Comparison of data acquisition modes with Orbitrap high-resolution mass spectrometry for targeted and non-targeted residue screening in aquacultured eel.

RATIONALE: A current trend in monitoring chemical contaminants in animal products is to use high-resolution mass spectrometry (HRMS). In this study, several HRMS data acquistion modes using Orbitrap MS for simultaneous full-scan MS in combination with MS2 analysis were evaulated for their effectiveness in detecting and identifying both targeted and non-targeted veterinary drug residues in aquacultured eel samples. METHODS: Sample preparation consisted of an acidic acetonitrile extraction with solid-phase extraction cleanup for analysis using LC/HRMS. Different data acquisition methods, including full-scan MS with non-targeted all ion fragmentation (AIF), multiplexed or variable data-independent analysis (mDIA or vDIA), targeted data-dependent MS2 (DDMS2), and parallel reaction monitoring (PRM) acquisition, were explored. The methods were evaluated with fortified eel tissue and imported eel samples to determine how many analytes could be detected and identified. RESULTS: For non-targeted data acquisition, the number of analytes detected using DIA methods matched the results obtained by AIF, but the resulting product ion scans were more diagnostic because characteristic ions were predominant in the DIA MS2 spectra. In targeted analysis for a limited list of 68 compounds, full-scan MS followed by PRM was advantageous compared with DDMS2 because high-quality MS2 spectra were generated for almost all the analytes at target testing levels. CONCLUSIONS: For residue screening, AIF has fast MS1 scan speed with adequate detection of product ions but may lead to false positive findings. DIA methods are better suited to monitor for both targeted and non-targeted compounds because they generate more characteristic MS2 spectra for retrospective library searching. For follow-up targeted analysis, PRM is prefered over DDMS2 when searching for a limited set of compounds.

Analytical methods for mixed organic chemical residues and contaminants in food.

Developing methods that can analyze multiple categories of organic chemical residues such as pesticides, veterinary drugs, mycotoxins, human drugs, and environmental contaminants in food with a single analytical procedure is a growing trend. These methods for mixed organic chemical residues and contaminants focus on the chemical properties of these analytes rather than how they are used and adulterate the food supply. This paper highlights recently published methods for mixed residue and contaminant methods in food including advances in technology (instrumental hardware, data processing programs, and sample cleanup) that allow for a larger number of compounds to be monitored simultaneously. The factors that determine the scope, or number and type of analytes in a given method, including needs for specific food commodities, complexity of the analytical procedure, and the intended purpose (qualitative vs quantitative analysis) will be examined. Although there are clear advantages to expanding the number of unwanted chemicals being monitored in the global food supply, challenges to developing and implementing mixed organic residue and contaminant methods will also be discussed. Going forward, it will be important to implement these methods to more thoroughly protect the food supply for a wide variety of targeted and non-targeted chemical residues and contaminants while also having the regulatory framework in place to effectively manage the results of these comprehensive analyses. Graphical abstract.

Application and evaluation of a high-resolution mass spectrometry screening method for veterinary drug residues in incurred fish and imported aquaculture samples.

The ability to detect chemical contaminants, including veterinary drug residues in animal products such as fish, is an important example of food safety analysis. In this paper, a liquid chromatography high-resolution mass spectrometry (LC-HRMS) screening method using a quadrupole-Orbitrap instrument was applied to the analysis of veterinary drug residues in incurred tissues from aquacultured channel catfish, rainbow trout, and Atlantic salmon and imported aquacultured products including European eel, yellow croaker, and tilapia. Compared to traditional MS methods, the use of HRMS with nontargeted data acquisition and exact mass measurement capability greatly increased the scope of compounds that could be monitored simultaneously. The fish samples were prepared for analysis using a simple efficient procedure that consisted of an acidic acetonitrile extraction followed by solid phase extraction cleanup. Two different HRMS acquisition programs were used to analyze the fish extracts. This method detected and identified veterinary drugs including quinolones, fluoroquinolones, avermectins, dyes, and aminopenicillins at residue levels in fish that had been dosed with those compounds. A metabolite of amoxicillin, amoxicillin diketone, was also found at high levels in catfish, trout, and salmon. The method was also used to characterize drug residues in imported fish. In addition to confirming findings of fluoroquinolone and sulfonamide residues that were found by traditional targeted MS methods, several new compounds including 2-amino mebendazole in eel and ofloxacin in croaker were detected and identified. Graphical Abstract Aquacultured samples are analyzed with a high-resolution mass spectrometry screening method to detect and identify unusual veterinary drug residues including ofloxacin in an imported fish.

