C. Gieseker, R. Reimschuessel, M. C. Carson, CVM, FDA
Background: MS-222 (tricaine methanesulfonate) is an approved anesthetic drug for aquaculture in the U.S. It was approved for use as a handling aid with a 3 week withdrawal time. The drug is rapidly metabolized and excreted, therefore it was approved without a regulatory method. However there are suspicions that the drug is used to sedate fish during transport to slaughter. A regulatory method will enable monitoring for unsafe residues of this drug resulting from extra label use. We present a quantitative analysis method using liquid chromatography (LC) at a target level of 100 ppb for three different species, salmon, tilapia and catfish.
Method: The assay begins with an extraction with acetonitrile, followed by filtration and mixed mode cation exchange solid-phase extraction cleanup of the sample. The extracts are analyzed by reversed-phase LC with UV detection at 320 nm.
Results: The method was validated using incurred fish fillets, control fish fillets, and fish fillets fortified at half the target, target and twice the target level. For all species, Accuracy is > 80% and Precision is < 10%.
Conclusion: The method complies with CVM performance criteria for the analysis of veterinary drug residues and can also be a useful starting point for future studies on the metabolism and depletion of MS-222.
G. O. Noonan, T. H. Begley, G. W. Diachenko, CFSAN, FDA, College Park, MD
Monofluoroacetic acid (MFA) , also referred to as compound 1080 is a rodenticide commonly used in the US and New Zealand and a naturally occurring toxic component of poisonous plants found in Australia, South Africa, and India. The availability, stability and proximity of MFA to agricultural products could potentially lead to an accidental or intentional contamination of food. Therefore a screening and confirmatory method for the detection of MFA in foods was needed. The screening method developed, utilized rapid sample cleanup and liquid chromatography mass spectrometry for detection. This method showed a high degree of specificity, with limits of detection (LOD) of 4 ppb and limits of quantitation (LOQ) of 13 ppb. Spike recoveries were matrix dependent and varied from 81 to 110% with comparable recoveries at low (2 ppm) and high (20 ppm) spiking levels. Reproducibility tests at the low spiking levels, had RSD's of less than 5% for all matrices analyzed. None of the unfortified samples showed detectable levels of MFA.
The qualitative confirmatory method developed is conceptually different from the screening method, ensuring that both methods would not be subject to the same interferences. The method uses the formation of the hydrazide of MFA through derivatization with 2-nitrophenylhydrazine. This derivatization is well established for the detection of carboxylic acids, but this is the first application to the detection of MFA. The derivatization yield was matrix dependent, however the LOD (<1 ppb) was sufficiently sensitive to confirm the presence of MFA in all spiked matrices. Reproducibility tests at the low spiking levels, had RSD's of approximately 6% for all matrices analyzed.
M. Hamed, L. Mello, M. Osman, Bioanalytical R&D, Wyeth Research, Collegeville, PA
Background: HKI-272 is an inhibitor of the Her2/neu receptor tyrosine kinase, thus blocking cell proliferation, and is being developed as a potent, irreversible and selective Her2/neu inhibitor for the treatment of cancer. An LC/MS/MS method for quantification of HKI-272 in human plasma has been evaluated for specificity, precision, accuracy and stability.
Methods: The method utilized 250 mL of human plasma. Following addition of internal standard (d6-HKI-272), samples were processed by protein precipitation with acetonitrile. Following centrifugation, the liquid supernatant was separated and evaporated to dryness. The residue was reconstituted in 0.05 M ammonium acetate buffer: methanol: acetonitrile (3:2:5), transferred to autosampler vials and injected on the LC column. Samples were separated on reversed phase C18 column.
Results: Endogenous components in control human plasma did not interfere with measurement of HKI-272 or the internal standard (D6-HKI-272). The assay was linear over the range of 3.0 ng/mL to 250 ng/mL. The intra-day and inter-day precision (CV) and inaccuracy (bias) for HKI-272 were within 15%. HKI-272 was found to be stable in human plasma for 4 hours at room temperature and after three freeze/thaw cycles. The processed samples were found to be stable at least 32 hours at 4oC.
Conclusions: An LC/MS/MS method for the determination of HKI-272 in human plasma has been validated. Based on a 250 mL plasma sample, the linear range of the method was 3.0 to 250 ng/mL. The method was utilized in the analysis of HKI-272 in human plasma generated from clinical studies in cancer patients.
L. Mello, S. Mathews, M. Khan, M. Osman, Bioanalytical R&D, Wyeth Research, Collegeville, PA
Background: PSI-697 is an inhibitor of P-selectin, a platelet and endothelial adhesion molecule that is critical for platelet and leukocyte recruitment to sites of vascular inflammation and injury. Inhibition of P-selectin offers the advantage of blocking a key initial step in the cell adhesion cascade and thereby preventing subsequent downstream activation and signaling events involved in thrombosis and inflammatory disorders. An LC/MS/MS method for quantification of PSI-697 in human plasma has been evaluated for specificity, precision, accuracy and stability.
