Determination of neomycin in animal tissues, using ion-pair liquid chromatography with fluorometric detection

A liquid chromatographic (LC) method is described for the determination of neomycin in animal tissues. Tissues are homogenized in 0.2M potassium phosphate buffer (pH 8.0); the homogenate is centrifuged, and the supernate is heated to precipitate the protein. The heat-deproteinated extract is acidified to pH 3.5-4 and directly analyzed by LC. The LC method consists of an ion-pairing mobile phase, a reverse phase ODS column, post-column derivatization with o-phthalaldehyde reagent, and fluorometric detection. The LC method uses paromomycin as an internal standard, and separates neomycin from streptomycin or dihydrostreptomycin because they have different retention times. The LC column separates neomycin in 25 min; the detection limit is about 3.5 ng neomycin. The overall recovery of neomycin from kidney tissues spiked at 1-30 ppm was 96% with a 9.0% coefficient of variation. The method was also applied to muscle tissue.     

Liquid chromatographic determination of monensin in chicken tissues with fluorometric detection and confirmation by gas chromatography-mass spectrometry

An accurate, sensitive method is described for the determination of monensin residue in chicken tissues by liquid chromatography (LC), in which monensin is derivatized with a fluorescent labeling reagent, 9-anthryldiazomethane (ADAM), to enable fluorometric detection. Samples are extracted with methanol-water (8 + 2), the extract is partitioned between CHCl3 and water, and the CHCl3 layer is cleaned up by silica gel column chromatography. Free monensin, obtained by treatment with phosphate buffer solution (pH 3) at 0 degrees C, is derivatized with ADAM and passed through a disposable silica cartridge. Monensin-ADAM is identified and quantitated by normal phase LC using fluorometric detection. The detection limit is 1 ppb in chicken tissues. Recoveries were 77.6 +/- 1.8% at 1 ppm, 56.7 +/- 7.1% at 100 ppb, and 46.5 +/- 3.7% at 10 ppb fortification levels in chicken. Gas chromatography-mass spectrometry is capable of confirming monensin methyl ester tris trimethylsilyl ether in samples containing residues greater than 5 ppm.     

Sensitive silica gel-packed flowcell for fluorometric detection of aflatoxins by high pressure liquid chromatography.

Aflatoxins B1, B2, G1, and G2 were quantitatively detected by high pressure liquid chromatography on a 5 micronm Lichrosorb column, using a Lichrosorb-packed flowcell in the fluorometric detector. The relationship between peak height and the amount injected was linear only up to about 2 ng but showed a linear loglog relationship. Methods for constructing and packing the flowcell are given. A guard column and venting valve were used to minimize deterioration of the analytical column and the adsorbent-packed flowcell. The method was applied to a peanut butter extract, although with the cleanup procedure used, the life expectancy of the flowcell is limited.     

Liquid chromatographic determination of amprolium in chicken tissues, using post-column reaction and fluorometric detection

A method is presented for determination of amprolium residues in chicken muscles by a liquid chromatographic post-column reaction system. The drug is extracted from muscles with methanol, and the extract is concentrated to 3-4 mL. This aqueous solution is rinsed with n-hexane and cleaned up by alumina column chromatography. The drug is separated from the interferences on a LiChrosorb RP-8 column, reacted with ferricyanide in alkaline solution, and quantitated by fluorometric detection at 367 nm (excitation) and 470 nm (emission). Recoveries of amprolium added to chicken muscles at levels of 0.1 and 0.2 ppm were 74.9 and 80.9%, respectively. The detection limit was 1 ng for amprolium standard and 0.01 ppm in chicken muscles.     

