Liquid chromatographic determination of hypoxanthine content in fish tissue

A liquid chromatography (LC) method for determining the hypoxanthine content in fish tissues has been developed. Hypoxanthine is extracted with 0.6M perchloric acid, and determined by LC on a reverse phase microparticulate column with UV absorbance detection. The mobile phase is 0.01M potassium phosphate buffer (pH 4.5). The percent relative standard deviation for measurements by the recommended method was less than 7% with a detection limit of 10 ng. Recoveries of hypoxanthine added to various fish tissues were better than 90%. The operational errors, interferences, and recoveries for spiked samples have been investigated and compare favorably with an established xanthine oxidase enzyme method. The described LC method is simple, rapid, and specific for measuring hypoxanthine content in various fish tissues. Some post-mortem studies have indicated the method may also be used for the determination of adenosine monophosphate, inosine monophosphate, and inosine.     

Liquid chromatographic determination of zoalene and its metabolites in chicken tissues with electrochemical detection

A procedure is described for the quantitation of Zoalene (3,5-dinitro-o-toluamide) and its 2 major monoamino metabolites in chicken tissues. The method includes blender extraction of tissue with chloroformethyl acetate (1 + 1), adsorption of the drug and metabolites on neutral alumina, and subsequent elution of the residues with pH 3.5 formate buffer-methanol (6.5 + 3.5). Recovered residues were separated on a 5 micron C18 column with the alumina eluting solvent as the LC mobile phase. The parent drug and metabolites were detected and quantitated with an electrochemical detector in the reductive mode with a minimum level of reliable measurement of 0.1 ppm. Overall mean recoveries greater than 85% were obtained with Zoalene and its 2 monoamino metabolites in breast, thigh, and liver tissues fortified with 0.25-2.00 ppm. The results on tissues from chickens fed a diet containing 0.0125% Zoalene are presented.     

Liquid chromatographic determination of monensin in chicken tissues with fluorometric detection and confirmation by gas chromatography-mass spectrometry

An accurate, sensitive method is described for the determination of monensin residue in chicken tissues by liquid chromatography (LC), in which monensin is derivatized with a fluorescent labeling reagent, 9-anthryldiazomethane (ADAM), to enable fluorometric detection. Samples are extracted with methanol-water (8 + 2), the extract is partitioned between CHCl3 and water, and the CHCl3 layer is cleaned up by silica gel column chromatography. Free monensin, obtained by treatment with phosphate buffer solution (pH 3) at 0 degrees C, is derivatized with ADAM and passed through a disposable silica cartridge. Monensin-ADAM is identified and quantitated by normal phase LC using fluorometric detection. The detection limit is 1 ppb in chicken tissues. Recoveries were 77.6 +/- 1.8% at 1 ppm, 56.7 +/- 7.1% at 100 ppb, and 46.5 +/- 3.7% at 10 ppb fortification levels in chicken. Gas chromatography-mass spectrometry is capable of confirming monensin methyl ester tris trimethylsilyl ether in samples containing residues greater than 5 ppm.     

Liquid chromatographic determination of medroxyprogesterone acetate in tablets

A reverse-phase liquid chromatographic method is described for the assay of medroxyprogesterone acetate in tablets. An octadecylsilane (C18) column with a mobile phase of methanol-0.01M dibasic ammonium phosphate (80 + 20 v/v, pH 7.2 +/- 0.1) and photometric detection at 254 nm separates medroxyprogesterone acetate from excipients. Detector responses were linear to concentrations of medroxyprogesterone acetate over the range 50-150 micrograms/mL (r = 0.999). Mean recovery of medroxyprogesterone acetate added to tablet excipients was 100.8%. Mean assay results were 101.3% (n = 3). The assay results are comparable to those obtained by the compendial liquid chromatographic method.     

Chromatographic methods for determination of macrolide antibiotic residues in tissues and milk of food-producing animals

Tylosin, an antibiotic developed specifically for agricultural use, and erythromycin are the main macrolide antibiotics used in animal production. Two-dimensional thin layer chromatography has been used for detection of tylosin in poultry meat, eggs, and milk and for erythromycin in poultry meat. Detection limits reported are, for tylosin, 0.1 ppm in poultry meat, 0.05 ppm in egg, and 0.01 ppm in milk, and for erythromycin, 0.25 ppm in poultry meat. Liquid chromatography (LC) has also been used for determination of tylosin in milk, blood, and tissues of animals. Samples (milk, blood serum, or tissue homogenates in water or pH 2.2 buffer) were deproteinized with acetonitrile, tylosin was partitioned into methylene chloride, and the extracts were concentrated and dissolved in acetonitrile. Chromatography was done on a reverse phase end-capped C18 column using 0.002-0.005 M ammonium dihydrogen phosphate-acetonitrile-methanol (10 + 60 + 30-5 + 80 + 15). Solvent composition was varied with the type of sample analyzed. The method will detect 0.1 ppm tylosin in tissues and less in milk and blood serum. The LC method was more sensitive than microbiological assays for detection of tylosin in tissues of treated swine; recoveries of tylosin by the LC method were frequently several-fold higher.     

