Screening method for the gas chromatographic/mass spectrometric determination of microgram/litre levels of bromate in bottled water.

Bromate can be formed as a by-product of ozone treatment that is sometimes used for the disinfection of municipal water supplies and bottled waters. The US Environmental Protection Agency has proposed a maximum contaminant level (MCL) of 10 micrograms/l for bromate in public drinking water. Should the proposed MCL for bromate become final, it may then be considered for adoption as a bottled water quality standard by the US Food and Drug Administration. This paper reports the development of a gas chromatographic/ mass spectrometric (GC/MS) method for the determination of parts-per-billion (microgram/l) levels of bromate (BrO3-) in bottled water. The GC/MS method was validated by using distilled and deionized Milli-Q water; detection limits, quantitation limits, and recoveries were determined and identities were confirmed by MS on the basis of analyses of test portions fortified with BrO3- at 0.8, 3.8, 7.7, 15, and 46 micrograms/l. The method also was evaluated on the basis of recoveries determined for two commercial brands of bottled water fortified with BrO3- at 3.8 and 7.7 micrograms/l and two commercial brands fortified at 0.8, 3.8, and 7.7 micrograms/l. For the Milli-Q water, recoveries ranged from 100 to 121%; for the fortified commercial products, recoveries ranged from 87 to 115%. The limits of detection and quantitation were determined to be 0.4 and 0.7 microgram/l, respectively. Several commercial brands of bottled water were analysed, and BrO3- was found in these products at levels ranging from none to 38 micrograms/l.

Measurement of bromate in bottled water by high-performance liquid chromatography with post-column flow reactor detection.

The objective of this work was to develop a reliable, rugged high-performance liquid chromatographic (HPLC) method for determination of trace levels of bromate (< 10 micrograms/l) in bottled water. HPLC separation was achieved by ion interaction chromatography using a C-18 reversed-phase column and a mobile phase consisting of methanol/water (20:80, v/v) with tetrabutylammonium acetate as the ion interaction reagent. A post-column reaction based on oxidation of o-dianisidine in acidic solution to a product detected at 500 nm provided selective measurement of the oxidants. The limit of detection and the limit of quantitation were 1 and 3 micrograms/l, respectively. Iodate, chlorite, and nitrite were chromatographically separated from bromate and measured by monitoring the post-column reaction. Chloride and chlorate at levels that might be found in bottled water did not interfere with the determination of bromate. Bromate was detected in bottled waters at concentrations up to 40 micrograms/l.

Determination of free glutamic acid in a variety of foods by high-performance liquid chromatography.

A survey of free glutamic acid levels in a variety of foods was conducted. The foods were analysed by high-performance liquid chromatography (HPLC). Free glutamic acid was extracted from the food with 0.02 M potassium phosphate with subsequent precipitation of other food components with acetone. Impurities were removed by passing the extract through a C-8 or C-18 reversed-phase solid-phase extraction column. The free glutamic acid was derivatized with phenylisothiocyanate, and the derivative was separated by HPLC with detection at 254 nm. Free glutamic acid levels ranged from not found (< 20 ppm) in some spices to as high as 89% in another spice.

Determination of ethyl carbamate in alcoholic beverages and soy sauce by gas chromatography with mass selective detection: collaborative study.

A method using gas chromatography with mass selective detection for the determination of ethyl carbamate (EC; also known as urethane) in alcoholic beverages and soy sauce was collaboratively studied by 17 laboratories including authors' laboratories. The method uses prepacked columns for extraction of liquids with methylene chloride, and n-propyl carbamate as the internal standard. A practice sample and 6 samples of distilled spirits, fortified wines, table wines, and soy sauces were analyzed by each collaborator. Each matrix included blind duplicates of incurred and fortified EC at 3 levels. Distilled spirits contained 50-330 ng EC/g (ppb), fortified wine 40-160 ppb, table wine 10-50 ppb, and soy sauce 15-70 ppb. The ranges of the repeatability relative standard deviations, excluding outliers, were 4.03-6.63% for distilled spirits, 4.01-5.05% for fortified wine, 3.94-6.73% for table wine, and 4.70-8.49% for soy sauce. The ranges of the reproducibility relative standard deviations, excluding outliers, were 8.53-9.49% for distilled spirits, 6.84-12.02% for fortified wine, 8.86-18.47% for table wine, and 13.87-27.37% for soy sauce. Recoveries of added EC ranged from 87 to 93%. Recoveries relative to reference values, labeled as the internal standard, obtained by using gas chromatography/tandem mass spectrometry with a triple quadrupole mass spectrometer ranged from 89 to 100%.

Comparison of two clean-up methodologies for the gas chromatographic/mass spectrometric determination of low nanogram/gram levels of polynuclear aromatic hydrocarbons in seafood.

