Determination of usnic acid in lichen toxic to elk by liquid chromatography with ultraviolet and tandem mass spectrometry detection

Usnic acid is unambiguously confirmed by tandem mass spectrometry (MS/MS) in tumbleweed shield lichen, Xanthoparmelia chlorochroa. The lichen contains 2% usnic acid by liquid chromatography with UV quantification at 282 nm. The UV linear range for usnic acid quantification is from its 4 ng limit of detection to 2 microg injected. UV signal saturation is recognized by distortion of the usnic acid UV spectrum. Positive ion electrospray-tandem mass spectrometry offers no similar means to recognize quantification data recorded above the linear range of electrospray. Electrospray ionization capacity and matrix effects limit the reliability of tandem mass spectrometry quantification. The combination of UV quantification and MS confirmation provides a reliable analytical method for measuring usnic acid levels in plant material.     

Multiresidue assay for benzimidazole anthelmintics by liquid chromatography and confirmation by gas chromatography/selected-ion monitoring electron impact mass spectrometry

A multiresidue method utilizing all-disposable labware has been developed for 8 benzimidazole anthelmintics from ovine, bovine, and swine muscle and liver tissues. After an initial extraction with ethyl acetate and subsequent evaporation, a 3-component extraction using hexane, ethanol, and 0.2N HCl was used for final cleanup. Clean extracts were produced for separation and determination by reverse-phase liquid chromatography at 298 nm, using methanol and aqueous buffer as mobile phase. A synthesized internal standard, 2-(n-butylmercapto)benzimidazole, was used for quantitation of all drugs. Results are included along with statistical information verifying the performance of the method. Spiked control tissues and incurred drug tissues were used for an intralaboratory study with a concentration range of 50-1470 ppb. A series of standard curves at 0, 50, 100, and 200 ppb were analyzed. Overall recovery at the 100 ppb level averaged 92% (CV 8%) in liver tissues, across all 3 species and 88% (CV 5%) in muscle tissues across all 3 species. Results were confirmed by gas chromatography/mass spectrometry with acid hydrolysis of the remaining extract in 2N HCl followed by re-extraction of the amine and derivatization to the tert-butyldimethylsilyl derivative. The anthelmintics were identified by gas chromatography/selected ion monitoring electron-impact mass spectrometry. Ion ratio measurements were taken and compared to standard material. CVs averaged 10% or less for all drugs tested.     

A rapid confirmatory method for analyzing tetracycline antibiotics in bovine, swine, and poultry muscle tissues: matrix solid-phase dispersion with heated water as extractant followed by liquid chromatography-tandem mass spectrometry

A rapid, specific, and sensitive procedure for determining four widely used tetracycline antibiotics and three related epimers in bovine, swine, and poultry muscle tissues is presented. The method is based on the matrix solid-phase dispersion technique with heated water as the extractant followed by liquid chromatography (LC)-tandem mass spectrometry (MS) equipped with an electrospray ion source. Target compounds were extracted from tissues with 5 mL of water heated at 70 degrees C. After acidification and filtration, 100 microL of the aqueous extract was injected in the LC column. MS data acquisition was performed in the multireaction monitoring mode, selecting two precursor ion to product ion transitions for each target compound. Heated water appeared to be an excellent extractant, since the absolute recovery data ranged between 70 and 78%. The accuracy of the method was determined at three spike levels, using minocycline as a surrogate analyte, in any different kind of muscle tissues considered and varied between 88 and 109% with relative standard deviations ranging between 3 and 11%. Limits of quantification were estimated to range between 1 (chlortetracycline) and 9 ng/g (4-epioxytetracycline), based on a signal-to-noise ratio of 10, and are well below the tolerance levels set by the European Union. The effects of the extraction temperature, volume of the extractant, and washing of the material supporting the biological matrix with ethylenediamine tetraacetic disodium salt on the analyte recovery were studied.     

