Extraction and analysis of an organophosphate salt nucleating agent from irradiated polypropylene resin.

Although it is well-established that irradiation of produce can reduce food-borne pathogens and spoilage organisms, data on the effect of irradiation on polymer additives in food packaging materials are limited, particularly for those additives used in packaging leafy greens or in current food packaging materials. We investigated the effects of irradiating a nucleating agent, aluminium, hydroxybis[2,4,8,10-tetrakis(1,1-dimethylethyl)-6-hydroxy-12H-dibenzo [d,g][1,3,2]dioxaphosphocin 6-oxidato]- (CAS Reg. No. 151841-65-5), at doses of 1-20 kGy in polypropylene. That nucleating agent was then extracted using accelerated solvent extraction and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography-photodiode array detection (LC-PDA), and solid-state nuclear magnetic resonance (SSNMR) spectroscopy. We found this nucleating agent was not significantly affected by radiation treatment up to 20 kGy. Therefore, this nucleating agent could potentially be useful in food packaging materials that will be irradiated at doses of 20 kGy or less. Establishing which additives are stable under anticipated irradiation doses will help support safety evaluation of food packaging materials.

Acute oral toxicity and tissue residues of saxitoxin in the mallard (Anas platyrhynchos).

Since 2014, widespread, annual mortality events involving multiple species of seabirds have occurred in the Gulf of Alaska, Bering Sea, and Chukchi Sea. Among these die-offs, emaciation was a common finding with starvation often identified as the cause of death. However, saxitoxin (STX) was detected in many carcasses, indicating exposure of these seabirds to STX in the marine environment. Few data are available that describe the effects of STX in birds, thus presenting challenges for determining its contributions to specific mortality events. To address these knowledge gaps, we conducted an acute oral toxicity trial in mallards (Anas platyrhynchos), a common laboratory avian model, using an up-and-down method to estimate the median lethal dose (LD50) for STX. Using an enzyme-linked immunosorbent assay (ELISA), we tested select tissues from all birds and feces from those individuals that survived initial dosing. Samples with an ELISA result that exceeded approximately 10 µg 100 g-1 STX and randomly selected ELISA negative samples were further tested by high-performance liquid chromatography (HPLC). Tissues collected from mallards were also examined grossly at necropsy and then later by microscopy to identify lesions attributable to STX. The estimated LD50 was 167 µg kg-1 (95% CI = 69-275 µg kg-1). Saxitoxin was detected in fecal samples of all mallards tested for up to 48 h after dosing and at the end of the sampling period (7 d) in three birds. In those individuals that died or were euthanized <2 h after dosing, STX was readily detected throughout the gastrointestinal tract but only infrequently in heart, kidney, liver, lung, and breast muscle. No gross or microscopic lesions were observed that could be attributable to STX exposure. Given its acute toxicity, limited detectability, and frequent occurrence in the Alaska marine environment, additional research on STX in seabirds is warranted.

Structural determination of four manufacturing impurities of D&C Red No. 33.

Four manufacturing impurities of D&C Red No. 33 isolated by counter-current chromatography were analyzed by NMR and ESI mass spectrometry. Three of these impurities were reported previously with minimal details of structural determination. All four are structurally related to the main component of the dye. The fourth exhibited an unusual discrepancy between the NMR structure and its chemical formula suggested by ESI-MS results. Structural determination and assignment of the main component and four impurities are discussed as well as resolution of the discrepancy between the NMR and ESI-MS results of the fourth impurity.

Identification, separation by spiral high-speed counter-current chromatography, and quantification of 7-chloro-5-methyl-2H-1,4-benzothiazin-3(4H)-one, an impurity in the thioindigoid color additive D&C Red No. 30 and its lakes.

