Determination of seven artificial sweeteners in diet food preparations by reverse-phase liquid chromatography with absorbance detection

The artificial sweeteners aspartame, saccharin, cyclamate, alitame, acesulfam-K, sucralose, and dulcin are determined in diet soft drinks and tabletop sweetener preparations. Samples are diluted, filtered, and analyzed directly by liquid chromatography on a C-18 reverse-phase column with a mobile phase gradient ranging from 3% acetonitrile in 0.02M KH2PO4 (pH 5) to 20% acetonitrile in 0.02M KH2PO4 (pH 3.5). Diet puddings and dessert toppings are extracted with ethanol, filtered, and diluted with mobile phase for analysis. The sweeteners, except sucralose and cyclamate, were detected by UV absorbance at either 200 or 210 nm. Sucralose was determined at 200 nm or by refractive index. Cyclamate was determined after post-column ion-pair extraction. The sweeteners stevioside and talin were not detected. Additives such as caffeine, sorbic acid, and benzoic acid did not interfere.     

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 aspartame in dry beverage bases and sweetener tablets with confirmation by thin layer chromatography

A liquid chromatographic method is described for the determination of aspartame in dry beverage bases and sweetener tablets. The sample was mixed with the mobile phase, the pH was adjusted to within +/- 0.1 pH unit of the mobile phase, and the sample was diluted to volume with the mobile phase. The solution was filtered and a 10 microL aliquot was injected onto a C18 reverse phase column. Aspartame was quantitated with an ultraviolet detector. Recoveries of aspartame ranged from 94 to 111%. The dry beverage bases contained 5-13% aspartame and the sweetener tablets contained 19% aspartame. The presence of aspartame was confirmed by using thin layer chromatography.     

Determination of phenol in honey by liquid chromatography with amperometric detection

A sensitive, selective analytical method has been developed for determination of phenol in honey by liquid chromotography (LC) with amperometric detection (AMD). Phenol is extracted with benzene from the distillate of honey. The benzene extract is washed with 1% sodium bicarbonate solution and then reextracted with 0.1N sodium hydroxide followed by cleanup on a C18 cartridge. Phenol is determined by reverse-phase LC with amperometric detection. An Inertsil ODS column (150 X 4.6 mm, 5 microns) is used in the determination. The mobile phase is a mixture (20 + 80 v/v) of acetonitrile and 0.01M sodium dihydrogen phosphate containing 2mM ethylenediaminetetraacetic acid, disodium salt (EDTA) with the pH adjusted to 5.0. The flow rate is 1 mL/min under ambient conditions. The applied potential of the AMD using a glassy carbon electrode is 0.7 V vs an Ag/AgCl reference electrode. Average recoveries of phenol added to honey were 79.8% at 0.01 ppm spiking level, 90.4% at 0.1 ppm, and 91.0% at 1.0 ppm. Repeatabilities were 3.4, 1.3, and 1.8%, respectively. The detection limit of phenol in honey was 0.002 ppm. For analysis of 112 commercial honey samples, the range and average values of 32 detected samples were 0.05-5.88 ppm and 0.71 ppm, respectively.     

Determination of the fluoroquinolone enrofloxacin in edible chicken muscle by supercritical fluid extraction and liquid chromatography with fluorescence detection

A supercritical fluid extraction (SFE) method for the extraction of enrofloxacin from a chicken breast muscle was examined. A liquid chromatograph, equipped with a fluorescence detector, was used for the detection of enrofloxacin. Optimal extraction parameters, such as extraction time, supercritical fluid volume, modifier concentration, pressure, and temperature, were determined by examining SFE recoveries from control muscle samples spiked with enrofloxacin at different levels. In all of the experiments, high recovery values were observed, ranging from 101 to 104%. The extraction of enrofloxacin from real muscle samples was examined in chickens that were treated orally with enrofloxacin. Extraction was carried out by the SFE method after each oral treatment and under optimal extraction conditions at set intervals over time. The SFE, combined with liquid chromatographic analysis, showed that the concentration of enrofloxacin in the chicken muscles decreased continuously with time, giving a negligible concentration 72 h after the treatment. These results suggest that SFE is a useful approach for the extraction of enrofloxacin from chicken breast muscles.     