Immunochemical screening and liquid chromatographic-tandem mass spectrometric confirmation of drug residues in edible tissues of calves injected with a therapeutic dose of the synthetic glucocorticoids dexamethasone and flumethasone

A field study was performed to assess the drug residue level in edible tissues after a therapeutic application of the synthetic glucocorticoids dexamethasone and flumethasone. Three diseased calves were injected intramuscularly with a commercial batch of dexamethasone esters and slaughtered 72 h after treatment. Another three calves were injected intramuscularly with an aqueous flumethasone preparation and slaughtered 24 h later. Residues of synthetic glucocorticoids in liver, muscle, kidney, and urine were assessed by competitive enzyme immunoassay. All dexamethasone concentrations exceeded the maximal residue level of 0.75 microg/kg in muscle and kidney and 2 microg/kg in the liver. The presence of both dexamethasone and flumethasone in the liver was confirmed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). These results indicate that liver tissue provides a suitable matrix to monitor the presence of illegal residues of synthetic glucocorticoids in slaughtered animals.     

Identification and quantification of astaxanthin esters in shrimp (Pandalus borealis) and in a microalga (Haematococcus pluvialis) by liquid chromatography-mass spectrometry using negative ion atmospheric pressure chemical ionization

Negative ion liquid chromatography-atmospheric pressure chemical ionization mass spectrometry [negative ion LC-(APCI)MS] was used for the identification of astaxanthin esters in extracts of commercial shrimp (Pandalus borealis) and dried microalga (Haematococcus pluvialis) samples. A cleanup step using a normal phase solid phase extraction (SPE) cartridge was applied prior to analysis. Recovery experiments with astaxanthin oleate as model compound proved the applicability of this step (98.5 +/- 7.6%; n = 4). The assignment of astaxanthin esters in negative ion LC-(APCI)MS was based on the detection of the molecular ion (M*-) and the formation of characteristic fragment ions, resulting from the loss of one or two fatty acids. Quantification of individual astaxanthin esters was performed using an astaxanthin calibration curve, which was found to be linear over the required range (1-51 micromol/L; r2 = 0.9996). Detection limits, based on the intensity of M*-, a signal-to-noise ratio of 3:1, and an injection volume of 20 microL, were estimated to be 0.05 microg/mL (free astaxanthin), 0.28 microg/mL (astaxanthin-C16:0), and 0.78 microg/mL (astaxanthin-C16:0/C16:0), respectively. This LC-(APCI)MS method allows for the first time the characterization of native astaxanthin esters in P. borealis and H. pluvialis without using time-consuming isolation steps with subsequent gas chromatographic analyses of fatty acid methyl esters. The results suggest that the pattern of astaxanthin-bound polyunsaturated fatty acids of P. borealis does not reflect the respective fatty acid pattern found in triacylglycerides. Application of the presented LC-(APCI)MS technique in common astaxanthin ester analysis will forestall erroneous xanthophyll ester assignment in natural sources.     

Direct determination of paclobutrazol residues in pear samples by liquid chromatography-electrospray tandem mass spectrometry

A rapid and sensitive liquid chromatography/electrospray ionization/tandem mass spectrometry (LC-ESI-MS-MS) method has been developed for the determination of the plant growth regulator paclobutrazol in pear samples. Extraction was performed with methanol by using a high-speed blender Ultra-Turrax, and 10 microL of pear extract was directly injected in the LC-ESI-MS-MS system without any previous sample treatment. The highest sensitivity of the method was achieved under MS-MS conditions obtaining a limit of detection of 0.7 microg/kg and a quantification limit of 5 microg/kg, with a run time of only 5.5 min. Recoveries for paclobutrazol from spiked pear samples at 0.005, 0.05, and 0.5 mg/kg were around 82-102% with relative standard deviations between 2 and 7%. The method was applied to real treated and untreated samples of pears, using quality control samples as a evaluation of the method reliability. Two MS-MS transitions were selected, one for quantification (294 > 70) and the other for confirmation of the analyte (296 > 70). All the experiments were performed in compliance with good laboratory practices.     