Liquid chromatographic determination of colchicine in pharmaceuticals: collaborative study

A liquid chromatographic (LC) method for the determination of colchicine in pharmaceutical dosage forms and the bulk drug was evaluated in an interlaboratory study which included 13 participating laboratories. The method involves extraction (or dissolution) of the active ingredient with methanol-water (1 + 1), followed by filtration of the extract and reverse phase LC using an octylsilane bonded phase column and UV detection at 254 nm. The mobile phase consists of a methanol-phosphate buffer mixture (pH = 5.5). Three commercial tablet formulations (0.5-0.6 mg colchicine/tablet), 1 synthetic injectable preparation (0.510 mg colchicine/mL), and 1 bulk drug sample were assayed in duplicate by the proposed method. The reproducibility and repeatability standard deviations based on nonpooled results for each sample ranged from 0.0062 mg/mL to 0.0147 mg/tablet and from 0.0037 mg/mL to 0.0127 mg/tablet, respectively; the corresponding coefficients of variation ranged from 1.21 to 2.54% and from 0.73 to 2.19%, respectively. The mean recovery from the synthetic injectable formulation was 100.0%. The method has been adopted official first action.     

Sensitive determination of ethopabate residues in chicken tissues by liquid chromatography with fluorometric detection

A liquid chromatographic (LC) method is described for determination of ethopabate residues in chicken tissues. The drug is extracted from tissues with acetonitrile, and the extract is concentrated to 2-3 mL. This aqueous solution is rinsed with ethyl acetate and cleaned up by Florisil column chromatography. LC analysis is carried out on a Zorbax ODS column, and ethopabate is quantitated by using a fluorometric detector set at 306 nm (excitation) and 350 nm (emission). Recoveries of ethopabate added to chicken tissues at levels of 0.01 and 0.05 ppm were 87.8 and 92.7%, respectively. The detection limit was 100 pg for ethopabate standard, and 0.5 ppb in chicken tissues.     

Determination of olaquindox residues in swine tissues by liquid chromatography

A liquid chromatographic (LC) method is described for determination of olaquindox residues in swine tissues. The drug is extracted from tissues with acetonitrile, and the extract is evaporated to dryness. This residue is cleaned up by alumina column chromatography. LC analysis is carried out on a Nucleosil C18 column, and olaquindox is quantitated by ultraviolet detection at 350 nm. The average recoveries of olaquindox added to tissues at levels of 0.2, 0.1, and 0.05 ppm were 74.0, 68.6, and 66.3%, respectively. The detection limit was 2 ng for olaquindox standard and 0.02 ppm in tissues.     

Liquid chromatographic determination of L-ascorbic acid in candies and soft drinks

The L-ascorbic acid (AsA) contents of candies and soft drinks available in the market were determined by liquid chromatography (LC). Samples are cleaned up on a disposable Sep-Pak C18 cartridge followed by reverse phase separation on an ODS column using a mobile phase of 0.1% phosphoric acid (pH 2.2). The AsA peak is detected on the basis of the UV absorption at 254 nm. The detection limit was 1 microgram/mL final concentration. Recoveries of AsA added at levels of 1-10 mg/g candy and 1-10 mg/10 mL soft drink were 99.2-101.7% with a coefficient of variation of 0.52-1.20% (n = 5). The present method allows rapid and accurate assays because it is a simple procedure compared with the official dye-titration method, and it is suitable for the routine analysis of AsA in selected candies and soft drinks.     

Simplified cleanup and liquid chromatographic ultraviolet determination of linuron and three metabolites in potatoes

A simple and efficient method is presented for the extraction, cleanup, and liquid chromatographic (LC) determination of linuron and 3 of its metabolites, 3-(3,4-dichlorophenyl)-1-methyl urea (DCPMU), 3-(3,4-dichlorophenyl) urea (DCPU), and 3,4-dichloroaniline (DCA), in potatoes. Samples are extracted with acetone, partitioned into dichloromethane-hexane (1 + 1), and cleaned up using disposable silica cartridges. LC determination is performed using a LiChrosorb NH2 5 microns column, with an isopropanol-isooctane gradient mobile phase and UV detection at 248 nm. Recoveries of linuron and 2 of the metabolites from untreated samples fortified at 0.02-2 micrograms/g ranged from 80 to 102%, while recoveries for the metabolite DCA ranged from 60 to 78%. The detection limit was 0.015 micrograms/g for linuron and each metabolite; the minimum quantitation level was 0.5 micrograms/g. The developed method was applied to potato samples from a field experiment.     