Determination of maduramicin by liquid chromatography with atomic absorption spectrometric detection

A liquid chromatograph was interfaced to an atomic absorption spectrometer for the detection and quantitation of maduramicin in feed matrixes at the 1-8 ppm level. Ionophores in general form strong 1:1 products with various metal cations, yielding complexes that are insoluble in water but very soluble in organic solvents. Maduramicin, a carboxylic, polyalcohol, polyether antibiotic, is labeled with the sodium cation and analyzed by atomic absorption spectroscopy (AAS). The lower limit of detection is approximately 100-200 ng maduramicin sodium salt. Feeds containing 1-8 ppm maduramicin are extracted with acetone, the extract is passed through an alumina column, the column is eluted with acetonitrile-water (90 + 10), and the eluate is analyzed for maduramicin by liquid chromatography-AAS after concentration and conversion of maduramicin to the sodium salt. Recoveries of maduramicin averaged 89.5%. Liquid chromatography with AAS detection has been shown to be a sensitive and highly specific technique for the determination of ionophores in general and maduramicin in particular.     

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 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%.     

Fluorometric determination of benzylideneacetone in fragrance products by liquid chromatography with post-column derivatization

A method is described for the liquid chromatographic (LC)-fluorometric determination of benzylideneacetone in fragrance products. Benzylideneacetone is first separated from other fragrance ingredients by LC and then reacted post-column with a methanolic solution of isonicotinic acid hydrazide and aluminum nitrate. The reactants are maintained at 65 degrees C for about 1.5 min to quantitatively form the fluorescent isonicotinoyl hydrazone derivative of benzylideneacetone. The aluminum ion forms a complex with the hydrazone to enhance the fluorescence of the derivative. The amount of benzylideneacetone is determined by measuring the intensity of the fluorescence emitted by the hydrazone derivative and comparing that value with those obtained for derivatized standards. Recovery studies were conducted by spiking commercial fragrances with benzylideneacetone at concentrations of 0.01, 0.05, and 0.1% (w/v). Recoveries ranged from 98 to 104% with a mean recovery of 100.2% and a standard deviation of 2.4%.     

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 terbutaline sulfate in dosage forms by liquid chromatography with electrochemical detection

A liquid chromatographic method with electrochemical detection has been developed for the determination of terbutaline sulfate in dosage forms. A cyanopropyl bonded-phase column is used with a mobile phase consisting of methanol-0.1 M monobasic potassium phosphate containing 0.1M sodium heptanesulfonate and 1mM disodium ethylenediaminetetraacetate (15 + 85). The compound of interest is detected at a glassy carbon electrode held at a potential of +0.9 V vs silver-silver chloride. The response is linear from 0 to 10 micrograms/mL terbutaline sulfate. The method is applicable to tablet composites, individual tablets, dissolution determinations, and injections. Results and supporting data are reported for the above analyses.     

Determination of nabam fungicide in crops by liquid chromatography with postcolumn reaction detection

The ethylenebisdithiocarbamate (EBDC) fungicide, nabam, was determined in several crop matrixes using liquid chromatography with postcolumn reaction detection. After separation by micellar liquid chromatography, nabam (EBDC sodium salt) was acid hydrolyzed to ethylenediamine and fluorigenically labeled with o-phthalaldehyde-mercaptoethanol (OPA-MERC). Standard curves were linear from the detection limit of ca 1 ng to 1000 ng. Nabam was recovered in high yield (89 plus or minus 7.7%) over a range of concentrations (0.1 to 20 ppm) from fortified samples of papaya, lettuce, cucumber, spinach, and applesauce using a simple extraction method. Efforts to convert the more popular EBDC fungicides, maneb and mancozeb, to nabam are discussed.     

Determination of xanthomegnin in grains and animal feeds by liquid chromatography with electrochemical detection

A sensitive, highly selective liquid chromatographic (LC) method is described which uses electrochemical (EC) reduction of the analyte in the determinative step. The method is capable of determining xanthomegnin in mixed animal feeds and grains at levels ranging from 15 to 1200 ng/g. The method can detect as little as 0.5 ng xanthomegnin injected on the LC column. Xanthomegnin is extracted with chloroform and 0.1M phosphoric acid. An aliquot of the crude extract is purified by silica gel column chromatography using a Sep-Pak silica gel cartridge. A novel feature of the method is that xanthomegnin is "backed off" the column by reversing the flow of the eluant through the column. LC is then used to separate xanthomegnin from other interfering substances. Xanthomegnin is detected by EC reduction at -0.16 V. Recoveries of xanthomegnin added to samples at levels ranging from 15 to 1200 ng/g averaged 79% with a coefficient of variation of 7.9%. Results also demonstrate that this LC system can separate the related metabolites viomellein and rubrosulphin from each other and from xanthomegnin and that the same EC detection system can be used to detect these metabolites.     