Rapid method for determination of leucogentian violet in chicken fat by liquid chromatography with electrochemical detection

The metabolite leucogentian violet (LGV) was found in chicken fat obtained from chickens dosed with gentian violet (GV); however, no residues of the parent compound, GV, and its oxidized metabolites were found. Therefore, a rapid method was developed for the specific determination of LGV in chicken fat. Chicken fat containing LGV is separated from the cellular protein with methylene chloride. LGV is then separated from the fat by partition extraction with an aqueous acid phase in which LGV is protonated, and the fat is discarded with the methylene chloride layer. The aqueous solution is neutralized, LGV is re-extracted into methylene chloride, and the methylene chloride is evaporated. An acetonitrile-water solution containing LGV is filtered before liquid chromatography using a cyano column, an acetate buffer-acetonitrile mobile phase, and an electrochemical detector set at a potential of +1.000 V. Average recoveries of LGV from chicken fat were 83.9% with a coefficient of variation (CV) of 12.9% for the 5 ppb level; 82.8% with a CV of 13.5% for the 10 ppb level; and 77.7% with a CV of 2.56% for the 20 ppb level. Levels of incurred LGV in chicken fat averaged 49.3 ppb with a CV of 2.43%.     

Determination of ampicillin residues in fish tissues by liquid chromatography

A liquid chromatographic (LC) method is described for determination of ampicillin residues in fish tissues. The drug is extracted from tissues with methanol, and the extract is evaporated to dryness. This residue is cleaned up by Florisil cartridge chromatography. LC analysis is carried out on a Nucleosil C18 column, and ampicillin is quantitated by ultraviolet detection at 222 nm. Recoveries of ampicillin added to tissues at levels of 0.2 and 0.1 ppm were 73.2 and 61.5%, respectively. The detection limit was 3 ng for ampicillin standard, and 0.03 ppm in tissues.     

Determination of olaquindox residues in swine tissues by liquid chromatography

A liquid chromatographic (LC) method is described for determination of olaquindox residues in swine tissues. The drug is extracted from tissues with acetonitrile, and the extract is evaporated to dryness. This residue is cleaned up by alumina column chromatography. LC analysis is carried out on a Nucleosil C18 column, and olaquindox is quantitated by ultraviolet detection at 350 nm. The average recoveries of olaquindox added to tissues at levels of 0.2, 0.1, and 0.05 ppm were 74.0, 68.6, and 66.3%, respectively. The detection limit was 2 ng for olaquindox standard and 0.02 ppm in tissues.     

Liquid chromatographic determination of spiramycin residues in chicken tissues

A liquid chromatographic (LC) method is described for determination of spiramycin residues in chicken muscles. The drug is extracted from muscles with acetonitrile, the extract is concentrated to 3-4 mL and rinsed with n-hexane followed by ethyl ether, and the drug is extracted with chloroform. LC analysis is carried out on a Zorbax BP-C8 column, and spiramycin is detected spectrophotometrically at 231 nm. Recoveries of spiramycin added to chicken muscles at 0.2 and 0.1 ppm were 93.9 and 89.0%, respectively. The detection limit was 5 ng for spiramycin standard, and 0.05 ppm in chicken muscles.     

Determination of tetracycline antibiotic residues in edible swine tissues by liquid chromatography with spectrofluorometric detection and confirmation by mass spectrometry

A sensitive and specific method is described for the simultaneous determination of oxytetracycline, tetracycline (TC), and chlortetracycline residues in edible swine tissues, by combining liquid chromatography with spectrofluorometric and mass spectrometry detection. The procedure involved a preliminary extraction with EDTA-McIlvaine buffer acidified at pH 4.0, followed by solid-phase extraction cleanup using a polymeric sorbent. The liquid chromatography analysis was performed with spectrofluorometric detection after postcolumn derivatization with magnesium ions. The limits of quantification were 50 microg/kg for muscle and 100 microg/kg for kidney tissues. The recovery values were greater than 77.8% for muscle and 65.1% for kidney. The method has been successfully used for the quantification of tetracyclines in swine tissues samples. The selective liquid chromatography mass spectrometric analysis for confirmation of oxytetracycline in one positive swine muscle sample was made by atmospheric pressure chemical ionization (APCI). The APCI mass spectra of the TCs gave the protonated molecular ion and two typical fragment ions, required for their confirmation in single ion monitoring scan mode in animal tissues.     