Development of an indirect competitive ELISA for the detection of furazolidone marker residue in animal edible tissues

Due to its carcinogenicity and mutagenicity, furazolidone has been prohibited completely from being used in food animal production in the world since 1995. To monitor the illegal abuse of furazolidone, a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the determination of tissue-bound furazolidone metabolite 3-amino-2-oxazolidone (AOZ). The highly specific antibody was targeted for PAOZ, the benzaldehyde derivative of AOZ. The 50% inhibition values (IC 50) of 0.91 microg/L for AOZ was achieved with the most sensitive antibody Ab-B1 by altering ELISA conditions. In the ELISA, sample extraction and cleanup were performed by an is MAX cartridge following combined hydrolysis of the tissue-bound AOZ and derivatization of the homogenized tissues with benzaldehyde. The limits of detection (LOD) calculated from the analysis of 20 known negative tissue samples (swine liver, swine muscle, chicken liver, chicken muscle,and fish muscle) were 0.3-0.4 microg/kg (mean+3 SD). Recoveries of AOZ fortified at the levels of 0.4, 1, and 5 microg/kg ranged from 55.8 to 96.6% in the tissues. The coefficients of variation were less than 20% over the range of AOZ concentrations studied. The linear detection range was between 0.1 and 25.6 microg/L. Validation of the ELISA method with swine muscle and liver from furazolidone-treated pigs was carried out using HPLC, resulting in a similar correlation in swine muscle (r=0.99) and in swine liver (r=0.98). The results suggest that this ELISA is a specific, accurate, and sensitive method of detecting AOZ residues in animal edible tissues.     

Analysis of alternariol and alternariol monomethyl ether on flavedo and albedo tissues of tangerines (Citrus reticulata) with symptoms of alternaria brown spot

A method was developed for the quantification of alternariol and alternariol monomethyl ether on tangerines with and without symptoms of Alternaria brown spot disease. The method employs solid-phase extraction for cleanup, followed by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) for detection. This method was validated on flavedo (exocarp or epicarp, exterior yellow peel) and on albedo tissue (mesocarp, interior white peel). An excellent linearity over a range of 0.50-20.0 mg/kg was achieved, with r2 >or= 0.997. The limits of detection (LOD) and quantification (LOQ) were fewer than 0.13 and 0.50 microg/kg, respectively. The relative standard deviations (RSDs) were 相似文献   

Simultaneous determination of trace levels of 10 quinolones in swine, chicken, and shrimp muscle tissues using HPLC with programmable fluorescence detection

A HPLC method using a modified sample preparation procedure was optimized and validated for the quantification of 10 quinolones (QNs), including marbofloxacin, ciprofloxacin, norfloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, and flumequine, in swine, chicken, and shrimp tissues. In this method, only a small mass (相似文献   

A rapid spectrofluorometric screening method for enrofloxacin in chicken muscle

A simple spectrofluorometric method was developed for screening enrofloxacin (ENRO) in chicken muscle. A single-step extraction with acidic acetonitrile gave the best results without further cleanup. Following centrifugation the supernatants were excited at 324 nm and the emission was measured at 442 nm. Using this procedure, 18 chicken breast samples from 3 producers were tested. The results showed background signal levels significantly lower than those corresponding to 300 microg/kg ENRO, the FDA approved tolerance level. Statistical treatment of these data established a threshold which can be used in subsequent screening of ENRO at the tolerance level. The calibration curve revealed a satisfactory linear relationship (R(2) = 0.9991) in a range of 0-700 microg/kg ENRO in fortified chicken breast. ENRO-incurred samples were examined using this approach, and the results agreed with those obtained from more extensive separation followed by high-performance liquid chromatography. Because the threshold can be set at the 3 sigma limit, reliable screening can be accomplished with an error rate of less than 0.26%. Based on this investigation, a high-throughput screening method for ENRO in chicken tissue is proposed.     

Determination of five macrolide antibiotic residues in eggs using liquid chromatography/electrospray ionization tandem mass spectrometry

A method using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) for the determination of trace levels of five macrolide antibiotics (spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin) in eggs is presented. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two or three fragment ion transitions to provide a high degree of sensitivity and specificity for both quantification and confirmation. Matrix-matched standard calibration curves were used to achieve the best accuracy of the method. A fully nested experimental design was used to study the measurement uncertainty arising from intermediate precision and trueness or proportional bias. The overall recoveries, that is, those determined by the nested experiments, of spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin at fortified levels of 60, 100, 200, and 300 microg/kg were 96.8, 98.2, 98.3, 98.8, and 95.4%, respectively. The LC/ESI-MS/MS method detection limits (S/N > or = 3:1) of five macrolides were <1.0 microg/kg.     

