Analysis of pharmaceutical dosage forms for oxfendazole: I. Reverse phase liquid chromatographic determination of oxfendazole in swine premix

A reverse phase liquid chromatographic (LC) procedure is described for quantitating oxfendazole (2-(methoxycarbonylamino)-5-phenylsulfinylbenzimidazole] in swine premix. Sample preparation consists of extracting oxfendazole with an acetone-methanol mixture. An aliquot of the extract is then centrifuged to separate undissolved premix excipients. Internal standard is added to the supernate and the sample is further diluted with water-acetonitrile-phosphoric acid (80 + 20 + 1). Oxfendazole is quantitatively determined using a Partisil-5-ODS-3 column with acetonitrile-0.01 M phosphate buffer (pH 6.0) as the mobile phase. The method is stability specific and yields a mean recovery of 101.1 +/- 0.4% for the 1.35% premix formulation. The dependence of chromatographic performance characteristics on mobile phase organic content, pH, and buffer concentration is also reported.     

Determination of sulfite in foods by headspace liquid chromatography

Sulfite was determined in a variety of foods by liquid chromatography (LC) after the samples were mixed with a solution containing mannitol, FeSO4, and Na2HPO4, adjusted to pH 11, and left to stand for 15 min at room temperature. An aliquot of the mixture was placed in a headspace vial and mixed with 50% H3PO4. After 15 min, a portion of the headspace was removed with a syringe containing LC mobile phase without acetonitrile. The syringe was shaken and an aliquot of the solution was analyzed on an anion exchange column with a mobile phase of 0.03M methane sulfonate (pH 10.8) containing 5% acetonitrile. Sulfite was detected amperometrically (glassy carbon electrode) at +0.7 V. The method was successfully compared to the FDA-modified Monier-Williams procedure for a variety of foods. Minimum detectable levels were about 1 microgram/g, based on a 15 g sample.     

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.     

Liquid chromatographic determination of desfuroylceftiofur metabolite of ceftiofur as residue in cattle plasma

A liquid chromatographic (LC) method has been developed for the determination of the desfuroylceftiofur metabolite of ceftiofur as a residue in the plasma of animals. Plasma sample in 0.1M pH 8.7 phosphate buffer containing dithioerythritol is incubated under nitrogen for 15 min at 50 degrees C. The sample is centrifuged, charged to a C18 cartridge, and washed with 0.1M ammonium acetate. The desfuroylceftiofur residue on the cartridge is derivatized by adding 0.1M ammonium acetate containing iodoacetamide and letting the cartridge stand in the dark for 30 min. The cartridge is then drained and rinsed, and the desfuroylceftiofur acetamide is eluted with methanol. The mixture is evaporated to dryness, dissolved in pH 10.6 sodium hydroxide, and charged to a SAX cartridge. The derivative is eluted with 2% acetic acid, reduced in volume, and dissolved in mobile phase for liquid chromatography. The LC system includes a C8 column and guard cartridge with UV detection at 254 nm. The gradient mobile phase (flow rate 1 mL/min) is 0.01M pH 5 ammonium acetate programmed to 29% methanol-water (60 + 40) in 25 min. Recoveries were 90-100% with a sensitivity of 0.1 ppm or less. The procedure has been applied to the plasma of cattle, rats, horses, pigs, and dogs.     

Simultaneous determination of nicotinic acid and nicotinamide in cooked sausages

A simple and sensitive method for determining simultaneously nicotinic acid and nicotinamide content in cooked sausages by ion-pair reversed-phase liquid chromatography is described. Samples are extracted with ultrapure water, centrifuged, deproteinized with zinc hydroxide, filtered, and chromatographed with UV detection at 261 nm on a 25 cm x 4 mm i.d. Spherisorb ODS-2 cartridge using as mobile phase a mixture consisting of 5 mM heptanesulfonic acid adjusted to pH 3.3 with phosphoric acid and acetonitrile (75:25, v/v). Both vitamins are measured on a reversed-phase column with a single ion-pair reagent. Precision of the method was 0.5 and 1.0% (within a day) and 2.3 and 4.5% (between days) for nicotinic acid and nicotinamide, respectively. The detection limit was 0.300 mg/100 g. The recovery was >92% of nicotinic acid and nicotinamide added to samples of meats. Twenty samples of six different products have been analyzed in duplicate. The mean value for nicotinic acid ranged between 0.908 and 1.267 mg/100 g of fresh weight and for nicotinamide between 1.968 and 2.880 mg/100 g of fresh weight.     

