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 ergotamine tartrate in tablets by liquid chromatography with fluorescence detection

A method is presented for the liquid chromatographic (LC) determination of ergotamine tartrate in tablets that is applicable even in the presence of other ingredients such as phenobarbital, belladonna alkaloids, and caffeine. The sample is transferred to a volumetric flask, a small volume of formic acid is added to dissolve and stabilize the ergotamine, and the solution is diluted to volume with methanol. The solution is mixed and filtered through paper. The LC system consists of a Rheodyne injector fitted with a 20 microL loop and a C18 reverse phase column; the mobile phase is acetonitrile-water-triethylamine (700 + 300 + 0.5). Ergotamine tartrate is determined fluorometrically at an excitation wavelength of 250 nm and an emission wavelength of 430 nm. Recovery studies were conducted at the 0.3 and 1.0 mg levels. Average recoveries were 99.6 and 100.8%, respectively; relative standard deviations (RSDs) were 1.08 and 2.21%, respectively. Some commercial preparations containing ergotamine tartrate in combination with other ingredients were also analyzed. The RSDs for 5 determinations of each of 2 ground composites were 0.09 and 0.34%.     

Determination of reserpine in commercial tablets by liquid chromatography with fluorescence detection

A procedure is presented for the determination of reserpine in commercial tablets by liquid chromatography (LC). The sample is extracted with methanol if only reserpine is present. If the sample contains other ingredients, CHCI3 is used for extraction from aqueous suspension; the CHCI3 is subsequently completely evaporated in the presence of methanol. For LC, a normal phase column, methanol as the eluting solvent, and a fluorometric detector are used. A recovery study indicated that no measurable degradation of reserpine occurs during evaporation of the CHCI3 extract. Several commercial tablets containing reserpine alone or in combination with other ingredients were analyzed by the proposed method, and the results were compared with those obtained by the current official USP methods for reserpine.     

Determination of seven glycidyl esters in edible oils by gel permeation chromatography extraction and liquid chromatography coupled to mass spectrometry detection

A method based on a gel permeation chromatography (GPC) extraction procedure combined with an additional cleanup by solid-phase extraction (SPE) on silica gel and liquid chromatography-mass spectrometry (LC-MS) detection has been validated for the analysis of seven glycidyl esters (GEs) including glycidyl laurate, myristate, palmitate, stearate, oleate, linoleate, and linolenate in various edible oils. This method was conjointly developed and validated by two different laboratories, using two different detection systems, a LC time of flight MS (LC-ToF-MS) and a LC triple-quadrupole MS (LC-MS/MS). The extraction procedure allowed targeting low contamination levels due to a highly efficient matrix removal from the 400 mg oil sample loaded on the GPC column and is suitable for routine analysis as 24 samples can be extracted in an automated and reproducible way every 12 h. GPC extraction combined with SPE cleanup and LC-MS/MS detection leads to a limit of quantification in oil samples between 50 and 100 μg/kg depending on the type of glycidyl ester. Recoveries ranged from 68 to 111% (average = 93%). Quantification was performed by automated standard addition on extracts to compensate matrix effects artifacts. To control recoveries of each sample four isotopically labeled GEs ((13)C(3) or (13)C(4)) were included in the method.     

Determination of aflatoxin M1 in milk by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method is proposed for the determination of aflatoxin M1 in milk. The method was successfully applied to both liquid whole and skim milk and also whole and skim milk powder. The samples are initially extracted with acetonitrile-water followed by purification using a silica gel cartridge and a C18 cartridge. Final analysis by LC was achieved using a radial compression module equipped with a 5 micron C18 column and a fluorescence detector. The method was successfully applied to samples at levels of 10 to 0.08 ppb added aflatoxin M1 with recoveries in the range of 70-98%.     

