Liquid chromatographic determination of sulfamethazine in milk

A simple, relatively rapid liquid chromatographic method has been developed for the determination of sulfamethazine (SMZ) in milk at levels in the low ppb range. The method is based on extracting SMZ from milk with chloroform, evaporating the chloroform, dissolving the residues in hexane, extracting into buffers, and chromatographing the buffer solution. The method has been shown to determine levels as low as 5 ppb reliably. Levels greater than or equal to 7 ppb have been confirmed by gas chromatography/mass spectrometry after derivatization of extracts from fortified, incurred, and shelf milk. Intralaboratory recoveries and percent coefficients of variation are satisfactory. Sulfadimethoxine and sulfaquinoxaline can also be determined by the method. Application of the method to other dairy products is being investigated.     

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 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.     

Liquid chromatographic determination of carbamazepine in tablets

A simple and rapid liquid chromatographic method is described for the qualitative and quantitative determination of carbamazepine in tablet composites and individual tablets, using the internal standard technique. Analyses were performed on a C-18 reverse-phase column with tetrahydrofuran-methanol-water (8 + 37 + 55) as the mobile phase. A linear relationship was obtained between detector responses at 254 nm and amounts of carbamazepine injected ranging from 0.2 to 1.7 micrograms. The coefficient of variation for 10 consecutive injections of a standard preparation was 0.4%. Recoveries of carbamazepine from 100 and 200 mg tablets averaged 101.4 and 99.7%, respectively. Assay results for commercial tablets analyzed by the proposed method agreed favorably with those obtained by the method of USP XXI. The assay results for individual tablets indicated that deviations from the average value and the range of individual values are much wider with the compendial method than with the proposed method.     

Liquid chromatographic determination of flucytosine in capsules

A liquid chromatographic method has been developed for determination of flucytosine in capsules. Flucytosine and p-aminobenzoic acid, the internal standard, are separated on a C18 reverse phase column using water-methanol-acetic acid mobile phase containing 1-octane-sulfonic acid sodium salt. Compounds are detected photometrically at 285 nm. Mean assay results for 250 and 500 mg commercial capsules were 101.5% (n = 5) of declared, respectively. Mean recovery of flucytosine added to commercial capsules was 99.3%.     

Liquid chromatographic determination of nicarbazin in feed

A liquid chromatographic method was developed for the determination of nicarbazin (4,4'-dinitrocarbanilide.2-hydroxy-4,6-dimethylpyrimidine) in chicken feed. Ground feed was extracted with hot dimethylformamide, filtered, and then cleaned up on an alumina column. The nicarbazin was eluted from the column with ethanol and quantitated using a reverse phase C-18 column, with a methanol-water mobile phase and ultraviolet detection at 344 nm. Recoveries at a typical use level of 100 micrograms/g feed averaged 98% with a standard deviation of 3%. Samples fortified at levels as low as 0.1 micrograms/g were analyzed with 92% recovery. The detection limit is 1 ng, and the response is linear between 4 and 1000 ng. Feed additives in combination with nicarbazin do not interfere with recovery.     

Liquid chromatographic determination of 2,4-dinitroaniline and 2-naphthol in D&C Orange No. 17

A method is described for the determination of the intermediates in D&C Orange No. 17 by reverse phase liquid chromatography. The pigment is dissolved in boiling dioxane and then precipitated. The filtrate is chromatographed by isocratic elution, which is followed by a wash and equilibration. Peak area calibrations were linear. At the provisional specification levels, 99% prediction limits were 0.200 +/- 0.0012% 2,4-dinitroaniline (2,4-DNA) and 0.200 +/- 0.006% 2-naphthol. The limits of determination were 0.0023% for 2,4-DNA and 0.013% for 2-naphthol at the 99.5% confidence level. Recoveries were 98-100% for 2,4-DNA added at the 0.005-2% level, and 93-103% for 2-naphthol added at the 0.025-2% level. A survey of certified D&C Orange No. 17 samples showed that the lots contained higher levels of the intermediates than were determined previously by a cellulose column method, in which the pigment is not dissolved.     

Liquid chromatographic determination of strychnine in stomach contents

A chloroform extract of stomach contents at basic pH is concentrated and then extracted with 0.1 M phosphoric acid. The acid extract is chromatographed on a 10 cm reverse phase column, using 0.005 M phosphate buffer (pH 3.0)-acetonitrile-tetrahydrofuran (750 + 135 + 115) containing 0.01 M octanesulfonic acid at a flow rate of 1.0 mL/min for elution. Strychnine eluted in 7.3 min. Recoveries from spiked stomach contents averaged 92%. The method can be used without modification for other alkaloids.     

Liquid chromatographic determination of narasin in feed premixes

A liquid chromatographic (LC) method has been developed to determine narasin in feed premixes. Narasin is extracted from the premix with a methanol-water solvent, and the extracted solution is assayed by using LC. Recovery of narasin from a 12.5 g/lb premix is quantitative (100%), with a relative standard deviation of 1.44%. The results correlated well (coefficient 0.92) with a turbimetric bioassay method.     

