Determination of daminozide in apples by gas chromatography/chemical ionization mass spectrometry

A method using gas chromatography/chemical ionization mass spectrometry (GC/CIMS) for the determination of daminozide residues in apples has been developed. Daminozide was separated from the sample matrix by water extraction and cation exchange, converted to the methyl ester by treatment with HCl-methanol, and determined by GC/CIMS using succinonitrile as an internal standard. The detection level was 0.05 ppm. Recoveries were 92-104% from apples spiked at the 0.05-0.5 ppm levels. Of the 25 apple samples analyzed, only 2 were positive for daminozide (1.04 and 0.32 ppm).     

Determination of N-nitrosodiethanolamine in dinoseb formulations by mass spectrometry and thermal energy analyzer detection

A new method is described to determine trace quantities of N-nitrosodiethanolamine (NDElA) in aqueous diethanolamine (DElA) formulations and in oil solutions of dinoseb. A formate anion-exchange column is used in series with a cation-exchange column if there is DElA in the formulation. The eluate is then passed through a Clin Elut column. Depending on the concentration of NDElA in the sample, a packed silica-gel column is used to purify the extract further. This extract is analyzed on a liquid chromatograph coupled with a thermal energy analyzer (LC/TEA), using a mixture of methanol-hexane-methylene chloride containing 0.1% acetic acid (8 + 56 + 35) as the mobile phase. This solvent system gives good separation of NDElA from trace quantities of dinoseb remaining in the extract. The NDElA is also converted to the trimethylsilyl derivative and analyzed by gas chromatograph coupled with a mass spectrometer (GC/MS). Analyses of 11 commercial samples of dinoseb diethanolamine salt showed NDElA levels of 116-2409 ppm expressed relative to the weight of dinoseb. In contrast, analyses of 2 samples of organic solutions of technical dinoseb showed NDElA levels to be nondetectable and 0.3 ppm, respectively. Limit of detection by LC/TEA is 6.5 ng (0.5 ppm), and by GC/MS it is 0.02 ng (0.15 ppm). Recoveries from samples spiked at 0.514-1664 ppm range from 92.2 to 105.2%.     

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.     

Ion-pair reverse-phase liquid chromatographic determination of amprolium in complete feeds and premixes

Amprolium is extracted with methanol-water (2 + 1) containing 5mM dioctylsulfosuccinate (DOSS) and 10mM CaCl2. The analyte is separated from coextracted materials by isocratic ion-pair reverse-phase liquid chromatography, following removal of late-eluting materials on an acid alumina cleanup column, and is detected at 270 nm. The mobile phase contains 4mM DOSS with 0.3% diethylamine and 1% acetic acid in 40% acetonitrile. Linearity is satisfactory over the range of 2.5-50 micrograms/mL. Mean recovery, as determined by standard addition to commercial samples, is 100.1%. Accuracy was further tested in studies comparing the LC method to the official AOAC colorimetric method, using commercial samples, and was found to be satisfactory. Studies show that common poultry feed additives, grass meals, and some pelletization aids do not interfere with the analysis; however, when bentonite is present, recovery is decreased. The precision of the method, measured over several experiments on commercial samples, is satisfactory as indicated by coefficients of variation ranging from less than 1 to 4.5. A ruggedness test resulted in an overall CV of 3.2%, indicating the probable success of the method in a collaborative study.     

High performance liquid chromatographic determination of clocapramine in feed and plasma

A high performance liquid chromatographic method is described for the determination of clocapramine in animal feed and plasma. Samples are made alkaline and then extracted with chloroform containing opipramol as internal standard. For plasma samples, the organic phase is evaporated to dryness under a stream of nitrogen, and the residue is dissolved in dichloromethane-methanol. Extracts are chromatographed on silica gel with dichloromethane-methanol-ammonia (100 + 10 + 0.25) as eluant, and quantitated using an internal standard. Within-day precision for plasma extracts (n = 15) was 3.39, 5.7, and 4.13% at 5, 10, and 15 mg clocapramine/L plasma, respectively, and day-to-day precision was 4.6, 6.8, and 4.4% at the same levels. The detection limit was 0,5 mg/L. Recovery from feed over the concentration range 2-6 g/kg was greater than 96%.     

