Preparative separation of isomeric 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione monosulfonic acids of the color additive D&C Yellow No. 10 (quinoline yellow) by pH-zone-refining counter-current chromatography.

The main components of the color additive D&C Yellow No. 10 (Quinoline Yellow, Color Index No. 47005), 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione-6'-sulfonic acid (6SA) and 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione-8'-sulfonic acid (8SA), were isolated from the dye mixture by pH-zone-refining counter-current chromatography (CCC) in the ion-exchange mode. These positional isomers were separated from a portion of dye using sulfuric acid as the retainer acid and dodecylamine as the ligand (ion exchanger). The added ligand enhanced the partitioning of the hydrophilic components in the organic stationary phase of the two-phase solvent system that consisted of isoamyl alcohol-methyl tert.-butyl ether-acetonitrile-water (3:1:1:5). Thus, separation of 1.8 g of D&C Yellow No. 10 using the above method resulted in 0.6 g of 6SA and 0.18 g of 8SA of over 99% purity. The isolated compounds were characterized by mass spectrometry and proton nuclear magnetic resonance with correlated spectroscopy assignments. The study exemplifies a new field of applications for pH-zone-refining CCC, to the separation of positional isomers of strongly hydrophylic compounds containing sulfonic acid groups.

Allium chemistry: identification of organosulfur compounds in ramp (Allium tricoccum) homogenates.

Supercritical fluid (SF) extracts of homogenized ramp (Allium tricoccum Ait.) were separated and characterized with liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometric identification. The profiles of SF extracts of aqueous homogenates of ramp bulbs from three different seasons and growing regions revealed that the thiosulfinates were major components. In addition, some of the cepaenes (alpha-sulfinyldisulfides) found in extracts of onion juice, as well as allyl containing cepaenes (2-propenyl l-(2-propenylsulfinyl)propyl disulfide), are present in the ramp extracts. The amount of allicin in ramp bulb homogenates ranged from approximately 10% to 50% of that found in extracts of aqueous garlic homogenates. The greater amount of the methyl 1-propenyl thiosulfinates in the ramp extracts relative to that found in the garlic extracts correlates with the flavor characteristics of ramp bulbs.

Confirmation of identities of propylene and ethylene glycols in anchovies by tandem mass spectrometry.

A gas chromatographic/tandem mass spectrometric (GC/MS/MS) method has been developed for confirming the identity of propylene and ethylene glycols added to bait fish for preservation. Bait fish are occasionally illegally diverted to human food use. The bait fish were extracted with methanol, the extract was centrifuged and filtered, and the filtrate was concentrated 10-fold and then analyzed by GC/MS/MS. The glycols were separated chromatographically without derivatization or preliminary cleanup. Isobutane positive ion chemical ionization was used to generate the protonated molecular ion species of each glycol. Product-ion MS/MS experiments were performed to obtain spectra to confirm the identities of propylene and ethylene glycols. The identities of these 2 compounds in anchovy extracts were successfully confirmed by this approach.

Liquid chromatographic determination and liquid chromatographic-thermospray mass spectrometric confirmation of nicarbazin in chicken tissues: interlaboratory study.

The U.S. Food and Drug Administration sponsored an interlaboratory study of a liquid chromatographic determination with ultraviolet detection of nicarbazin in chicken liver and muscle tissues. The method determined the 4,4'-dinitrocarbanilide (DNC) portion of nicarbazin. The interlaboratory study of the determinative method was successful for nicarbazin at the 4 ppm level. Results showed good reproducibility for the fortified liver and muscle samples. Mean interlaboratory recoveries and percent coefficients of variation at about 4 ppm were 87.1 and 10.9%, respectively, for muscle and 87.4 and 7.5%, respectively, for liver. The interlaboratory analyses of the dosed liver and muscle tissues produced concentration levels similar to those obtained by the sponsor. The confirmatory procedure, which identified DNC in purified tissue extracts, used liquid chromatography-thermospray/mass spectrometry. The confirmatory procedure was successfully evaluated by one FDA laboratory.

beta-Carotene uptake, metabolism, and distribution in BALB/c 3T3 cells.

