LC-MS/MS Determination of Antibiotic Residues in Distillers Grains: Method Modification.

BACKGROUND: Antibiotics are used in ethanol production to discourage undesirable bacteria growth. To determine if antibiotic residues remain in the distillers grain (DG) byproduct, which is used as an animal food ingredient, the U.S. Food and Drug Administration/Center for Veterinary Medicine previously developed an LC-MS/MS method to detect residues of erythromycin A, penicillin G, virginiamycin M1, and virginiamycin S1 in DG to enable regulatory decision-making. OBJECTIVE: Erythromycin and penicillin G were quantitated using the stable isotope dilution technique with their isotopically labeled compounds, which are considered optimal internal standards (ISTDs) for quantitative mass spectrometry. With the commercial availability of virginiamycin M1-d2 since then, the objectives of this study were to evaluate the feasibility of its use as it is only doubly deuterated, and to incorporate it in the method to enhance method performance. METHOD: Antibiotic residues were solvent-extracted from DG; the extract was cleaned up by a hexane wash and solid phase extraction (SPE) and analyzed by LC-MS/MS. RESULTS: We established suitability of virginiamycin M1-d2 as an ISTD and incorporated it in the method. For all analytes, accuracy and precision ranged 90 to 102% and 3.8 to 6.8, respectively. CONCLUSIONS: We modified a previously developed LC-MS/MS method that uses virginiamycin M1-d2 as an ISTD to support surveillance studies to determine several drugs in DG. HIGHLIGHTS: Virginiamycin M1-d2 was successfully incorporated into the method for better virginiamycin M1 quantitation. This addition also allowed calibration curves for all analytes to be constructed in solvent, thereby simplifying the method.

A Dilute-and-Shoot UHPLC-MS/MS Isotope Dilution Method for Simultaneous Determination and Confirmation of 11 Mycotoxins in Dried Distiller's Grains with Solubles.

BACKGROUND: Natural contamination with mycotoxins in dried distiller's grains with solubles (DDGS) as a mainstream animal feed ingredient poses a risk to animal health. OBJECTIVE: A regulatory method was needed for the agency to simultaneously detect 11 mycotoxins of high regulatory priority in DDGS. METHODS: A DDGS sample (10 g) was extracted twice with acetonitrile-water under mildly acidic condition. Two aliquots from the combined crude extract were taken and processed separately: (1) diluted 400-fold with solvent for analysis of deoxynivalenol and fumonisins B1 and B2; and (2) with the pH adjusted to 7.5, and then diluted 15.7-fold for analysis of aflatoxins B1, B2, G1, and G2, ochratoxin A, zearalenone, and T-2 and HT-2 toxins. Uniformly labeled 13C-isotopologues of these mycotoxins were added as internal standards to the diluted extracts for quantitative analysis by ultra-high-performance LC-tandem MS. RESULTS: The linear quantitation ranges (µg/kg) were: aflatoxins B1, B2, G1, and G2, 1.57-105; zearalenone, 16.3-1090; T-2 toxin, 3.14-208; HT-2 toxin, 48.2-3220; ochratoxin A, 0.47-31.4; deoxynivalenol, 240-16 000; fumonisin B1 and B2, 320-21 200. Accuracies for these analytes at each of three fortification levels ranged from 70.7 to 100%, with corresponding RSDs between 1.4 and 10.5%. True recoveries were all higher than 83%. CONCLUSION: This method was successfully validated to meet the agency's performance guidelines for regulatory methods. HIGHLIGHTS: This method is easy, quick, and robust to simultaneously quantify and confirm the presence of 11 regulated mycotoxins in DDGS.

An LC-MS/MS Method for the Determination of Antibiotic Residues in Distillers Grains: Collaborative Study.

