Liquid chromatographic determination of multiresidue fluorescent derivatives of ionophore compounds, monensin, salinomycin, narasin, and lasalocid, in beef liver tissue

A liquid chromatographic (LC) method with fluorometric detection was developed to quantitatively determine residue levels of monensin, salinomycin, narasin, and lasalocid in beef liver tissue. The ionophores are extracted from the tissue, purified by both alumina and Sephadex LH-20 column chromatography, and then derivatized. Lasalocid was directly esterified with 9-anthryldiazomethane (ADAM), but monensin, salinomycin, and narasin were first acetylated with acetic anhydride and then esterified with ADAM. The ADAM derivatives were purified on a silica gel column and separated by LC using an RP C-8 5 micron column. A fluorescence detector set at 365 nm (excitation) and 418 nm (emission) was used to monitor the column effluent. The detection limits were 0.15 ppm, and the calibration curves were linear between 0.5 and 5.0 ppm for all 4 ionophores. Mean recoveries were 57, 70, 75, and 90% for lasalocid (5 ppm), monensin (2.5 ppm), salinomycin (2.5 ppm), and narasin (2.5 ppm), respectively. The ionophores were also separated and semiquantitated by using bioautography and thin layer chromatography with a vanillin spray.     

Determination of natamycin in cheese and cheese rind: interlaboratory collaborative study

A collaborative test on the determination of natamycin in cheese and cheese rind was conducted. Participants were from 37 laboratories in 13 countries. Eight samples, consisting of 4 duplicates, were investigated by a spectrometric method and a liquid chromatographic (LC) method. The spectrometric method gave good results (coefficient of variation [CV] = 12%) and the LC method with ultraviolet detection gave reasonable results (CV = 25%) for levels down to 15 mg/kg (0.9 mg/dm2). For very low levels, a preconcentration step is necessary, but even then quantitation is poor (CV = 35-37%) for both methods at 1.7 mg/kg, although the presence of natamycin can be detected qualitatively. For a level of 0.3 mg/kg, quantitation is poor (CV = 39%) for the LC method and impossible (CV = 60%) for the spectrometric method.     

Liquid chromatographic determination of lasalocid sodium in chicken skin: interlaboratory study

The U.S. Food and Drug Administration (FDA) sponsored an interlaboratory study of a liquid chromatographic determinative procedure for lasalocid sodium in chicken skin with adhering fat. Four laboratories analyzed 35 dosed tissue samples and 82 fortified tissue samples containing lasalocid at levels ranging from 0.1 to 0.6 ppm. Samples were homogenized with acetonitrile, washed with hexane, and partitioned into the mobile phase prior to analysis liquid chromatography with fluorescence detection. The results of the interlaboratory study showed good reproducibility for fortified samples. Fortification levels, average recoveries, and interlaboratory percent coefficients of variation were as follows: 0.6 ppm, 0.57 ppm, and 9.7; 0.3 ppm, 0.25 ppm, and 9.1; and 0.15 ppm, 0.14 ppm, and 7.0, respectively. Data for analysis of the dosed tissue also showed good agreement among the laboratories.     

Rapid reverse phase liquid chromatographic determination of aflatoxin M1 in milk

A rapid, economical, and reliable liquid chromatographic (LC) method is described for determination of aflatoxin M1 in milk. The method includes an improved AOAC extraction procedure, cleanup of the extract on a silica cartridge, and LC quantitation. Alternatively, a rapid column cleanup procedure can be used. Milk artificially spiked with aflatoxin M1 at 0.05, 0.1, and 0.5 ppb was analyzed using both new approaches as well as an AOAC method coupled with LC for quantitation of the toxin. Recovery of aflatoxin M1 by the first approach of the new method ranged between 93.4 and 99.1%, and for the alternative procedure between 92.4 and 96.8%. The AOAC method gave lower recovery (85.6-90.7%) of toxin, but the results from this method had a somewhat smaller standard deviation for replicate analyses than did results of the new method.     

Problems of official methods and new techniques for analysis of foods and feeds for vitamin A

The official methods for measuring vitamin A and carotene in foods and feeds have evolved over several decades. New procedures are needed to permit the use of modern analytical techniques such as liquid chromatography (LC), and to expand the application of official methods to a wide variety of foods. Recent improvements in methodology are reviewed. Emphasis is often placed on the separations achieved by LC, but all factors that affect quantitation, such as sampling, preparation of standards, and protection of the vitamin against oxidation, must be considered in the development of new methods. Many approaches to analysis are possible because of the versatility of LC but not all of them can be used in official methods. Methods for milk, margarine, and feeds can be prepared now for collaborative assays. Although straightforward methods can be developed for the measurement of beta-carotene in certain foods, a full examination of provitamins will probably continue to require a flexible approach and special expertise.     