Methods: The method utilized 200 mL of human plasma. Following addition of internal standard (d6-PSI-697), samples were processed by protein precipitation with acetonitrile. Following centrifugation, the liquid supernatant was separated and evaporated to dryness. The residue was reconstituted in acetonitrile: HPLC water: triethylamine (50:50:0.1), transferred to autosampler vials and injected on the LC column. Samples were separated on reversed phase C8 column.
Results: Endogenous components in control human plasma did not interfere with measurement of PSI-697 or the internal standard. The assay was linear over the range of 4.0 ng/mL to 1000 ng/mL. The intra-day and inter-day precision (CV) and inaccuracy (bias) for PSI-697 were within 15%. PSI-697 was found to be stable in human plasma for 24 hours at room temperature and after three freeze/thaw cycles. The processed samples were found to be stable at least 96 hours at 4oC.
Conclusions: An LC/MS/MS method for the determination of PSI-697 in human plasma has been validated. The method was utilized in the analysis of PSI-697 in human plasma generated from clinical studies in healthy humans.
S. H. Atwell, P. A. White, S. Z. Wahab, United States Pharmacopeia
Background: Two formulae for glycopyrrolate oral suspensions were approved by the USP Compounding Pharmacy Expert Committee. A stability-indicating assay and appropriate beyond-use-date for both formulae were needed to develop compounded monographs. This study covers the development and validation of an HPLC stability-indicating method for sugar and sugar-free Glycopyrrolate Oral Suspension formulae.
Methods: Sugar and sugar-free suspensions were prepared from glycopyrrolate and Vehicles specified in NF. An HPLC method with UV detection at 222 nm was developed to quantitate the content of glycopyrrolate. An L1 column (Phenomenex Gemini C18, 25 cm x 4.6 mm, 5 µm) with 1.0 mL/min flow rate and 20-µL injection was used. The mobile phase consisted of a mixture of 0.1 M monobasic potassium phosphate, 0.1 M dibasic potassium phosphate, acetonitrile, and methanol (26:28:24:16,v/v/v/v).
Results: Glycopyrrolate peak area responses were linear from 0.019 to 0.0 57 mg/mL (r > 0.999). Recoveries of glycopyrrolate from accuracy preparations were not significantly different from theory. Recoveries ranged between 98.4% to 102.8%. The relative standard deviations for intermediate precision were less than 1.2%. Uniformity of glycopyrrolate in samples from both suspensions was less than or equal to 1.0% RSD for triplicate preparations from different locations of the suspensions.
Conclusion: The HPLC procedure was stability-indicating, specific, precise, accurate, linear, and suitable to monitor the stability of gylcopyrrolate in sugar and sugar-free oral suspensions.
A. Ashley, R. Maheswaran, J. L. Belsky, S. Z. Wahab, United States Pharmacopeia
Background: A stability-indicating assay and appropriate beyond-use-dates were needed for two formulas for compounded trimethoprim oral suspensions. This study covers the development and validation of an HPLC stability-indicating method for regular and sugar-free Trimethoprim Oral Suspension formulae.
Method: Trimethoprim Oral Suspensions of two different formulae were prepared from trimethoprim drug substance and Vehicles specified in NF. An HPLC method with UV detection at 271 nm was developed to quantitate the content of trimethoprim in the compounded preparations. A Phenomenex Luna C18(2), 4.6-mm x 100-mm, 5-mm column with 1.0 mL/min flow rate and a 25 mL injection were used. The mobile phase consisted of a mixture of 0.1% triethylamine in water, pH 5.7 and acetonitrile, (850:150, v/v).
Results: Trimethoprim peak area responses were linear from 70% to 130% of the nominal sample concentration. Recoveries of trimethoprim from accuracy preparations were not significantly different from theory. Uniformity of trimethoprim in samples from both suspensions was < 3.0% RSD for triplicate preparations from different locations of the suspensions.
Conclusion: The HPLC method was stability-indicating, specific, precise, accurate and linear and is suitable to monitor the stability of Trimethoprim Oral Suspension, Formula 1 (regular) and Formula 2 (sugar-free).
R. Maheswaran, P. A. White, S. Z. Wahab, United States Pharmacopeia
Background: Formulas for Flecainide Oral Suspensions were approved by the USP Compounding Pharmacy Expert Committee. Stability-indicating assays and appropriate beyond-use-dates were needed to develop these compounded monographs. This study covers the development and validation of an HPLC stability-indicating method for regular and sugar-free Flecainide Oral Suspension formulae.
