Performance of a proposed determinative method for p-TSA in rainbow trout fillet tissue and bridging the proposed method with a method for total chloramine-T residues in rainbow trout fillet tissue

Chloramine-T is an effective drug for controlling fish mortality caused by bacterial gill disease. As part of the data required for approval of chloramine-T use in aquaculture, depletion of the chloramine-T marker residue (para-toluenesulfonamide; p-TSA) from edible fillet tissue of fish must be characterized. Declaration of p-TSA as the marker residue for chloramine-T in rainbow trout was based on total residue depletion studies using a method that used time consuming and cumbersome techniques. A simple and robust method recently developed is being proposed as a determinative method for p-TSA in fish fillet tissue. The proposed determinative method was evaluated by comparing accuracy and precision data with U.S. Food and Drug Administration criteria and by bridging the method to the former method for chloramine-T residues. The method accuracy and precision fulfilled the criteria for determinative methods; accuracy was 92.6, 93.4, and 94.6% with samples fortified at 0.5X, 1X, and 2X the expected 1000 ng/g tolerance limit for p-TSA, respectively. Method precision with tissue containing incurred p-TSA at a nominal concentration of 1000 ng/g ranged from 0.80 to 8.4%. The proposed determinative method was successfully bridged with the former method. The concentrations of p-TSA developed with the proposed method were not statistically different at p < 0.05 from p-TSA concentrations developed with the former method.     

Liquid chromatographic determination of para-toluenesulfonamide in edible fillet tissues from three species of fish.

Chloramine-T (N-sodium-N-chloro-p-toluene-sulfonamide) is a candidate therapeutic drug for treating bacterial gill disease, a predominant disease of a variety of fish species. Research has been initiated to obtain the U.S. Food and Drug Administration's (FDA) approval for the use of chloramine-T on a variety of fish species. An attribute of a therapeutic aquaculture drug that must be characterized before the FDA approves its use is depletion of the drug's marker residue (the drug's parent compound or metabolite of highest concentration in an edible tissue). para-Toluenesulfonamide (p-TSA) is the primary degradation product and marker residue for chloramine-T in rainbow trout. To conduct residue depletion studies for chloramine-T in fish, a robust analytical method sensitive and specific for p-TSA residues in edible fillet tissue from a variety of fish was required. Homogenized fillet tissues from rainbow trout (Oncorhynchus mykiss), walleye (Stizostedion vitreum), and channel catfish (Ictalurus punctatus) were fortified at nominal p-TSA concentrations of 17, 67, 200, 333, and 1000 ng/g. Samples were analyzed by isocratic reversed-phase liquid chromatography (LC) with absorbance detection at 226 nm. Mean recoveries of p-TSA ranged from 77 to 93.17%; relative standard deviations ranged from 1.5 to 14%; method quantitation limits ranged from 13 to 18 ng/g; and method detection limits ranged from 3.8 to 5.2 ng/g. The LC parameters produced p-TSA peaks without coelution of endogenous compounds and excluded chromatographic interference from at least 20 chemicals and drugs of potential use in aquaculture.     

Simultaneous analysis of select pharmaceuticals and personal care products in fish tissue using pressurized liquid extraction combined with silica gel cleanup

Analytical improvements were developed and validated for measuring select personal care products (PCPs) and two pharmaceuticals in fish tissue. The method was validated using fortified fillet tissue for twelve PCPs including fragrance materials, alkylphenols, photo initiators, and triclosan as well as two pharmaceuticals including carbamazepine (anti-seizure) and diazepam (anti-convulsant). The analytical method utilized pressurized liquid extraction (PLE) combined with silica gel cleanup, gel permeation chromatography, and gas chromatography ion-trap tandem mass spectrometry. Silica gel cleanup was combined with the PLE to produce one automated extraction/cleanup technique. This analytical improvement served to reduce the incurred cost, time, and loss of potential target analytes associated with independent cleanup steps. The combined extraction/cleanup technique resulted in an average increase of 10% in analyte recoveries. Average triplicate recoveries and relative standard deviations for the entire method, using 2.5 g of fish fillet tissue, were 92 ± 9% (recoveries ranged from 64 to 131%). The sensitivity of the analytical methods was improved by optimizing the resonant collision induced dissociation energy to the hundredths place (0.01 V). Improvements in ion production range from 24 to 122% for six of the 12 PCPs. Statistically derived method detection limits (MDLs) were also lowered on average by a factor of 8 and ranged from 1.2 to 38 ng/g wet weight. MDLs for carbamazepine and diazepam were 18 and 3.7 ng/g wet weight, respectively. Galaxolide and tonalide were measured in an environmental sample at concentrations of 81 and 5.5 ng/g wet weight, respectively.     

