Microstructure of Seeds,Flours, and Starches of Legumes

The microstructure of cotyledons and flours of both garbanzo beans and peas were examined by a scanning electron microscope (SEM) at low and high magnification. While the cells in the outer layer of the cotyledon were elongated and tightly packed in both garbanzo beans and peas, they were rounder and more loosely packed in the central part of the cotyledon, with many intercellular spaces. Cotyledon cells of garbanzo beans were smaller than those of the pea cultivars. Flour fractions from the inner layer of the cotyledon of garbanzo beans and pea cultivars had much finer particles; were lower in protein, lipid, ash and fiber; and contained more starch than those from the outer layer of the cotyledon. In prime starches isolated from garbanzo beans and smooth peas, protein, free lipid, and ash were lower than 0.41, 0.11, and 0.17%, respectively, indicating that the starches are highly pure. Garbanzo bean starch had smaller and smoother granules than those of smooth peas. Starch granules of wrinkled pea cv. Scout had a unique shape, with deep fissures and grooves, which could be partly responsible for difficulties encountered during the wet fractionation process.     

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.     

Composition,Thermal Behavior,and Gel Texture of Prime and Tailings Starches from Garbanzo Beans and Peas

Prime and tailings starches of garbanzo beans and peas were separated and the chemical composition, physical properties, thermal behavior, and gel properties were determined. Starch granules <35 μm were 85% in garbanzo beans, 66.8% in a smooth pea cv. Latah, and only 18.4% in a smooth pea cv. SS Alaska. Amylose content of prime starch was 35.9% in garbanzo beans, 44.5–48.8% in smooth peas, and 86.0% in wrinkled pea cv. Scout. Tailings starch amylose content was at least 8% higher than the corresponding prime starch. The endothermic enthalpy value of garbanzo bean and two smooth pea prime starches ranged from 12.1 to 14.2 J/g, while prime starch from wrinkled peas gave a distinctly lower enthalpy value of 1.1 J/g. Differential scanning calorimetry endothermic enthalpy and amylograph pasting properties of prime starch were significantly related to its amylose content (P < 0.05). Prime starches of garbanzo beans and smooth peas produced highly cohesive elastic gels. Wrinkled pea prime starch formed the strongest (though brittle) gel, as indicated by high hardness (21.8 N), low cohesiveness (0.29), and low springiness (0.82). Hardness of gel stored at 22°C and at 4°C was positively correlated with amylose content of starch.     

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%.     

Criteria for determining purity of Fusarium mycotoxins

Physical and chemical properties that may be used to determine the purity of several Fusarium mycotoxins have been investigated. A combination of analytical procedures, which include high performance thin-layer chromatography (HPTLC), liquid chromatography (LC), gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS), ultraviolet spectrometry (UV), and nuclear magnetic resonance (NMR) spectrometry have been used to examine mycotoxin standards obtained from commercial sources and from laboratory fermentations. Results of this investigation indicate that commercially available standards are greater than 90% pure, but the label weight of purchased reference standards in individual containers should be verified. Mycotoxin standards, determined to be greater than 98% pure by HPTLC, LC, and GC/MS, were examined by UV spectrometry and the coefficients of extinction were determined. An interlaboratory study, involving 5 collaborators who determined coefficients of extinction (in methanol) for identical samples, gave the following results: alpha-zearalenol (lambda 236 = 28 538 +/- 558); beta-zearalenol (lambda 238 = 24 963 +/- 747); deoxynivalenol (lambda 219 = 6395 +/- 349, lot 1), (6020 +/- 228, lot 2); and T-2 toxin (lambda 202 = 3681 +/- 255). UV maxima and coefficients of extinction are also reported for HT-2 toxin (lambda 202 = 1959), diacetoxyscirpenol (lambda 203 = 2487), neosolaniol (lambda 203 = 2644), nivalenol (lambda 220 = 5142), and fusarenon-X (lambda 217 = 5997).     