Liquid chromatographic determination of seven antioxidants in dry food

The liquid chromatographic determinative step of the official method for propyl gallate, trihydroxybutyrophenone, tert-butylhydroquinone, nordihydroguaiaretic acid, butylated hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxymethylphenol, and butylated hydroxytoluene (BHT) in fats and oils has been applied to their determination in a number of dry foods. A representative sample (10 g) is homogenized first with hexane (25 mL), then with 5 mL added water, and finally with 75 mL added acetonitrile. The hexane and acetonitrile are decanted, filtered, and separated; the hexane and rehydrated food are reextracted with 2 additional portions of acetonitrile, and the combined acetonitrile extracts are concentrated and diluted to 10 mL. An aliquot is analyzed as described in the official method, using a 150 x 4.6 mm 5 microns C-18 column. The need for rehydration to maximize the recovery of BHA and other antioxidants from marketplace dry food samples such as potato flakes, dry coffee whiteners, and dessert topping mixes was demonstrated. Rehydration was not required for cheese snacks, breakfast cereals, cake mixes, and some other foods. The need for rehydration should be determined by analyzing other foods with and without the addition of water. Potato and corn chips, popcorn and cheese snacks, breakfast cereals, dry beverage mixes, rice, potato flakes, french fried potatoes, and cake mixes were spiked with the above antioxidants at 10-50 ppm. Overall recoveries ranged from 64.3 to 105.6% and repeatabilities ranged from 0.7 to 10.8%. A total of 109 samples of the above foods were analyzed, and 64% contained detectable (greater than 1-2 ppm) antioxidants, mainly BHA and BHT.     

Determination of total riboflavin in cooked sausages.

A simple and rapid method for determining riboflavin content in cooked sausages by ion-pair reversed-phase liquid chromatography has been set up. Samples were subjected to acid and enzymatic hydrolysis. Sample extracts were directly chromatographed, avoiding purification and concentration treatment. Final determination was performed by high-performance liquid chromatography with fluorescence detector (excitation, 227 nm; emission, 520 nm), on a 25 cm x 4 mm i.d. Spherisorb ODS-2 cartridge using a mixture of 5 mM heptanesulfonic acid adjusted to pH 2.7 with phosphoric acid and acetonitrile (75:25, v/v) as mobile phase. Precision of the method was 1.3% (within a day) and 2.6% (between days). The detection limit was 0.015 mg/100 g. The recovery was >95%.     

Determination of albendazole and its major metabolites in the muscle tissues of Atlantic salmon,tilapia, and rainbow trout by high performance liquid chromatography with fluorometric detection

A liquid chromatographic procedure for the determination of albendazole ([5-(propylthio)-1H-benzimidazol-2yl]carbamic acid methyl ester) and its major metabolites, albendazole sulfoxide, albendazole sulfone, and albendazole-2- aminosulfone in rainbow trout, tilapia, and salmon muscle with adhering skin tissue is described. The muscle tissue samples are made alkaline with potassium carbonate and extracted with ethyl acetate. The extracts are further subjected to cleanup by utilizing a number of liquid-liquid extraction steps. After solvent evaporation, the residue is reconstituted in mobile phase and chromatographed. The chromatography is carried out on a reversed phase Luna C(18) column, using acetonitrile/methanol/buffer as a mobile phase and a fluorescence detector. The average recoveries from the fortified muscle tissue of the three fish species for albendazole (25-100 ppb), albendazole sulfoxide (15.5-62 ppb), albendazole sulfone (1-10 ppb), and albendazole-2- aminosulfone (10-100 ppb) were 94, 77, 82, and 67%, respectively. The average CV for each compound was < or =10%. The procedure was validated and then applied to the determination of albendazole and its three major metabolites in the muscle tissue of the three fish species obtained after orally dosing with albendazole.     

Liquid chromatographic determination of sulfamethazine in feeds

A reverse-phase liquid chromatographic method for the assay of sulfamethazine (SMZ) in feeds is described. Feed samples are extracted with 50% methanol solution, centrifuged, filtered, and diluted when necessary, and chromatographed on a C-18 column. Samples are eluted with a mobile phase of 20% methanol and 80% of a solution containing acetic acid and tetramethylammonium chloride. The average recovery from spiked samples was 97.2% with a coefficient of variation of 1.2%. Linearity was very good (correlation coefficient 0.9997). Within-day and between-day coefficients of variation averaged 1.3 and 2.6%, respectively. The results for samples assayed by this method compared closely with the results from the same extracts assayed by the AOAC colorimetric method.     

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.     

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%).     

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.     

Analysis of pharmaceutical dosage forms for oxfendazole: II. Simultaneous liquid chromatographic determination of oxfendazole and trichlorfon in equine paste

A reverse phase liquid chromatographic procedure is described for the simultaneous determination of oxfendazole [2-(methoxycarbonylamino)-5-phenylsulfinylbenzimidazole] and trichlorfon [(2,2,2-trichloro-1-hydroxyethyl)phosphonic acid dimethyl ester] in equine paste. The sample is extracted by sonication in methanol. Insoluble excipients are removed by centrifugation and an aliquot plus internal standard are diluted with dilution solvent (water-acetonitrile-phosphoric acid, 80 + 20 + 1). The samples are filtered and injected onto a Partisil-5 ODS-3 column with acetonitrile-0.01 M phosphate buffer pH 6.0 (20 + 80) as mobile phase. Method specificity is confirmed using an absorbance rationing technique. The method yields mean recoveries of 100.9 and 100.0% for trichlorfon and oxfendazole, respectively. Dependence of chromatographic performance characteristics on mobile phase organic content, pH, and buffer concentration is also reported.     

