Liquid chromatographic determination of multiple sulfonamide residues in bovine milk

A liquid chromatographic method has been developed for simultaneous determination of residues of 10 sulfonamide drugs at 10 ppb and above in raw bovine milk. The method is based on a chloroform-acetone extraction, evaporation of organic phase, dissolution of residues in an aqueous potassium phosphate solution, and extraction of fatty residue into hexane. The aqueous layer is collected, filtered, injected onto an LC system, and detected by ultraviolet absorption at 265 nm. To elute all 10 sulfonamides isocratically, 2 chromatographic conditions are required. Seven sulfonamides can be quantitated with 12% methanol in the mobile phase; 4 sulfonamides can be quantitated with 30% methanol. Sulfamethazine, the most widely used sulfonamide, is detected on both systems. Recoveries are 44-87% for individual sulfonamides, with only 2 below 60%. Coefficients of variation are 3-13% at 10 ppb.     

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 carbadox residues in animal feed

A liquid chromatographic (LC) method for determining residues of carbadox in the 0.01-10 ppm range in swine feed is described. Carbadox is extracted from ground feed with 25% acidified methanol-CHCl3, removed from emulsion-forming coextractables via an alumina column, separated from highly colored pigments by acid-base liquid-liquid partitioning, and finally isolated from interferences on a second alumina column. Isocratic reverse phase LC at 305 nm is used for quantitation. The average overall recovery at the 0.1, 0.5, and 1.0 ppm spike levels was 83.0% with a standard deviation of 2.04% and a coefficient of variation of 2.46%.     

Liquid chromatographic determination of tetracycline residues in animal feeds

A liquid chromatographic method for the multiresidue determination of tetracyclines (TCs) in feeds is described. The levels of quantitation were 10 ppm each for tetracycline-HCl (TC), oxytetracycline (OTC), and chlortetracycline-HCl (CTC); the detection limit was 40 ppb for each. The calibration curves were linear between 2.5 and 100 ppm. The procedure involved double extraction with pH 2.0 and pH 4.5 McIlvain buffers, cleanup on a Sephadex LH-20 column, separation on a Nova-Pak C18 column, and detection at 370 nm. Recoveries of 10 micrograms/g of each TC in multiresidue feed samples ranged from 55.8 to 75.5% for OTC, 71.6 to 100% for TC, and 22.4 to 60.6% for CTC. The identities of the TCs were confirmed by thin layer chromatography.     

Liquid chromatographic determination of spiramycin residues in chicken tissues

A liquid chromatographic (LC) method is described for determination of spiramycin residues in chicken muscles. The drug is extracted from muscles with acetonitrile, the extract is concentrated to 3-4 mL and rinsed with n-hexane followed by ethyl ether, and the drug is extracted with chloroform. LC analysis is carried out on a Zorbax BP-C8 column, and spiramycin is detected spectrophotometrically at 231 nm. Recoveries of spiramycin added to chicken muscles at 0.2 and 0.1 ppm were 93.9 and 89.0%, respectively. The detection limit was 5 ng for spiramycin standard, and 0.05 ppm in chicken muscles.     

Liquid chromatographic determination of aflatoxin M1 in milk

The official AOAC method for aflatoxin M1 in milk was modified by replacing cellulose column chromatography with cartridge chromatographic cleanup and replacing thin layer chromatographic (TLC) determination with liquid chromatographic (LC) quantitation to yield a new method for bovine and porcine milk. An acetone extract of milk is treated with lead acetate and defatted with hexane, and M1 is partitioned into chloroform as in the AOAC method. Chloroform is removed by evaporation under a stream of nitrogen at 50 degrees C. The residue is dissolved in chloroform, the vessel is rinsed with hexane, and the 2 solutions are applied in sequence to a hexane-activated silica Sep-Pak cartridge. Less polar impurities are removed with hexane-ethyl ether, and M1 is eluted with chloroform-methanol, and determined by C18 reverse phase LC using fluorescence detection. Recoveries of M1 added to bovine milk at 0.25, 0.50, and 1.0 ng/mL were 90.8, 93.4, and 94.1%, respectively. The limit of detection was less than 0.1 ng M1/mL for both bovine and porcine milk.     

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 chloramphenicol residues in meat: interlaboratory study

A liquid chromatographic (LC) method for the determination of chloramphenicol (CAP) residues in meat at the 10 microgram/kg level was tested in an interlaboratory study. The method used, based on aqueous extraction and sample cleanup with a cartridge containing Extrelut, was published earlier. A prestudy to familiarize collaborators with the method was performed before the actual interlaboratory precision study. The meat samples used in the precision study were prepared by diluting dosed chicken and pig muscle tissues with blank tissues from other species. Fourteen laboratories received 20 meat samples; 13 laboratories actually participated in the study. Two blank samples and 2 positive samples each of pig, calf, chicken, lamb, and cow meat were tested. The chloramphenicol concentrations in the positive samples ranged from 6.5 to 21 micrograms/kg. The overall mean reproducibility coefficient of variation was 17.9% after the results per laboratory were corrected for the mean recovery obtained within each sample series. The overall mean recovery was 55.1% with a coefficient of variation of 18.0% at the 10 micrograms/kg level. The limit of detection, based on chromatograms of blank samples, was estimated to be 1.5 micrograms/kg of chloramphenicol. No false positives or false negatives were observed in the concentration range tested; only 2 false positive results above the detection limit (1.7 and 6 micrograms/kg) on a total number of 60 blank analyses (3.3%) were observed.     