Determination of ampicillin residues in fish tissues by liquid chromatography

A liquid chromatographic (LC) method is described for determination of ampicillin residues in fish tissues. The drug is extracted from tissues with methanol, and the extract is evaporated to dryness. This residue is cleaned up by Florisil cartridge chromatography. LC analysis is carried out on a Nucleosil C18 column, and ampicillin is quantitated by ultraviolet detection at 222 nm. Recoveries of ampicillin added to tissues at levels of 0.2 and 0.1 ppm were 73.2 and 61.5%, respectively. The detection limit was 3 ng for ampicillin standard, and 0.03 ppm in tissues.     

Liquid chromatographic determination of taurine in vitamin premix formulations

A liquid chromatographic (LC) method is described for the determination of taurine in vitamin and vitamin-mineral premix formulations. The method involves extraction of taurine with 0.1 M bicarbonate buffer, followed by precolumn derivatization with dansyl chloride and LC using fluorescence detection. 6-Aminocaproic acid is used as an internal standard. A reverse phase analytical column and a mobile phase of 0.1 M acetate buffer solution (pH 7.2)-acetonitrile (75 + 25) are used. Vitamins, minerals, and other excipients in the premix formulations do not interfere in the determination. The method is simple, precise, and accurate.     

Liquid chromatographic methodology for the characterization of orange juice

Liquid chromatographic (LC) methodology potentially useful for the characterization of orange juice, with particular regard to detecting adulteration of orange juice by computer pattern recognition analysis, has been developed. After dilution with methanol the juice is extracted with hexane to remove the carotenoids, which are chromatographed on a C18 column with an acetonitrile-methanol-methylene chloride mobile phase and detection at 450 nm. Further extraction of the juice with methylene chloride isolates the methoxylated flavones, which are chromatographed by reverse phase LC with an acetonitrile-methanol-water mobile phase and detection at 280 nm. The flavanone glycosides remaining in solution are chromatographed on a C18 column with an acetonitrile-water mobile phase and detection at 280 nm. The precisions of the heights of the 32 LC peaks selected for pattern recognition analysis were determined from 5 replicate analyses of a single juice. Coefficients of variation of the replicates ranged from 0.3 to 4.5%, with an average of 2.1%. Adulteration of products with sodium benzoate-fortified pulpwash or grapefruit juice can be detected by this method. Pattern recognition analysis of the data obtained for 80 authentic and 19 adulterated orange juices showed that the method is potentially useful for distinguishing between authentic and adulterated products.     

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.     

Analysis of fat-soluble vitamins. XXIX. Liquid chromatographic determination of vitamin D in AD concentrates: collaborative study

A simplified liquid chromatographic (LC) method for determining vitamin D in vitamin AD concentrates (greater than or equal to 5000 IU vitamin D/g) was collaboratively studied. In the simplified method, the 2 columns specified in AOAC LC method 43.101-43.109 are replaced by a single column, which separates the vitamin D isomers and the vitamin A esters. The procedure for oils includes dissolution and quantitation by normal phase LC. Dry multivitamin concentrates and aqueous dispersions are treated with an enzyme system and the vitamins are extracted with n-pentane. Six coded samples were distributed to 16 laboratories; 15 collaborators returned their results. Estimates of repeatability and reproducibility for the oil samples were 1.1 and 3.1%, respectively; for the high-level concentrated dry preparation 1.4 and 3.9% and for the low-level concentrated dry preparation 1.3 and 11.4%, respectively. These values are a considerable improvement over the results obtained in the 1979 multivitamin collaborative study. The method has been adopted official first action for determination of vitamin D in vitamin AD concentrates containing greater than or equal to 5000 IU vitamin D/g.     

Liquid chromatographic method for determining the macrolide antibiotic sedecamycin and its major metabolites in swine plasma and tissues

A liquid chromatographic (LC) method was developed to determine sedecamycin, a 17-membered macrolide antibiotic used for treating swine dysentery, and its major metabolites (lankacidin C, lankacidinol A, and lankacidinol) in swine plasma and tissues. Plasma is directly extracted with ethyl acetate and analyzed by liquid chromatography without purification. Tissues are homogenized in a phosphate buffer containing sodium chloride, and then extracted with ethyl acetate. The extracts are subjected to silica gel-Florisil, double-layered column chromatography to remove endogenous interfering substances. The LC determination uses silica gel and ODS-silica as a stationary phase. The detection limits for sedecamycin and its metabolites were less than or equal to 0.05 ppm, and average recoveries and coefficients of variation (0.2-1 ppm range) were greater than 75% and less than 10%, respectively.     