The March 1989 oil spill in Alaska prompted the Food and Drug Administration (FDA) to conduct a thorough investigation of clean-up methodologies aimed at determining low ng/g (ppb) levels of polynuclear aromatic hydrocarbons (PAHs) in seafood. The clean-ups from a modified FDA method and a National Marine Fisheries Service (NMFS) method were evaluated on the basis of the determination of 18 PAHs at levels ranging from 1 to 5 ppb by gas chromatography/mass spectrometry. In the modified FDA method, seafood extracts were purified by a liquid-liquid partition followed by a three-step elution through silica, alumina, and C18 solid-phase extraction cartridges. In the NMFS method, seafood extracts were purified by column chromatography through a deactivated silica gel/alumina column and a gel permeation high performance liquid chromatography column. Both methods quantitated 18 PAHs at levels ranging from 1 to 5 ppb. With the exception of naphthalene, average recoveries based on internal deuterated standards ranged from 73 to 144% for the modified FDA method and 63 to 106% for the NMFS method.

Survey of sulphites determined in a variety of foods by the optimized Monier-Williams method.

A survey of sulphite levels in a variety of foods including dried fruit, dried mashed potatoes, shrimp, canned mushrooms and fruit juices has been conducted. The levels found ranged from 0 ppm in orange juice to 3722 ppm as sulphur dioxide in dried fruit. The foods were analysed by the optimized Monier-Williams procedure.

Determination of free and reversibly bound sulphite in foods by reverse-phase, ion-pairing high-performance liquid chromatography.

The reaction of sulphite with formaldehyde to form hydroxymethylsulphonate (HMS), which is very stable under the controlled conditions of this assay, was used as the first step in an analytical procedure to determine foodborne sulphite. The effect of mobile-phase pH on the stability of HMS during high-performance liquid chromatography was studied. It was found that on-column HMS dissociation to formaldehyde and bisulphite increased with the pH of the mobile phase; therefore the relatively low pH 4.7, at which the dissociation of HMS was approximately 2%, was selected for the analysis. In addition, the release of sulphite from its reversibly bound forms in wine and other foods was examined as a function of the pH of the extraction medium by following the appearance of HMS formed from the reaction of the freed sulphite with formaldehyde. The rate of dissociation of the reversibly bound sulphite was relatively slow at pH 3 but very rapid at pH 7. This difference in kinetics was exploited to develop a procedure to determine free and reversibly bound sulphite in food. The method was challenged by post-reagent spiking studies, i.e. adding the sulphite spike after the food has been blended with the sulphite-protective formaldehyde solution but before proceeding with the remainder of the assay. An average recovery of 100% with a standard deviation of 5.2% (n = 45) was realized at levels of 5, 10 and 20 ppm by weight as sulphur dioxide. Recovery of the sulphite added as the bisulphite addition product of acetaldehyde, a model compound for reversibly bound sulphite, was 95%.

Ethyl carbamate levels in selected fermented foods and beverages.

Ethyl carbamate (EC), also known as urethane, is an animal carcinogen and a by-product of fermentation. Because EC has been found in distilled spirits and wines, a variety of fermented foods and beverages were analyzed to assess its occurrence in other products. Previously described methods using a gas chromatograph-thermal energy analyzer with a nitrogen converter were modified for each matrix and gave recoveries of greater than 80%, with a limit of detection in the 1-2 micrograms/kg (ppb) range. A total of 152 test samples were analyzed; EC levels ranged from none found to 3 ppb in 15 cheeses, 6 teas, 12 yogurts, and 8 ciders; from none found to 13 ppb in 30 breads and 69 malt beverages; and from none found to 84 ppb in 12 soy sauces. Gas chromatography/mass spectrometry/mass spectrometry was used to confirm EC identity and to quantitate EC in selected food extracts.

Liquid chromatographic determination of sulfite in grapes and selected grape products.

A liquid chromatographic (LC) method is described for the determination of sulfite in grapes and certain grape products. Sulfite is extracted from grapes with aqueous formaldehyde solution buffered at pH 5; free sulfite is converted to hydroxymethylsulfonate (HMS), which is extremely stable at pH 3-7. Subsequent heating to 80 degrees C for 30 min converts reversibly bound forms of sulfite to HMS. The extract is then analyzed by reverse-phase ion-pairing liquid chromatography, using a C18 column and a mobile phase of aqueous 0.005 M tetrabutylammonium ion in 0.05 M acetate, pH 4.7, and a flow rate of 1 mL/min. Aqueous KOH is added to the eluate to convert HMS to free sulfite, which is then treated with 5,5'-dithiobis[2-nitrobenzoic acid]. This reaction produces the 3-carboxy-4-nitrothiophenolate anion, which is determined by measurement of electronic absorption at 450 nm. For grapes spiked with HMS at 5-20 ppm (as SO2), recoveries ranged from 92 to 112%, with a coefficient of variation of 4.6%. The method was also used to determine sulfite in various grape products. Results were comparable to those obtained by the AOAC official Monier-Williams method.

