Multiclass, Multiresidue Method for the Quantification and Confirmation of 112 Veterinary Drugs in Game Meat (Bison, Deer, Elk, and Rabbit) by Rapid Polarity Switching Liquid Chromatography-Tandem Mass Spectrometry.

An analytical program for multiclass, multiresidue residue analysis to qualitatively and quantitatively determine veterinary drug compounds in game meats by LC-MS/MS has been developed and validated. The method was validated for the analysis of muscle from bison, deer, elk, and rabbit to test for 112 veterinary drug residues from the following drug classes: ß-agonists, anthelmintics, anti-inflammatory drugs, corticosteroids, fluoroquinolones, ß-lactams, macrolides, nitroimidazoles, phenicols, polypeptides, sulfonamides, tetracyclines, thyreostats, and tranquilizers. Muscle was extracted using a simple and quick procedure based on a solvent extraction with 80% ACN/water and sample cleanup with dispersive solid-phase extraction. The compounds of interest were separated using a Waters HSS T3 column and detected by tandem mass spectrometry with rapid polarity switching to detect both negatively and positively charged ions in a single run. Recoveries were calculated using extracted matrix-matched calibration curves for each type of matrix. The average accuracy of fortified compounds ranged from 95.6 to 101% at the target quantitative validation level in the four matrices. The method was also validated as a qualitative screening method where all sample responses were compared with a single extracted matrix-matched calibrant at the target testing level (5 or 25 ng/g). Samples demonstrating a presumptive positive above the threshold value were re-extracted and analyzed with a five-point matrix-matching extracted calibration curve. Since the beginning of this survey program, 360 samples have been analyzed for veterinary drug residues in game meats. Antibiotic or tranquilizer residues have been identified in deer (chlortetracycline, haloperidol, and tulathromycin) and rabbit (sulfadiazine).

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.

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.

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.

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.

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.

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.

Determination and Confirmation of the Antiviral Drug Amantadine and Its Analogues in Chicken Jerky Pet Treats.

In this study, we investigated two methods for the detection of antiviral compounds in chicken jerky pet treats. Initially, a screening method developed to detect many different chemical contaminants indicated the presence of amantadine, 1, in some pet treats analyzed. A second antiviral-specific method was then developed for amantadine and its analogues, rimantadine, 2, and memantine, 3. Both methods used an acidic water/acetonitrile extraction. The antiviral-specific method also included a dispersive sorbent cleanup. Analytes were detected and identified by LC-MS (ion trap and Orbitrap) instruments. The antiviral-specific method was validated by analyzing matrix blanks and fortified samples (2.5-50 µg/kg levels). Average recoveries for amantadine (using a deuterated internal standard) in fortified samples ranged from 76 to 123% with relative standard deviations of ≤12%. Amantadine was detected and identified in suspect chicken jerky pet treat samples at levels ranging from <2.5 µg/kg to over 600 µg/kg. Rimantadine and memantine were not detected in any samples.

Determination of Triphenylmethane Dyes and Their Metabolites in Salmon, Catfish, and Shrimp by LC-MS/MS Using AOAC First Action Method 2012.25: Collaborative Study.

A collaborative study was conducted to evaluate the 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. Fourteen laboratories from the United States, Canada, and the European Union member states participated in the study including national and state regulatory laboratories, university and national research laboratories, and private analytical testing laboratories. A variety of LC-MS/MS instruments were used for the analysis. Each participating laboratory received blinded test samples in duplicate of salmon, catfish, and shrimp consisting of negative control matrix; matrix fortified with residues at 0.42, 0.90, and 1.75 µg/kg; and samples of incurred matrix. The analytical results from each participating laboratory were evaluated for both quantitative residue determination and qualitative identification of targeted analytes. Results from statistical analysis showed that this method provided excellent trueness (generally ≥90% recovery) and precision (RSDr generally ≤10%, HorRat<1). The Study Directors recommend Method 2012.25 for Final Action status.

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.

Application of single-stage Orbitrap mass spectrometry and differential analysis software to nontargeted analysis of contaminants in dog food: detection, identification, and quantification of glycoalkaloids.

The objective of this study was to perform a preliminary investigation of the nontargeted search and quantitative capabilities of a single-stage Exactive High-Resolution Mass Spectrometer (HRMS). To do this, the instrument and its associated software performed a non-targeted search for deleterious substances in a dog food sample suspected of causing gastrointestinal problems in dogs. A single-stage Orbitrap/high-performance liquid chromatography method and differential expression analysis software (Sieve) was used to detect and identify, and subsequently quantify, nontargeted compounds occurring only in the suspect dog food sample. When combined with an online database (ChemSpider), a preliminary identification of one of the nontargeted compounds was determined to be potato glycoalkaloids. The diagnostic product ion ratios and quantitative data accuracy generated by the single-stage Orbitrap MS were shown to be similar to results obtained using a triple quadrupole LC-MS/MS. Additionally, the ability of the single-stage Orbitrap instrument to provide precursor and product ion accurate masses and isotope patterns was also investigated.

A rapid liquid chromatography determination of free formaldehyde in cod.

A rapid method for the determination of free formaldehyde in cod is described. It uses a simple water extraction of formaldehyde which is then derivatised with 2,4-dinitrophenylhydrazine (DNPH) to form a sensitive and specific chromophore for high-performance liquid chromatography (HPLC) detection. Although this formaldehyde derivative has been widely used in past tissue analysis, this paper describes an improved derivatisation procedure. The formation of the DNPH formaldehyde derivative has been shortened to 2 min and a stabilising buffer has been added to the derivative to increase its stability. The average recovery of free formaldehyde in spiked cod was 63% with an RSD of 15% over the range of 25-200 mg kg(-1) (n = 48). The HPLC procedure described here was also compared to a commercial qualitative procedure - a swab test for the determination of free formaldehyde in fish. Several positive samples were compared by both methods.