Determination of methylglyoxal in ruminal fluid by high-performance liquid chromatography using fluorometric detection

There is no reported method for the quantification of methylglyoxal in ruminal fluid. The method reported here is based on the conversion of methylglyoxal to 6-methylpterin, followed by quantification of the resulting pteridinic compound by fluormetric detection using liquid chromatography. Ruminal fluid was collected and preserved with 1 M HCl at -20 degrees C. Cation exchange prior to derivatization was used to eliminate possible interfering peaks. The detection limit of 0.125 microg/mL was calculated. The recoveries were >80%, and the coefficients of variation were <15%. This method has proven to be rugged and accurate for the detection of methylglyoxal concentration in ruminal fluid collected from cows fed diets deficient in degradable intake protein as a marker. Methylglyoxal is produced by ruminal bacteria in response to low nitrogen levels in the rumen. The ruminal methylglyoxal concentration has the potential to be a useful marker to assess ruminal nitrogen status to aid in more accurate diet formulation.     

Ultrasensitive determination of jasmonic acid in plant tissues using high-performance liquid chromatography with fluorescence detection

An ultrasensitive and selective high-performance liquid chromatographic method for the volatile signaling hormone, jasmonic acid, has been developed based on precolumn derivatization with 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene (BODIPY-aminozide). The derivatization reaction was carried out at 60 °C for 30 min in the presence of phosphoric acid. The formed jasmonic acid derivative was eluted using a mobile phase of methanol/pH 6.50 ammonium formate buffer/tetrahydrofuran (67:30:3, v/v/v) in 10 min on a C(18) column and detected with fluorescence detection at excitation and emission wavelengths of 495 and 505 nm, respectively. The detection limit (signal-to-noise ratio = 4) reached 1.14 × 10(-10) M or 2.29 fmol per injection (20 μL), which is the lowest of the existing methods. The proposed method has been successfully applied to the direct determination of trace jasmonic acid in the crude extracts of soybean leaves from soybean mosaic virus-infected and normal plants with recoveries of 95-104%.     

Rapid procedure for determination of nicarbazin residues in chicken tissues

A procedure is described for the quantitation of nicarbazin residues in chicken tissues. The method includes extraction of tissue with chloroform-ethyl acetate-dimethyl sulfoxide (50 + 50 + 0.8), adsorption on neutral alumina, and subsequent elution of the residues with methanol-pH 6.0 phosphate buffer (1 + 1). Extracts are separated on a 15 cm, 5 micron C18 column with methanol-pH 6.0 phosphate buffer (6.5 + 3.5) as the mobile phase. The dinitrocarbanilide portion of the complex is detected and quantitated with an electrochemical detector in the reductive mode. Recoveries, based on dinitrocarbanilide, were greater than 95% in liver, breast, and thigh muscle tissues fortified with 0.25-8.0 ppm nicarbazin.     

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).     

Determination of nicarbazin in chicken tissues by liquid chromatography and confirmation of identity by thermospray liquid chromatography/mass spectrometry

A liquid chromatographic method is described for the quantitative measurement of nicarbazin in chicken liver, fat, muscle, and skin tissues. The 4,4'-dinitrocarbanilide (DNC) portion of nicarbazin is extracted from tissues with ethyl acetate. After filtration and evaporation, the extract is purified by liquid-liquid partitioning with acetonitrile-hexane and alumina cartridge chromatography. DNC is separated and measured by reverse-phase liquid chromatography (RP-LC) with an octadecylsilyl (ODS) column and a UV detector set at 340 nm. The overall average recovery of DNC added to tissues was 83.4 +/- 3.1%. The lowest level validated in tissues by this procedure was 0.10 ppm. The limit of detection was estimated to be 0.020 ppm. This method provides a sensitive, selective, rapid, and reproducible alternative to existing purification, separation, and detection techniques, such as differential pulse polarography and colorimetry, for determination of nicarbazin in chicken tissues. Identity of DNC is confirmed by subjecting the purified extracts to thermospray-LC/mass spectrometric analysis using negative-ion detection and selected ion monitoring. Three structural-indicating ions at m/z 302, 272, and 164 are monitored in the thermospray-mass spectrum which are characteristic of the DNC molecule.     