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.

Rapid gas chromatographic method for determining ethyl carbamate in alcoholic beverages with thermal energy analyzer detection.

A rapid column elution method has been developed for the determination of ethyl carbamate (EC) in alcoholic beverages. The beverage is mixed with Celite and packed in a column containing deactivated alumina capped with a layer of sodium sulfate. EC is then eluted with methylene chloride. The method, using a gas chromatograph-thermal energy analyzer with a nitrogen converter for detection and quantitation of EC, has been applied to a variety of alcoholic beverages. Recoveries +/- standard deviations of EC in wine and whisky fortified at the 20 and 133 micrograms/kg (ppb) levels averaged 87.3 +/- 5.3 and 88.7 +/- 3.6%, respectively. The method has a limit of detection of 1.5 ppb. Gas chromatography/mass spectrometry/mass spectrometry was used to confirm the identity and quantitation of EC in selected beverage extracts.

Sulphite stabilization and high-performance liquid chromatographic determination: a reference method for free and reversibly bound sulphite in food.

A high-performance liquid chromatographic (HPLC) post-column reactor combination has been developed for the determination of sulphite (free and reversibly bound) in 15 different foods. The foods were treated with a pH 5.1 buffered aqueous 2% formaldehyde solution to convert the labile sulphite to the relatively stable hydroxymethylsulphonate. Reverse-phase ion-pairing HPLC with a post-column detector consisting of an initial reaction with KOH followed by colorimetric reaction with Ellman's reagent buffered at pH 6.3 were used for separation and quantitation. The photometric detector at 412 nm quantitated the strongly absorbing 3-carboxy-4-nitrothiophenolate anion, the product of the sulphite reaction with Ellman's reagent. The recovery at levels of 5-100 ppm as sulphur dioxide was greater than 90% by reverse isotope dilution assay. Repetitive analysis of a single grape juice extract containing 20 ppm SO2 showed a relative standard deviation of 2.2%.

Current trends in levels of volatile N-nitrosamines in fried bacon and fried-out bacon fat.

Commercially processed bacon samples purchased from the Washington, DC, retail market have been periodically analyzed since 1971 for the presence of volatile N-nitrosamines in the fried product. During that time, a downward trend in the concentration of N-nitrosopyrrolidine has been observed, and between 1978 and 1980 it plateaued at 4-30 ppb, with an average of 11 ppb. A recent survey, however, indicates a change in this downward trend, with N-nitrosopyrrolidine found at levels ranging from 1 to 65 ppb, average 21 ppb. Volatile N-nitrosamines were found at levels up to 110 ppb in the fried product and up to 85 ppb in the fried-out bacon fat.

Reactions of antioxidants in foods.

The chemical transformations of BHT and BHA in the deep-fat frying of potatoes were studied. Approximately 80% of the BHT and its associated decomposition products was lost from the lard after six batches of french fries. This may have been due to steam distillation caused by the water boiled out of the potatoes. In contrast, 80% of the radiocarbon introduced as radiolabelled BHA was retained by the frying medium even after 12 batches of french fries. The concentration of intact BHA decreased to undetectable levels after four batches. Because of the complexity of the products formed in the heated fats, studies were undertaken on the thermolysis of a hydroperoxy derivative of BHT, 2,6-di-tert-butyl-4-hydroperoxy-4-methylcyclohexa-2,3-dienone++ + (HBHT). The product mixture, which was identified by mass spectrometry and gas chromatography-mass spectrometry, included BHT, 2,6-di-tert-butyl-4-hydroxy-4-methylcyclohexa-2,5-dienone and 3,5-di-tert-butyl-4-hydroxybenzaldehyde. Elemental compositions of other unidentified products were determined by high-resolution mass spectrometry.

Gas chromatographic determination of fatty acids and sterols in orange juice.

A gas chromatographic (GC) method has been developed for the simultaneous quantitation of fatty acids and sterols in orange juice, using a bonded phase fused silica capillary column of intermediate polarity, splitless automatic injection, and flame ionization detection. Sample preparation has been simplified by using 1 g C-18 adsorbent in a disposable minicolumn to extract 2 mL orange juice. Methylation of fatty acids and silylation of the sterols were carried out in the eluted extract (low polarity lipid fraction). The method precision was 7%; recoveries ranged from 83 to 113%. The precision of the injection technique was 2%. Seven major fatty acids and 5 sterols in orange juice were quantitated by the GC method and identified by GC/mass spectrometry. Quantitative data for several orange juice samples indicated that the levels of the compounds of interest were in the 1.3-72.0 mg/L range. The results demonstrate that bonded phase fused silica capillary GC has great versatility and potential for the quantitative determination of fatty acids and sterols.