Liquid chromatographic determination of benzocaine and N-acetylbenzocaine in the edible fillet tissue from rainbow trout.

A method was developed for determining benzocaine and N-acetylbenzocaine concentrations in fillet tissue of rainbow trout. The method involves extracting the analytes with acetonitrile, removing lipids or hydrophobic compounds from the extract with hexane, and providing additional clean-up with solid-phase extraction techniques. Analyte concentrations are determined using reversed-phase high-performance liquid chromatographic techniques with an isocratic mobile phase and UV detection. The accuracy (range, 92 to 121%), precision (R.S.D., < 14%), and sensitivity (method quantitation limit, < 24 ng/g) for each analyte indicate the usefulness of this method for studies characterizing the depletion of benzocaine residues from fish exposed to benzocaine.     

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

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

Multilaboratory trial for determination of ceftiofur residues in bovine and swine kidney and muscle,and bovine milk

A multilaboratory trial for determining ceftiofur-related residues in bovine and swine kidney and muscle, and bovine milk was conducted following regulatory guidelines of the U.S. Food and Drug Administration, Center for Veterinary Medicine. The methods convert all desfuroylceftiofur-related residues containing the intact beta-lactam ring to desfuroylceftiofur acetamide to establish ceftiofur residues in tissues. Four laboratories analyzed 5 sets of samples for each tissue. Each sample set consisted of a control/blank sample and 3 control samples fortified with ceftiofur at 0.5 Rm, Rm, and 2 Rm, respectively, where Rm is the U.S. tolerance assigned for ceftiofur residue in each tissue/matrix: 0.100 microg/mL for milk, 8.0 microg/g for kidney (both species), 1.0 microg/g for bovine muscle, and 2.0 microg/g for swine muscle. Each sample set also contained 2 samples of incurred-residue tissues (one > Rm and one < Rm) from animals treated with ceftiofur hydrochloride. All laboratories completed the method trial after a familiarization phase and test of system suitability in which they demonstrated > 80% recovery in pretrial fortified test samples. Results showed that the methods met all acceptable performance criteria for recovery, accuracy, and precision. Although sample preparation was easy, solid-phase extraction cartridge performance must be carefully evaluated before samples are processed. The liquid chromatography detection system was easily set up; however, the elution profile may require slight modifications. The procedures could clearly differentiate between violative (> Rm) and nonviolative (< Rm) ceftiofur residues. Participating laboratories found the procedures suitable for ceftiofur residue determination.     

Normal-phase high-performance liquid chromatographic determination and identification of aldrin, dieldrin, and DDTs in eggs

A method for the routine monitoring of residual aldrin, dieldrin, pp'-DDT, op'-DDT, pp'-DDE, and pp'-DDD in eggs by high-performance liquid chromatography (HPLC) with a photo-diode array (PDA) detector is described. The lipids extracted from a whole egg are cleaned by a solid-phase extraction (SPE) ISOLUTE NH2 column with a diethyl ether-hexane (5:95, v/v) eluent. The HPLC separation is carried out using a normal-phase (LiChrosorb NH2) column, a heptane-hexane eluent (97:3, v/v), and a PDA detector. The average recoveries from fortified target compounds (0.1, 0.2, 0.3, and 0.4 microg/g, respectively) are > or = 83%, with standard deviations (SDs) between 2 and 5%. The interassay variabilities and their SDs are < or = 4.8% and < or =0.7%, with intra-assay variabilities of 2.1-3.3%. The limits of determination for these compounds range from 0.04 to 0.08 microg/g.     