Rapid, sensitive liquid chromatographic method for determination of zearalenone and alpha- and beta-zearalenol in wheat

A rapid, sensitive liquid chromatographic (LC) method is described for quantitative determination of zearalenone and alpha- and beta-zearalenol in wheat. The procedure incorporates an internal standard, zearalenone oxime, to facilitate quantitation and automated analysis. A sample, buffered with pH 7.8 phosphate, is extracted with water-ethanol-chloroform (2 + 50 + 75) and cleaned up. The final residue is dissolved in LC mobile phase and injected onto a reverse phase RP-18 column under the following conditions: water-methanol-acetonitrile (5 + 3 + 2) mobile phase; fluorescence (excitation wavelength 236 nm, 418 nm cut-off emission filter) and UV (254 nm, range 0.0025 AU) detectors. The limit of detectability (twice background) is 0.5 ng for zearalenone and alpha-zearalenol standards on the fluorescence detector and 4 ng for beta-zearalenol on the UV detector, which is equivalent to 20 micrograms zearalenone and 20 micrograms alpha-zearalenol/kg, and 160 micrograms beta-zearalenol/kg feed. Standard curves are linear over the range 0-35 ng zearalenone and alpha-zearalenol on the fluorescence detector and 0-50 ng beta-zearalenol on the UV detector. Recoveries of all compounds are 87.5-101% in the range 0.1-3.0 mg/kg (ppm).     

Multiresidue method for isolation and liquid chromatographic determination of seven benzimidazole anthelmintics in milk

A multiresidue method for the isolation and liquid chromatographic determination of 7 benzimidazole anthelmintics (thiabendazole, oxfendazole, para-hydroxyfenbendazole, fenbendazole sulfone, mebendazole, albendazole, and fenbendazole) in milk is presented. Blank or benzimidazole-spiked milk samples (0.5 mL) were blended with octadecylsilyl (C-18, 18% load, end-capped) derivatized silica packing material. A column made from the C-18/milk matrix was first washed with hexane (8 mL), and then the benzimidazoles were eluted with methylene chloride-ethyl acetate (1 + 2, v/v; 8 mL). The eluate contained benzimidazole analytes which were free from interfering compounds as determined by UV detection (photodiode array, 290 nm). Correlation coefficients of standard curves for individual benzimidazoles isolated from spiked samples were linear (0.989 +/- 0.003 to 0.998 +/- 0.001) with recoveries ranging from 70 +/- 9% to 107 +/- 2% for the concentration range (62.5-2000 ng/mL) examined. The inter-assay variabilities ranged from 4 +/- 1% to 9 +/- 7% with intra-assay variabilities of 3-6%.     

Rapid confirmatory assay for determining 12 sulfonamide antimicrobials in milk and eggs by matrix solid-phase dispersion and liquid chromatography-mass spectrometry

A rapid confirmatory method for determining 12 sulfonamide (SAs) antibacterials in whole milk and eggs is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography (LC)-mass spectrometry (MS). The LC-MS instrument was equipped with an electrospray ion source and a single quadrupole. After 4 mL of a milk sample containing the analytes had been deposited on sand (crystobalite), this material was packed into an extraction cell. SAs were extracted by flowing 4 mL of water through the cell heated at 75 degrees C. With some modifications, this procedure was applied also to eggs. After pH adjustment and filtration, 0.5 mL of the final extracts was then injected into the LC column. MS data acquisition was performed in the positive-ion mode and by monitoring at least three ions for each target compound. The in-source collision-induced dissociation process produced confirmatory ions. At the 50 ng/g level, recovery of the analytes in milk and eggs was 77-92% with relative standard deviations ranging between 1 and 11%. Estimated limits of quantification (S/N = 10) were 1-3 ng/g of SAs in milk and 2-6 ng/g in eggs. With both matrices, attempts to reduce the analysis time by using a short chromatographic run time caused severe ion signal suppression for the early-eluted SAs. This effect was traced to competition effects by polar endogenous coextractives, maybe proteinaceous species, which are eluted in the first part of the chromatographic run. This unwelcome effect was almost completely removed by simply adopting more selective chromatographic conditions.     