Method: Oral suspensions were prepared from Flecainide Acetate tablets and Vehicles specified in NF. An HPLC method with UV detection at 298 nm was developed to quantitate the content of flecainide acetate in the compounded preparations. A Zorbax C8, 4.6-mm x 150-mm, 5-mm column with 1.0 mL/min flow rate and a 10-mL injection were used. The mobile phase is a mixture of water, acetonitrile, acetic acid and 1 N tetrabutylammonium hydroxide, (710:290:10:5, v/v/v/v) with an apparent pH of 5.8. The method was validated for both formulae (10.0 mg/mL strength).
Results: Peak area responses were linear from 70% to 140% of the nominal assay concentration of 1.0 mg/mL. Recoveries of flecainide acetate from accuracy preparations were not significantly different from theory. Uniformity of flecainide acetate in samples from both suspensions was < 3.0% RSD for triplicate preparations from different locations in the suspensions.
Conclusion: The HPLC method was proven to be stability-indicating, specific, precise, accurate and linear and suitable for its intended purpose.
E. Biba, L. R. Strauch, S. Z. Wahab, P. A. White, United States Pharmacopeia
Background: Ginger (Zingiber Officinale Roscoe), a well known dietary supplement, has been used in Asian, Indian, and Arabic herbal traditional medicine. Today it is readily available in many avdifferent forms: extracts, tinctures, capsules and oils. Powdered ginger root is the main ingredient of Ginger Capsules
Methods: Three commercial brands of Ginger Capsules were assayed by HPLC to determine the Ginger Capsules Content of gingerols, gingerdiones, and shogaols USP 29, 2333. Dissolution profiles were evaluated per Ginger Capsules Dissolution using the major gingerol component, 6-gingerol, as a marker.
Results: The total amount of actives, individual weight percentages, and the chromatographic profile of the alcohol soluble extractives were brand dependent. Each brand met the USP requirement of not less than 0.8% for the content of gingerols and gingerdiones. Only one brand had a label value for the content of gingerols. The dissolution profiles differed by brand. One brand which contained excipients failed the USP dissolution test.
Conclusions: The lack of labeling raises an issue for use because of reported dose-dependent side effects. The different dissolution profiles of the tested Ginger Capsules represent another concern for standardization.
G. Deng1 , A. Ashley1 , W. Brown1 , J. Eaton1 , M. Liddell1 , L. Kikwai-Mutua1 , R. Manning1 , J. Munoz1 , B. Ning1 , P. Nithyanandan1 , H. Rowe1 , S. Tan1 , S. Z. Wahab1 , W. Hauck2 , 1United States Pharmacopeia, Rockville, MD, 2Thomas Jefferson University, Philadelphia, PA
Background: USP Prednisone Tablets Lot P are disintegrating type tablets. Each tablet weights about 220 mg with a prednisone content of 10 mg. Dissolution process of prednisone tablets is sensitive to experimental parameters. This study developed an experimental protocol to generate data for statistical variance analysis to evaluate and identify the key dissolution variables.
Methods: Prednisone tablets were tested on five dissolution testers (α, β, γ, δ, ε) by at least four analysts (A, B, C, D, E, F) on each tester. Six to eight tablets were tested in each run. Six dissolution runs were performed by each analyst on each tester. Prednisone release percentages on USP apparatus 2 (rotating paddles) were determined at 30 minutes in deaerated water at 37°C.
Results: A total of 132 dissolution runs were performed with near 1000 prednisone tablets. The overall average prednisone release percentage was 48.3% with a range of 37.8% to 76.9%. Significant variances in prednisone release between testers were observed. On Tester α, the average prednisone release percentages were 53.7% to 61.2%, and the average RSD were 13.8% to 17.5%. On Tester γ, the corresponding ranges were 41.4% to 46.5%, with 3.8% to 4.9% RSD. Further studies by switching vessels on α and γ indicated that vessels were one of the major sources of the varibility.
Conclusions: Prednisone Tablets Lot P showed a broad range of prednisone release. Dissolution tester particularly dissolution vessels were one of the major source of dissolution variance. Variances from tablet, analyst, and analytical procedure were low.
G. Deng, W. Brown, B. Chow, J. Eaton, M. Glasgow, M. Goede, L. Kikwai-Mutua, R. Manning, L. Wang, S. Z. Wahab, United States Pharmacopeia
Background: The USP Prednisone Tablets Lot P were recently developed for USP dissolution Apparatus Suitability Test. The tablet has an average weight of 220 mg with a prednisone content of 10 mg. This work examined the key chemical and physical characteristics of the tablets that can contibute to dissolution variability of the tablets.
Methods: USP Prednisone Tablets Lot P have been examined for their appearance, assay, content uniformities, disintegration, weight variation, hardness and friability.