Liquid chromatographic determination of basic nitrogen-containing polynuclear aromatic hydrocarbons in smoked foods.

Several smoked foods were analyzed for basic nitrogen-containing polynuclear aromatic hydrocarbon (NPAH) content by a relatively rapid liquid chromatographic (LC) technique. The analyzed products included both domestic and imported market basket commodities. Nanogram quantities of NPAH standards were detected by UV and fluorescence detectors connected in series. The NPAHs were extracted from basic aqueous ethanolic solution into cyclohexane, extracted from cyclohexane into 6N HCl, and extracted back into cyclohexane after neutralization of the acid. The NPAHs were then purified by filtering the extract through deactivated basic alumina. The eluate from this step was concentrated to dryness, and the residue was dissolved in 95% ethanol and analyzed by LC, using a Vydac C-18 column and acetonitrile-water (9 + 1) as the mobile phase. Recoveries of 3 NPAHs, 5,7-dimethylbenz(a)acridine, dibenz(a,j)acridine, and dibenz(a,h)acridine, each added to salmon and sausage at the 5 ppb level, ranged from 62 to 101% by fluorescence measurement and from 64 to 106% by UV measurement. None of the NPAHs used as standards were found by either fluorescence or UV detection at levels greater than or equal to 5 ppb in any of the foods analyzed.

Reevaluation of Monier-Williams method for determining sulfite in food.

The Monier-Williams distillation procedure has a long history of successful use for determining sulfite in fruit products and wine; however, a systematic evaluation of its accuracy and precision with other food matrices has not been undertaken. We found that the Monier-Williams distillation yielded greater than 90% recovery of sulfite added to foods such as table grapes, hominy, dried mangoes, and lemon juice. Less than 85% recovery was obtained with broccoli, soda crackers, cheese-peanut butter crackers, mushrooms, and potato chips. These results may, in fact, accurately reflect the residual levels of sulfite if a portion of the sulfite undergoes irreversible reaction with some food components. Analysis of commercial food products gave sulfite levels ranging from 1400 ppm in dried apple slices to 25 ppm in cream sherry.

Liquid chromatographic determination of trace residues of polynuclear aromatic hydrocarbons in smoked foods.

A liquid chromatographic (LC) method was developed and applied to the determination of polynuclear aromatic hydrocarbons (PAHs) in a variety of smoked, market basket commodities. The PAHs are extracted with 1,1,2-trichloro-1,2,2-trifluoroethane (Freon 113) from alcoholic KOH digests of the commodities. The extracts are purified by column chromatography through a deactivated silica gel/alumina column and by liquid-liquid partitioning between dimethyl sulfoxide and cyclohexane before separation of the PAHs by LC. Both fluorescence and UV detectors are used to monitor the LC column effluent to detect nanogram quantities of PAHs. Trace levels of carcinogenic and non-carcinogenic PAHs were found in all samples analyzed, although generally at less than 1 ppb levels.

Liquid chromatographic determination of aspartame in dry beverage bases and sweetener tablets with confirmation by thin layer chromatography.

A liquid chromatographic method is described for the determination of aspartame in dry beverage bases and sweetener tablets. The sample was mixed with the mobile phase, the pH was adjusted to within +/- 0.1 pH unit of the mobile phase, and the sample was diluted to volume with the mobile phase. The solution was filtered and a 10 microL aliquot was injected onto a C18 reverse phase column. Aspartame was quantitated with an ultraviolet detector. Recoveries of aspartame ranged from 94 to 111%. The dry beverage bases contained 5-13% aspartame and the sweetener tablets contained 19% aspartame. The presence of aspartame was confirmed by using thin layer chromatography.

Reverse phase high pressure liquid chromatography and fluorescence detection of ethoxyquin in milk.

A high pressure liquid chromatographic (HPLC) method has been developed for the determination of ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethyl-quinoline) in milk. Milk solids are precipitated by adding acetonitrile, and the water-acetonitrile supernate is washed with hexane to remove fat. Addition of sodium chloride causes the water-acetonitrile solution to separate into an aqueous phase and an acetonitrile phase, thus separating ethoxyquin from most water-soluble impurities. A large volume of water is then added to the acetonitrile layer and ethoxyquin is partitioned into hexane, which is removed at reduced pressure. The residue is dissolved in the mobile phase and analyzed on a 4.6 mm id X 250 mm Ultrasphere ODS column using fluorescence detection (excitation 230 nm; 418 nm cutoff filter). Water-acetonitrile with a diethylamine-acetic acid buffer is the mobile phase. Recoveries from samples fortified at 1, 5, and 10 ppb averaged 78% with a coefficient of variation of 5.0%. Low levels (less than 1 ppb) of apparent ethoxyquin were found in commercial milk samples that were analyzed by using the method.