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.

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.

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.

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.

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.

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.

The 49th annual Florida Pesticide Residue Workshop.

The papers in this special issue of Journal of Agricultural and Food Chemistry were originally presented at the 49th annual Florida Pesticide Residue Workshop (FPRW). The FPRW is an annual meeting for scientists specializing in trace level analysis of pesticides, veterinary drug residues, and other chemical contaminants in food, animal feed, and environmental samples.