Rapid fluorescence screening assay for enrofloxacin and tetracyclines in chicken muscle

A simple, rapid fluorescence assay was developed for screening both enrofloxacin (ENRO) and tetracyclines in chicken muscle at the U.S. tolerance levels (300 ng/g and 2 microg/g, respectively). Screening for both classes of antibiotics is accomplished using one extraction, thus simplifying and expediting the process. The method requires an initial extraction of chicken muscle with 1% acetic acid in acetonitrile, centrifugation, and analysis of the supernatant for ENRO fluorescence. After addition of ammonium hydroxide, magnesium chloride, and methanol, followed by centrifugation and filtration, the supernatant can be measured for tetracycline fluorescence. Chlortetracycline (CTC) was chosen as a representative tetracycline to demonstrate the method, as it displays intermediate sensitivity among the three tetracyclines approved in the U.S. Comparison of the fluorescence of control and tolerance-level-fortified samples of both ENRO and CTC shows no overlap. Setting a threshold as the average fortified fluorescence minus 3sigma allows for successful screening, as illustrated with blind samples as controls or fortified with ENRO and/or CTC over a range of concentrations. This method can provide an alternative or supplemental approach to currently used microbial screening assays.     

Determination of reserpine and rescinnamine in Rauwolfia serpentina preparations by liquid chromatography with fluorescence detection

A method is presented for the determination of reserpine and rescinnamine in Rauwolfia serpentina powder or tablets by liquid chromatography (LC) with fluorescence detection. The sample is dispersed in CH3OH, 0.5N H2SO4 is added, and the mixture is extracted with five 30 mL portions of CHCl3. The extracts are separated from interfering materials on a Celite-0.1N NaOH column, and the eluates are collected in 50 mL CH3OH. After complete removal of the CHCl3, reserpine and rescinnamine are determined by liquid chromatography on a normal phase column with CH3OH as the mobile phase. Because reserpine and rescinnamine produce a single peak, chromatograms are obtained at different wavelengths. Reserpine is determined at an excitation wavelength of 280 nm and an emission wavelength of 360 nm. Rescinnamine is determined at an excitation wavelength of 330 nm and an emission wavelength of 435 nm. Recovery studies were conducted at 2 different levels to simulate 100 and 50 mg Rauwolfia serpentina tablets. Samples of Rauwolfia serpentina powders and tablets were also examined, and the results were compared with those obtained by the current AOAC official method. The proposed method is applicable to the analysis of ground composites and individual tablets.     

Determination of p-hydroxybenzoic acid esters in cosmetics by liquid chromatography with ultraviolet and fluorescence detection

A simple, precise, and accurate liquid chromatographic method with both ultraviolet (UV) and fluorescence detection is described for the determination of methyl, ethyl, propyl, and butyl p-hydroxybenzoates (PHBA-esters) in cosmetics. sec-Butyl p-hydroxybenzoate is added to the sample as an internal standard. Then the PHBA-esters are extracted with ether, the ether is evaporated to dryness, and the residue is dissolved in 60% (v/v) acetonitrile. The acetonitrile solution is passed through a Sep-Pak C18 cartridge to remove co-extracted lipids. PHBA-esters are determined by reverse-phase liquid chromatography with UV detection at 254 nm and fluorescence detection at ex 280 nm, em 305 nm. The mobile phase is acetonitrile-water (35 + 65). The method was linear over the concentration range of 0.005-0.15 mg/mL. Mean recoveries of each PHBA-ester were 98.9-102.7% (coefficients of variation less than or equal to 2.0%).     

Determination of small quantities of atropine in commercial preparations by liquid chromatography with fluorescence detection

A method is presented for the determination of small quantities of atropine in commercial preparations by liquid chromatography (LC) with fluorescence detection. The sample is extracted with CHCl3 from basic suspension, the CHCl3 is evaporated on the steam bath, and the dry residue is dissolved in a small volume of CH3OH. A reverse phase column is used for the LC analysis; the eluting solvent is prepared by mixing 950 mL CH3OH with 50 mL water containing 1 g of the sodium salt of 1-pentanesulfonic acid. The fluorescence detector is set at an excitation wavelength of 255 nm and an emission wavelength of 285 nm. Several commercial tablets and injections containing atropine in combination with other ingredients and a commercial sample of belladonna extract were analyzed by the proposed method. Recoveries of atropine sulfate from aqueous solutions averaged 100.7% with a relative standard deviation (RSD) of 3.35% for atropine sulfate levels of 0.12 mg. Recoveries of atropine sulfate from synthetic injection formulations were 99.8 and 100.0% with RSDs of 2.03 and 2.35%, respectively; the atropine sulfate concentrations of commercial injections with the same formulations were found to be 97.0 and 100.0% of the labeled amounts with RSDs of 0.53 and 1.46%, respectively.     