Review: Application of High Resolution Mass Spectrometry to Monitor Veterinary Drug Residues in Aquacultured Products.

High resolution MS (HRMS) instruments provide accurate mass measurements. With HRMS, virtually an unlimited number of compounds can be analyzed simultaneously because full-scan data are collected, rather than preselected ion transitions corresponding to specific compounds. This enables the development of methods that can monitor for a wide scope of residues and contaminants in aquacultured fish and shellfish including antibiotics, metabolites, and emerging contaminants. Applications of HRMS to the analysis of veterinary drug residues in aquacultured products are summarized in this review including methods for screening, quantifying, and identifying drug residues in these matrixes. The use of targeted, semi-targeted, and nontargeted analysis of HRMS data and the implications to the global aquaculture industry are also reviewed.

Expansion of the Scope of AOAC First Action Method 2012.25--Single-Laboratory Validation of Triphenylmethane Dye and Leuco Metabolite Analysis in Shrimp, Tilapia, Catfish, and Salmon by LC-MS/MS.

Prior to conducting a collaborative study of AOAC First Action 2012.25 LC-MS/MS analytical method for the determination of residues of three triphenylmethane dyes (malachite green, crystal violet, and brilliant green) and their metabolites (leucomalachite green and leucocrystal violet) in seafood, a single-laboratory validation of method 2012.25 was performed to expand the scope of the method to other seafood matrixes including salmon, catfish, tilapia, and shrimp. The validation included the analysis of fortified and incurred residues over multiple weeks to assess analyte stability in matrix at -80°C, a comparison of calibration methods over the range 0.25 to 4 µg/kg, study of matrix effects for analyte quantification, and qualitative identification of targeted analytes. Method accuracy ranged from 88 to 112% with 13% RSD or less for samples fortified at 0.5, 1.0, and 2.0 µg/kg. Analyte identification and determination limits were determined by procedures recommended both by the U. S. Food and Drug Administration and the European Commission. Method detection limits and decision limits ranged from 0.05 to 0.24 µg/kg and 0.08 to 0.54 µg/kg, respectively. AOAC First Action Method 2012.25 with an extracted matrix calibration curve and internal standard correction is suitable for the determination of triphenylmethane dyes and leuco metabolites in salmon, catfish, tilapia, and shrimp by LC-MS/MS at a residue determination level of 0.5 µg/kg or below.

Analysis of chemical contaminants in fish using high resolution mass spectrometry - A review.

High resolution mass spectrometry (HRMS) has become an important tool in environmental and food safety analysis. This review highlights how HRMS has been used to analyze chemical contaminants in fish. Measuring and documenting chemical contaminants in fish serves not only as an indicator of environmental conditions but can also monitor the health of these animals and help protect an important source of human food. The incidence and significance of contaminants including veterinary drugs, human drugs and personal care products, pesticides, persistent organic pollutants, per- and poly fluorinated substances, and marine toxins will be reviewed. The advantage of HRMS over traditional MS is its ability to expand the number of compounds that can be detected and identified. This is true whether HRMS is used for targeted analytes, or more broadly for suspect screening and nontargeted analyses. The classes of compounds, types of fish or seafood, options for data acquisition and analysis, and reports of unexpected findings from recent HMRS methods for chemical contaminants in fish are summarized.

Determination and Identification of Antibiotic Residues in Fruits Using Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS).