Improved liquid chromatography methods for the separation and quantification of biotin in NIST standard reference material 3280: multivitamin/multielement tablets

Two independently developed liquid chromatography (LC) methods for the quantitative determination of biotin in multivitamin/multielement tablets (NIST Standard Reference Material 3280 (SRM 3280)) are described. The methods use distinctly different tablet extraction solvents (methanol vs 1.5% aqueous formic acid) and analyte detection principles (mass spectrometry (MS) versus evaporative light-scattering detection (ELSD)) to ensure quantitative reliability. The use of different extraction and detection procedures allows cross-validation of the methods and enhances confidence in the final quantitative results. Both methods yield highly comparable results for the mean level of biotin (LC/MS = 26.5 mg/kg +/- 0.3 mg/kg (n = 12); LC/ELSD = 24.7 mg/kg +/- 1.7 mg/kg (n = 12)) in SRM 3280, yet the methods differ considerably in their analytical characteristics. The isotope-dilution LC/MS method exhibits excellent linearity from 0.02 ng to 77 ng biotin on-column with a method limit of detection (LOD) and limit of quantification (LOQ) of 0.02 ng (S/N > 3) and 0.06 ng (S/N > 10) biotin on-column, respectively. The LC/ELSD method exhibits good linearity from 155 ng to 9900 ng biotin on-column with a method LOD and LOQ of 155 ng (S/N > 3) and 310 ng (S/N > 10) biotin on-column, respectively. Method performance data indicates that the LC/MS method is analytically superior to the LC/ELSD method; however, either method in combination with SRM 3280 should provide quality assurance, accuracy, and traceability for biotin levels in multivitamin/multielement dietary supplements.     

Liquid chromatographic determination of paraquat and diquat in crops using a silica column with aqueous ionic mobile phase.

A method was developed for the determination of paraquat (PQ) and diquat (DQ) in high moisture food crops. Samples were digested with 6M HCl, and the herbicides were isolated from the digest using pH-controlled silica solid phase extraction. The analytes were then determined by ion-pairing liquid chromatography with a silica analytical column, sodium chloride as the ion-pairing reagent, and acetonitrile as the organic modifier. A diode array UV absorbance detector was used to simultaneously quantify PQ and DQ at their respective maximum absorbance wavelengths, 257 and 310 nm. Average recoveries of PQ and DQ standards from 4 different crops fortified at 0.01-0.50 ppm levels ranged from 79.3 to 104.8%.     

Matrix solid-phase dispersion (MSPD) isolation and liquid chromatographic determination of oxytetracycline, tetracycline, and chlortetracycline in milk

A multiresidue method for the isolation and liquid chromatographic determination of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) antibiotics in milk is presented. Blank and tetracycline (OTC, TC, and CTC) fortified milk samples (0.5 mL) were blended with octadecylsilyl (C18, 40 microns, 18% load, endcapped, 2 g) derivatized silica packing material containing 0.05 g each of oxalic acid and disodium ethylenediaminetetraacetic. A column made from the C18/milk matrix was first washed with hexane (8 mL), following which the tetracyclines were eluted with ethyl acetate-acetonitrile (1 + 3; v/v). The eluate contained tetracycline analytes that were free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array, 365 nm). Correlation coefficients of standards curves for individual tetracycline isolated from fortified samples were linear (from 0.982 +/- 0.009 to 0.996 +/- 0.004) with average percentage recoveries from 63.5 to 93.3 for the concentration range (100, 200, 400, 800, 1600, and 3200 ng/mL) examined. The inter-assay variability ranged from 8.5 +/- 2.4% to 20.7 +/- 13.0% with an intra-assay variability of 1.0-9.3%.     