BACKGROUND: Antibiotics are used in ethanol production to discourage the growth of bacteria that would lower the yield of the product. Any antibiotic residues remaining in distillers grain (DG) co-product could lead to antimicrobial resistance, which is a public health concern. The U.S. Food and Drug Administration (FDA) Center for Veterinary Medicine (CVM) previously developed an LC-MS/MS analytical method to detect residues of erythromycin A, penicillin G, virginiamycin M1, and virginiamycin S1 in DG to enable regulatory decision making. OBJECTIVE: The objective of this study was to ensure the method's robustness by carrying out a multi-laboratory validation of the method. METHOD: Test portions were extracted with a mixture of acetonitrile and buffer. The extract was cleaned by solid phase extraction. The concentrated eluant was reconstituted and analyzed by LC-MS/MS. Eight laboratories participated in the study. RESULTS: Average accuracies for the combined three matrixes for all four compounds at all fortification levels ranged from 83->109% with repeatability relative standard deviation (RSDr; within laboratory) ≤17% and reproducibility relative standard deviation (RSDR; between laboratory) ≤21%. The Horwitz ration (HorRat) values ranged 0.4-1.0 indicating that method reproducibility is acceptable. CONCLUSIONS: An interlaboratory study was successfully conducted to evaluate an LC-MS/MS method for the determination of the drugs of interest in DG. The results demonstrate that the method is fit for purpose to determine the drugs in DG and could serve as a regulatory method capable of being used for compliance actions for DG containing these antibiotic contaminants. HIGHLIGHTS: The method was posted to the FDA/Foods Program Compendium of Analytical Laboratory Methods.

Reproductive toxicity in rats with crystal nephropathy following high doses of oral melamine or cyanuric acid.

The industrial chemical melamine was used in 2007 and 2008 to raise the apparent protein content in pet feed and watered down milk, respectively. Because humans may be exposed to melamine via several different routes into the human diet as well as deliberate contamination, this study was designed to characterize the effect of high dose melamine or cyanuric acid oral exposure on the pregnant animal and developing fetus, including placental transfer. Clear rectangular crystals formed following a single triazine exposure which is a different morphology from the golden spherulites caused by combined exposure or the calculi formed when melamine combines with endogenous uric acid. Crystal nephropathy, regardless of cause, induces renal failure which in turn has reproductive sequelae. Specifically, melamine alone-treated dams had increased numbers of early and late fetal deaths compared to controls or cyanuric acid-treated dams. As melamine was found in the amniotic fluid, this study confirms transfer of melamine from mammalian mother to fetus and our study provides evidence that cyanuric acid also appears in the amniotic fluid if mothers are exposed to high doses.

Depletion of melamine and cyanuric acid in serum from catfish Ictalurus punctatus and rainbow trout Onchorhynchus mykiss.

Melamine and its triazine analogs, such as cyanuric acid, have been used to artificially inflate protein content both in animal feed ingredients, as well as in milk products produced for human consumption. We report here a LC-MS/MS method to quantify and confirm melamine and cyanuric acid in serum from channel catfish and rainbow trout with a limit of quantification of 0.8 µg/mL. The method was applied to serum samples from a residue depletion study in which fish were given a single oral dose of 20 mg/kg body weight melamine, cyanuric acid, or both compounds together. Samples were taken at 1, 3, 7, 14, and 28 days (an additional 42 day was added for trout). When given alone or in combination with cyanuric acid, melamine residues were highest on day 1 in both catfish and trout. Cyanuric acid was only quantifiable at day 1 in trout when given alone, and not at all in catfish. The serum half life of melamine in catfish was 1.50-1.62 days and 3.09-3.67 days in trout. This work highlights the differences of depletion kinetics in fish, which can be measured in days, as compared to the depletion in mammals, measured in hours.

Bioaccumulation of melamine in catfish muscle following continuous, low-dose, oral administration.

In this study, catfish muscle was analyzed for melamine (MEL) and cyanuric acid (CYA) residues following experimental feeding with low doses of MEL and MEL and CYA (MEL+CYA) and with the insoluble melamine-cyanurate complex (MEL=CYA). Catfish were daily fed 0.1 mg/kg BW of MEL for 15, 28, or 42 days, 0.1 mg/kg BW of MEL+CYA for 28 days, 2.5 mg/kg BW of MEL+CYA for 14 days, or 400 mg/kg BW of MEL=CYA for 3 days. Residues in the tissue were determined by LC-MS/MS. MEL was extracted with acidic acetonitrile, followed by defatting with dichloromethane, and isolated with cation exchange solid phase extraction (SPE). For CYA analysis, fish were extracted with dilute acetic acid, defatted with hexane, and cleaned up with a graphitic carbon SPE. Catfish fed 0.1 mg/kg BW of MEL reached a maximum muscle residue concentration of 0.33 ± 0.04 mg/kg (ppm) after 28 days of continuous feeding. The same concentration was found for MEL+CYA feeding at the 0.1 mg/kg BW level for 28 days. Feeding at 2.5 mg/kg BW of MEL+CYA yielded muscle concentrations above the 2.5 mg/kg level of concern for most of the study fish. Finally, catfish fed high levels of the MEL=CYA complex (400 mg/kg BW) accumulated relatively little MEL in the muscle (0.14 ± 0.07 mg/kg) and, unlike treatment with MEL+CYA, did not form renal melamine-cyanurate crystals. Appreciable concentrations of CYA were not detected in any of the muscles tested. These studies provide data to model the bioaccumulation of triazine residues into edible fish tissue as a result of the continuous consumption of adulterated feed.

