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.

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.

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.

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.

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 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.

Determination of cyanuric acid residues in catfish, trout, tilapia, salmon and shrimp by liquid chromatography-tandem mass spectrometry.

In May 2007, investigators discovered that waste material from the pet food manufacturing process contaminated with melamine (MEL) and/or cyanuric acid (CYA) had been added to hog and chicken feeds. At this time, investigators also learned that adulterated wheat gluten had been used in the manufacture of aquaculture feeds. Concern that the contaminated feed had been used in aquaculture and could enter the human food supply prompted the development of a method for the determination of CYA residues in the edible tissues of fish and shrimp. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed as a sensitive technique for the analysis of CYA in catfish, tilapia, salmon, trout and shrimp tissue. CYA was extracted from ground fish or shrimp with an acetic acid solution, defatted with hexane, and isolated with a graphitic carbon black solid-phase extraction column. Residues were separated from matrix components using a porous graphitic carbon LC column, and then analyzed with electrospray ionization in negative ion mode on a triple quadrupole mass spectrometer. Selective reaction monitoring was performed on the [M-H](-)m/z 128 ion resulting in the product ions m/z 85 and 42. Recoveries from catfish, tilapia and trout fortified with 10-100 microgkg(-1) of CYA averaged 67% with a relative standard deviation (R.S.D.) of 18% (n=107). The average method detection limit (MDL) for catfish, tilapia and trout is 3.5 microgkg(-1). An internal standard, (13)C(3)-labeled CYA, was used in the salmon and shrimp extractions. Average recovery of CYA from salmon was 91% (R.S.D.=15%, n=18) with an MDL of 7.4 microgkg(-1). Average recovery of CYA from shrimp was 85% (R.S.D.=10%, n=13) with an MDL of 3.5 microgkg(-1).

Multiresidue method for the triphenylmethane dyes in fish: Malachite green, crystal (gentian) violet, and brilliant green.

Liquid chromatographic methods are presented for the quantitative and confirmatory determination of crystal violet (CV; also known as gentian violet), leucocrystal violet (LCV), brilliant green (BG), and leucobrilliant green (LBG) in catfish. LCV and LBG were oxidized to the chromic CV and BG by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and residues were measured as the combined CV+/-LCV and BG+/-LBG. These methods are extensions of published methods for malachite green (MG) analysis to allow simultaneous determination of MG, CV, and BG. Residues were extracted from muscle with ammonium acetate buffer and acetonitrile, and extracts cleaned up using dichloromethane partitioning and solid-phase extraction. Extracts were analyzed by liquid chromatography with visible detection (LC-VIS). The method was validated for catfish fortified with LCV over the range 0.25-10 ngg(-1) and CV at 2 ngg(-1). Average recoveries were 90.6% (+/-8.1% R.S.D., n=45) for LCV and 84.4% (+/-4.2% R.S.D., n=6) for CV. The average recovery for samples fortified with BG or LBG over the range 0.5-10 ngg(-1) was 67.2% (+/-14.8% R.S.D., n=31). CV and BG were confirmed in fish extracts by ion trap LC-mass spectrometry (LC-MS(n)) with no discharge-atmospheric pressure chemical ionization. Average LC-MS(n) recoveries were 96.5, 96.6, and 70.2% for samples fortified with CV, LCV, and BG or LBG. The limits of detection for CV, BG, and MG were in the range of 0.07-0.24 ngg(-1) (ppb) for the two different instrumental methods. This methodology was applied to the analysis of catfish treated with CV and BG.

Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass spectrometry.

Pet and food animal (hogs, chicken, and fish) feeds were recently found to be contaminated with melamine (MEL). A quantitative and confirmatory method is presented to determine MEL residues in edible tissues from fish fed this contaminant. Edible tissues were extracted with acidic acetonitrile, defatted with dichloromethane, and cleaned up using mixed-mode cation exchange solid-phase extraction cartridges. Extracts were analyzed by liquid chromatography with tandem mass spectrometry with hydrophilic interaction chromatography and electrospray ionization in positive ion mode. Fish and shrimp tissues were fortified with 10-500 microg/kg (ppb) of MEL with an average recovery of 63.8% (21.5% relative standard deviation, n = 121). Incurred fish tissues were generated by feeding fish up to 400 mg/kg of MEL or a combination of MEL and the related triazine cyanuric acid (CYA). MEL and CYA are known to form an insoluble complex in the kidneys, which may lead to renal failure. Fifty-five treated catfish, trout, tilapia, and salmon were analyzed after withdrawal times of 1-14 days. MEL residues were found in edible tissues from all of the fish with concentrations ranging from 0.011 to 210 mg/kg (ppm). Incurred shrimp and a survey of market seafood products were also analyzed as part of this study.