Residue depletion of cefquinome in swine tissues after intramuscular administration

A high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed for the detection of cefquinome (CEQ) residues in swine tissues. The limit of detection (LOD) of the method was 5 ng g(-1) for muscle and 10 ng g(-1) for fat, liver, and kidney. Mean recoveries of CEQ in all fortified samples at a concentration range of 20-500 ng g(-1) were 80.5-86.0% with coefficient of variation (CV) below 10.3%. Residue depletion study of CEQ in swine was conducted after five intramuscular injections at a dose of 2 mg kg(-1) of body weight with 24 h intervals. CEQ residue concentrations were detected in muscle, fat, liver, and kidney using the HPLC-UV method at 265 nm. The highest CEQ concentration was measured in kidney tissue during the study period, indicating that kidney was the target tissue for CEQ. CEQ concentrations in all examined tissues were below the accepted maximum residue limit (MRL) recommended by the Committee for Veterinary Medical Products of European Medical Evaluation Agency (EMEA) at 3 days post-treatment.     

Quantitative confirmation of dimetridazole and ipronidazole in swine feed by capillary gas chromatography/mass spectrometry with multiple ion detection

Extracts from 4 types of swine feed containing 0.11 ppm each of dimetridazole (DMZ) and ipronidazole (IPR) were analyzed by capillary gas chromatography/mass spectrometry (GC/MS) using multiple ion detection (MID) techniques. We demonstrate in this paper that the quantitative results obtained by capillary GC/MS with MID are comparable for both compounds to results obtained by liquid chromatography and have a lower coefficient of variation for DMZ. Moreover, consistency in the ion ratios (5 ions in DMZ and 6 ions in IPR) permits identification of these compounds by electron ionization MS.     

Confirmatory method for sulfamethazine residues in cattle and swine tissues, using gas chromatography--chemical ionization mass spectrometry

A gas chromatographic (GC) method has been reported for the determination of sulfamethazine residues in cattle and swine tissues. The extracts from this procedure were found to be directly amenable to examination by gas chromatography-mass spectrometry (GC-MS), allowing positive confirmation of an apparent residue of sulfamethazine. Chemical ionization mass spectrometry (CIMS) was chosen as the MS technique because it generated an ion indicative of intact sulfamethazine and fragment ions indicative of the amine functionality and sulfanil moiety. Positive ion (PI) chemical ionization mass spectrometry was adequate by itself for a confirmatory technique. Negative ion (NI) chemical ionization mass spectrometry alone could not be used for the confirmatory analysis of sulfamethazine, but it did offer a means to check the quantitative data from the positive ion analyses and provided complementary confirmatory data. Satisfactory recoveries were obtained for sulfamethazine in swine and cattle tissues at the tolerance level of 0.1 ppm. Apparent sulfamethazine residues in control tissues were less than 0.01 ppm.     

Determination of pesticides in apple-based infant foods using liquid chromatography electrospray ionization tandem mass spectrometry

A liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated to quantify and confirm trace levels of 13 pesticides including aldicarb sulfoxide, aldicarb sulfone, oxamyl, methomyl, formetanate, 3-hydroxycarbofuran, carbendazim, thiabendazole, aldicarb, propoxur, carbofuran, carbaryl, and methiocarb in apple-based infant foods such as apple sauces, apples and strawberries, apples and blueberries, and apples and plums. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring of two fragment ion transitions to provide a high degree of sensitivity and selectivity for both quantification and confirmation. LC/ESI-MS/MS quantitative results were significantly affected by matrices, and thus, the standard addition was employed to compensate for the matrix effects to achieve the best accuracy of the method. Recoveries of 13 pesticides, spiked at 5.0, 25.0, and 45.0 microg/kg, were around 100% using the LC/ESI-MS/MS standard addition. The method detection limits (S/N > or = 3:1) of 13 pesticides were less than 0.2 microg/kg.     

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.     

Quantitative determination and structure elucidation of type A- and B-trichothecenes by HPLC/ion trap multiple mass spectrometry.

