Liquid chromatographic determination of saccharin in beverages and sweets: NMKL Collaborative Study

A reverse phase liquid chromatographic method for the determination of saccharin in a soft drink and a juice was collaboratively studied in 8 laboratories. Collaborators were supplied with 3 samples of the soft drink and 3 samples of the juice containing sodium saccharin levels of 40-100 mg/L. Average recoveries of sodium saccharin were 95.3% for the soft drink and 98.0% for the juice. The reproducibility coefficients of variation were 16.9% for the soft drink and 10.4% for the juice. In addition, a mini-collaborative study was conducted for the determination of saccharin in 3 samples of sweets produced commercially. Five collaborators analyzed the samples, which contained saccharin at levels of 100-600 mg/kg according to the maker's specifications. Saccharin was extracted with water and ethanol and chromatographed using a modified liquid chromatographic method. The reproducibility coefficient of variation was 12.4% for the sweets.     

Liquid chromatographic determination of saccharin in beverages and desserts: complementary collaborative study

A complementary collaborative study was conducted on a liquid chromatographic method for determination of saccharin in accordance with the latest international recommendations. One industrial and 6 official food control laboratories analyzed 3 samples of a juice, a soft drink, and a dessert at concentration levels of 26-90 mg/L, 33-73 mg/L, and 56-147 mg/kg, respectively. Blind duplicates and a blank were supplied for each type of material at each concentration level. The beverage was chromatographed directly and the dessert was extracted with ethanol before chromatography. Average recoveries were 95-107%. The reproducibility relative standard deviations were 6.4-7.3% for the juice, 9.2-20.6% for the soft drink, and 13.4-16.2% for the dessert. The outlier percentage was 14.3%. The results were compared with those of an earlier collaborative study by Nordic laboratories and with general collaborative results obtained by AOAC.     

Liquid chromatographic determination of quinine, hydroquinine, saccharin, and sodium benzoate in quinine beverages

A liquid chromatographic (LC) method is described for the determination of quinine, hydroquinine, sodium saccharin, and sodium benzoate in soft drinks. The method involves simple sample preparation, direct injection onto an octadecylsilane column, and elution with a methanol-acetonitrile-water-acetic acid (20 + 10 + 70 + 1) mobile phase. Eluted constituents are measured spectrophotometrically at 254 nm. The relationship between peak height and concentration was linear between 20 and 120 micrograms/mL for quinine. A relative standard deviation of 0.82% was obtained for commercial samples spiked with quinine, and the average recovery was 100.3%. The proposed procedure is accurate and rapid and can also detect hydroquinine (a natural contaminant of quinine), sodium saccharin, and sodium benzoate. Linear responses ranged from 0.45 to 20 micrograms/mL for hydroquinine, from 54.8 to 219 micrograms/mL for sodium saccharin, and from 10.1 to 145.1 micrograms/mL for sodium benzoate. The reproducibility of the LC method was evaluated with standard solutions of hydroquinine, sodium saccharin, and sodium benzoate, which produced relative standard deviations of 0.42, 0.46, and 1.13%, respectively. The average recoveries for sodium saccharin and sodium benzoate from spiked samples were 99.4 and 100.2%, respectively.     

Liquid chromatographic determination of scopoletin in hydroalcoholic extract of oak wood and in matured distilled alcoholic beverages

A liquid chromatographic (LC) method is described for determination of the coumarins esculin, umbelliferone, scopoletin, and 4-methyl umbelliferone in hydroalcoholic extracts of oak wood and in matured distilled alcoholic beverages. Samples were injected directly into the LC column (30 cm, 5 micron C18) and detected by fluorescence detector. Under these experimental conditions, only scopoletin (detection limit, 200 pg) was found in hydroalcoholic oak wood extracts and in spirits matured in oak wood. Applications of this method to spirits distilled from wine, grain, and sugar cane aged in oak barrels showed that amounts varied from 0.026 to 1.57 ppm.     

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 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 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 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 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.     

Gas chromatographic determination of N-nitrosamines in beverages following automatic solid-phase extraction

A semiautomatic method for the determination of seven N-nitrosamines in beverages by gas chromatography with nitrogen-phosphorus detection is proposed. Beverage samples are aspirated into a solid-phase extraction module for preconcentration and cleanup. The influence of the experimental conditions was examined by using various sorbents among which LiChrolut EN was found to provide quantitative elution and the highest preconcentration factors of all. The proposed method is sensitive, with limits of detection between 7 and 33 ng/kg, and precise, with relative standard deviations from 4.3% to 6.0%. The recoveries of N-nitrosamines from beverage samples spiked with 0.5 or 1 microg/kg concentrations of these compounds ranged from 95% to 102%. The method was successfully applied to the determination of residues of the studied N-nitrosamines in beverages including beer, wine, liquor, whisky, cognac, rum, vodka, grape juice, cider, tonic water, and soft drinks. The analytes were only detected in beer samples, positives being confirmed by gas chromatography coupled with impact ionization mass spectrometry.     

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 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 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 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 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 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 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 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.