Results: USP Prednisone Tablets Lot P were round and white in color. There were no observable defects on any of the tablets tested. The average weight was 222.0 mg per tablet (n = 108, SD = ± 2.4). The average hardness was 64 Newtons. The weight loss was 0.04% in the friability test. The average disintegration time was 18 seconds. Five portions of a powder composite of twenty tablets were assayed to determine the prednisone content. The average prednisone content was 96.2% (n = 5, SD = ± 0.8) of the label claim. Thirty tablets were assayed individually to determine the content uniformity. The average content of prednisone was 9.7 mg per tablet (n = 30, SD = ± 0.2). Ten tablets were assayed individually to determine the content uniformity of the disintegrant. The average weight of disintegrant per tablet was 4.5 mg (n = 10, SD = ± 0.1).
Conclusions: Chemical and physical properties of the tablets were well maintained in the manufacture process. Low variations of less than 3% RSD were observed.
J. Eaton, G. Deng, W. Brown, R. Manning, S. Z. Wahab, United States Pharmacopeia
Background: USP Prednisone Tablets Lot P are disintegrating type tablets. This study examined the usefulness of prednisone tablets for performance testing using flow-through cell apparatus.
Methods: Both open system and closed system configurations were used at 3 different flow rates (8, 12, and 16 mL/min) using 22.6-mm cells. Sampling time points were 1, 2, 3, 4, 5, 10, 15, 20, 30 and 45 min. 1-mL samples were collected and analyzed by HPLC.
Results: For open system runs, data was reported as percent of label claim dissolved per minute. For each of the 3 flow rates, the highest percent dissolved per minute value occurred at either 1 minute or 2 minutes and decreased at each subsequent time point. At 30 minutes, the value was less than 1 percent dissolved per minute for each flow rate. An approximation of total percent dissolved yielded values of about 85% dissolved after 45 minutes at 8 mL/min and about 100% dissolved after 45 minutes at 12 and 16 mL/min. For closed system runs, data was reported as cumulative percent dissolved at each time point. At a flow rate of 8 mL/min, the cumulative percent dissolved was 52.9% at 15 minutes. At flow rates of 12 and 16 mL/min, the cumulative percent dissolved was 50.5% and 58.8%, respectively, at 10 minutes.
Conclusions: Prednisone tablets disintegrated very quickly, and the onset of dissolution appears to be immediate. It appears that the very beginning of the test could be critical in obtaining reproducible percentage released values.
J. L. Perry, M. P. Yurawecz, CFSAN, FDA, College Park, MD
Background: Total carbohydrate in foods is currently calculated by difference. The calculation involves subtracting the sum of the crude protein, total fat, moisture, and ash from the total weight of the food. Calculation of total carbohydrate by difference creates problems because of inclusion of non-carbohydrate compounds and a dependence on accurate measurements of other food components. Such problems demonstrate the need for more direct methods of analysis.
Method: Foods with various carbohydrate claims were analyzed to determine the level of sugars and /or sugar alcohols in each. The products were selected to test an improved sample cleanup procedure. The matrices varied from different types of bread and cereals to high fat products such as chocolate cookies, ranch and olive oil salad dressings. All samples were composited and extracted in eighty percent (80 %) methanol. Aliquots were withdrawn, dried, and reconstituted with hydroxylamine HCl in pyridine containing phenyl-B-D-glucoside as the internal standard. The oximes were converted to silyl derivatives with hexamethyldisilazane(HMDS) and trifluroacetic acid(TFA) treatment. The derivatives were then analyzed by gas chromatography (GC) using flame ionization detection.
Results: The analyzed concentrations of sugars and / or sugar alcohols agreed closely with amounts of these components declared on the products' labels. Spike recoveries ranged from 92-100 %. The limit of quantification was 10 ug/g.
Conclusions: The analytical procedures worked well for the foods analyzed. The GC runtime of 57 minutes is somewhat lengthy. Work is in progress to reduce overall analysis time.
B. R. Petigara, A. L. Scher, CFSAN, FDA, College Park, MD
Background: A reversed-phase high performance liquid chromatography (RP-HPLC) method was developed to determine parts-per-million levels of Sudan I, 1-(phenylazo)-2-naphthalenol, the unsulfonated subsidiary color in FD&C Yellow No. 6. The color additive used in food, drugs, and cosmetics is subject to FDA batch certification to ensure that each lot meets the published identity, specifications, and other requirements, including a limit of <10 ppm of Sudan I (21CFR 74.706).
Method: FD&C Yellow No. 6 is dissolved in water and methanol, and the filtered solution is directly analyzed by RP-HPLC using a 150 x 2.1 mm i.d. XTerra RP-18 column with gradient elution. Sudan I is identified by retention time and diode-array spectrum and is quantitated from the peak area at 485 nm. For simplicity, calibrations are performed in the absence of the dye matrix.