Liquid chromatographic determination of tolnaftate in commercial products

A liquid chromatographic (LC) method for the determination of the antifungal agent tolnaftate was developed. Isolation of the analyte was achieved by direct extraction or dilution with acetonitrile-water (80 + 20) followed by reverse-phase liquid chromatography using a C18 column. The mobile phase was acetonitrile-water (80 + 20) acidified with phosphoric acid. Detection was by UV absorption at a wavelength of 257 nm. The proposed procedure was applied to 20 consumer products comprising 6 formulation types, including solutions, powders, liquid and power aerosols, creams, and gels. The precision (RSD) for the products ranged from 0.23 to 1.16% (n = 5), and recoveries via fortification ranged from 98.1 to 103.0%. Six different brands of C18 columns were evaluated for use with the method. The overall simplicity and versatility of the method suggest possible adaptations to both regulatory and quality-control situations.     

Liquid chromatographic determination of ergot alkaloids in wheat

A method is described for the determination of individual ergot alkaloids in wheat. The sample is extracted with ethyl acetate-4% ammonium hydroxide (100 + 10), and the extract is cleaned up by liquid-liquid partition. The ergot alkaloids are resolved by liquid chromatography (LC), using a porous cross-linked polystyrene-divinylbenzene resin column and a mobile phase consisting of acetonitrile-0.05 M dibasic ammonium phosphate (55 + 45) buffered at pH 10.0. The ergot alkaloids ergonovine, ergonovinine, ergotamine, ergotaminine, alpha-ergocryptine, alpha-ergocryptinine, ergocristine, and ergocristinine are separated by LC and detected with a fluorescence detector. Recovery of ergot alkaloids added to wheat at levels of 16-760 ng/g averaged 85.6% with a coefficient of variation of 11.1%.     

Liquid chromatographic determination of oxfendazole in swine feeds

A relatively simple analytical method is presented for determination of oxfendazole (2-(methoxycarbonylamino)-5-phenylsulfinyl-benzimidazole) at levels as low as 0.012% in swine feeds, using cation exchange liquid chromatography (LC). The sample was extracted with a solvent mixture of methanol-glacial acetic acid (90 + 10) at 45 degrees C, using a gyrorotory shaker. Plant pigments and other feed excipients were removed using zinc acetate treatment and pH-controlled extraction. Oxfendazole was further separated from the remaining interferences and quantitatively determined by LC on a Partisil SCX column with acetonitrile-0.01M phosphate buffer as mobile phase. The method is stability-specific, linear, precise, and accurate at 80-120% labeled strength (relative standard deviation 0.9-1.7 with mean recovery of 98-99%). Supporting data at a level of 0.0135% oxfendazole in swine feed indicated that this method is capable of complete recovery of oxfendazole from medicated swine feeds.     

Liquid chromatographic determination of glibenclamide in human plasma

The oral hypoglycemic agent glibenclamide was determined in human plasma by liquid chromatography (LC). Samples, with internal standard added, are extracted with dichloromethane. The organic phase is evaporated, and the residue is reconstituted in mobile phase for injection onto the LC column. Intra- and inter-day variability of the method was assessed at high and low levels of the drug. Although coefficients of variation were similar for both intra- and inter-day studies at both levels, CVs were smaller at the higher concentration level. Recovery of the drug was good at both high and low levels. The minimum level of detection was 5 ng/mL.     

Liquid chromatographic determination of aflatoxicol in porcine liver

A liquid chromatographic (LC) method for determination of aflatoxicol in porcine liver was developed. Liver sample is homogenized with water, diluted with saturated Na2SO4 solution, and extracted with acetone. After filtration, less polar interferences are removed by partition with isooctane. Aflatoxicol in the aqueous fraction is partitioned into CHCl3. The extract is dried over anhydrous Na2SO4 and evaporated nearly to dryness at 35 degrees C under a gentle flow of dry filtered air or nitrogen. Residue is dissolved in CHCl3-hexane and applied to a hexane-activated silica cartridge. The cartridge is washed with hexane-CHCl3, then aflatoxicol is eluted with CHCl3-acetone. Purified extract is evaporated to dryness, dissolved in methanol, and analyzed by C18 reverse phase liquid chromatography using a water-CH3CN-acetic acid mobile phase and fluorescence detection. Recovery of aflatoxicol from spiked liver samples at levels ranging from 0.25 to 4.0 ng aflatoxicol/g wet tissue averaged 92% with a limit of detection of about 0.1 ng aflatoxicol/g liver.     