Determination of florfenicol amine in channel catfish muscle by liquid chromatography

A method for quantifying florfenicol amine (FFA) in channel catfish muscle was validated according to U.S. Food and Drug Administration guidelines. FFA is the proposed marker residue for the veterinary antibiotic florfenicol in catfish muscle for regulatory surveillance purposes. The method includes acid hydrolysis followed by sample cleanup with ethyl acetate extraction, basification, solid-phase extraction, and quantitation by liquid chromatography with UV detection. The assay was validated at 5 concentrations in the range of 0.075-35 microg/g muscle. The overall mean recovery of FFA from fish tissues fortified at these concentrations ranged from 85.7 to 92.3%, 4.8-17.2% relative standard deviation (RSD). The assay limit of detection was 0.044 microg/g muscle based on analysis of control muscle. Catfish muscle samples containing incurred florfenicol residues were analyzed in quintuplicate with RSD < 5%. Acid hydrolysis has previously been demonstrated to convert florfenicol and its known metabolites to FFA and to release a significant amount of FFA from nonextractable florfenicol residues in tissues containing incurred residues in other species. By using acid hydrolysis, this method should yield a more accurate estimate of the total florfenicol-related residue level in muscle tissue from florfenicol-treated catfish than could be achieved by solvent extraction alone.     

Determination of putrescine and cadaverine in seafood (finfish and shellfish) by liquid chromatography using pyrene excimer fluorescence

A liquid chromatography (LC) method is described for the easy determination of the biogenic diamines putrescine (PUT) and cadaverine (CAD) in canned tuna, frozen tuna loin, fresh mahimahi fillet, frozen raw shrimp, cooked lump crabmeat, and fresh and cold-smoked salmon. The method is also a useful screen for histamine (HTA). The method involves homogenization of fish tissue, extraction of biogenic amines into borate-trichloroacetic acid solution, centrifugation, and derivatization of supernatant with 1-pyrenebutanoic acid succinimidyl ester. The derivatized diamine species allow for the intramolecular excimer fluorescence of the pyrene moiety at a higher emission wavelength than is possible for the endogenous tissue monoamines, thus providing visual specificity of detection. All seafood species were fortified with 0.5, 1.0, 5.0, 10.0, and 15.0 microg/g (ppm) of PUT and CAD. Determination was based on standard graphs for PUT and CAD using peak areas with standard solutions equivalent to 0.375, 1.0, 5.0, 10.0, and 20.0 ppm in tissue. A set of five matrix controls (unfortified seafood tissue) were also analyzed; endogenous PUT was found in all samples except the canned tuna, and CAD found only in the shrimp, crab, and cold-smoked salmon. The background amines were thus subtracted prior to determining spike recovery. The intra-assay average recoveries ranged from 71 to 94% across species and spike levels.     

Determination of perfluorinated compounds in fish fillet homogenates: method validation and application to fillet homogenates from the Mississippi River

We report herein a simple protein precipitation extraction-liquid chromatography tandem mass spectrometry (LC/MS/MS) method, validation, and application for the analysis of perfluorinated carboxylic acids (C7–C12), perfluorinated sulfonic acids (C4, C6, and C8), and perfluorooctane sulfonamide (FOSA) in fish fillet tissue. The method combines a rapid homogenization and protein precipitation tissue extraction procedure using stable-isotope internal standard (IS) calibration. Method validation in bluegill (Lepomis macrochirus) fillet tissue evaluated the following: (1) method accuracy and precision in both extracted matrix-matched calibration and solvent (unextracted) calibration, (2) quantitation of mixed branched and linear isomers of perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) with linear isomer calibration, (3) quantitation of low level (ppb) perfluorinated compounds (PFCs) in the presence of high level (ppm) PFOS, and (4) specificity from matrix interferences. Both calibration techniques produced method accuracy of at least 100 ± 13% with a precision (%RSD) ≤18% for all target analytes. Method accuracy and precision results for fillet samples from nine different fish species taken from the Mississippi River in 2008 and 2009 are also presented.     

Multiresidue determination of pesticides in drinking and related waters by gas chromatography/mass spectrometry after solid-phase extraction: interlaboratory study