Analysis of sulfonamides, trimethoprim, fluoroquinolones, quinolones, triphenylmethane dyes and methyltestosterone in fish and shrimp using liquid chromatography-mass spectrometry.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method is described for the detection and identification of 26 veterinary drugs in fish and other aquaculture products. The analytes include: 13 sulfonamides, trimethoprim, 3 fluoroquinolones, 3 quinolones, 3 triphenylmethane dyes, 2 leuco dye metabolites, and 1 hormone. In this method, tissue is mixed with EDTA-McIlvaine buffer, double-extracted with acetonitrile, p-toluenesulfonic (p-TSA) acid and N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD), and analyzed using LC-MS/MS. Inclusion of p-TSA and TMPD in the extraction procedure was critical for simultaneous analysis of dyes with the other groups of veterinary drugs. The proposed procedure was validated as both a quantitative analysis method and as a semi-quantitative screening method for multiple fish and shrimp matrices. The method was applied to eight types of fish (catfish, eel, pangasius, sablefish, tilapia, swai, salmon, and trout) and shrimp at the appropriate level of concern: 10ng/g for sulfonamides, trimethoprim, and quinolones, 5ng/g for fluoroquinolones, 1ng/g for dyes and their metabolites, and 0.4ng/g for methyltestosterone.

Challenges in implementing a screening method for veterinary drugs in milk using liquid chromatography quadrupole time-of-flight mass spectrometry.

High resolution mass spectrometry (HRMS) is a valuable tool for the analysis of chemical contaminants in food. Our laboratory has successfully developed methods to screen for veterinary drug residues using liquid chromatography quadrupole time-of-flight (Q-TOF). There have been, however, significant challenges as methods are transferred from the development stage to routine regulatory analysis. Having experimental retention time and product ion information for analytes greatly facilitates the ability to determine if residues found by the HRMS searching software are false detects. These data were collected for over 200 veterinary drug residues using LC Q-TOF MS. The screening levels of detection for over 150 veterinary drug residues in milk were determined, and over half of those tested can be detected at concentrations of 10 ng/mL or less; 72% can be found in milk when present at 100 ng/mL. Tentative identification of the product ions from these analytes is also presented.

Analysis of stilbene residues in aquacultured finfish using LC-MS/MS.

This analytical method was developed for the determination of three stilbene residues, diethylstilbestrol (DES), dienestrol (DEN), and hexestrol (HEX), in edible tissues of finfish including catfish, salmon, trout, and tilapia. Fortified fish samples were extracted with acetonitrile and further cleaned up using silica solid phase extraction columns. Stilbene residues were separated from matrix components by reversed phase high-performance liquid chromatography on a C8 column and analyzed using a tandem mass spectrometer with negative electrospray ionization. The overall average residue recoveries using post-fortified matrix-matched calibrants were 119, 99, and 104% with %RSDs of 18, 11, and 15% for DEN, DES, and HEX, respectively. Method detection limits of DEN, DES, and HEX in each matrix were found to be at or below 0.21 ng/g, and the limit of quantification averaged 0.3 ng/g and ranged from 0.18 to 0.65 ng/g for all analytes in all matrices.

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.

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.

Bioaccumulation of cyanuric acid in edible tissues of shrimp following experimental feeding.

Due to concerns that cyanuric acid (CYA)-contaminated feed had been used in aquaculture and could enter the human food chain, a method to quantify CYA residues in the edible tissues of fish and shrimp was previously developed and validated. This paper provides further data on the deliberate feeding of CYA to shrimp to determine the extent of residue accumulation in edible tissue. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for the analysis of CYA in shrimp tissue. Edible tissue of shrimp fed 1666 or 3333 mg kg⁻¹ CYA in their diet (approximately 55 and 124 mg kg⁻¹ body weight) contained 0.767 and 0.406 mg kg⁻¹ CYA, respectively. The residue levels are below the World Health Organization (WHO) tolerable daily intake level for CYA and are generally considered unlikely to pose a human health risk.

Analysis of aminoglycoside residues in bovine milk by liquid chromatography electrospray ion trap mass spectrometry after derivatization with phenyl isocyanate.

A derivatization procedure using phenyl isocyanate was adapted to liquid chromatography ion trap mass spectrometry (LC-MS(n)) for confirmation and quantification of aminoglycoside residues in milk. Aminoglycoside residues were extracted from milk with acid and isolated from the matrix with a weak cation exchange solid-phase extraction cartridge. After isolating the compounds from the milk, derivatives of gentamicin, neomycin, and tobramycin were formed by reacting the drugs with phenyl isocyanate in the presence of triethylamine. The analytes were separated using a dilute formic acid/acetonitrile gradient on a reversed-phase LC column. The derivatized compounds were analyzed using positive ion electrospray LC-MS(n) with ion trap detection. Product ion spectra were generated from the derivatized protonated molecules. Specific ion transitions were evaluated for quantitative determination and qualitative confirmation of residues in milk. Using this procedure, residues were qualitatively confirmed in milk samples fortified with gentamicin and neomycin at levels ranging from 15 to 300 ng mL(-1). Gentamicin has four major components that were successfully separated and confirmed independently; for quantitative determination the peak areas from the four analogs were summed. Tobramycin was added as an internal standard for quantitation to mitigate the effects of matrix ion suppression and variable recoveries. Overall recoveries for this method ranged from 80% to 120% with relative standard deviations of less than 25%. The method detection limits are 9.8 ng mL(-1) for NEO and 12.8 ng mL(-1) for total GEN residues.