High pressure liquid chromatographic determination of zearalenone in chicken tissues

A method is reported for the extraction and analysis of zearalenone in chicken fat, heart muscle, and kidney tissue by using high pressure liquid chromatography (HPLC). Zearalenone is extracted with acetonitrile, cleaned up with hexane, and extracted further with ethyl acetate. Zearalenone is determined by HPLC using a reverse phase radial compression separation system, an ultraviolet absorbance detector, and a mobile phase of acetonitrile-water (60 + 40) (v/v). Recoveries of zearalenone added at levels from 50 to 200 ng/g are in the range 82.6-95.1%.     

Evaluation of phenolic compounds in virgin olive oil by direct injection in high-performance liquid chromatography with fluorometric detection

Hydrophilic phenols are the most abundant natural antioxidants of virgin olive oil (VOO), in which tocopherols and carotenes are also present. The prevalent classes of hydrophilic phenols found in VOO are phenyl alcohols, phenolic acids, secoiridoids such as the dialdehydic form of decarboxymethyl elenolic acid linked to (3,4-dihydroxyphenyl)ethanol or (p-hydroxypheny1)ethanol (3,4-DHPEA-EDA or p-HPEA-EDA) and an isomer of the oleuropein aglycon (3,4-DHPEA-EA), lignans such as (+)-1-acetoxypinoresinol and (+)-pinoresinol, and flavonoids. A new method for the analysis of VOO hydrophilic phenols by direct injection in high-performance liquid chromatography (HPLC) with the use of a fluorescence detector (FLD) has been proposed and compared with the traditional liquid-liquid extraction technique followed by the HPLC analysis utilizing a diode array detector (DAD) and a FLD. Results show that the most important classes of phenolic compounds occurring in VOO can be evaluated using HPLC direct injection. The efficiency of the new method, as compared to the liquid-liquid extraction, was higher to quantify phenyl alcohols, lignans, and 3,4-DHPEA-EA and lower for the evaluation of 3,4-DHPEA-EDA and p-HPEA-EDA.     

Determination of oxolinic acid residues in salmon muscle tissue by liquid chromatography with fluorescence detection

The present paper describes a method for determination of oxolinic acid in salmon muscle tissue. Tissue (0.5-2 g) mixed with 2 g anhydrous sodium sulfate is extracted twice with ethyl acetate, centrifuged, and the extract evaporated. The residue is partitioned in a mixture of hexane and 0.01M oxalic acid and the aqueous phase chromatographed using fluorescence detection at 327 nm excitation and 369 nm emission. Calibration and standard curves are linear from 10-200 ppb and 100-2000 ppb at different sensitivity settings. Recoveries ranged from 71-83% in spiked blanks, with a CV of 4-10.3% over a 2-week period. Preliminary results in treated salmon were variable, possibly because some fish refused to eat medicated feed.     

Determination of novobiocin residues in milk, blood, and tissues by liquid chromatography

Residues of novobiocin in milk, blood, and tissues can be detected by microbiological tests but cannot be distinguished from other antibiotics. A simple liquid chromatographic (LC) method was developed for identification of residues. Tissues were blended and milk and blood serum were mixed with 0.2M NH4H2PO4. The mixture was deproteinized by adding aqueous methanol and filtering. The LC apparatus consisted of a variable wavelength detector, set at 340 nm, an automatic loop injector, and a C18 column with guard cartridge. The flow rate was 1 mL/min and the solvent mixture of 0.01M H3PO4-acetonitrile-methanol was programmed from 50 + 0 + 50 (0-1 min) to 20 + 80 + 0 (20 min). Novobiocin was concentrated directly by solid-phase extraction on the analytical column. Five or more 200 microL aliquots of the filtrate in water-methanol (1 + 1) (adjusted if necessary) were injected with the column solvent at 50 + 0 + 50. After the final injection, the program was run to completion. Recoveries were 90-100% with sensitivities of 0.05 ppm or less. The procedure should be adaptable for use with formulations and feeds.