Determination of tricaine residues in fish by liquid chromatography.

Tricaine methanesulfonate (MS-222) is approved by the U.S. Food and Drug Administration, Center for Veterinary Medicine (CVM), as an anesthetic drug for select aquaculture species. It was approved for use as a handling aid with a 3 week withdrawal time. The drug is rapidly metabolized and excreted; therefore, CVM approved its use without requiring a regulatory method for drug residues in tissues. However, there are concerns that the drug may be used to sedate fish during transport to slaughter. A regulatory method will enable monitoring for unsafe residues of this drug resulting from extralabel use. We present a quantitative method, using LC at a target level of 0.1 mg/kg (ppm), for three different farmed species: salmon (Salmo salar); tilapia (Oreochromis spp.); and catfish (Ictalurus punctatus). The assay begins with an acetonitrile extraction, followed by filtration and mixed-mode cation-exchange solid-phase extraction cleanup. The extracts are analyzed by reversed-phase LC with UV detection at 320 nm. The method was validated by using fish fillets with incurred residues, control fish fillets, and fish fillets fortified at half the target level, the target level, and twice the target level (0.05, 0.1, and 0.2 ppm, respectively). For all species, accuracy is > or =80% and the RSD is < or =10%. The method complies with CVM performance criteria for the determination of veterinary drug residues.

Simultaneous determination and confirmation of melamine and cyanuric acid in animal feed by zwitterionic hydrophilic interaction chromatography and tandem mass spectrometry.

We developed a new method to analyze animal feed and feed ingredients for melamine and cyanuric acid. The method is capable of extracting and detecting both melamine and cyanuric acid in a single procedure, whether present as free compounds or bound together as the melamine:cyanurate complex. A novel chromatographic system based on zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) columns enables separation and detection of both compounds in one run. Samples are extracted with a strong aqueous acid which is then diluted to bring the concentration within the working range of the method. The method is applicable over the range of 0.5 to 50 micrograms/gram (microg/g). Samples at higher concentrations may be diluted into this range, which is equivalent to 3.6-360 ng/mL in the injection solvent. Analytes are detected using liquid chromatography/tandem mass spectrometry. The data confirm the presence of both compounds according to criteria recommended by the US FDA Center for Veterinary Medicine. The LC/MS/MS method provides an alternative to derivatization and gas chromatography-mass spectrometry for regulatory analysis of feed samples. Published in 2008 by John Wiley & Sons, Ltd.

Evaluation of the renal effects of experimental feeding of melamine and cyanuric acid to fish and pigs.

OBJECTIVE: To determine whether renal crystals can be experimentally induced in animals fed melamine or the related triazine compound cyanuric acid, separately or in combination, and to compare experimentally induced crystals with those from a cat with triazine-related renal failure. ANIMALS: 75 fish (21 tilapia, 24 rainbow trout, 15 channel catfish, and 15 Atlantic salmon), 4 pigs, and 1 cat that was euthanatized because of renal failure. PROCEDURES: Fish and pigs were fed a target dosage of melamine (400 mg/kg), cyanuric acid (400 mg/kg), or melamine and cyanuric acid (400 mg of each compound/kg) daily for 3 days and were euthanatized 1, 3, 6, 10, or 14 days after administration ceased. Fresh, frozen, and formalin-fixed kidneys were examined for crystals. Edible tissues were collected for residue analysis. Crystals were examined for composition via Raman spectroscopy and hydrophilic-interaction liquid chromatography-tandem mass spectrometry. RESULTS: All animals fed the combination of melamine and cyanuric acid developed goldbrown renal crystals arranged in radial spheres (spherulites), similar to those detected in the cat. Spectral analyses of crystals from the cat, pigs, and fish were consistent with melamine-cyanurate complex crystals. Melamine and cyanuric acid residues were identified in edible tissues of fish. CONCLUSIONS AND CLINICAL RELEVANCE: Although melamine and cyanuric acid appeared to have low toxicity when administered separately, they induced extensive renal crystal formation when administered together. The subsequent renal failure may be similar to acute uric acid nephropathy in humans, in which crystal spherulites obstruct renal tubules.

Development of multiclass methods for drug residues in eggs: hydrophilic solid-phase extraction cleanup and liquid chromatography/tandem mass spectrometry analysis of tetracycline, fluoroquinolone, sulfonamide, and beta-lactam residues.