Determination of theanine in commercial tea by liquid chromatography with fluorescence and diode array ultraviolet detection

Two liquid chromatographic methods that involve precolumn derivatization with o-phthaladehyde (OPA) and phenylisothiocyanate (PITC) with fluorescence and diode array UV detection for the determination of theanine have been developed. The chromatographic separations were achieved by reverse-phase high-performance liquid chromatography using octadecyl columns and gradient elution. The methods were applied to evaluate the theanine content of commercial tea leaves. The coefficient of variation of the peak area repeatability for within day (n = 8) and between day (n = 8 over 10 days) was lower than 3% for both of the methods. The estimated limit of detection (LOD) and limit of quantitation (LOQ) for the OPA method was 0.12 and 0.35 microg theanine, respectively. The PITC method was 500-fold more sensitive with LOD and LOQ values of 0.25 and 0.75 ng, respectively. The theanine content of the commercial tea samples varied from 2-5 mg/g leaf. The overall % recoveries for these methods ranged from 93-99.3. The sensitivity and simplicity of the method render them suitable for use in quality control laboratories.     

Determination of ochratoxin A in animal feed and cereal grains by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method is described for determination of ochratoxin A in animal feeds and cereal grains. Samples are initially extracted with chloroform-ethanol (8 + 2) and 5% acetic acid in water. Extracts are purified using a silica gel cartridge followed by a cyano cartridge. The samples are evaporated, diluted to a known volume, and analyzed using a 10 cm column of 3 micron C18 and a fluorescence detector. The method was applied to a variety of animal feeds and cereal grains at levels of 1.0-0.005 ppm added ochratoxin A. The overall recovery was 90.6% +/- 3.6.     

Improved solid-phase extraction procedure in the analysis of paralytic shellfish poisoning toxins by liquid chromatography with fluorescence detection

The analysis of shellfish extracts for the determination of paralytic shellfish poisoning (PSP) toxins by liquid chromatography with fluorescence detection repeatedly showed the presence of a compound suspected to interfere with gonyautoxin 4. The first aim of this study was to confirm by liquid chromatography coupled to tandem mass spectrometry that this compound was not gonyautoxin 4. The second part of this work was to improve a nonvolumetric C(18) solid-phase extraction (SPE) procedure to evaluate the removal of the interference associated with the recovery of PSP toxins. The cleanup procedure was modified into a volumetric SPE procedure and proved to efficiently and totally remove the interference while recovering from 78 to 85% of the PSP toxins available as commercial standards (saxitoxin, neosaxitoxin, gonyautoxins 1-4) and considered as major PSP toxins in human intoxication, with 85% recovery for gonyautoxin 4. The efficiency of this cleanup procedure was checked on shellfish extracts containing this interference and originating from France and Turkey.     

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.     

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.     

Multiresidue analysis of pesticides in soil by supercritical fluid extraction/gas chromatography with electron-capture detection and confirmation by gas chromatography-mass spectrometry

The applicability of supercritical fluid extraction (SFE) in pesticide multiresidue analysis (organohalogen, organonitrogen, organophosphorus, and pyrethroid) in soil samples was investigated. Fortification experiments were conducted to test the conventional extraction (solid-liquid) and to optimize the extraction procedure in SFE by varying the CO2 modifier, temperature, extraction time, and pressure. The best efficiency was achieved at 400 bar using methanol as modifier at 60 degrees C. For the SFE method, C-18 cartridges were used for the cleanup. The analytical screening was performed by gas chromatography equipped with electron-capture detection (ECD). Recoveries for the majority of pesticides from spiked samples of soil at different residence times were 1, 20, and 40 days at the fortification level of 0.04-0.10 mg/kg ranging from 70 to 97% for both methods. The detection limits found were <0.01 mg/kg for ECD, and the confirmation of pesticide identity was performed by gas chromatography-mass spectrometry in a selected-ion monitoring mode. Multiresidue methods were applied in real soil samples, and the results of the methods developed were compared.     