Liquid chromatographic determination of zearalenone and alpha- and beta-zearalenols in milk

Previous research has demonstrated transmission of zearalenone and alpha- and beta-zearalenols into the milk of cows and other animals. Since human intake of zearalenone and its metabolites via milk is an unknown factor in risk assessment of zearalenone and because appropriate methodology for their determination in milk is not available, a rapid and sensitive analytical method has been developed. Essentially, the method includes extraction with basic acetonitrile, acidification, partition into methylene chloride on a hydrophilic matrix, cleanup on an aminopropyl solid phase extraction column, and reverse-phase liquid chromatography with fluorescence detection. Recoveries from milk averaged 84% for zearalenone, 93% for alpha-zearalenol, and 90% for beta-zearalenol at spiking levels of 0.5 to 20 ng/mL. As little as 0.2 ng/mL of zearalenone and alpha-zearalenol and 2 ng/mL of beta-zearalenol can be detected in milk. These 3 compounds are stable in refrigerated milk for at least 2 weeks and in milk brought to boiling. Enzymes (beta-glucuronidase and aryl sulfatase) may be added to milk prior to extraction to hydrolyze any conjugates.     

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.     

Gas-liquid chromatographic determination of pentachlorophenol in milk

A gas-liquid chromatographic (GLC) method developed by other workers for determining pentachlorophenol (PCP) in water has been adapted for determining PCP in raw and homogenized milk. PCP is extracted from milk with benzene and from the benzene into a potassium carbonate solution. Acetic anhydride is added to the aqueous solution to form PCP acetate, which is extracted into hexane and then determined by electron capture GLC. Duplicate determinations of PCP in milk fortified at levels of 0.02, 0.2, and 2.0 ppm gave respective average recoveries of 80.0, 87.2, and 85.0%. PCP levels as low as 0.005 ppm can be detected in 20 g milk.     

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 depletion of bithionol sulfoxide and its two major metabolites in bovine milk

A liquid chromatographic method is described for determining bithionol sulfoxide and its metabolites, bithionol and bithionol sulfone, in milk. Samples are treated with HCl to precipitate proteins and to permit extraction of bithionol sulfoxide in nonionized form. Tetrahydrofuran is added to the organic phase to facilitate extraction in diethyl ether; the dried residue is dissolved in chloroform, hexane, and sodium hydroxide and subjected to LC analysis. Residues of bithionol sulfoxide and its 2 metabolites were determined in milk of lactating cows. Holstein-Friesian dairy cows were administered a single oral dose of bithionol sulfoxide (50 mg/kg). Milk samples were analyzed with a reliable detection level of 0.025 microgram/mL for each compound. Residues of bithionol sulfoxide and bithionol were detected during 30 and 16 milkings, respectively; bithionol sulfone was never present at detectable levels.     

Liquid chromatographic determination of vitamins D2 and D3 in fortified milk and infant formulas

A liquid chromatographic (LC) method was developed for determining vitamins D2 and D3 in fortified milk and infant formulas. The lipid-soluble components were extracted from the aqueous phase by homogenizing in isopropanol-methylene chloride with magnesium sulfate added to remove water. The vitamins were fractionated from the lipid material by using gel permeation chromatography (GPC) followed by further cleanup of the combined GPC fractions on a muBondapak/NH2 column. Four muStyragel (100 A) columns connected in series were used for GPC fractionation of sample extracts in methylene chloride. Injection and collection were repeated 3 times to collect enough vitamin D for quantitation. The muBondapak/NH2 column, using a mobile phase of methylene chloride-isooctane-isopropanol (600 + 400 + 1), resolved vitamin D from other UV-absorbing compounds and soy sterols in infant formula and from cholesterol in milk. Vitamins D2 and D3 coeluted as one peak, with the resolution and vitamin level sufficient for visual monitoring (280 nm/0.02 absorbance unit full scale) in a collection time of 22-26 min. A Zorbax ODS (6 micron) column and a methylene chloride-acetonitrile-methanol (300 + 700 + 2) mobile phase were used for LC quantitation; vitamins D2 and D3 were baseline resolved in about 11 min. The infant formula samples included ready-to-use and concentrated liquids prepared in nonfat milk base or soy base fortified with vitamins D2 or D3 at 400 IU/qt or L (10 micrograms). The mean percent recovery of added vitamin D3 (400-500 IU/qt) from infant formula (n = 7) was 89.6 +/- 6.7 (coefficient of variation (CV) 7.5%).(ABSTRACT TRUNCATED AT 250 WORDS)     

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%.