A method is presented for the quantification and structure confirmation of trichothecenes in wheat by high-performance liquid chromatography combined with multiple mass spectrometry (MS(n)()). Nine type A- and B-trichothecenes were determined (nivalenol, deoxynivalenol, fusarenon-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin). Extraction was carried out with acetonitrile/water. The extract was purified on a MycoSep column. Quantification was based on an internal standard and atmospheric pressure chemical ionization in the positive ion mode. Recoveries from spiked wheat were in the range of 80-106% at levels of 500 ppb. The limits of quantification for the whole method were between 10 and 100 ppb. Ion adduct formation with ammonium and acetate ions and MS(n) experiments provided information about substitution and fragmentation behavior of the mycotoxins. A scheme has been established for the partial structure elucidation of type A- and B-trichothecenes in fungal cultures.     

Simultaneous detection of residues of 25 β₂-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry

A sensitive method has been developed for the simultaneous determination of residues of 25 β?-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry (HPLC-LIT-MS). This method is based on a new procedure of hydrolysis and extraction by 5% trichloracetic acid, and then cleaned up by mixed strong cation exchange (MCX) cartridges coupled with a novelty cleanup step by methanol. Methanol and 0.1% formic acid were used as mobile phases for gradient elution, while a Supelco Ascentis Express Rp-Amide column was used for LC separation. ESI positive ion scan mode was used with consecutive reaction monitoring (CRM, MS3). Nine β?-agonists labeled by the deuterium isotope were used as internal standards for quantification. The linear ranges of 48 analytes were from 5 to 200 μg/L; the coefficient of correlation was not less than 0.995. Blank pork muscle, blank liver, and blank kidney were selected as representative matrix for spiked standard recovery test. The recoveries of each compound were in the range of 46.6-118.9%, and the relative standard deviations were in the range of 1.9-28.2%. Decision limits (CCα, α = 0.01) of 48 analytes in muscles, liver, and kidney samples ranged from 0.05 to 0.49 μg/kg, and the detection capability (CCβ, β = 0.05) ranged from 0.13 to 1.64 μg/kg. This method was successfully applied to 110 real animal origin food samples including meat, liver, and kidney of pig and chicken samples.     

A simple and selective method for the measurement of azadirachtin and related azadirachtoid levels in fruits and vegetables using liquid chromatography electrospray ionization tandem mass spectrometry

Neem-based insecticides containing azadirachtin and related azadirachtoids are widely used in agriculture. Here, we report an analytical method for the rapid and accurate quantification of the insecticide azadirachtin A and B and other azadirachtoids such as salannin, nimbin, and their deacetylated analogues on tomatoes and peaches. Azadirachtoids were extracted from fruits and vegetables with acetonitrile. Using high-performance liquid chromatography/electrospray ionization tandem mass spectrometer, azadirachtoids were selectively detected monitoring the multiple reaction transitions of sodium adduct precursor ions. For azadirachtin A, calibration was linear over a working range of 1-1000 microg/L with r > 0.996. The limit of detection and limit of quantification for azadirachtin A were 0.4 and 0.8 microg/kg, respectively. The presence of interfering compounds in the peach and tomato extracts was evaluated and found to be minimal. Because of the linear behavior, it was concluded that the multiple reaction transitions of sodium adduct ions can be used for analytical purposes, that is, for the identification and quantification of azadirachtin A and B and related azadirachtoids in fruit and vegetable extracts at trace levels.     

Determination of penicillin G residues in edible animal tissues by liquid chromatography

An improved method has been developed for the determination of benzyl penicillin in animal tissues. Tissues are fortified with a known amount of penicillin V (internal standard) and extracted with water. The extract is deproteinized with sulfuric acid and sodium tungstate, filtered, and concentrated on a conditioned C18 solid phase extraction column. Penicillin V and benzyl penicillin are then eluted from the column with 1 mL 60% acetonitrile-35% water-5% 0.2M phosphate buffer solution and derivatized with 1 mL 1,2,4-triazole-mercuric chloride solution at 65 degrees C for 30 min. An aliquot of this sample is analyzed by reverse phase liquid chromatography with UV detection at 325 nm. The limit of detection is 5 micrograms/kg (ppb) penicillin G (8.4 IU/kg) in liver, kidney, and muscle tissues).