As part of a project funded by the European Commission (EC) for the development and evaluation of multiresidue methods for analysis of drinking and related waters, 15 European laboratories evaluated a method using styrene-divinylbenzene co-polymer solid-phase extraction followed by gas chromatography/mass spectrometry. The main aim of the study was to evaluate whether the method meets the requirements of EC Directive 98/83 in terms of accuracy, precision, and detection limit for 22 pesticides according to the following requirements: limit of detection, < or = 0.025 microg/L; accuracy, expressed as recovery between 75 and 125%; and precision, expressed as repeatability relative standard deviation of the method of < 12.5% and as reproducibility relative standard deviation of the method of < 25%. Analyses for unknown concentrations were performed with fortified commercial bottled and tap waters. All laboratories were able to achieve detection limits of 0.01 microg/L for all pesticides except dimethoate and desisopropylatrazine (0.02 microg/L). The criteria for repeatability were met for all compounds except trifluralin, dimethoate, and lindane in bottled water and chlorpyrifos, dimethoate, and lindane in tap water. The criteria for reproducibility were met for all compounds except trifluralin, dimethoate, and lindane in bottled water and pendimethalin, chlorpyrifos, dimethoate, terbutryn, and lindane in tap water. In terms of accuracy, the method meets the requirements for all pesticides in both matrixes, except for lindane in bottled water and lindane and chlorpyrifos in tap water.     

A bridging study for oxytetracycline in the edible fillet of rainbow trout: analysis by a liquid chromatographic method and the official microbial inhibition assay.

Oxytetracycline (OTC) is a drug approved by the U.S. Food and Drug Administration (FDA) to control certain diseases in salmonids and catfish. OTC is also a likely control agent for diseases of other fish species and for other diseases of salmonids and catfish not currently on the label. One requirement for FDA to extend and expand the approval of this antibacterial agent to other fish species is residue depletion studies. The current regulatory method for OTC in fish tissue, based on microbial inhibition, lacks sensitivity and specificity. To conduct residue depletion studies for OTC in fish with a liquid chromatographic method, a bridging study was required to determine its relationship with the official microbial inhibition assay. Triplicate samples of rainbow trout fillet tissue fortified with OTC at 0.3, 0.6, 1.2, 2.4, 4.8, and 9.6 ppm and fillet tissue with incurred OTC at approximately 0.75, 1.5, and 3.75 ppm were analyzed by high-performance liquid chromatography (HPLC) and the microbial inhibition assay. The results indicated that the 2 methods are essentially identical in the tested range, with mean coefficients of variation of 1.05% for the HPLC method and 3.94% for the microbial inhibition assay.     

Multiresidue determination of organochlorine pesticides and polychlorinated biphenyls in milk by gas chromatography with electron-capture detection after extraction by matrix solid-phase dispersion

A multiresidue analytical method based on matrix solid-phase dispersion was developed to analyze liquid milk for 22 organochlorine pesticides (OCPs) and 6 polychlorinated biphenyls (PCBs). Initial extraction is performed by loading 3 mL milk onto a 2.0 g octadecyl (C18)-bonded silica cartridge with n-hexane as the eluant. Neutral alumina column chromatography with sodium sulfate as the drying agent is used for further cleanup. The eluate is concentrated to 0.5 mL, and target analytes are determined by capillary gas chromatography with electron-capture detection. The optimized method was validated by determining accuracy (recovery percentages), precision (repeatability and reproducibility), and sensitivity (detection and quantitation limits) from analyses of milk samples fortified at 10 and 1 microg/L levels. Average recoveries were between 74 and 106% for all residues except beta-HCH, beta-endosulfan, and endosulfan sulfate. Both repeatability and reproducibility relative standard deviation values were < 22% for all residues. Detection limits ranged from 0.02 to 0.12 microg/L and quantitation limits were between 0.02 and 0.62 microg/L. The proposed analytical method may be used as a fast and simple procedure in routine determinations of OCPs and PCBs in milk.     

Determination of polycyclic aromatic compounds in fish tissue

A method is presented for the analysis of polycyclic aromatic hydrocarbons (PAHs), polycyclic aromatic sulfur heterocycles (PASHs), and basic polycyclic aromatic nitrogen heterocycles (PANHs) in fish. The analytical procedure includes Soxhlet extraction of prepared fish tissue with methylene chloride followed by gel permeation chromatography (GPC) using Bio-beads SX-3. For PAHs/PASHs, further cleanup is performed using adsorption chromatography on Florisil (5% water deactivated) and elution with hexane. For basic PANHs further cleanup of the fish extracts after GPC is achieved using liquid-liquid partitioning with 6 M hydrochloric acid and chloroform and then basifying the aqueous phase and extracting it with chloroform. Analysis of fortified fish samples was performed using capillary gas chromatography with flame ionization detection and capillary gas chromatography-mass spectrometry. Good agreement was observed for both methods of analysis when applied to fish samples fortified with PAHs, PASHs and basic PANHs at 0.1 to 1 microgram/g, suggesting that the method is effective at removing interfering biogenic compounds prior to analysis. Average recovery of PAHs/PASHs from fortified fish tissue was 87% and 70% for fish tissue fortified at 0.24-1.1 and 0.024-0.11 microgram/g, respectively. Average recovery for basic PANHs was 97% for fish fortified at 1.2-1.4 micrograms/g.     