Liquid chromatographic-electrochemical detection screening procedure for six nitro-containing drugs in chicken tissues at low ppb level

A screening procedure is described for the detection of furazolidone, nitrofurazone, aklomide, zoalene, nitromide, and sulfanitran residues in a single extract of chicken liver, breast, or thigh muscle at the low ppb level. The method includes extraction of tissue with chloroformethyl acetate-dimethyl sulfoxide (50 + 50 + 0.8), adsorption on neutral alumina, and subsequent elution of the residues with pH 6.0 phosphate buffer-methanol (1 + 1). Eluants are separated on a 25 cm, 5 microns C18 column with pH 6.0 phosphate buffer-methanol (57.5 + 42.5) as mobile phase. The drugs are detected with an electrochemical detector in the reductive mode at -0.8 V. Mean recoveries from all tissues ranged from 76.5% for nitrofurazone to 97.1% for zoalene.     

Reverse phase liquid chromatographic assay for calcium pantothenate in multivitamin preparations and raw materials

A reverse phase liquid chromatographic (LC) method has been developed for the assay of calcium pantothenate in commercial multivitamin tablet formulations and raw materials. The assay was validated according to the Pharmaceutical Manufacturers Association Quality Control HPLC Committee guidelines. The chromatographic system includes a C-18 column and a mobile phase consisting of 0.25M sodium phosphate buffer, pH 2.5, and acetonitrile (97 + 3 v/v). The column effluent is monitored by UV detection at 205 nm. The sample preparation involves only extraction in water followed by filtration. The method is stability-indicating with a detection limit of approximately 50 ng/mL of the calcium pantothenate in the samples. The system is linear from at least 0.02 to 0.10 mg/mL. The mean recovery of spiked placebos ranged from 98.7 to 99.8%. The within-day precision of the assay on finished products (N = 6) ranged from 0.3 to 2.0% CV. A system suitability criterion for resolution is based on the separation between calcium pantothenate and 2 closely eluting compounds, saccharin and a saccharin degradation product, 2-sulfamoylbenzoic acid.     

Determination of fluazifop-butyl and fluazifop acid in soybeans and soybean oil using liquid chromatography with oxidative amperometric detection

A new method is described for the determination of the herbicide fluazifop-butyl, and its metabolite fluazifop acid, in soybeans and soybean oil as fluazifop acid. Liquid chromatography with amperometric detection (LC/AD) is used to determine fluazifop acid produced from the metabolism or base hydrolysis of fluazifop-butyl in soybeans and soybean oil. These foods were spiked with fluazifopbutyl at 0.05, 0.10, and 0.50 ppm and hydrolyzed with 0.2N NaOH in methanol. The hydrolysate (adjusted to pH less than or equal to 1) is extracted with dichloromethane and the extract is washed with 1.0% NaHCO3. The NaHCO3 is acidified to pH less than or equal to 1 and extracted with dichloromethane; the partitioning is repeated 2 more times. The dichloromethane is removed, mobile phase solvent is added, and aliquots are injected onto a PRP-1 liquid chromatographic column; fluazifop acid is separated from coextracted compounds and detected at an applied potential of + 1.25 V, using an amperometric electrochemical detector in the oxidation mode. Recoveries ranged from 69 +/- 6.5 to 101 +/- 18% and from 72 +/- 7.5 to 88 +/- 11% for soybeans and soybean oil, respectively. Accuracy of these recoveries was confirmed by use of 14C-radiolabeled fluazifop-butyl and by liquid scintillation spectrometry of the 14C-fluazifop acid released.     

Reverse-phase liquid chromatographic determination of lysine in complete feeds and premixes, using manual precolumn derivatization

A sample portion is hydrolyzed with 6N HCl for 23 h and cooled, the pH is adjusted to 7.7 with NaOH, and the solution is diluted with pH 7.7 borate buffer. An aliquot of the sample extract is derivatized with 9-fluorenylmethyl chloroformate (9-FMC). Lysine is separated from other amino acids by isocratic reverse-phase liquid chromatography (LC) using fluorescence detection: 260 nm excitation and 313 nm emission. The mobile phase is acetonitrile-0.1M acetic acid (pH 4.2) buffer (53 + 47). Linearity is satisfactory over a range of 0.4-24 micrograms/mL. Results from 9 feed samples (1.1-2.7% lysine) analyzed by both the LC method and an amino acid analyzer were not significantly different statistically. Recovery of standard lysine, spiked just before derivatization on these same 9 samples (in duplicate), was 100.9% with a coefficient of variation (CV) of 2.4%. A study of within-day and between-day method precision resulted in CVs of 1.1 and 1.8%, respectively. The variation of results was negligible when the method was tested for ruggedness on 7 factors.