Liquid chromatographic determination of coumarin anticoagulants in tablets

A simple and rapid liquid chromatographic method is described for the qualitative and quantitative determination of 5 coumarin anticoagulants in tablet composites and individual tablets. Analyses are carried out on a C18 reverse phase column using tetrahydrofuran-methanol-water-acetic acid (35 + 10 + 65 + 0.1) as mobile phase and photometric detection at 311 nm. The coefficients of variation for 10 consecutive injections of a mixed standards solution ranged from 0.28% for ethyl biscoumacetate to 0.78% for acenocoumarol. Standard recoveries were as follows: acenocoumarol, 99.3%; dicumarol, 99.6%; phenprocoumon, 101.6%; and warfarin sodium, 99.0%. The method was linear between 2 and 8 micrograms of drug injected. Assay results agreed favorably with those of the USP XX methods for dicumarol, phenprocoumon, and warfarin, and the NF XIV method for acenocoumarol. In addition, close correspondence was found with the results previously reported for the same drugs by a semiautomated spectrophotometric method. The content uniformity testing of individual 50 mg dicumarol tablets and 5 mg warfarin sodium tablets by the proposed method gave average (SD) values of 100.32% (0.64) and 101.00% (0.14), respectively, whereas these values were 101.60% (1.81) and 101.80% (0.18), respectively, by the method of USP XX.     

Liquid chromatographic determination of cholecalciferol in rodent baits

Cholecalciferol (vitamin D3) is extracted in acetonitrile on a Goldfisch apparatus, diluted to volume, and determined by reverse phase liquid chromatography (LC). The sum of the peak heights of pre-vitamin D3 and cis-vitamin D3 is used for quantitation. The method was tested for precision, linearity, and recovery. Quadruplicate analyses of 5 formulation samples gave relative standard deviations of 1.56-2.65%. Linearity was excellent with regression and correlation coefficients of 0.9997 and 0.9998, respectively. Recovery was 98.0 +/- 2.7%. The method is applicable to 0.075% cholecalciferol rodent baits.     

Liquid chromatographic determination of zoalene in medicated feeds

A rapid, reliable separation and quantitation of zoalene (3,5-dinitro-o-toluamide) from feeds is accomplished by using reverse phase liquid chromatography (LC) and ultraviolet detection. An extraction technique which is similar to the present AOAC official colorimetric method is used before chromatographic analysis. This extraction is followed by an activated alumina cleanup and LC to separate zoalene from feed matrix. The methodology was applied to a variety of spiked feed matrices, and yielded good recoveries. Liquid chromatographic results were shown to correlate with colorimetric determinations.     

Liquid chromatographic determination of cephapirin residues in milk

A liquid chromatographic (LC) method was developed for quantitative determination of cephapirin residues in milk that also resolved cephapirin from ampicillin, cloxacillin, and penicillin G. Diluted milk was passed through a C18 cartridge on which the cephapirin was adsorbed; then, interfering material was removed by washing with water and methylene chloride and cephapirin residues were eluted with methanol-acetonitrile (25 + 75). After drying, residues were dissolved in the mobile phase for injection. The LC system had an ultrasphere-ODS column with RP-18 Spheri-10 guard column and a UV detector with a 254 nm filter. The mobile phase was 85% sodium acetate (0.01M) and 15% methanol-acetonitrile (25 + 75) with a flow rate of 1 mL/min. Sensitivity was 20 ppb or less with a recovery of 61-80% in the range studied. Other beta-lactam antibiotics tested did not interfere with detection of cephapirin. Analysis of 30 samples of commercial homogenized milk obtained for a survey of antibiotics in consumer milk in Canada revealed no detectable cephapirin residues.     

Liquid chromatographic determination of sulfamoyldapsone in swine tissues

A liquid chromatographic (LC) method is described for the quantitative determination of sulfamoyldapsone (2-sulfamoyl-4,4'-diaminodiphenyl sulfone) in swine muscle, liver, kidney, and fat. Sulfamoyldapsone was extracted from tissues with acetonitrile saturated with n-hexane. The extract was washed with n-hexane saturated with acetonitrile, concentrated, and cleaned up by alumina column chromatography. Sulfamoyldapsone was separated on an ODS column by using acetonitrile-methanol-water (6 + 18 + 76) and was detected at 292 nm. Overall average recovery of sulfamoyldapsone added to tissues at levels of 0.1 and 0.5 microgram/g was 93.3% +/- 6.0. Detection limit was 0.02 microgram/g in these tissues.     

Liquid chromatographic determination of polydextrose in food matrixes

A liquid chromatographic (LC) method has been developed to determine the content of polydextrose, a water-soluble 1 calorie/g bulking agent, in food matrixes such as cookies, cakes, fruit spreads, and chocolate toppings. This analysis, which requires use of a blank matrix, provides a feasible means to control the manufacture of foods containing this additive and provides a component for the accurate determination of the caloric value of a particular food product. The method involves aqueous extraction of the polydextrose from the food matrix followed by separation on a carbohydrate analysis column. The LC system uses a mobile phase of 0.005M CaSO4.2H2O and a refractive index detector for quantitation. Polydextrose recoveries from the food matrixes varied from 91.5 to 100.9% with assay precision, expressed as coefficient of variation, ranging from 0.7 to 4.3%. Each error estimate was derived from 5 parallel determinations. The present methodology is precise and selective in contrast to the modified classical phenol-sulfuric acid colorimetric method for assaying carbohydrates, which had been used for polydextrose determination in food matrixes in the past. Because the coefficient of variation frequently exceeded 10%, replicate analyses were necessary to achieve quantitation.