An impurity in the color additives D&C Red No. 30 (R30) and D&C Red No. 30 lakes (R30L) was newly identified and characterized as 7-chloro-5-methyl-2H-1,4-benzothiazin-3(4H)-one (BTZ), and its extent and level in certified batches of these color additives was determined. BTZ was extracted from the dye with ethanol, resulting in a crude extract enriched to a concentration of over 60%. BTZ was then separated from a portion of the enriched extract by high-speed counter-current chromatography using a spiral-tube assembly column with intermittently pressed tubing of 60 ml capacity. It was the first reported use of such a column to separate a small, moderately hydrophobic compound. The two-phase solvent system was also moderately hydrophobic, consisting of hexane-ethyl acetate-methanol-water (5:2:5:2), and the retention of the organic stationary phase measured after the separation was 83.3%. The separation yielded BTZ of two purity grades, the higher of which (~95.5%) was used as a standard to quantify the impurity in 37 batches of R30 and R30L using an HPLC method developed and validated for that purpose. Analyses revealed a wide range of BTZ levels across batches, <0.05 - 0.84%, and suggested that BTZ contamination could be reduced by appropriate adjustments in the manufacturing process. An explanation of the likely source of BTZ - as a side-reaction product in a particular step of the manufacturing process - was also presented.

Separation and characterization of products from acidic methanolysis of plasmalogenic lipids by two-dimensional gas chromatography with online reduction.

The complexity of determining the composition of animal tissue lipids is greatly increased by the presence of plasmalogens in which the alkyl chain is linked to glycerol by an enol ether bond instead of being esterified. Acidic methanolysis of animal tissue lipids provides the simultaneous scission of acyl and alkenyl ether moieties, but the complexity of the products of reaction poses a great challenge in their gas chromatographic analysis. Two-dimensional gas chromatography with online reduction (GC-OR × GC) provided the resolution of all components contained in acid methanolyzed animal lipids, taking advantage of the selective hydrogenation of alkenyl ether methanolysis products prior to the second-dimension separation (2D). In this study, we also studied the chemical transformations occurring during the acidic methanolysis of animal lipids and the subsequent gas chromatographic analysis. In particular, we observed that using methanolysis reagents contaminated with water resulted in the undesired formation of fatty aldehydes, and we made recommendations on how to avoid these side reactions using proper methanolysis conditions. Products of acidic methanolysis were studied by GC-OR × GC, GC-MS, NMR spectroscopy, and GC with flame ionization detection (GC-FID). We defined the GC-FID elution order of animal lipid acidic methanolysis products using 100 m × 0.25 mm 100% bis(cyanopropyl)siloxane columns and two different set of elution conditions: isothermal elution at 180°C, and a temperature program optimized for dairy fats. A simple procedure for isolating dimethyl acetals (DMA) prior to GC analysis is also described.

Elucidation and partial NMR assignment of monosulfated maitotoxins from the Caribbean.

Compounds similar to maitotoxin (MTX) have been isolated from several laboratory strains of the dinoflagellate Gambierdiscus spp. from the Caribbean. Mass spectral results suggest that these compounds differ from MTX by the loss of one sulfate group and, in some cases, the loss of one methyl group with the addition of one degree of unsaturation. NMR experiments, using approximately 50 nmol of one of these compounds, have demonstrated that the 9-sulfo group of MTX is still present, suggesting that these compounds are 40-desulfo congeners of MTX.

Separation using high-speed counter-current chromatography and identification of 1,3-bis(4-phenylazophenyl)triazene, an impurity in the color additive D&C Red No. 17 (Sudan III).

The present work describes the application of high-speed counter-current chromatography to the preparative separation of a previously unreported impurity in the color additive D&C Red No. 17 (R17, Colour Index No. 26100, Sudan III). Due to the hydrophobic nature of the impurity, a hydrophobic two-phase solvent system (hexane-ethanol-water, 5:4:1) was used for its separation. The separated impurity was chemically characterized by spectroscopic methods as a disazo triazene, 1,3-bis(4-phenylazophenyl)triazene (PAPT). This impurity was synthesized and used as a reference material to quantify it in 15 batches of the color additive produced by various domestic and foreign manufacturers and certified by the U.S. Food and Drug Administration (FDA). Analysis of test portions by high-performance liquid chromatography showed a range of PAPT levels, from "not detected" (<0.006%) to 0.70%, across batches. The variability suggests that contamination by PAPT can be decreased or eliminated through manufacturing modifications. A chemical pathway for PAPT formation and an associated adjustment to minimize it during the process of manufacturing R17 are proposed.