Multi-class, multi-residue liquid chromatography/tandem mass spectrometry screening and confirmation methods for drug residues in milk.

This paper describes the development and optimization of a multi-residue veterinary drug screening method for whole milk. The drug residues of regulatory interest in milk include beta-lactams, sulfonamides, tetracyclines, fluoroquinolones, and macrolides. Milk samples were extracted with acetonitrile and the samples were then subjected to a clean-up procedure using a bonded solid-phase extraction cartridge and a molecular weight cut-off filter. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) triple quadrupole electrospray methods were developed to monitor for the drugs in milk. Since established tolerance levels are set for most of these drugs in milk, the initial screening procedure was semi-quantitative, where samples were compared to the response of a positive control. The positive control, consisting of an extract from a portion of milk fortified with the drugs at half their allowed levels, was used to set the laboratory's minimum response criteria for unknown samples. Confirmatory analyses, with additional ion transitions for each residue, were performed on the same extracts.

Confirmation of sulfamethazine, sulfathiazole, and sulfadimethoxine residues in condensed milk and soft-cheese products by liquid chromatography/tandem mass spectrometry.

A liquid chromatography/tandem mass spectrometry method (LC/MS/MS) is described for the simultaneous detection of 3 sulfonamide drug residues at 1.25 ppb in condensed milk and soft-cheese products. The 3 sulfonamide drugs of interest are sulfathiazole (STZ), sulfamethazine (SMZ), and sulfadimethoxine (SDM). The method includes extraction of the product with phosphate buffer, centrifugation of the diluted product, and application of a portion of the extract onto a polymeric solid-phase extraction cartridge. The cartridge is washed with water, and the sulfonamides are eluted with methanol. After evaporation, the residue is dissolved in 0.1% formic acid solution, and the solution is filtered before analysis by LC/MS/MS. The LC/MS/MS program involved a series of time-scheduled selected-reaction monitoring transitions. The transitions of MH+ to the common product ions at m/z 156, 108, and 92 were monitored for each residue. In addition, SMZ and SDM had a fourth significant and unique product ion transition that could be measured. Validation was performed with control and fortified-control condensed bovine milk with 2.5, 5, and 10 ppb sulfonamides. This method was applied to imported flavored and unflavored condensed milk and cream cheese bars. The presence of STZ and SMZ residues was confirmed in 3 out of 6 products.

Interlaboratory comparison of methods for the determination of incurred tilmicosin residues in bovine liver.

The objective of this study was to compare 2 methods for the determination of tilmicosin residues in bovine liver samples. Three laboratories participated in the comparison of the 2 methods. The first method was described in a New Animal Drug Application (NADA 140-929), and the second was a modification of that method in which hexane was substituted for carbon tetrachloride in one cleanup step. Each of the 3 laboratories analyzed subsamples of 10 bovine livers containing incurred tilmicosin. Residues ranged from 2.3 to 81 ppm tilmicosin in the 10 liver samples with an 11.8% relative standard deviation obtained by using both methods. In addition, fortified-control liver tissue samples were analyzed concurrently with tissues containing incurred residues by using the modified method in one of the laboratories. The fortification levels ranged from 0.3 to 112 ppm, with recoveries ranging from 76 to 92%. The results from the 3 laboratories were comparable, indicating that the modified method was not only as effective as the original NADA method, but also more desirable because of the change to a less hazardous solvent.

Confirmation of phenylbutazone residues in bovine kidney by liquid chromatography/mass spectrometry.

A confirmatory method is described for phenylbutazone (PB) residues in bovine kidney tissue. Ground kidney tissue is diluted with water, and the mixture is made basic with 25% ammonium hydroxide in water; the lipids are extracted with ethyl and petroleum ethers. The ether layer is discarded, and the tissue is acidified with 6N HCl. PB residues are extracted with tetrahydrofuranhexane (1 + 4). The extract is passed through a silica solid-phase extraction column, and the eluate is evaporated to dryness. The residue is dissolved in acidified acetonitrile-water-acetic acid (50 + 49.4 + 0.6). A single quadrupole mass spectrometer coupled to a liquid chromatograph with an electrospray interface is used to confirm the identity of the PB residues in the kidney extract. Negative-ion detection with selected-ion monitoring of 4 ions is used. Sets of control and fortified-control kidney tissues (at 50, 100, and 200 ppb PB) and several kidney tissue field samples were analyzed for method validation. The method was tested further during the course of a survey to determine the incidence of PB residues in bovine kidney samples obtained from slaughterhouses across the country. In addition, the method was tested for use with an ion-trap mass spectrometer coupled to a liquid chromatograph, which allowed confirmation of PB at lower levels (5-10 ppb) in kidney tissue.