Antibiotic residues may be present in fruit products from trees that were treated to combat bacterial diseases such as citrus greening or blight. A liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was developed for the simultaneous determination and identification of streptomycin, kasugamycin, penicillin, and oxytetracycline residues in fruit. Samples were extracted with acidic methanol and separation was optimized for a hydrophilic interaction LC column. A Q-Exactive HRMS instrument was used to obtain product ion spectra for analyte identification. Quantitation was performed with matrix-extracted calibration curves and internal standard correction. The method was tested on many different types of fruit. In general, fortified samples demonstrated acceptable recoveries (82-116%) and reproducibility (<15% RSD). Method detection limits for these analytes were well below the established US EPA tolerance levels. It was also possible to analyze the fruit extracts prepared using this method for additional chemical contaminants using LC-HRMS.

Analysis and Stability Study of Isoeugenol in Aquaculture Products by Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry.

Headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME-GC-MS) offers an alternative analysis method for isoeugenol (an active ingredient in fish sedatives) that avoids the use of organic solvents, simplifies sample preparation, and can be fully automated. This work focuses on developing and evaluating an HS-SPME-GC-MS method for isoeugenol in aquaculture samples and testing the stability of isoeugenol itself. Because of isoeugenol's relatively low volatility, more polar SPME fiber coatings (polyacrylate and polydimethylsiloxane/divinylbenzene) had better performance and the headspace extractions took over 30 min to reach equilibrium. Additionally, it was found that isoeugenol was relatively unstable compared to a deuterated standard (d3-eugenol) in the presence of water. To address this, after the fish samples were homogenized with water, they were heated at 50 °C for 1 h prior to analysis for equilibration. By using the method developed in this work, isoeugenol's detection limits in multiple aquaculture matrices (shrimp, tilapia, and salmon) were in the low ng/g range (<15 ng/g), well below the target testing level (200 ng/g). Additionally, by adding d3-eugenol as an internal standard, excellent linearity (R2 > 0.98), accuracy (97-99% recoveries), and precision (5-13% RSDs) were all achieved.

Laser diode thermal desorption mass spectrometry for the analysis of quinolone antibiotic residues in aquacultured seafood.

RATIONALE: Veterinary drug residue analysis of meat and seafood products is an important part of national regulatory agency food safety programs to ensure that consumers are not exposed to potentially dangerous substances. Complex tissue matrices often require lengthy extraction and analysis procedures to identify improper animal drug treatment. Direct and rapid analysis mass spectrometry techniques have the potential to increase regulatory sample analysis speed by eliminating liquid chromatographic separation. METHODS: Flumequine, oxolinic acid, and nalidixic acid were extracted from catfish, shrimp, and salmon using acidified acetonitrile. Extracts were concentrated, dried onto metal sample wells, then rapidly desorbed (6 s) with an infrared diode laser for analysis by laser diode thermal desorption atmospheric pressure chemical ionization with tandem mass spectrometry (LDTD-MS/MS). Analysis was conducted in selected reaction monitoring mode using piromidic acid as internal standard. RESULTS: Six-point calibration curves for each compound in extracted matrix were linear with r(2) correlation greater than 0.99. The method was validated by analyzing 23 negative samples and 116 fortified samples at concentrations of 10, 20, 50, 100, and 600 ng/g. Average recoveries of fortified samples were greater than 77% with method detection levels ranging from 2 to 7 /g. Three product ion transitions were acquired per analyte to identify each residue. CONCLUSIONS: A rapid method for quinolone analysis in fish muscle was developed using LDTD-MS/MS. The total analysis time was less than 30 s per sample; quinolone residues were detected below 10 ng/g and in most cases residue identity was confirmed. This represents the first application of LDTD to tissue extract analysis. Published 2012. This article is a US Government work and is in the public domain in the USA.

Optimization and validation of a multiclass screening and confirmation method for drug residues in milk using high-performance liquid chromatography/tandem mass spectrometry.