Analysis of antioxidants from orange juice obtained by countercurrent supercritical fluid extraction,using micellar electrokinetic chromatography and reverse-phase liquid chromatography

Antioxidants from orange juice were determined by the combined use of countercurrent supercritical fluid extraction (CC-SFE) prior to reverse-phase liquic chromatography (RP-LC) or micellar electrokinetic chromatography (MEKC). The separation of antioxidants found in the SFE fractions was achieved by using a new MEKC method and a published LC procedure, both using diode array detection. The characterization of the different antioxidants was further done by LC-mass spectrometry. Advantages and drawbacks of LC and MEKC for analyzing the antioxidants found in the different orange extracts are discussed. Although LC yields higher peak area and slightly better reproducibility than MEKC, the latter technique provides information about the CC-SFE extracts in analysis times 7 times faster than by LC. This analysis advantage can be used for the quick adjustment of CC-SFE conditions, thus providing a fast way to obtain orange fractions of specific composition.     

Determination of cinnamyl anthranilate in perfume, cologne, and toilet water by liquid chromatography with fluorescence detection

A liquid chromatographic method with fluorescence detection was developed for the determination of cinnamyl anthranilate in perfumes and other fragrance compositions. The method was evaluated by conducting recovery studies of 10 different commercial fragrance compositions to which cinnamyl anthranilate had been added at levels of 0.1, 0.5, and 1.0 mg/mL. Recoveries ranged from 91 to 103% with a mean of 97% and a standard deviation of +/- 3.3%.     

Analysis of sulfonamides in animal feeds by liquid chromatography with fluorescence detection

Two analytical methodologies for the simultaneous analysis of eight sulfonamide antibiotics in animal feeds were developed. Analytes were extracted in a simple and rapid procedure by manual shaking with an ethyl acetate/ultrapure water mixture (99:1, v/v) without further sample cleanup. Mean recoveries ranging from 72.7% to 99.4% with relative standard deviations below 9% were achieved from spiked animal feed samples. Determination was carried out by high-performance liquid chromatography using fluorometric detection with precolumn derivatization. The separation of the derivatized compounds was performed using two different chromatographic columns: a conventional C(18) column and a recently available core-shell particle Kinetex C(18) column. Both methods were validated in-house in six different feed matrices, and the two approaches were compared. The experiments showed that the method using the Kinetex column was superior with regard to speed of analysis and precision, both under repeatability and intermediate reproducibility conditions. The limits of detection and quantification were also greatly improved, below 0.10 and 0.34 μg/g, respectively. Finally, this novel approach was successfully applied to the analysis of real feed samples.     

Assay of oxolinic acid residues in salmon muscle by liquid chromatography with fluorescence detection: interlaboratory study.

A previously developed method that uses a simplified sample preparation and fluorometric detection of liquid chromatographic eluates for the determination of oxolinic acid in salmon muscle has been collaboratively studied. Five laboratories participated in the study to analyze, in quintuplicate, blank salmon muscle fortified at 10, 20, 50, and 100 micrograms/kg (ppb), and 2 incurred samples from salmon given feed with medicated oxolinic acid. The tissue, 2 g mixed with 2 g Na2SO4, is extracted with ethyl acetate and centrifuged, and the solvent is evaporated. The residue is partitioned in a mixture of hexane and 0.01 M oxalic acid, and the aqueous phase is chromatographed using fluorescence detection at 327 nm excitation and 369 nm emission. Mean recoveries ranged from 77.2 to 84.5% in spiked samples with reproducibility relative standard deviation (RSDR) ranging from 11.5 to 18.3%. Treated salmon were found to contain 8.71 and 53.8 micrograms/kg with RSDR of 18.6 and 16.7%, respectively. The corresponding repeatability relative standard deviations (RSDR) were 5.8-12.2%, and 7.7 and 6.2%. The method is recommended for regulatory purposes in Canada.