Screening method for vitamins A and D in fortified skim milk, chocolate milk, and vitamin D liquid concentrates

Vitamin A was determined in fortified chocolate milk and skim milk; vitamin D was determined in fortified chocolate milk, skim milk, and vitamin D concentrates, using reverse phase high pressure liquid chromatography (HPLC). The sample is saponified, extracted with hexane, and chromatographed in an HPLC system on a 10 micron Vydac TP reverse phase C18 column, using acetonitrile-methanol (9+1) as the mobile phase. For 6 replicates, the recoveries of vitamins A and D, using this procedure, were 99 and 98%, respectively.     

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.     

High pressure liquid chromatographic determination of nitrofurazone in milk.

A rapid high pressure liquid chromatographic (HPLC) screening method for the quantitative determination of nitrofurazone in milk has been developed. The drug is extracted with ethyl acetate from a 2.0 ml milk serum sample, the organic layer is evaporated to dryness, and the residue is dissolved in the mobile phase and injected into the liquid chromatogarph. A reverse phase muBondapak C18 column is used with monitoring at 365 nm. The detection limit is 5 ppb and recoveries are 57--67%. Mass spectroscopic confirmation of the HPLC nitrofuran peak is described.     

Liquid chromatographic determination of melamine in beverages

A liquid chromatographic method is described for the determination of melamine in beverages. Melamine is separated by column chromatography using cation and anion exchange resin and determined by ion-pair liquid chromatography using an ODS column and a mixture of acetonitrile and 0.05M phosphate buffer (pH 3.0) containing 0.005M sodium 1-laurylsulfate (1 + 4, v/v) as mobile phase. Recoveries of melamine ranged between 90.3 +/- 7.8 and 102.1 +/- 5.6% at levels of 0.6 to 2.4 ppm in 4 kinds of beverages. The quantitation limit was 2.5 micrograms melamine in 50 mL beverage. The method was applied to the migration test of melamine from melamine-formaldehyde resin products to the beverages.     

Liquid chromatographic determination of total niacin in beef, semolina, and cottage cheese

A sensitive and precise liquid chromatographic method has been developed for the determination of total niacin in beef, semolina, and cottage cheese. The samples are extracted with Ca(OH)2, cleaned up using C18 Sep-Pak cartridges, and chromatographed on a C18 column using a mobile phase consisting of 23% acetonitrile, 0.10% H3PO4, and 0.10% sodium dodecyl sulfate in water. Niacin is detected using an absorbance detector operating at 254 nm. This chromatographic system produces an unusually sharp peak for niacin, enabling a detection limit of approximately 0.05 mg/100 g. The mean recovery was 99.5% and the coefficients of variation for beef, semolina, and cottage cheese were 3.58, 1.02, and 10.0%, respectively.     

Steviol quantification at the picomole level by high-performance liquid chromatography

A simple and highly sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of steviol (SV) using dihydroisosteviol (DHISV) as an internal standard (IS). SV and DHISV were derivatized by reaction of the acids with 4-(bromomethyl)-7-methoxycoumarin in an aprotic solvent (DMF or acetone). The resulting ester derivatives were separated on an ODS column (250 x 4.6 mm i.d., 5 microm particle size) using fluorescence detection with excitation at 321 nm and emission at 391 nm. The mobile phase consisted of acetonitrile/water (80:20 v/v) with a flow rate of 1 mL min(-)(1). A linear relationship was observed for concentrations between 0.5 and 50 microg/mL of SV, and the detection limit was 100 pg. For application of this method to samples of beer fortified with stevioside, a simple procedure for extraction of the beer with diethyl ether and derivatization in DMF was applied. Whereas beer samples spiked with SV gave a linear response over the range 0.1-15 microg/mL beer, no SV could be detected in beer samples enriched in stevioside that had been stored for over 3 years. The application of the method to plant samples involved preparation of an acid fraction containing the SV analyte, derivatization, and sample cleanup using small silica columns and thin-layer chromatography. A sensitive determination of 594 ng of steviol present in 100 mg of dry plant material was performed with high precision and accuracy.     

High pressure liquid chromatographic determination of naphthaleneacetic acid residues in apples.

An ion-suppression reverse phase high pressure liquid chromatographic method is described for determining naphthaleneacetic acid (NAA) residues in apples. Samples are extracted with acidic chloroform, filtered through pre-acidified Hy-Flo Supercel, and cleaned up by acid-base partitioning. The extract can be successfully chromatographed on either a muLiChrosorb NH2 or muBondapak C18 column and quantitated by using a variable wavelength ultraviolet detector set at 220 nm. The mobile phase is acetonitrile-water (20 + 80) buffered to pH 3.5 (MULiChrosorb column) or pH 5.2 (MUBondapak column) and flowing at 1.0--2.0 ml/min. Recoveries ranged from 86 to 98%. The minimum detectable amount was 0.5 ng, which easily permitted the quantitation of 0.01 ppm NAA in 50 g sample. A fluorometric detector was 4 times as sensitive, using an excitation wavelength of 220 mm and monitoring the emission at 340 nm. For this detector, the minimum detectable amount was 0.12 ng NAA.