Rapid determination and confirmation of biogenic amines in tuna loin by gas chromatography/mass spectrometry using ethylchloroformate derivative

A gas chromatography-mass spectrometry (GC/MS) method is described for the easy rapid determination and simultaneous confirmation of the biogenic amines putrescine (PUT), cadaverine (CAD), histamine (HTA), and spermidine (SPD) in fresh frozen tuna loin. The method can also be used to monitor tyramine (TYR). The method involves homogenization of fish tissue, extraction of biogenic amines into trichloroacetic acid solution, centrifugation, alkalization, and derivatization of supernatant with ethylchloroformate. All seafood species were fortified to contain 2.5, 5.0, 10.0, 12.5, and 25.0 microg/g (ppm) PUT, CAD, and SPD; and 10.0, 20.0, 40.0, 50.0, and 100.0 microg/g (ppm) HTA. Determination was based on standard curves for each analyte using peak areas with matrix standards equivalent to a concentration range bracketing the spike level. A set of 5 matrix controls (unfortified tuna tissue) was also analyzed; only endogenous SPD was found in all samples. The interassay average recoveries ranged from 57 to 79% across analytes and spike levels.     

Analysis of paroxetine, fluoxetine and norfluoxetine in fish tissues using pressurized liquid extraction, mixed mode solid phase extraction cleanup and liquid chromatography-tandem mass spectrometry

Recent studies have shown that selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine are accumulated in the tissues of fish as a result of discharges of pharmaceuticals into surface waters from municipal wastewater treatment plants. In this study, an analytical method based on liquid chromatography with atmospheric pressure chemical ionization and tandem mass spectrometry (LC-APCI-MS/MS) was developed and validated for the determination of residues of paroxetine, fluoxetine and its active metabolite, norfluoxetine, in fish tissue. The procedure for sample preparation includes extraction of tissue by pressurized liquid extraction (PLE), followed by cleanup on a mixed-mode solid phase extraction (SPE) cartridge, Oasis MCX. With the optimized method, matrix interferences were reduced and recoveries >85% were obtained. The limits of quantitation (LOQ) determined by analysis of spiked fish tissue were 0.24, 0.07, and 0.14 ng/g wet weight for paroxetine, fluoxetine and norfluoxetine, respectively. This method was successfully applied to the analysis of samples of fish collected from Hamilton Harbour in Ontario, Canada, which is an urbanized and industrialized embayment of Lake Ontario. These analyses showed that the three analytes were present in fish tissues at concentrations up to approximately 1 microg/kg wet weight.     

Response factor considerations for the quantitative analysis of western redcedar (Thuja plicata) foliar monoterpenes

A method is described for quantitative analysis of monoterpenes in western redcedar (Thuja plicata) foliage by gas chromatography with flame ionization detection. Response factors for monoterpenes identified in redcedar are evaluated to determine similarities among monoterpene responses. Evaluation demonstrates that redcedar monoterpenes yield detector responses that fall into two groups. One monoterpene from each group is used as a standard for quantitative analysis. Redcedar monoterpenes are quantitated by comparing analyte response with the response factor of one of the standards in single-point calibrations. Homogenized foliage samples are extracted with ethyl acetate and the extracts passed through a solid phase extraction column of graphitized carbon to remove plant pigments. Method bias and repeatability are evaluated by fortifying foliage samples with (1S)-(+)-carvone and (1S)-(+)-2-carene and subjecting the samples to the extraction and analysis procedures. Detection limits are also assessed from fortified samples. Excellent recovery (> 95.0%) and precision (< 5%) are obtained from the analysis of 2-carene from fortified samples. Carvone recovery is approximately 80% with excellent precision (< 4%). The method limits of detection obtained from 2-carene and carvone fortified samples are 4.7 and 13.5 microg/g, respectively.     