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%.     

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 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 glucosinolates in canola seeds using anion exchange membrane extraction combined with the high-pressure liquid chromatography detection

A rapid, simple, and reliable method for the determination of individual glucosinolates in canola seeds was developed using a semiquantitative extraction of glucosinolates with anion exchange membranes and HPLC detection. In this one-step extraction procedure, a membrane (7 cm(2)) is placed in the seed suspension prepared by grinding and boiling 0.8 g of seeds in 20 mL of water. After 10 min of shaking on the mechanical shaker, the membrane is removed from the suspension, washed, and transferred to a vial containing 5 mL of 1 N tetramethylammonium chloride. The glucosinolates are eluted from the membrane by shaking the membrane for 10 min with the eluting solvent. The glucosinolate content in membrane eluates is determined by HPLC using sinigrin standards. A coefficient of variation ranging from 1.9 to 7.6% for aliphatic glucosinolates indicated very good reproducibility of the method. Because of the instability of 4-hydroxyglucobrassicin, the coefficient of variation for the determination of this indolyl glucosinolate was 13.9%. To verify the results of the membrane extraction/HPLC detection, this new method was compared with the existing colorimetric and GC procedures. Very good correlation (R(2) = 0.98) was obtained between the total glucosinolates determined by the membrane extraction/HPLC method and the palladate colorimetric procedure for 17 canola varieties. Concentrations of individual glucosinolates in five canola varieties were compared with the GC data. Very good agreement between these two methods was obtained for aliphatic glucosinolates. However, the membrane extraction/HPLC method yielded slightly higher values for 4-hydroxyglucobrassicin than the GC method, possibly indicating that the decomposition of this glucosinolate was reduced during the sample extraction with the membranes. The simplicity and low cost of the membrane extraction/HPLC method make it an attractive alternative to the existing procedures for glucosinolate analysis in canola seeds.     

Determination of fluoroquinolones in milk samples by postcolumn derivatization liquid chromatography with luminescence detection

A liquid chromatography (LC) method with luminescence detection for the determination of eight quinolone antibiotics is reported. The system encompasses three consecutive steps: (a) chromatographic separation using reverse-phase mode (RP-LC), (b) postcolumn derivatization reaction, and (c) luminescence detection by monitoring fluorescence (FL) and time-resolved (TR) signals. The derivatization step is based on the reaction between quinolones and terbium(III) to form luminescent chelates, which were determined at lambda(ex) 340 and lambda(em) 545 nm (FL mode) or at lambda(ex) 281 and lambda(em) 545 nm (TR mode). Dynamic ranges of the calibration graphs, obtained with standard solutions of analytes and FL and TR modes, respectively, were 190-3500 and 316-2000 ng mL-1 for marbofloxacin, 8-3500 and 8.1-1500 ng mL-1 for ciprofloxacin, 6.2-3500 and 13-1500 ng mL-1 for danofloxacin, 7.4-3500 and 8.4-1500 ng mL-1 for enrofloxacin, 14-3500 and 20-2000 ng mL-1 for sarafloxacin, 12.5-3500 and 13.9-1200 ng mL-1 for difloxacin, 7.6-3500 and 13-3000 ng mL-1 for oxolinic acid, and 9-2000 and 130-3000 ng mL-1 for flumequine. Limit of detection values obtained using FL and TR modes, respectively, were 60 and 95 ng mL-1 for marbofloxacin, 2 and 2.4 ng mL-1 for ciprofloxacin, 1.9 and 3.9 ng mL-1 for danofloxacin, 2.2 and 2.5 ng mL-1 for enrofloxacin, 3.8 and 7 ng mL-1 for sarafloxacin, 4 and 4.2 ng mL-1 for difloxacin, 2.3 and 4 ng mL-1 for oxolinic acid, and 2.7 and 40 ng mL-1 for flumequine. The precision was established at two concentration levels of each analyte and expressed as the percentage of relative standard deviation with values ranging between 1.9 and 7.8%. The validation procedure for the analysis of samples was carried out using European Community recommendations, and the decision limit and detection capability were calculated for bovine whole milk. The method was applied to whole, semiskimmed, and skimmed milk samples spiked with the target analytes, and the recoveries ranged between 93.3 and 106.0%.     

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.