The further optimization and validation of a multiresidue veterinary drug screening method for milk is described. The drug residues of regulatory interest in milk include beta-lactams, sulfonamides, tetracyclines, fluoroquinolones, and macrolides. A previously published procedure has been modified to incorporate new compounds and to collect both screening and confirmatory ion transitions in one acquisition method. Milk samples were extracted with an equal volume of acetonitrile. The samples were then subjected to cleanup with a bonded SPE cartridge and a MW cutoff filter. The SPE protocol was modified to effectively recover a metabolite of flunixin. Established tolerance levels are set for most of these drugs in milk; thus, the screening procedure was semiquantitative, using positive controls for comparison. The positive controls, consisting of extracts from milk fortified with the drugs at their tolerance or safe level, were used to set statistically valid minimum response criteria for unknown samples. This updated method was validated with fortified milk, as well as with milk samples from animals administered veterinary drugs.

Analysis of peptide antibiotic residues in milk using liquid chromatography-high resolution mass spectrometry (LC-HRMS).

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed and validated for the determination of residual peptide antibiotics (bacitracin A, colistin A and B, enramycin A and B, virginiamycin M1 and S1) in bovine milk. LC-HRMS accurate mass data provided the necessary selectivity and sensitivity to quantitate and identify these important antibiotics in milk at residue levels without extensive sample preparation. Milk samples were extracted using 0.3% formic acid in acetonitrile with 0.06% trifluoroacetic acid added to improve peptide recoveries. Sample clean-up was minimal with an aliquot of the extract evaporated and reconstituted in a formic acid/water-acetonitrile mixture and then filtered. LC separation was performed with 0.3% formic acid in the gradient to improve the peak shape and reproducibility of the peptide analytes. A Quadruple-Orbitrap HRMS instrument with full-scan MS1 data collection followed by all-ion-fragmentation was used to obtain the exact mass of the precursor and confirmatory product ions. One advantage of LC-HRMS is that a combination of multiple precursor ions, including different charge states or adducts, can be used for quantification. The method was validated at four concentration levels ranging from 12.5 to 200 ng/g in three types of bovine milk. For bacitracin A, colistins and enramycins, the average recoveries compared to solvent standards ranged between 70% and 120%. Average recoveries for virginiamycin residues in milk extracts were unacceptably high (up to 138%) using solvent standards, but recoveries using matrix-matched calibration were determined to be 90-115%. Matrix effects were found to be less than 25% for the other analytes when internal standard correction was used for the colistins. Intra-day relative standard deviations were generally below 15%. The method detection limits for the peptide antibiotic residues in milk (0.5 to 5.5 ng/g) were well below regulatory levels of concern.

Fast analysis of caffeinated beverages using laser diode thermal desorption mass spectrometry (LDTD-MS/MS).

A rapid method for quantitative caffeine analysis in carbonated and non-carbonated beverages and liquid dietary supplement products was developed based on the direct sample introduction technique of laser diode thermal desorption atmospheric pressure chemical ionisation with tandem mass spectrometry (LDTD-MS/MS). Product samples were diluted with a mixture of methanol, water, and d3-caffeine internal standard. Sample aliquots were filtered, spotted on a metal-lined LDTD microtitre plate, dried, and thermally desorbed for subsequent ionisation and analysis by MS/MS analysis. Each sample required a 6 s desorption, and sample-to-sample analysis time of less than 30 s per sample. Caffeine yielded a linear calibration curve over the range 0.5-100 µg mL-1 (R2 > 0.995). Caffeine recoveries from fortified samples ranged from 97% to 107% with <5% RSD. The caffeine determination was not affected by matrix interferences despite the large range of ingredients, vitamins, sweeteners, extracts, and additives present in the products tested, even though LDTD-MS/MS is a whole-sample desorption technique with no separation of matrix background. The method detection limit was below 0.12 µg mL-1. The method was applied to 33 caffeinated products and LDTD-MS/MS quantitative results closely correlated (R2 > 0.998) with the regulatory standard HPLC-UV method (AOAC Official Method 979.08).