Results: Linear calibration curves (R = 0.9999) were obtained in both the presence and the absence of the dye. The limit of determination for Sudan I was 0.4 ppm, and the confidence interval was 10.1+ 0.1 ppm (99% confidence level) at the specification level for Sudan I. FD&C Yellow No. 6 batches from 17 different manufacturers were found to contain Sudan I at undetected levels (16 samples), 0.5-9.7 ppm (11 samples, including the pharmacology batch) and >10 ppm (2 samples).
Conclusions: This RP-HPLC method to determine Sudan I in FD&C Yellow No. 6 by direct injection is simple, fast, and reproducible. The use of a narrow-bore column reduces solvent waste. The results agree with those obtained using a more complex method that requires a preliminary extraction.
B. R. Petigara, A. L. Scher, CFSAN, FDA, College Park, MD
Background: A reversed-phase high performance liquid chromatography (RP-HPLC) method was developed to determine part-per-million to percent levels of Sudan I, 1-(phenylazo)-2-naphthalenol, the unsulfonated subsidiary color in D&C Orange No. 4.The color additive, used in externally applied drugs and cosmetics, is subject to FDA batch certification to ensure that each lot meets the published identity, specifications, and other requirements, including a limit of <3% of subsidiary colors (21CFR 74.1304).
Method: D&C Orange No. 4 is dissolved in water and methanol, and the filtered solution is directly analyzed by RP-HPLC using a 150 x 2.1 mm i.d. XTerra RP-18 column with gradient elution. Sudan I is identified by its retention time and diode-array spectrum and is quantitated using the peak area at 485 nm. For simplicity, calibrations are performed in the absence of the dye matrix.
Results: Linear calibration curves (R = 0.9998-0.9999) were obtained in both the presence and the absence of the dye matrix.The limit of determination for Sudan I was 0.0001%, and the confidence interval near the highest level found was 0.2 + 0.002% (99% confidence level).D&C Orange No. 4 batches from 8 different manufacturers were found to contain Sudan I at undetected levels (8 samples), 0.0005 to <0.005% (3 samples, including a pharmacology batch), 0.005 to <0.05% (9 samples), and 0.18% (1 sample).
Conclusions: This RP-HPLC method to determine Sudan I in D&C Orange No. 4 by direct injection is simple, fast, and reproducible. The use of a narrow-bore column reduces solvent waste.
L. Qi1 , Y. Ito2 , 1ONDQA, CDER, FDA, 2Center for Biochemistry and Biophysics, NHLBI, NIH
Background: Centrifugal Precipitation Chromatography has been introduced to replace the traditional differential ammonium sulfate protein precipitation. Enzymes, such as ketosteroid isomerase and hyaluronidase, have been purified using this method. The purpose of this pilot study is to demonstrate that immuno-affinity centrifugal precipitation chromatography is capable of isolating an antigen by exploiting antigen-antibody binding.
Methods and Results: The separation is initiated by filling the sample channel with antibody (rabbit anti-human serum albumin IgG) solution, followed by elution with 40% saturated ammonium sulfate (40% AS) to precipitate the antibody under centrifugal force field. Antigen (Human Serum Albumin) is introduced into the sample channel at a flow rate of 0.06 mL/min. After an excess antigen and other proteins are eluted out, a releasing agent (0.5 M Glycine, pH 10 in 40% AS) is introduced into the sample channel to harvest the isolated target antigen leaving the antibody inside the channel. The human serum albumin was isolated and identified by SDS-PAGE.
Conclusion: The present method does not require immobilizing the antibody onto a matrix, which is used by the conventional immuno-affinity chromatography. This method ensures full recovery of the antigen and antibody.L. Caputo1 , N. Barnoy1 , J. Kim1 , J. Park1 , K. Senthil1 , P. Stahl1 , P. Delmonte2 , 1U.Md., 2CFSAN, FDA, College Park, Md.
There has been a significant proliferation of dietary supplements in the marketplace in the last 10 years. This has occurred in part because ofincreased interest in alternative medicines and beliefs that use of supplements may help reduce health care costs. Ginseng is a popular supplement which is used as a general health tonic; it is believed to increase energy.Preparations of the dried root are the most common commercially available form. The primary active components are thought to be ginsenosides.The mechanisms of action of these and other componentsarenot completely understood.
Standards for 6 of the ginsenosidesare commercially available, as are analytical methods for their determination. We analysed the content of 6 ginsenosides in 10 ginseng products.Samples of each product were extracted using accelerated solvent extraction and analyzed by HPLC.Extraction conditions were optimized considering different extraction solvent compositions, temperature and pressure.HPLC-PDA was used for the analysis of extracts because of its applicability to routine analysis.
Data from the HPLC analysis will allow the calculation of concentrations of specific ginsenosides.A cost comparison analysis of the 10 products will be conducted to determine whether a correlation exists between costand ginsenoside content (e.g., do higher priced supplements provide more ginsenosides than lower priced products?).Comparisons will utilize information provided on the product labels.Label information will also be used with the analytical data to calculate the amounts of ginsenosides that might be ingested if a consumer followed label directions for use.