A method was developed for detection of a variety of polar drug residues in eggs via liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). A total of twenty-nine target analytes from four drug classes-sulfonamides, tetracyclines, fluoroquinolones, and beta-lactams-were extracted from eggs using a hydrophilic-lipophilic balance polymer solid-phase extraction (SPE) cartridge. The extraction technique was developed for use at a target concentration of 100 ng/mL (ppb), and it was applied to eggs containing incurred residues from dosed laying hens. The ESI source was tuned using a single, generic set of tuning parameters, and analytes were separated with a phenyl-bonded silica cartridge column using an LC gradient. In a related study, residues of beta-lactam drugs were not found by LC/MS/MS in eggs from hens dosed orally with beta-lactam drugs. LC/MS/MS performance was evaluated on two generations of ion trap mass spectrometers, and key operational parameters were identified for each instrument. The ion trap acquisition methods could be set up for screening (a single product ion) or confirmation (multiple product ions). The lower limit of detection for screening purposes was 10-50 ppb (sulfonamides), 10-20 ppb (fluoroquinolones), and 10-50 ppb (tetracyclines), depending on the drug, instrument, and acquisition method. Development of this method demonstrates the feasibility of generic SPE, LC, and MS conditions for multiclass LC/MS residue screening.

Peer validation of a method to confirm chloramphenicol in honey by liquid chromatography-tandem mass spectrometry.

Chloramphenicol (CAP) is extracted from an aqueous dilution of honey using ethyl acetate. The extracts are evaporated and redissolved in water. CAP is then extracted from the aqueous solutions using reversed-phase solid-phase extraction cartridges. CAP is eluted from the reversed-phase cartridges with acetonitrile-water and re-extracted into ethyl acetate. The ethyl acetate is evaporated, and the residue is reconstituted in an aqueous solution. Extracts are chromatographed using a reversed-phase column and analyzed by electrospray negative mode tandem mass spectrometry. Four product ions of precursors m/z 321 or 323 are monitored. The method meets confirmation criteria recommended by the U.S. Food and Drug Administration and 4-point identification criteria established by the European Union. With slight modifications to accommodate different equipment, the method was validated in 2 laboratories.

LC/MS/MS measurement of gentamicin in bovine plasma, urine, milk, and biopsy samples taken from kidneys of standing animals.

Methods for the measurement of gentamicin concentration in several bovine tissues were developed and validated. A novel liquid chromatographic (LC) technique employed trifluoroacetic acid in the mobile phase so that all gentamicin components co-eluted. Analytes were ionized by positive-ion pneumatically assisted electrospray and detected by selected reaction monitoring (SRM) with an LC-tandem mass spectrometer (LC/MS/MS). Calibration of plasma and urine samples was based on tobramycin internal standard. Calibration of milk and kidney samples was based on external standard, due to variability of tobramycin response in these matrices. The extraction technique employed treatment with aqueous trichloroacetic acid to both precipitate protein and liberate gentamicin from the matrix. Milk samples had to be defatted by centrifugation prior to extraction. Urine samples were further cleaned up with C-18 solid phase extraction (SPE). These methods were validated for use in several residue depletion studies (reported elsewhere) to monitor the depletion of gentamicin in tissues under various dosing conditions. The plasma method was calibrated from 1 to 5000 ng/mL in two ranges, with a limit of quantitation (LOQ) in the low range calculated at 3.3 ng/mL. The milk method was calibrated from 2.5 to 2500 ng/mL with an LOQ calculated at 4.5 ng/mL. The urine method was designed for use at low levels, and was calibrated from 1 to 100 ng/mL with an LOQ of 3.8 ng/mL. The kidney method was primarily designed for analysis of small samples (approximately 100mg). This method was calibrated from 10 to 50,000 ng/g with an LOQ of 26 ng/g.

Development of multiclass methods for drug residues in eggs: silica SPE cleanup and LC-MS/MS analysis of ionophore and macrolide residues.