Bioautography and chemical characterization of antimicrobial compound(s) in commercial water-soluble annatto extracts

Annatto preparations based on extracts of the seed of tropical bush Bixa orellana L consist of carotenoid-type pigments. Previous reports indicate that commercial annatto extracts have biological activities against microorganisms of significance to food fermentation, preservation, and safety. The objective of this study was to separate and identify the compound(s) responsible for the antimicrobial activity of annatto preparations. Commercial water-soluble annatto extracts were screened by thin-layer chromatography and bioautography followed by liquid chromatography/photodiode array/mass spectrometry (LC/PDA/MS) analysis of active fractions. Bioautography revealed two fractions with antimicrobial activity against Staphylococcus aureus. LC/PDA/MS analysis of both fractions revealed 9'-cis-norbixin (UV(max) 460 and 489 nm) and all-trans-norbixin (UV(max) 287, 470, and 494 nm) as the major components. Structure confirmation was achieved by (1)H NMR spectroscopy. Results indicate that 9'-cis-norbixin and all-trans-norbixin are responsible for the antimicrobial properties of annatto.     

Sample preparation and liquid chromatographic determination of vitamin D in food products

Vitamin D in different fortified foods is determined by using liquid chromatography (LC). Sample preparation is described for fortified skim milk, infant formulas, chocolate drink powder, and diet food. The procedure involves 2 main steps: saponification of the sample followed by extraction, and quantitation by LC analysis. Depending on the sample matrix, additional steps are necessary, i.e., enzymatic digestion for hydrolyzing the starch in the sample and cartridge purification before LC injection. An isocratic system consisting of 0.5% water in methanol (v/v) on two 5 microns ODS Hypersil, 12 X 0.4 cm id columns is used. Recovery of vitamin D added to unfortified skim milk is 98%. The results of vitamin D determination in homogenized skim milk, fortified milk powder, fortified milk powder with soybean, chocolate drink powder, and sports diet food are given.     

Development of multiclass methods for drug residues in eggs: silica SPE cleanup and LC-MS/MS analysis of ionophore and macrolide residues

A method was developed that is suitable for screening eggs for a variety of nonpolar residues in a single procedure. Residues are extracted by silica solid-phase extraction (SPE). Analysis is conducted via reverse-phase gradient liquid chromatography, electrospray ionization, and tandem ion trap mass spectrometry. For screening purposes (based on a single precursor-product ion transition) the method can detect ionophore (lasalocid, monensin, salinomycin, narasin) and macrolide (erythromycin, tylosin) residues in egg at approximately 1 ng/mL (ppb) and above and novobiocin residues at approximately 3 ppb and above. Conditions are described for confirmatory analysis based on multiple ions in the product ion spectrum. The extraction efficiency for ionophores was estimated at 60-85%, depending on drug. Recovery of macrolides and novobiocin was not as good (estimated at 40-55% after a hexane wash of the final extract was included), but the method consistently screened and confirmed these residues at concentrations below the target of 10 ppb. The method was applied to eggs from hens dosed with each drug individually. Lasalocid was found to have the highest probability of detection in eggs based on its high ionization efficiency and higher rate of deposition relative to the other drugs. The method is part of a larger scheme to provide surveillance methods for a wide variety of drug residues in eggs.     

Identification and quantitation of sulfamethazine metabolites by liquid chromatography and gas chromatography-mass spectrometry

Methods for the identification and quantitation of carbon-14 labeled sulfamethazine [4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide], N4-acetylsulfamethazine, the N4-glucose conjugate of sulfamethazine, and desaminosulfamethazine in swine tissue are described. Tissues are ground and extracted with methanol, and the 14C-labeled compounds are purified by XAD-2 column chromatography and C-18 reverse phase liquid chromatography (LC) and the 14C-labeled compounds are then methylated and identified by gas chromatography-mass spectrometry analysis. Quantitation is accomplished by measuring the amount of 14C-activity that cochromatographs (C-18 reverse phase LC) with reference compounds.     

Rapid determination of aflatoxins in raw peanuts by liquid chromatography with postcolumn iodination and modified minicolumn cleanup

A method is described for rapid cleanup followed by reverse-phase liquid chromatographic (LC) quantitation of aflatoxins in raw peanuts. A modified minicolumn cleanup is used for sample preparation, and a preliminary estimation of aflatoxin content by minicolumn can be made so that highly contaminated samples can be diluted before LC analysis. The use of the simple, quick minicolumn cleanup eliminates the need for further column or cartridge cleanup, thus greatly reducing sample preparation time. Sensitive quantitation is achieved using a phenyl column, a mobile phase of water-tetrahydrofuran (80 + 20, v/v), and postcolumn derivatization with water-saturated iodine followed by fluorescence detection. The recoveries of aflatoxins B1, B2, G1, and G2 from peanut meal spiked at 3 levels ranged from 71.7 to 88.3% (average 80%) with coefficients of variation from 2.7 to 10.4%.     