J. C. Reepmeyer, J. T. Woodruff, Division of Pharmaceutical Analysis, CDER, FDA, St. Louis, MO
Background: An herbal dietary supplement, marketed as a natural product for the enhancement of sexual function, was purchased covertly over the internet.Preliminary analysis suggested that the product contained a compound related to synthetic phosphodiesterase 5 (PDE-5) inhibitors.
Methods:Standards of the three FDA-approved erectile dysfunction drugs (sildenafil, vardenafil and tadalafil) and an extract of the herbal product were analyzed by liquid chromatography on a reversed-phase C18 stationary phase using a photodiode array detector and a single quadrapole mass spectrometer in tandem and by MSn ion trap mass spectrometry. Sildenafil, vardenafil, and the herbal component were subjected to hydrolysis in order to cleave the sulfonamide bond, splitting the molecule into two pieces, which were analyzed by LC-MS and GC-MS.Results: The unknown compound generated a pseudomolecular ion via electrospray positive ion mode MS at 460 Da.Its UV spectrum was practically superimposable with that of vardenafil.Sildenafil, vardenafil and the unknown each generate a mass spectral product ion with a common mass accompanied by loss of dissimilar neutral fragments. The structure of the compound was tentatively identified as a designer drug of vardenafil in which the N-ethylpiperazine ring had been replaced by a piperidine ring.This structure was unambiguously confirmed by analysis of the hydrolysis products of the unknown ("piperidenafil"), sildenafil and vardenafil.
Conclusion: A component of an herbal product, marketed as a dietary supplement to enhance sexual function, was found to contain a vardenafil designer drug. The dosage level was estimated at 41 mg per capsule.
A. Grinev1 , S. Daniel1 , M. Laassri2 , K. Chumakov2 , V. E. Chizhikov2 , I. K. Hewlett1 , M. Rios1 , 1LMV/DETTD/OBRR/CBER/FDA, Rockville, MD, 2OVRR/CBER/FDA, Rockville, MD
BACKGROUND: Since the first outbreak of West Nile Virus (WNV) in the US in 1999, and in the six subsequent years. To date 7 subsequent epidemics have occurred causing more than 19,000 known human cases, and at least 750 human deaths. WNV is primarily transmitted by mosquito bites, but in 2002 additional modes of transmission were identified, including human-to-human by blood transfusion, breast-feeding, transplacental and by organ transplants. WNV has been a great public health concern and as an attempt to prevent viral spread by blood transfusion, screening for WNV by NAT was implemented under FDA approved INDs in July 2003. The reoccurrence of epidemics could suggest viral adaptation through mutations in the viral genome. In addition, genetic mutations can potentially interfere with the performance of diagnostic and screening assays, viral pathogenesis and potential therapeutic approaches. Therefore, the investigation, identification and detection of variants that may appear in the course of WNV outbreaks are extremely important and can only be achieved by the genomic characterization of new WNV isolates in a timely fashion. METHODS: We have developed a microarray-based assay that can rapidly detect mutations in the WNV structural region (5'NCR, Core, preM, M and Env). Glass slides containing 263 covalently immobilized oligoprobes were produced. The oligoprobes were designed and synthesized based on the reference strain NY99-flamingo 382-99 (AF196835). This new microarray-based-assay was standardized using the reference isolate. WNV amplified products containing the T7 promoter were used to generate fluorescently labeled Cy3 WNV-RNA for hybridization with the microarray, as described in Chizhikov V., et al., J Clin Microbiol 2002; 40:2398-2407. Microchip images were taken using the fluorescent scanner ScanArray 5000 and analyzed using the ScanExpress software. RESULTS: Assay validation was performed with 18 previously sequenced WNV isolates obtained from the last four US epidemics 2002 - 2005. The microarray assay detected unambiguously all mutations previously identified in the structural region of each one of the isolates by traditional sequencing analysis. The assay was sensitive and specific for all isolates tested. CONCLUSION: We have developed a microarray-based assay capable of rapid and simultaneous detection of multiple mutations in a large genomic segment of WNV. The assay is far more suitable for the high throughput analysis needed in large epidemiological studies than other methods (e.g. RFLP or DNA sequencing). We are in the process of expanding the microarray-based assay to cover the entire WNV genome.
J.A. Roach, CFSAN, FDA, College Park, MD
Background
Usnic acid is unambiguously confirmed by tandem mass spectrometry (MS/MS) in tumbleweed shield lichen, Xanthoparmelia chlorochroa, shown to have caused a mass die-off of at least 328 elk within the Wyoming Red Rim wildlife management area in the spring of 2004. The lichen contains 2% usnic acid by liquid chromatography with UV quantification at 282 nm.