A method was developed that is suitable for screening eggs for a variety of nonpolar residues in a single procedure. Residues are extracted by silica solid-phase extraction (SPE). Analysis is conducted via reverse-phase gradient liquid chromatography, electrospray ionization, and tandem ion trap mass spectrometry. For screening purposes (based on a single precursor-product ion transition) the method can detect ionophore (lasalocid, monensin, salinomycin, narasin) and macrolide (erythromycin, tylosin) residues in egg at approximately 1 ng/mL (ppb) and above and novobiocin residues at approximately 3 ppb and above. Conditions are described for confirmatory analysis based on multiple ions in the product ion spectrum. The extraction efficiency for ionophores was estimated at 60-85%, depending on drug. Recovery of macrolides and novobiocin was not as good (estimated at 40-55% after a hexane wash of the final extract was included), but the method consistently screened and confirmed these residues at concentrations below the target of 10 ppb. The method was applied to eggs from hens dosed with each drug individually. Lasalocid was found to have the highest probability of detection in eggs based on its high ionization efficiency and higher rate of deposition relative to the other drugs. The method is part of a larger scheme to provide surveillance methods for a wide variety of drug residues in eggs.

Multilaboratory validation of a method to confirm chloramphenicol in shrimp and crabmeat by liquid chromatography-tandem mass spectrometry.

An existing method for chloramphenicol (CAP) determination in shrimp using a gas chromatograph with electron capture detector was adapted for confirmation of CAP with a liquid chromatograph interfaced to a triple quadrupole mass spectrometer. CAP residues are extracted from tissue with ethyl acetate, isolated via liquid-liquid extraction, and concentrated by evaporation. Extracts are chromatographed by using a reversed-phased column and analyzed by electrospray negative mode tandem mass spectrometry. Four product ions (m/z 152, 176, 194, and 257) of precursor m/z 321 were monitored. Moving from gas chromatography to liquid chromatography-tandem mass spectrometry improved the sensitivity of the method greatly, enabling reliable confirmation of CAP residues at 0.3 microg/kg (ppb). The method meets confirmation criteria recommended by the U.S. Food and Drug Administration and 4-point identification criteria established by the European Union. With slight modifications to accommodate different equipment, the method was validated in 3 laboratories.

Volatilization of trifluralin, atrazine, metolachlor, chlorpyrifos, alpha-endosulfan, and beta-endosulfan from freshly tilled soil.

The volatile and soil loss profiles of six agricultural pesticides were measured for 20 days following treatment to freshly tilled soil at the Beltsville Agricultural Research Center. The volatile fluxes were determined using the Theoretical Profile Shape (TPS) method. Polyurethane foam plugs were used to collect the gas-phase levels of the pesticides at the TPS-defined critical height above a treated field. Surface-soil (0-8 cm) samples were collected on each day of air sampling. The order of the volatile flux losses was trifluralin > alpha-endosulfan > chlorpyrifos > metolachlor > atrazine > beta-endosulfan. The magnitude of the losses ranged from 14.1% of nominal applied amounts of trifluralin to 2.5% of beta-endosulfan. The daily loss profiles were typical of those observed by others for volatile flux of pesticides from moist soil. Even though heavy rains occurred from the first to third day after treatment, the majority of the losses took place within 4 days of treatment, that is, 59% of the total applied atrazine and metolachlor and >78% of the other pesticides. Soil losses generally followed pseudo-first-order kinetics; however, leaching due to heavy rainfall caused significant errors in these results. The portion of soil losses that were accounted for by the volatile fluxes was ordered as follows: alpha-endosulfan, 34.5%; trifluralin, 26.5%; chlorpyrifos, 23.3%; beta-endosulfan, 14.5%; metolachlor, 12.4%; and atrazine, 7.5%.

Gas-phase analysis of trimethylamine, propionic and butyric acids, and sulfur compounds using solid-phase microextraction.

Complaints due to odors are an important problem for the wastewater, composting, and animal agriculture industries. Accurate, objective measurement techniques are needed to monitor emissions, to develop new waste handling procedures, and to reduce the production of these volatile gases. Solid-phase microextraction was investigated as a technique for the determination of representative odorous gases. A flow-through Teflon chamber was used to expose the fibers to certified gas standards. A 75-microm carboxen-poly(dimethylsiloxane) (Car-PDMS) coating was used for trimethylamine (TMA), carbon disulfide (CS2), dimethylsulfide (DMS), and dimethyl disulfide (DMDS), and an 85-microm polyacrylate coating was used for propionic acid (PA) and butyric acid (BA). Using a 1-h fiber exposure time and a flow rate through the chamber of 72 mL/min, method detection limits were 2.38, 0.074, 0.150, 0.063, 1.85, and 1.32 ppbv for TMA, DMS, CS2, DMDS, PA, and BA, respectively. Enhanced detector signal was observed for all analytes under flow conditions, as compared to static conditions, and the porous nature of the Car-PDMS coating appears to increase the time needed for analytes to reach equilibrium under flow conditions.