Improved Storherr tube for assisted and sweep co-distillation cleanup of pesticides, polychlorinated biphenyls, and pentachlorophenol from animal fats

A procedure is presented for the simultaneous extraction from animal fat and quantitation of 15 pesticide residues, 4 polychlorinated biphenyl (PCB) formulations, and pentachlorophenol (PCP). Silanization of the Storherr tube packing reduces thermal decomposition and allows for residue recoveries in excess of 88% from pork, beef, chicken, sheep, and rabbit fat. This Storherr tube performs well in either the assisted distillation or sweep co-distillation mode of operation. Detection limits of 2-5 ppb for the pesticides and 10 ppb for the PCBs are readily attained.     

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%.     

Determination of flunixin in edible bovine tissues using liquid chromatography coupled with tandem mass spectrometry

An accurate, reliable, and reproducible assay was developed and validated to determine flunixin in bovine liver, kidney, muscle, and fat. The overall recovery and percent coefficient of variation (%CV) of twenty-eight determinations in each tissue for flunixin free acid were 85.9% (5.9% CV) for liver, 94.6% (9.9% CV) for kidney, 87.4% (4.7% CV) for muscle, and 87.6% (4.4% CV) for fat. The theoretical limit of detection was 0.1 microg/kg (ppb, ng/g) for liver and kidney, and 0.2 ppb for muscle and fat. The theoretical limit of quantitation was 0.3, 0.2, 0.6, and 0.4 ppb for liver, kidney, muscle, and fat, respectively. The validated lower limit of quantitation was 1 ppb for edible tissues with the upper limit of 400 ppb for liver and kidney, 100 ppb for fat, and 40 ppb for muscle. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. Briefly, the method involves an initial acid hydrolysis, followed by pH adjustment ( approximately 9.5) and partitioning with ethyl acetate. A portion of the ethyl acetate extract was purified by solid-phase extraction using a strong cation exchange cartridge. The eluate was then evaporated to dryness, reconstituted, and analyzed using LC/MS/MS. The validated method is sensitive and specific for flunixin in edible bovine tissue.     

Acute bioassays with control mortality

When substantial mortality occurs in the control group of an acute bioassay, probit analysis and other methods to estimate the LC50 are usually supplemented with Abbott's correction (Abbott, 1925). This paper discusses the probit model with this adaption. Estimates of the LC50 obtained by probit analysis ignoring natural mortality or by probit analysis applied to data corrected with Abbott's formula beforehand are compared with maximum likelihood estimates based on the model. It is demonstrated that ignoring control mortality can lead to biased estimates of the LC50 even at low percentages of control mortality. For interpretation of the LC50 a distinction is made between control mortality caused by natural processes and control mortality caused by experimental manipulation.     

Determination of oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with tandem mass spectrometry

A new methodology is described for rapidly determining the herbicide oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with negative ion electrospray ionization tandem mass spectrometry (LC/MS/MS). Oryzalin is extracted from water using a polymeric sorbent solid phase extraction (SPE) column and from fruit using methanol. The water samples require no further purification, but an aliquot of the fruit sample extracts is diluted with water and purified using a polymeric 96 well SPE plate. Purified extracts are concentrated prior to determination by LC/MS/MS at m/z 345 (Q1) and m/z 281 (Q3) using an external standard for calibration. The validated limits of quantitation were 0.05 microg/L in water (drinking water, surface water, and groundwater) and 0.01 microg/g in citrus fruits (oranges and lemons) and stone fruits (peaches and cherries). Recoveries averaged 102% for water samples and 85-89% for the various types of fruit samples. For all fortification levels combined, the relative standard deviations ranged from 4 to 6% for water and from 2 to 4% for fruit.     

Liquid chromatographic determination of tenuazonic acid and alternariol methyl ether in tomatoes and tomato products

A liquid chromatographic (LC) method for determining tenuazonic acid (TA) and alternariol methyl ether (AME) in tomatoes and tomato products is described. The Alternaria metabolites are extracted from a water slurry of the sample with CHCl3, the mixture is centrifuged, and the extract is fractionated on a silica gel column. Reverse phase LC with an ultraviolet detector (for TA) and a fluorescence detector (for AME) connected in series is used for final separation and determination. The limit of determination for TA and AME is 25 and 3 ng/g, respectively, with average recoveries from catsup of 83 and 68%, respectively. The LC method also detects alternariol, but interfering peaks in some samples prevent accurate quantitation. Chemical ionization mass spectrometry (CIMS) is used to confirm TA. Samples (142) of tomatoes collected from commercial processing lines were analyzed; TA was found in 73 samples (0.4-70 micrograms/g).     