Extended liquid chromatography high resolution mass spectrometry screening method for veterinary drug, pesticide and human pharmaceutical residues in aquaculture fish.

A liquid chromatography high resolution mass spectrometry (LC-HRMS) screening method was developed previously to analyze for veterinary drug residues commonly found in different types of aquaculture products. This method has been further evaluated for its feasibility to detect several other classes of compounds that might also be a concern as possible contaminants in farmed tilapia, salmon, eel and shrimp. Some chemicals could contaminate water sources used in aquaculture production through agricultural run-off. These compounds include several widely used triazine herbicides, organophosphate and carbamate pesticides, as well as various discarded human pharmaceuticals. Other possible contaminants investigated were selected disinfectants, some newer antibiotics, growth promoters, and various parasiticides. The sample preparation consisted of an acidic acetonitrile extraction followed by solid-phase extraction clean-up. Data were collected with a quadrupole-Orbitrap MS using both non-targeted and targeted acquisition. This rapid clean-up procedure and HRMS detection method described previously for veterinary drug residues also worked well for many other types of compounds. Most analytes had screening limit levels between 0.5-10 ng/g in the matrices examined using exact mass identification criteria. The strategy described in this paper for testing the performance of additional analytes will help expand the applicability of the HRMS procedure as aquaculture samples can now be analyzed for a wider range of contaminants.

Determination of oxytocin in a dilute IV solution by LC-MS(n).

The most common drug prescribed to induce labor in the United States is oxytocin, a peptide hormone composed of nine amino acids. Oxytocin is often reconstituted in intravenous (IV) saline solutions at less than 0.05 units ml(-1) (125 ng ml(-1)) to be delivered at 1-4 drops per minute. Existing LC-UV methods for oxytocin do not have sufficient detection limits to quantitate and/or confirm oxytocin in IV solutions without sample concentration. A determinative and confirmatory method for oxytocin was developed using an LC-MS(n) ion trap instrument with an electrospray ionization (ESI) interface in positive ion mode. Separation was achieved on a C-18 column using an isocratic elution of water with 50% acetonitrile (v/v) and water with 0.05% formic acid (v/v) at a flow rate of 250 microl min(-1). Data was acquired from the selected ion monitoring (SIM) of the precursor ion (m/z 1007.3) and MS(2) scans from the collision induced dissociation of m/z 1007.3 at 30% collision energy. In this method, MS(2) full scans were utilized to obtain three structurally significant ions for the unambiguous identification of oxytocin. Calibration standards, prepared in de-ionized water from 0.006 to 0.046 units ml(-1), were linear with an R(2) value of 0.9983. The methods LOD and LOQ were 0.00084 and 0.0029 units ml(-1) (2 and 7 ng ml(-1)), respectively. This LC-MS(n) method was used to determine the amount of oxytocin in a 0.04 units ml(-1) clinical sample that was prepared in 0.9% sodium chloride IV solution.

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.

Dye Residue Analysis in Raw and Processed Aquaculture Products: Matrix Extension of AOAC INTERNATIONAL Official Method 2012.25.

Background: Triphenylmethane dyes and metabolites are known or suspected mutagens and are prohibited in animals intended for human consumption. Despite toxicity, triphenylmethane dyes are used illegally as inexpensive treatments for fungal and parasite infections in aquatic animals. Objective: AOAC INTERNTIONAL Official Method 2012.25 for the LC-MS/MS determination of malachite green, crystal violet, brilliant green, and metabolites leucomalachite green and leucocrystal violet in seafood products was previously validated for finfish (trout, salmon, catfish, and tilapia) and shrimp, but had not been fully validated for other types of aquacultured products such as eel, molluscan shellfish, or frog or for processed seafoods. Methods: Method 2012.25 was applied to a wide scope of raw and processed aquaculture products including Arctic char, barramundi, eel, frog legs, hybrid striped bass, pompano, scallops, seabream, smoked trout, dried shrimp, and highly processed canned eel and dace products. The canned products contained oil, salt, sugar, flavorings, spices, sauces, and/or preservatives. Results: Dyes and metabolites were recovered with >85% accuracy and precision generally <20% relative standard deviation. The method detection limit was ≤0.60 µg/kg and LOQ was <1.0 µg/kg. Compounds were identified in 99% of 330 fortified and incurred samples. Conclusions: This study supports the use of Method 2012.25 for triphenylmethane dye residue analysis in a wide variety of aquacultured and seafood products. Highlights: Method 2012.25 performed well with results consistent with previous validation studies, regardless of presence of additional food ingredients or the type of processing.