Methods
Grind lichen to pass 40 mesh screen. Extract between 20 and 400 mg lichen powder with 2 x 8 mL acetonitrile by vigorous shaking for 20 min. Spin filter to remove solids. Dilute extract to 100 mL with acetonitrile. Mix 100 µL extract portions with aliquots of standard usnic acid solution making total test portion volume 1 mL with acetonitrile. Quantify by method of standard additions by UV at 282 nm. Confirm usnic acid by comparison of MRM data for test portions with MRM data for usnic acid standard.
CAUTION: Usnic acid is a strong hepatotoxin associated with human liver failure.Acetonitrile is a contact and inhalation hazard.
Results
Quantifications by UV and electrospray ionization (ESI) MS/MS disagree.The respective chromatographic data show that usnic acid is the principal compound detected in the extracts by both techniques.The quantification disparity in the data decreases when the extracts are diluted into the linear operating range of the ESI-MS/MS. However, the MS/MS data do not provide a means to cull MS/MS data recorded outside the linear range of the instrument.In contrast, the recorded diode array UV spectra can be used to exclude saturated data points from the UV quantification data.
Conclusions
Quantification by mass spectrometry has its limitations. Quantification by UV with confirmation by mass spectrometry is a more reliable assay for usnic acid in lichen.
W. D. Rowe1 , M. R. Madson1 , J. N. Sofos1 , F. J. Schenck2 , V. A. Vega2 , L. H. Lagman3 , D. M. Altwein4 , 1FDA, Denver, CO, 2FDA, Atlanta, GA, 3FDA, Jefferson, AR, 4FDA, Bothell, WA
Background: FDA uses Laboratory Information Bulletin (LIB) #3461 to monitor for ivermectin (IVR), a potent antiparasitic agent, in raw milk.Lagman, et al. demonstrated in LIB #4155 a faster, more sensitive method for IVR and for three related compounds, doramectin (DOR), abamectin (ABA), and moxidectin (MOX). All four may be administered to cattle.
Method: A modified version of LIB #4155 was validated by a two-laboratory method trial with additional validation by the Denver lab. Method extracts the avermectins from milk with ammonia, ethanol, ethyl acetate and iso-octane. The ethyl acetate - iso-octane layer is evaporated to an oily residue.The oily residue is partitioned between hexane and acetonitrile and the acetonitrile is evaporated to near dryness. The residue is dissolved in acetonitrile and derivatized with trifluoroacetic anhydride in the presence of N-methylimidazole.The derivatized extracts are injected on an LC (C-18 column, fluorescence detector).
Results: Average recoveries are typically greater than 60% with CV's less than 20%.MDL calculates to less than 0.1 ppb. Incurred milk samples were positive for the expected analytes at the expected ranges, 0.5 to 4 ppb.Ruggedness was demonstrated by varying extraction volume (± 20%), extending drying times, and substituting glassware with plasticware.
Conclusion: This method, published as LIB #4347 and LIB #4348, is faster than LIB #3461, sensitive to 0.2 ppb and rugged for the determination of MOX, ABA, DOR and IVR residues in milk.
J. M. Betz1 , L. G. Saldanha1 , M. J. Smith2 , B. J. Cañas2 , Y. Tokiwa2 , L. C. Sander3 , K. E. Sharpless3 , S. A. Wise3 , 1Office of Dietary Supplements, NIH, Bethesda, MD, 2CFSAN, College Park, MD, 3National Institute of Standards and Technology , Gaithersburg, MD
Background: Validated analytical methods and reference materials to ensure the identity, purity, quality, and strength of constituents in dietary supplements are essential.Researchers need them to characterize materials used in safety and efficacy studies and ensure that these materials are of sufficient quality that the studies can be reproduced.Regulators and industry need them in dealing with regulatory, safety, labeling, quality control, and manufacturing issues.In FY02, the U.S. Congress directed ODS to accelerate an ongoing methods validation process.The Dietary Supplements Methods and Reference Materials Program was created and through this program the funding of initiatives at FDA and NIST.
Program Administration: The program is stakeholder driven, with participation by government agencies, non-governmental organizations, academia, and the private sector.The analytical methods validation program is conducted through an Interagency Agreement with FDA to provide funding to AOAC International.The Certified Reference Materials (CRM) development program is through an Interagency Agreement with NIST.
Results: About 38 methods are in various stages of validation at AOAC. Methods for ephedra, ginkgo flavones, beta-carotene, and glucosamine are now available. Suites of Standard Reference Materials (SRM) for ephedra are now available. Suites of SRM for beta-carotene, bilberries, bitter orange, black cohosh, blueberries, cranberries, ginkgo, green tea, oils containing omega-3 fatty acids, saw palmetto, St. John's wort, tocopherol, and multivitamin/multiminerals are in preparation.