Normal phase liquid chromatographic determination of nanogram quantities of ivermectin in cattle blood or plasma

A method has been developed for determining ivermectin in 5 mL samples of cattle blood by a 2-step process: cleanup solvent extraction followed by direct injection onto a normal phase liquid chromatography (LC) system with UV detection. Recovery was 77-80% +/- 5.5% standard deviation. Endogenous interference that may be present caused the lower limit of detection to be set at 4-5 ng/mL. The method was used to show that in blood the distribution of ivermectin favors plasma in a fixed proportion over cellular material, and further to provide a time-course profile of ivermectin in the whole blood of injected cattle. In whole blood, ivermectin concentration peaked between 3 and 5 days and dissipated slowly with a half-life of 3 days.     

Simultaneous determination of ochratoxin A and cyclopiazonic,mycophenolic, and tenuazonic acids in cornflakes by solid-phase microextraction coupled to high-performance liquid chromatography

A solid-phase microextraction (SPME) method, coupled to liquid chromatography with diode array UV detection (LC-UV/DAD), for the simultaneous determination of cyclopiazonic acid, mycophenolic acid, tenuazonic acid, and ochratoxin A is described. Chromatographic separation was achieved on a propylamino-bonded silica gel stationary phase using acetonitrile/methanol/ammonium acetate buffer mixture (78:2:20, v/v/v) as mobile phase. SPME adsorption and desorption conditions were optimized using a silica fiber coated with a 60 microm thick polydimethylsiloxane/divinylbenzene film. Estimated limits of detection and limits of quantitation ranged from 3 to 12 ng/mL and from 7 to 29 ng/mL, respectively. The method has been applied to cornflake samples. Samples were subjected to a preliminary short sonication in MeOH/2% KHCO(3) (70:30, v/v); the mixture was evaporated to near dryness and reconstituted in 1.5 mL of 5 mM phosphate buffer (pH 3) for SPME followed by LC-UV/DAD. The overall procedure had recoveries (evaluated on samples spiked at 200 ng/g level) ranging from 74 +/- 4 to 103 +/- 9%. Samples naturally contaminated with cyclopiazonic and tenuazonic acids were found; estimated concentrations were 72 +/- 9 and 25 +/- 6 ng/g, respectively.     

Matrix solid phase dispersion isolation and liquid chromatographic determination of five benzimidazole anthelmintics in fortified beef liver

A multiresidue method for isolation and liquid chromatographic determination of 5 benzimidazole anthelmintics (thiabendazole, oxfendazole, mebendazole, albendazole, and fenbendazole) in beef liver tissue is presented. Blank or benzimidazole-fortified liver samples (0.5 g) were blended with octadecylsilyl derivatized silica packing material (C18, 18% load, endcapped, 2 g). A column made from the C18/liver matrix was first washed with hexane (8 mL), following which the benzimidazoles were eluted with acetonitrile. The acetonitrile extract was then passed through an activated alumina column. The eluate contained benzimidazole analytes that were free from interfering compounds as determined by UV detection (photodiode array, 290 nm). Correlation coefficients of standard curves for individual benzimidazoles isolated from fortified samples, using internal standardization, were linear (0.996 +/- 0.002 to 0.999 +/- 0.001) with average relative percentage recoveries from 62.0 +/- 6.7 to 86.8 +/- 8.6% for the concentration range (100-3200 ng/g) examined. The interassay variability was 7.0 +/- 4.1 to 12.9 +/- 10.2% with an intra-assay variability from 2.2 to 4.0%.     

Development of multiclass methods for drug residues in eggs: hydrophilic solid-phase extraction cleanup and liquid chromatography/tandem mass spectrometry analysis of tetracycline, fluoroquinolone, sulfonamide, and beta-lactam residues

A method was developed for detection of a variety of polar drug residues in eggs via liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). A total of twenty-nine target analytes from four drug classes-sulfonamides, tetracyclines, fluoroquinolones, and beta-lactams-were extracted from eggs using a hydrophilic-lipophilic balance polymer solid-phase extraction (SPE) cartridge. The extraction technique was developed for use at a target concentration of 100 ng/mL (ppb), and it was applied to eggs containing incurred residues from dosed laying hens. The ESI source was tuned using a single, generic set of tuning parameters, and analytes were separated with a phenyl-bonded silica cartridge column using an LC gradient. In a related study, residues of beta-lactam drugs were not found by LC/MS/MS in eggs from hens dosed orally with beta-lactam drugs. LC/MS/MS performance was evaluated on two generations of ion trap mass spectrometers, and key operational parameters were identified for each instrument. The ion trap acquisition methods could be set up for screening (a single product ion) or confirmation (multiple product ions). The lower limit of detection for screening purposes was 10-50 ppb (sulfonamides), 10-20 ppb (fluoroquinolones), and 10-50 ppb (tetracyclines), depending on the drug, instrument, and acquisition method. Development of this method demonstrates the feasibility of generic SPE, LC, and MS conditions for multiclass LC/MS residue screening.     