Determination of N-nitrosodimethylamine levels in some Canadian 2,4-D amine formulations

Levels of N-nitrosodimethylamine (NDMA) were determined in 112 samples of 2,4-dichlorophenoxyacetic acid, (2,4-D), formulated as the dimethylamine salt, collected over a 2 year period from products on the Canadian market. A sample aliquot is partitioned with dichloromethane, and the co-extracted dimethylamine is removed by cleanup on a silica gel column. The eluates containing NDMA are concentrated, an internal standard of N-nitrosodipropylamine is added, and nitrosamine levels are determined using a gas chromatograph interfaced with a thermal energy analyzer. Recoveries of NDMA and N-nitrosodiethylamine spiked into samples were 103 +/- 16 and 96.3 +/- 9.8%, respectively. Of the 112 samples analyzed, 92 were below 1 part per million (ppm) relative to the amount of 2,4-D in the samples, 16 were between 1 and 5 ppm, and 4 were greater than 5 ppm. The gas chromatographic column used is compared to a conventional packing material for volatile nitrosamine analysis. Formation of NDMA during cleanup and analysis was shown not to occur.     

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).     

Determination of N-nitrosodipropylamine in trifluralin emulsifiable concentrates using minicolumn cleanup and gas chromatography with thermal energy analyzer

A quick method for determining N-nitrosodipropylamine (NDPA) levels in trifluralin emulsifiable concentrate formulations is described. At least 18 samples can be analyzed at one time in a minimum of fumehood space, with up to 90% savings on solvents and materials. A sample aliquot is mixed with a solution containing nitrosamine recovery standards, and nitrosamines are separated by minicolumn cleanup. Internal standard is added directly to the eluate containing the nitrosamines, and levels are determined by gas chromatography with thermal energy analyzer. Recoveries of spiked nitrosamines ranged from 98 to 102%. Coefficients of variation for samples containing less than 0.5 ppm NDPA are less than 13%. Minimum detectable limit, calculated as 3 times the noise, is 0.06 ppm. Comparison with the method formerly used by this laboratory shows no significant difference in the analytical results at 95% confidence limits, and control experiments were performed to ensure that there was no artifact formation of NDPA.     

Detection and quantification of protein residues in food grade amino acids and nucleic acids using a dot-blot fluorescent staining method

Food allergies represent an important health problem in industrialized countries, such that detection and quantitative analysis of the protein considered to be the main allergen is crucial. A dot-blot fluorescent staining method for the detection and quantitative analysis of protein residues in food grade amino acids and nucleic acids is presented. This method combines fluorescence staining with dot-blotting onto PVDF membrane. Several standard proteins, such as bovine serum albumin (66 kDa), lysozyme (14 kDa), ubiquitin (8.6 kDa), bovine insulin (5.7 kDa), and oxidized insulin B chain (3.5 kDa), were detectable at 0.1 ppm using SYPRO Ruby blot stain. Twenty-five different amino acids and two different nucleic acids of food grade were analyzed using this method combined with an internal standard addition method using BSA as an internal standard. All amino acids and nucleic acids were dissolved in 3.6% aqueous HCl and dot-blotted onto PVDF membrane before a large amount of amino acids and nucleic acid were removed. Protein residues and the internal standard protein immobilized on the membrane were stained using SYPRO ruby blot stain. The internal standard in all samples was detectable at 0.1 ppm. Samples were dissolved at 120 or 70 mg/mL, according to their solubility under acidic conditions. The detection limit of protein residues per weight was 0.8-1.4 ppm in amino acids and nucleic acids; residual protein was not detected in any sample.     

Determination of ivermectin in medicated swine feeds at the 2 ppm concentration level

An analytical method has been developed that is applicable to the determination of Ivermectin in medicated feeds at the 2 ppm concentration level. It is based upon liquid chromatographic analysis with a reverse-phase column and ultraviolet detection. After the drug is extracted from the feed into methanol, an analytical sample is prepared by the consecutive use of column chromatography on alumina and solid-phase extraction on Sep-Pak C18 and silica cartridges. This procedure has been applied to the concentration range 0.50-3.0 ppm of Ivermectin in feed with an accuracy of +/- 2% mean relative error and a precision of +/- 2% relative standard deviation at the 2 ppm concentration level.     

Simultaneous quantitation of 2-acetyl-4-tetrahydroxybutylimidazole, 2- and 4-methylimidazoles, and 5-hydroxymethylfurfural in beverages by ultrahigh-performance liquid chromatography-tandem mass spectrometry