Saldanha LG, Betz JM, and Coates PM. Development of the Analytical Methods and Reference Materials Program for Dietary Supplements at the National Institutes of Health.Journal of AOAC INTERNATIONAL, 2004 87:162-65.
R.D. Satzger, FCC, ORA, FDA, Cincinnati, OH
The mission of the Forensic Chemistry Center (FCC) is to develop and maintain the capability to respond immediately to incidents involving adulterated products and illegal or counterfeit products. The threat of terrorist events has placed an even higher level of importance on the ability to rapidly develop approaches that can be used to screen products for the presence of highly toxic chemical adulterants. Similar approaches have been used to identify differences between counterfeit/adulterated products. Scientists at the FCC have developed a series of analytical screening procedures to detect priority chemicals at levels that would pose a significant health threat to the consumer. These procedures have been applied to selected foods and pharmaceuticals.F. J. Schenck, S. E. McMullen, Southeast Regional Laboratory, FDA, Atlanta, GA
Chlorothalonil is a widely used pesticide, which accounts for a large number of violative samples in our laboratory each year.Unfortunately, there are difficulties involved in the analysis of fruit and vegetable samples for chlorothalonil residues.One of the most effective solid phase extraction (SPE) sorbents for the cleanup of fruit and vegetable sample extracts is primary secondary amine (PSA). Using PSA SPE columns, which are used in many multiresidue methods (MRMs), will result in the degradation of chlorothalonil residues.Consequently, chlorothalonil residues in the sample may be destroyed during sample preparation. There is an MRM that uses a buffered acidic extraction, which will stabilize chlorothalonil so that a PSA SPE cleanup can be used. However, this results in a diminished cleanup resulting in chromatographic interferences and the need for greater instrument maintenance. This study showed that simply using binary solvent mixtures containing toluene during the PSA SPE cleanup will stabilize the chlorothalonil.It also showed that the chlorothalonil residues in sample extracts were stable if the extracts were in toluene, but they were very unstable if they were in acetone or acetonitrile.K. J. Shefcheck, J. H. Callahan, S. M. Musser, CFSAN, FDA, College Park, MD
Peanut allergies are a serious public health issue.Peanut-adulterated foods can potentially be very harmful to sensitive individuals.Testing for the presence of peanut allergens in foods is most commonly performed with enzyme-linked immunosorbent assay (ELISA) based analytical methods, but false positives due to cross reactivity are a concern with ELISA.Mass spectrometry has the sensitivity and specificity to be used as a confirmatory method for positive ELISA tests. However, quantitation is difficult using a mass spectrometer since many variables, such as ionization efficiency, can affect the response of the species in question.Isotopically labeled internal standards can reduce this problem since the isotopic standard should have the same response as the sample.We use tryptic digestion products of the peanut allergen Ara h 1 as markers for peanut.Trypsin can also be used to label synthetic peptide standards, using 18O water to make isotopically labeled internal standards.We propose to use standard peptides labeled with 18O to effectively quantitate the peanut allergens in food with mass spectrometry.T. L. Williams1 , R. Singh2 , H. C. Harbottle3 , D. Andrezjewski1 , D. N. Heller3 , R. D. Walker3 , S. M. Musser1 , 1CFSAN, FDA, College Park, MD 20740, 2CVM, FDA, Rockville, MD 20855, 3CVM, FDA, Rockville, MD 20708
Background: Salmonella enterica serotypes cause approximately 1.4 million illnesses each year, with 95% of these illnesses contracted through contamination of food.Given the impact of diarrheal disease in the US, it is imperative to develop effective intervention strategies to ensure the safety of consumers against foodborne diseases. In this study we have developed a method for generating intact protein profiles from the LC/ESI/MS chromatogram of Salmonella Newport, in order to determine unique protein biomarkers indicative of food animal origin.
Materials and Methods: Thirty Salmonella serotype Newport isolates were chosen to represent a variety of animal sources of infection and a variety of PFGE (Pulsed-Field Gel Electrophoresis) subtypes.These isolates were analyzed in a blinded fashion.The method and supporting software translates the chromatographic and multiply-charged protein information into one comprehensive mass versus intensity spectrum.After the data is converted, a number of software tools can be used to compare bacterial protein profiles and monitor changes in protein expression that may be linked to specific traits such as thermal tolerance and pathogenicity.When significant changes in protein expression profiles are identified, the proteins of interest can be purified, sequenced, and examined for alterations in primary sequence composition and/or posttranslational modifications.Differences in protein sequence translates to a difference in nucleotide sequence that can be used to make PCR primers for rapid diagnostic detection.
Results: Two unique proteins were found at a molecular weight of 11236 Da and 11222 Da which corresponded to avian sources of infection (turkey and chicken) and quadruped sources of infection (cattle and swine), respectively.Upon further comparison with PFGE, these unique