Although a growing number of epidemiological studies indicate that dietary beta-carotene has anticarcinogenic activity, the mechanism(s) of beta-carotene protection remains to be definitively established. In this context, in vitro studies of beta-carotene have been, and continue to be, valuable. We examined the following critical features in designing an in vitro system for studying the protection action of beta-carotene: 1) form of beta-carotene used for cellular uptake, 2) cellular metabolism of beta-carotene, and 3) subcellular distribution of beta-carotene. It was determined that beta-carotene added to medium in a water-dispersible formulation is readily taken up by BALB/c 3T3 cells and is located predominantly in cellular membranes. Cellular uptake of beta-carotene added to medium in an organic solvent is greatly reduced. It was also found that intracellular retinol increased significantly after a three-day exposure of BALB/c 3T3 cells to media containing beta-carotene. This result suggests that the ability to metabolize beta-carotene to retinoids is not limited to cells of intestinal origin. The results and methodology described here will be useful in the rational design of in vitro assays for elucidating the mechanism(s) of beta-carotene protective effects at the cellular level.

Confirmation of identity by gas chromatography/tandem mass spectrometry of sulfathiazole, sulfamethazine, sulfachloropyridazine, and sulfadimethoxine from bovine or swine liver extracts after quantitation by gas chromatography/electron-capture detection.

Four sulfonamide veterinary drug residues were quantitated by electron-capture detection (ECD) after separation by gas chromatography (GC). The identities of sulfathiazole (ST), sulfamethazine (SM), sulfachloropyridazine (SCP), and sulfadimethoxine (SDM) were confirmed in bovine or swine liver residues by tandem mass spectrometry (MS/MS). Bovine or swine liver tissues were extracted by using either the Tishler or the Manuel-Steller cleanup. The methylated residues containing ST, SM, SCP, and SDM were separated by GC prior to MS/MS daughter ion analysis. Control tissue, control tissue fortified at 0.1 ppm, and incurred tissue residues at approximately 0.1 ppm were analyzed for these 4 sulfonamides. A Finnigan Model TSQ-46 operating in the chemical ionization mode was used to perform the MS/MS daughter ion experiments. The identities of all 4 sulfonamides were confirmed in a single GC/MS/MS analysis.

Gas chromatographic/mass spectrometric confirmation of identity of coprostanol in Mercenaria mercenaria (Bivalvia) taken from sewage-polluted water.

Coprostanol is a major fecal sterol in humans and may, therefore, be a good indicator of sewage-polluted waters. Some types of edible seafood, such as clams, that live in these waters may be contaminated with coprostanol. Coprostanol from clam tissue extracts had been previously quantitated by gas chromatography (GC). In the present work, capillary column GC was used to separate coprostanol, and electron ionization mass spectrometry was used to confirm its identity. Confirmation of identity of coprostanol at the 75 ng level was obtained by comparing the spectrum of the authentic standard with spectra of the clam tissue extract obtained under the same instrumental conditions. Various other compounds can be eliminated as potential interferences by virtue of either their different GC retention times or their spectra.

Determination of desaminosulfamethazine, sulfamethazine, and N4-acetylsulfamethazine by gas chromatography with electron capture detection and confirmation by gas chromatography-chemical ionization mass spectrometry.

An electron capture gas chromatographic method for the determination of sulfamethazine was modified to separate and quantitate simultaneously sulfamethazine and 2 of its metabolites, N4-acetylsulfamethazine and desaminosulfamethazine. The modified method was applied to incurred residues in a veal calf depletion study and to incurred residues in swine tissues. With capillary column gas chromatography-positive ion chemical ionization mass spectrometry, confirmation of the identities of incurred desaminosulfamethazine, N4-acetylsulfamethazine, and sulfamethazine in tissues was obtained from a single injection.

Evaluation of three methods for recovery of sulfamethazine metabolites from swine tissue.

Recent investigations have clearly established the presence of desaminosulfamethazine (I), N4-acetylsulfamethazine (II), and N4-D-glucosyl sulfamethazine (III) in the tissues of swine which have been dosed with sulfamethazine (V). N4(1-Deoxy-D-glucuronyl) sulfamethazine (IV) has frequently been found in the urine and feces of animals. These metabolites have generally not been investigated in current sulfamethazine methods; therefore, it is not clear to what extent they might be measured by these procedures. Metabolites I, II, III, and IV were synthesized and characterized by several chemical and physical measurements. These metabolites and their parent, V, were added to swine tissue and recoveries were measured by a gas-liquid chromatographic (GLC), a colorimetric, and a gas chromatographic/mass spectrometric method with electron ionization-selected ion monitoring (GC/MS EI-SIM). With modifications of the gas chromatographic parameters, the GLC method can quantitate compounds, I, II, and V simultaneously. If a hydrolysis step is added, all 3 methods can quantitate compound II as V and, in addition, the GC/MS EI-SIM and colorimetric methods can quantitate III as V. In none of these methods do metabolites I, II, III, or IV interfere in the determination of V.