Quantification of the mycotoxin patulin by a stable isotope dilution assay.

Two stable isotope dilution assays for the quantification of patulin [4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one] in foods were developed using (13)C-labeled patulin as the internal standard. One method was performed by means of LC/MS in negative electrospray ionization mode without derivatization; the other used HRGC/HRMS after trimethylsilylation of the patulin isotopomers. In comparison with previously reported methods based on high-performance liquid chromatography with UV detection, HRGC/HRMS of the derivatized samples showed better repeatability, higher recovery rates (96% at a spike level of 200 ng/L), and a 100 times lower detection limit (12 ng/L). In contrast, LC/MS showed a much lower performance as compared to HPLC/UV or HRGC/HRMS. Using HRGC/HRMS, the mycotoxin was quantified in many different fruit products and in molded wheat bread.     

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.     

Comparison of an ELISA-based screening test with liquid chromatography for the determination of aflatoxins in corn.

An enzyme-linked immunosorbent assay (ELISA) screening test (CITE PROBE) was compared to liquid chromatography (LC) for the determination of aflatoxins in naturally contaminated corn samples. The CITE PROBE, with a positive/negative cutoff of 5 ng/g aflatoxin B1, was correct (based on LC results) on 47 of 51 samples. Two of the incorrect responses by the CITE PROBE were false positives on samples containing 4.4 ng/g and 4.1 ng/g aflatoxins by LC. Another incorrect response was a false negative on a sample containing 5.5 ng/g aflatoxins by LC. The fourth incorrect response was a false positive on a sample containing 1.9 ng/g aflatoxins by LC. On the basis of these results, the CITE PROBE was determined to be a reliable screening method for the detection of greater than or equal to 5 ng/g aflatoxins in corn.     

Determination of 4,4'-dinitrocarbanilide (DNC), the active component of the antifertility agent nicarbazin, in chicken, duck, and goose plasma

4,4'-Dinitrocarbanilide (DNC) was extracted from chicken, duck, and goose plasma and isolated by reversed-phase high-performance liquid chromatography. DNC was detected by ultraviolet absorbance at 347 nm and quantified by comparison to a calibration standard. Recovery data were determined by analyzing DNC-fortified control plasma. The mean recovery of DNC in fortified chicken plasma samples was 99.7 +/- 1.9% for 0.18 and 9.1 ppm DNC, and in fortified duck and goose plasma samples was 99.5 +/- 4.9% and 101.4 +/- 4.5%, respectively, for 0.18, 9.1, and 18 ppm DNC.     

Comparison of liquid chromatographic and bioassay procedures for determining depletion of intramuscularly injected tylosin

Crossbred pigs weighing 80-110 kg were injected intramuscularly in the ham with 8.8 mg/kg tylosin. Animals were slaughtered in groups of 3 at intervals of 4 h, and 1, 2, 4, and 8 days after injection, and samples of blood, injected muscle, uninjected muscle, liver, and kidney were analyzed by liquid chromatography (LC) and by bioassay using Sarcina lutea as the test organism. The LC method was far more sensitive with a detection limit of less than 0.1 ppm, while the detection limit by bioassay was about 0.5 ppm in tissue. Results by bioassay and LC sometimes differed considerably for tissue samples. Residues in all tissues were below the tolerance limit of 0.2 ppm at 24 h, except in the injected muscle in one animal. Residues were not detected in any tissue of any animal at 48 h after treatment.