Wide-Scope Screening Method for Multiclass Veterinary Drug Residues in Fish, Shrimp, and Eel Using Liquid Chromatography-Quadrupole High-Resolution Mass Spectrometry.

A screening method for veterinary drug residues in fish, shrimp, and eel using LC with a high-resolution MS instrument has been developed and validated. The method was optimized for over 70 test compounds representing a variety of veterinary drug classes. Tissues were extracted by vortex mixing with acetonitrile acidified with 2% acetic acid and 0.2% p-toluenesulfonic acid. A centrifuged portion of the extract was passed through a novel solid phase extraction cartridge designed to remove interfering matrix components from tissue extracts. The eluent was then evaporated and reconstituted for analysis. Data were collected with a quadrupole-Orbitrap high-resolution mass spectrometer using both nontargeted and targeted acquisition methods. Residues were detected on the basis of the exact mass of the precursor and a product ion along with isotope pattern and retention time matching. Semiquantitative data analysis compared MS1 signal to a one-point extracted matrix standard at a target testing level. The test compounds were detected and identified in salmon, tilapia, catfish, shrimp, and eel extracts fortified at the target testing levels. Fish dosed with selected analytes and aquaculture samples previously found to contain residues were also analyzed. The screening method can be expanded to monitor for an additional >260 veterinary drugs on the basis of exact mass measurements and retention times.

Analysis of veterinary drug residues in shrimp: a multi-class method by liquid chromatography-quadrupole ion trap mass spectrometry.

A liquid chromatography-mass spectrometry (LC-MS) method was developed to screen and confirm veterinary drug residues in raw shrimp meat. This method simultaneously monitors 18 drugs of different classes, including oxytetracycline (OTC), sulfonamides, quinolones, cationic dyes, and toltrazuril sulfone (TOLS). The homogenized shrimp meat is extracted with 5% trichloroacetic acid. The extract is further cleaned using polymer-based SPE. A 50 mm phenyl column separates the analytes, prior to analysis with an ion trap mass spectrometer interfaced with an atmospheric pressure chemical ionization source. This method is able to confirm oxytetracycline residues at 200 ng/g, toltrazuril sulfone at 50 ng/g, sulfaquinoxaline at 20 ng/g, and the other 15 drugs at 10 ng/g or lower levels. An estimate of the level of residues can also be made so that only confirmed samples above action levels will be sent for quantitation. The method is validated with both fortified and incurred samples, using multiple shrimp species as well. This multi-class method can provide a means to simultaneously monitor for a wide range of illegal drug residues in shrimp.

Confirmation of diminazene diaceturate in bovine plasma using electrospray liquid chromatography-mass spectrometry.

Diminazene diaceturate is used as a trypanocide for cattle in tropical regions. This paper describes a LC-MS(n) method to confirm the presence of diminazene in bovine plasma. Bound diminazene in plasma samples was freed with dilute phosphoric acid, then concentrated on a bonded C(18) SPE cartridge. The LC-MS(n) method utilized electrospray ionization coupled with an ion trap mass spectrometer. Ions observed in MS(2) and MS(3) product ion spectra, as well as those from the MS(1) spectrum, were monitored. The method was validated with plasma samples fortified with diminazene diaceturate (4-100ng/mL). Diminazene was confirmed in samples fortified with diminazene diaceturate at levels of 6.4ng/mL or higher.