An ultrahigh-performance liquid chromatography (UHPLC) tandem mass spectrometric (MS/MS) method was developed for the simultaneous quantification of 2-acetyl-4-tetrahydroxybutylimidazole (THI), 2- and 4-methylimidazoles (2-MI and 4-MI), and 5-hydroxymethylfurfural (HMF) in beverage samples. A C30 reversed-phase column was used in this method, providing sufficient retention and total resolution for all targeted analytes, with an MS/MS instrument operated in selected reaction monitoring (SRM) mode for sensitive and selective detection using isotope-labeled 4-methyl-d(3)-imidazole (4-MI-d(3)) as the internal standard (IS). This method demonstrates lower limit of quantification (LLOQ) at 1 ng/mL and coefficient of determination (r(2)) >0.999 for each analyte with a calibration range established from 1 to 500 ng/mL. This method also demonstrates excellent quantification accuracy (84.6-105% at 5 ng/mL, n = 7), precision (RSD < 7% at 5 ng/mL, n = 7), and recovery (88.8-99.5% at 10, 100, and 200 ng/mL, n = 3). Seventeen carbonated beverage samples were tested (n = 2) in this study including 13 dark-colored beverage samples with different flavors and varieties and 4 light-colored beverage samples. Three target analytes were quantified in these samples with concentrations in the range from 284 to 644 ng/mL for 4-MI and from 706 to 4940 ng/mL for HMF. THI was detected in only one sample at 6.35 ng/mL.     

Chlorate analyses in matrices of animal origin

Sodium chlorate is being developed as a potential food-safety tool for use in the livestock industry because of its effectiveness in decreasing concentrations of certain Gram-negative pathogens in the gastrointestinal tracts of food animals. A number of studies with sodium chlorate in animals have demonstrated that concentrations of chlorate in meat, milk, wastes, and gastrointestinal contents range from parts per billion to parts per thousand, depending upon chlorate dose, matrix, and time lapse after dosing. Although a number of analytical methods exist for chlorate salts, very few were developed for use in animal-derived matrices, and none have anticipated the range of chlorate concentrations that have been observed in animal wastes and products. To meet the analytical needs of this development work, LC-MS, ion chromatographic, and colorimetric methods were developed to measure chlorate residues in a variety of matrices. The LC-MS method utilizes a Cl(18)O(3)(-) internal standard, is applicable to a variety of matrices, and provides quantitative assessment of samples from 0.050 to 2.5 ppm. Due to ion suppression, matrix-matched standard curves are appropriate when using LC-MS to measure chlorate in animal-derived matrices. A colorimetric assay based on the acid-catalyzed oxidation of o-tolidine proved valuable for measuring ≥20 ppm quantities of chlorate in blood serum and milk, but not urine, samples. Ion chromatography was useful for measuring chlorate residues in urine and in feces when chlorate concentrations exceeded 100 ppm, but no effort was made to maximize ion chromatographic sensitivity. Collectively, these methods offer the utility of measuring chlorate in a variety of animal-derived matrices over a wide range of chlorate concentrations.     

Nitrate determination in baby food, using the nitrate ion selective electrode.

The nitrate electrode has been utilized in the determination of nitrate content in food products. The AOAC xylenol method was employed for comparative results. A reasonable correlation (r=0.91) was found between the 2 methods in the analysis of 49 samples containing 30-350 ppm nitrate. At the average nitrate content (100 ppm) of these foods, the standard error was 4.3 ppm. The electrode responds directly to the ionic activity of the nitrate ion. It has a linear concentration range of 1-6000 ppm nitrate and can be used over a wide pH range. The electrode does respond to some extent to anions other than nitrate, and some interferences do occur. These interferences are easily controlled by the use of cation exchange resins. The Corning known addition (spiking) method is used on all samples to insure correct electrode response in solutions containing variable background ionic composition. The electrode has the advantage of simplicity, speed, reproducibility, and accuracy. Work time saved using the electrode as opposed to the xylenol method is about 7 hr for the analysis of 20 samples. Error, and the need for repeating analysis, is much less frequent.     

Headspace gas chromatographic method for determination of ethanol in canned salmon: collaborative study

Six laboratories collaboratively studied a headspace gas chromatographic method for determination of ethanol in the aqueous phase of canned salmon. Ethanol is determined by a headspace sampling technique with tert-butanol as the internal standard, using a gas chromatograph equipped with a Super Q column and a flame ionization detector. With outliers excluded, the mean recoveries from samples spiked with 25.1 and 78.4 ppm ethanol were 112 and 110%, respectively. For the 4 sample pairs quantitated, repeatability coefficients of variation ranged from 1.42 to 4.25% and reproducibility coefficients of variation from 2.55 to 8.09%, with 3 of the 4 reported values less than 5%. The method has been adopted official first action.     

Quantitative conformation of dimethoate residues in wheat plants by single ion mass spectrometry.

A gas chromatographic-single ion mass spectrometric method was developed for determining dimethoate residues in wheat plants. The base peak (m/e 37) of dimethoate was chosen as the single ion peak, and methyl stearate was used as an internal standard for this analysis. The minimum detectable concentration of dimethoate by this method was about 0.1 ppm for a 20 g wheat plant sample. The recoveries of dimethoate were about 89% at 0.13 ppm and greater than 96% at 0.5-1 ppm.     