Determination of arsenic species in marine samples by HPLC-ICP-MS.

Arsenic speciation analysis in marine samples was performed using high performance liquid chromatography (HPLC) with ICP-MS detection. The separation of eight arsenic species viz. arsenite (As(III)), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenate (As(V)), arsenobetaine, trimethylarsine oxide (TMAO), arsenocholine and tetramethylarsonium ion (TeMAs) was achieved on a Shiseido Capcell Pak C18 column by using an isocratic eluent (pH 3.0), in which condition As(III) and MMA were co-eluted. The entire separation was accomplished in 15 min. The detection limits for 8 arsenic species by HPLC/ICP-MS were in the range of 0.02 - 0.10 microg L(-1) based on 3sigma of blank response (n=9). The precision was calculated to be 3.1-7.3% (RSD) for all eight species. The method then successfully applied to several marine samples e.g., oyster, scallop, fish, and shrimps. For the extraction of arsenic species from seafood products, the low power microwave digestion was employed. The extraction efficiency was in the range of 52.9 - 112.3%. Total arsenic concentrations were analyzed by using the microwave acid digestion. The total arsenics in the certified reference materials (DORM-2 and TORT-2) were analyzed and agreed with the certified values. The concentrations of arsenics in marine samples were in the range 6.6 - 35.1 microg g(-1).     

Determination of lead and cadmium in ceramicware leach solutions by graphite furnace atomic absorption spectroscopy: method development and interlaboratory trial.

This method was developed to improve sensitivity and eliminate time consuming, evaporative pre-concentration in AOAC Method 973.82 and American Society for Testing and Materials method C738 for testing foodware. The method was developed using leach solutions obtained by leaching 9 differently decorated ceramic vessels with 4% acetic acid for 24 h at room temperature. Lead and cadmium concentrations in leach solutions were 0.005-17,600 and 0.0004-0.500 microg/mL, respectively. Concentrations were determined using peak area, phosphate chemical modifier (8.3 microg PO4(-3)), and a standard curve for quantitation. Optimized pre-atomization and atomization temperatures were 1,300 and 1,800 degrees C, respectively, for Pb and 1,100 and 1,700 degrees C, respectively, for Cd. Characteristic masses (mo) were 10 and 0.4 pg for Pb and Cd, respectively. Precision of repeated analyses of calibration solutions was < or =3% relative standard deviation. Precision of duplicate leach solution analyses on different days was 0-9% relative difference. Recovery from fortified leach solutions was 96-106%. Results obtained by this method agreed 92-110% with those of confirmatory analyses. Results of certified reference material solutions agreed 94-100% with certificate values. Pb and Cd limits of quantitation (LOQ) were 0.005 and 0.0005 microg/mL, respectively. Results from 3 trial laboratories for 4 leach solutions containing Pb and Cd concentrations of 0.017-1.47 and <0.0005-0.0864 microg/mL, respectively, agreed 89-102% with results of the author. Two attributes of this method were noteworthy: (1) Background absorbance due to organic matter was entirely absent from atomization profiles, making the use of short pre-atomization hold times (2 s) possible. (2) Instrument precision was excellent and only one determination per solution was needed. Acceptance criteria for quality control measurements and a practical procedure for estimating the method LOQ during routine regulatory analyses are described.     

Determination of melamine in pet food by enzyme immunoassay, high-performance liquid chromatography with diode array detection, and ultra-performance liquid chromatography with tandem mass spectrometry

Melamine in pet food (fortified or originally contaminated) was determined by enzyme immunoassay (EIA), high-performance liquid chromatography with diode array detection (HPLC-DAD), and ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The limits of detection (LOD) for EIA and HPLC-DAD were 0.02 and 0.1 microg/mL, respectively. The linear ranges of the calibration curves for EIA and HPLC-DAD were 0.02-0.5 and 0.1-500 microg/mL, respectively. The coefficient of determinations (r2) of the standard curves for EIA and HPLC were 0.9991 and 0.9999, respectively. Coefficient of variations from both inter- and intra-assay were <9.31%, and recovery range for all concentrations was between 71 and 105%. The r2 values between the EIA and HPLC-DAD methods for melamine analysis of the fortified and originally contaminated samples were 0.9973 and 0.9885. The r2 values for UPLC-MS/MS with HPLC-DAD and with EIA were 0.9566 and 0.9489, respectively.