Liquid chromatographic determination of vitamin B-6 in foods

Chromatographic analysis for vitamin B-6 in complex samples imposes certain requirements on the analyst, who must extract completely the bound, unstable vitamers without loss, remove interfering compounds, and provide clean extracts for analysis. The analyst also has to contend with the problems inherent in all methods, such as sample collection, storage, preparation, and homogenization. However, chromatography provides a means of identifying and quantitating all forms of the vitamin, and thus provides the possibility of addressing the problem of the bioavailability of specific vitamers. It also allows automation, which is absolutely essential in coping with the large numbers of samples that are generated in areas such as quality control. These factors are all addressed here, and chromatographic results for various meat and other food products are presented to illustrate the variations in vitamin content that occur from sample to sample, the agreement with microbiological results, and that liquid chromatography (LC) has come of age in dealing with complex biological samples, such as food and food products.     

Liquid chromatographic determination of vitamin D in fortified milk

A method for the determination of vitamins D2 + D3 in fortified milk is described. Vitamins D2 and D3 are extracted from the saponified sample and converted to isotachysterols with antimony trichloride. The isotachysterols are quantitated using liquid chromatography with ultraviolet detection at 301 nm, which is the absorption maximum. At this wavelength other materials present in the sample do not interfere with the analysis of isotachysterols and therefore a cleanup step is avoided. Recoveries of vitamin D added to skim milk were 98.1% (SD 5.3), 96.7% (SD 3.3), and 96.0% (SD 5.1) for samples fortified with 200, 400, and 600 IU/quart, respectively. For whole milk, recoveries were 102.0% (SD 6.5) and 97.1% (SD 3.5) in samples fortified with vitamin D equivalent to 200 and 400 IU/quart, respectively. The detection limit for vitamin D is 40 IU/quart.     

Liquid chromatographic determination of vitamin D in infant formula

A method is described for the determination of vitamins D2 + D3 in milk- and soy-based infant formula. Vitamins D2 and D3 are extracted from the saponified sample and converted to isotachysterols with acidified butanol. Reverse-phase liquid chromatography (LC) is used to remove interferences, and total vitamin D is quantitated using normal-phase LC. Recoveries of spiked samples averaged 97.6% for milk-based infant formula, and 98.8% for soy-based infant formula. This method quantitates vitamin D2 + D3 in infant formulas containing as low as 40 IU/qt when prepared according to label direction.     

Liquid chromatographic determination of vitamin D in milk and infant formula

Vitamin D2 or vitamin D3 is determined by liquid chromatography (LC) in milk and infant formula. Vitamin D is extracted from the saponified sample, passed through an amino-cyano LC cleanup column to remove major interferences, and quantitated using normal phase LC. Within-day precision is 4.5% relative standard deviation (RSD); the overall method RSD (reflecting technician-to-technician, day-to-day, and within-day variability) is 7.7%. Overspike recoveries averaged 97% for milk, 98% for milk-based infant formula, and 93% for soy-based infant formula. The performance of the method is compared with that of the official AOAC vitamin D method (rat bioassay). The method is applicable to the determination of vitamin D in milk and in the major milk- and soy-based infant formulas available in the United States. The method can quantitate (but not distinguish) either vitamin D2 or vitamin D3. The method is applicable to milk and infant formula samples containing between 100 and 1500 IU vitamin D/L. Sample throughput is between 4 and 8 replicates per day.     

Liquid chromatographic determination of vitamin K1 trans- and cis-isomers in infant formula

The normal phase liquid chromatographic (LC) method for determination of trans- and cis-isomers of vitamin K1 (phylloquinone) in infant formula described here uses an Apex silica column, isocratic elution, and UV absorption detection at 254 nm. Vitamin K1 is extracted quantitatively from the product matrix by pretreating the as-fed liquid with concentrated ammonium hydroxide and methanol, and then extracting it with a 2:1 mixture of dichloromethane and isooctane. The extract is cleaned up by silica open-column chromatography and concentrated for LC analysis. For trans-vitamin K1, the method precision is less than or equal to 3.3% RSD (relative standard deviation), and the spike recovery is 98 +/- 4%. For cis-vitamin K1, the precision is less than or equal to 12% RSD, determined at levels near the detection limit, and the spike recovery is 95 +/- 9%. The detection limit is 0.3 ng for both isomers at signal/noise = 3.     

Liquid chromatographic determination of bendiocarb in technical materials and wettable powder formulations: collaborative study

A liquid chromatographic method for determination of bendiocarb in technical materials and wettable powders was tested by 12 collaborators. Bendiocarb is dissolved in acetonitrile containing 0.1% propiophenone as internal standard. This solution is analyzed on a liquid chromatograph utilizing a reverse phase (C18) column. The compound is detected at 254 nm and peak area is used for quantitation. The 3 different materials studied contained 20, 80, and nominally 100% bendiocarb. Each was examined in duplicate to provide the necessary matched pairs. Collaborators approved of the ease and simplicity of the method and, in particular, the way the method can be applied to automatic injection assemblies. The statistical data show acceptable precision of the method: Reproducibility coefficients of variation were 20% material, 2.04%; 80% material, 1.02%; and nominal 100% material (technical product), 0.64%. The method has been adopted official first action.     

Liquid chromatographic cleanup and determination of low levels of vitamin D3 in sheep plasma

A liquid chromatographic (LC) method is described for the determination of vitamin D3 in sheep plasma. Samples are extracted by one of 2 different methods, depending on the concentration of vitamin D3. The samples are purified by using either a Sep-Pak silica cartridge or a small alumina column, followed by additional cleanup on a Metalsorb LC column. Final analysis was carried out on a 5 micron C18 column using a radial compression separation system with an acetonitrile-methanol solvent system. Vitamin D3 was completely resolved from any interfering compounds in the plasma; total run time was less than 15 min, using a variable wavelength detector set at 264 nm. The method was successfully applied to samples at levels of 1-10 ng added vitamin D3 mL sheep plasma, with recoveries in the range 90-97%.     

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 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 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 method for analysis of elemental sulfur in pesticide formulations

A nonaqueous reverse-phase liquid chromatographic (LC) method has been developed to determine elemental sulfur in pesticide formulations. Samples were extracted in 50 mL of stabilized tetrahydrofuran (THF) by gentle swirling while sonicating for 1 min. A 5 microL aliquot was injected into the LC instrument equipped with a Vydac 218 TP 54 column. The mobile phase was methanol-acetonitrile-stabilized tetrahydrofuran (58.5 + 40 + 1.5). Sulfur was monitored at 280 nm. Retention time was approximately 5 min with total analysis time of 7 min. For 6 different products analyzed 12 times each, the coefficients of variations were all less than 3.5%. Purity of each sulfur peak was checked by using a photodiode array detector in the spectrum and absorbance ratio modes. No impurities were observed at the monitoring wavelength.     

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 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 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.