Liquid chromatographic determination of three sulfonamides in animal tissue and egg

Sulfonamides are widely used as a feed additive in animal production in Japan. The present paper is a determination of 3 sulfonamides: sulfamethazine (SMZ), sulfamonomethoxine [SMX, 4-amino-N-(3-methoxypyrazinyl)-benzenesulfonamide], and sulfadimethoxine (SDX) in animal tissue and egg by liquid chromatography (LC). Tissues were extracted with acetonitrile and fat was removed by liquid/liquid partition. The sulfonamides were purified by an ODS cartridge column; then each compound was separated by an ODS LC column and detected at 268 nm. Quantification levels were 0.02 ppm for SMZ and SMX, and 0.04 ppm for SDX; detection limits were 0.01 ppm for SMZ and SMX, and 0.02 ppm for SDX. Calibration curves were linear between 2 and 40 ng for SMZ and SMX, and between 4 and 80 ng for SDX. Recoveries from muscle and egg samples spiked with 1-2 micrograms/10 g were 81-98%.     

Antibiotic identification by high voltage electrophoresis bioautography

The high voltage electrophoresis bioautography method is applicable to meat, milk, and animal feeds. Meat is freeze-dried, powdered, and extracted with acetonitrile-water (9 + 1), and the extract is concentrated by evaporation at room temperature. Milk is examined directly or following acetonitrile-water extraction. Feed is extracted with acetonitrile-water. Samples or extracts are applied to preliminary assay plates of antibiotic medium No. 1 at pH 6 and 8, seeded with Micrococcus luteus (ATCC 9341), M. luteus DHSR (ATCC 9341A), Bacillus cereus (ATCC 11778), or B. cereus K250 TR (NCIB 11183), and nutrient agar at pH 7 seeded with B. subtilis BGA. Inactivation of penicillinase indicates beta-lactam antibiotics. Addition of trimethoprim increases sensitivity to sulfonamides. After 18-24 h incubation at 30 degrees C, plates yielding clear inhibition zones guide selection of conditions for subsequent electrophoresis bioautography. Extracts are applied (5-100 microL) to 10 mm diameter wells on electrophoresis plates 60 cm long and 40 cm wide, with a gel depth of 1.6 mm. The support medium is 1% agar and 1% agarose in Tris/succinic acid buffers pH 6 and pH 8. A potential of 1500 V is applied for 1.5 h at 15 degrees C. Following electrophoresis, the migrated antibiotics are visualized by over-layering with antibiotic medium No. 1, pH 6 or 8, seeded with M. luteus or B. cereus spore suspension; plates are incubated for 18-24 h at 30 degrees C. Identification is based on results of preliminary screening together with electrophoretic migration distances and inhibition zone appearances compared with standards.     

Determination and confirmation of 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry

A method was developed and validated to determine 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean recovery and percentage coefficient of variation (%CV) of 35 determinations for 5-hydroxyflunixin was 101% (5% CV). The theoretical limit of detection was 0.2 ppb with a validated lower limit of quantitation of 1 ppb and an upper limit of 150 ppb. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. A LC/MS/MS confirmatory method using the extraction steps of the determinative method was developed and validated for 5-hydroxyflunixin in milk from cattle. Briefly, the determinative and confirmatory methods were based on an initial solvent (acetone/ethyl acetate) precipitation/extraction of acidified whole milk. The solvent precipitation/extraction effectively removed incurred ((14)C) residues from milk samples. The organic extract was then purified by solid phase extraction (SPE) using a strong cation exchange cartridge (sulfonic acid). The final SPE-purified sample was analyzed using LC/MS/MS. The methods are rapid, sensitive, and selective and provide for the determination and confirmation of 5-hydroxyflunixin at the 1 and 2 ppb levels, respectively.     

Analysis of illegally distributed anabolic steroid products by liquid chromatography with identity confirmation by mass spectrometry or infrared spectrophotometry

Anabolic steroid products found in the illegal market are primarily oil-based injectables or tablets and often do not contain the ingredients declared on the label. An analytical scheme based on a reverse-phase liquid chromatographic (LC) system for screening, tentative identification, and quantitation is presented. Methanolic sample extracts are chromatographed on an octadecyl column using 2 mobile phases (methanol and (75 + 25) methanol-water) and tracked at 3 wavelengths (240, 210, and 280 nm) or with a photodiode array UV detector. Retention time ratios (RR) relative to testosterone and UV data are used for tentative identification. The same LC system serves as a cleanup and isolation step for identity confirmation by direct insertion probe mass spectrometry (MS) or Fourier transform infrared spectrophotometry (FTIR). Recoveries range from 96.2 to 100.2% for 11 different steroids extracted from sesame oil. LC RR values for over 40 steroids, analytical results for typical products, and MS and FTIR spectra for selected compounds are presented.