Direct determination of lead in evaporated milk and apple juice by anodic stripping voltammetry: collaborative study

A method for the direct determination of lead in evaporated milk and in fruit juice with no prior sample digestion was successfully collaborated by 13 laboratories. The anodic stripping voltammetric (ASV) method studied consisted of adding 0.2 mL aliquots of evaporated milk or 0.3 mL aliquots of fruit juice to 2.9 mL of a dechelating reagent, Metexchange. The reagent-sample mixture is then analyzed for lead by ASV with no further sample preparation. Each collaborator received 24 samples, 2 each at 5 different levels (0.07-0.70 ppm for spiked evaporated milk and 0.09-0.87 ppm for spiked apple juice) along with duplicate practice samples of labeled lead content at each of 2 levels for each sample type. All unknowns were coded with random numbers. Approximately 69% of the reporting laboratories had never analyzed either evaporated milk or fruit juice for lead. Average time between receipt of samples and reporting of results was 1.6 days for all laboratories. The pooled variations between duplicate determinations for apple juice and evaporated milk were 0.00059 and 0.00043, respectively. The method was adopted official first action for both fruit juice and evaporated milk.     

Liquid chromatographic analysis of dehydroacetic acid and its application to wines

A simple liquid chromatographic procedure is described for determination of the preservative dehydroacetic acid (DHA) in wine. No cleanup procedure is necessary; the sample is injected onto an NH2 column without further pretreatment. The mobile phase is a 9 + 1 mixture of acetonitrile-sodium acetate buffer adjusted to pH 4 with acetic acid. Total run time is less than 10 min. Dehydroacetic acid is determined by using the absorbance at 307 nm; the detection limit is estimated to be 0.2 ppm. The method may also be suitable for detecting DHA in a variety of foodstuffs in low concentrations.     

Liquid chromatographic determination of the processing aid 4-hexylresorcinol in shrimp.

A rapid, sensitive, liquid chromatographic (LC) method has been developed for determination of residuals of the processing aid, 4-hexylresorcinol, on shrimp meat. An aqueous homogenate of shrimp meat is extracted with ethyl acetate followed by precolumn preparation on a silica Sep-Pak cartridge. LC determination is preformed with a Nova-Pak C18 column, with UV detection at 214 nm. Sensitivity was 0.006 micrograms, and recovery from shrimp meat samples of known 4-hexylresorcinol addition was 94%. Shrimp treated with 4-hexylresorcinol under the recommended dip protocol had mean residuals of 1.18 ppm, with a standard deviation of 0.13 ppm.     

Determination of linuron in potatoes using capillary column gas chromatography/mass spectrometry

A convenient method for the determination of the N-methyl,N-methoxy-phenylurea herbicide (linuron) in potatoes has been developed. The herbicide is extracted from potatoes using a slightly modified Luke multiresidue procedure. The extract is analyzed directly by gas chromatography with cold on-column injection, using an ion trap mass spectrometer in the chemical ionization mode as the detector. Quantitation is performed using p-bromonitrobenzene as the internal standard. The limit of detection is 0.1 ppm. Recoveries of linuron in potatoes averaged 112 +/- 6% at the 0.5 ppm level, and 110 +/- 2% at the 0.2 ppm level. No linuron residues were found in 25 potato samples that were analyzed by this method. Two other N-methyl,N-methoxy-phenylurea herbicides, metobromuron and chlorbromuron, are also sufficiently stable to be determined by this method, but the N,N-dialkyl-phenylurea herbicides neburon, diuron, and monuron are too thermally unstable and degrade in the gas chromatograph.     

Determination of caffeine, theobromine, and theophylline in standard reference material 2384, baking chocolate, using reversed-phase liquid chromatography

A rapid and selective isocratic reversed-phase liquid chromatographic method has been developed at the National Institute of Standards and Technology to simultaneously measure caffeine, theobromine, and theophylline in a food-matrix standard reference material (SRM) 2384, Baking Chocolate. The method uses isocratic elution with a mobile phase composition (volume fractions) of 10% acetronitrile/90% water (pH adjusted to 2.5 using acetic acid) at a flow rate of 1.5 mL/min with ultraviolet absorbance detection (274 nm). Total elution time for these analytes is less than 15 min. Concentration levels of caffeine, theobromine, and theophylline were measured in single 1-g samples taken from each of eight bars of chocolate over an eight-day period. Samples were defatted with hexane, and beta-hydroxyethyltheophylline was added as the internal standard. The repeatability for the caffeine, theobromine, and theophylline measurements was 5.1, 2.3, and 1.9%, respectively. The limit of quantitation for all analytes was <100 ng/mL. The measurements from this method were used in the value-assignment of caffeine, theobromine, and theophylline in SRM 2384.