Identification and quantification of the decarboxylated analogue of Pigments Red 57 and 57:1 in the color additives D&C Red No. 6, D&C Red No. 7, and their lakes, using a chelating agent and UHPLC.

Pigment Red 57 (Colour Index No. 15850, PR57) and Pigment Red 57:1 (Colour Index No. 15850:1, PR57:1) are certifiable in the USA as the color additives D&C Red No. 6 (R6) and D&C Red No. 7 (R7) for use in drugs and cosmetics. In the EU, PR57:1 is permitted in cosmetics and also as a food additive (E180) for colouring edible cheese rinds. The USFDA batch-certifies R6, R7, and their lakes in accordance with limiting specifications for impurities stated in the Code of Federal Regulations (CFR). In the current work, an impurity not specified in the CFR was studied because of its consistent presence in samples of R6 and R7 submitted for certification. Using spectroscopic methods, the impurity was tentatively identified as 4-[(4-methyl-2-sulfophenyl)azo]-3-naphthalenol (DPR57), the decarboxylated analogue of PR57 and PR57:1. Its identity was confirmed by synthesising DPR57 and determining that the UHPLC retention time, UV/visible spectrum and mass spectrum of the synthetic material were identical to those of the impurity. Using the synthesised DPR57 as a reference material, the impurity was quantified in 43 batches of R6, R7, and lakes produced by eight different manufacturers. Calibration curves ranging from 0.02% to 1.00% (w/w) were prepared by plotting the UHPLC area of DPR57 at 485 nm against its concentration. DPR57 levels ranged from < 0.02% to 0.50%. To facilitate dissolution of the color additive samples for DPR57 analysis, a relatively simple procedure was developed by adapting a previously published method that involves use of a basic solution of the chelating agent EDTA and the organic solvent N,N'-dimethylformamide. A source for DPR57 contamination of the color additives is also proposed.

Identification of 1',5'-naphthyridinophthalone and its quantification in the color additive D&C Yellow No. 10 (Quinoline Yellow) using high-performance liquid chromatography.

The present work reports the identification and characterization of a contaminant, 2-(2'-(1,5-naphthyridinyl))-1,3-indanedione (1',5'-naphthyridinophthalone, 1,5NP), in the color additive D&C Yellow No. 10 (U.S.-certifiable form of Quinoline Yellow), together with its quantification in batches of the color additive certified by the U.S. Food and Drug Administration (USFDA). The impurity, which is a compound not previously reported in the literature, was synthesised and characterised for use as a reference material. Test portions from 26 certified batches of D&C Yellow No. 10 submitted to USFDA by four domestic and four foreign manufacturers were analyzed for 1,5NP using high-performance liquid chromatography. The results revealed a wide range of 1,5NP levels across batches, with 18 (69.2%) of the test portions containing amounts from 0.32 to 169.94 µg g-1 while the remaining test portions contained no detectable (<0.07 µg g-1) amounts. Samples of the European and Japanese forms of Quinoline Yellow were also analyzed and found to contain a wide range of 1,5NP levels. The varying levels of 1,5NP in all three forms of Quinoline Yellow suggest that contamination can be significantly decreased or eliminated through manufacturing adjustments. Since 1,5NP is closely related to a D&C Yellow No. 10 contaminant (quinophthalone) that has a USFDA-specified limit of 4 µg g-1 and is a known allergen, assessment of the possible allergenicity of 1,5NP is warranted.

Profiling hydroxycinnamic acid glycosides, iridoid glycosides, and phenylethanoid glycosides in baobab fruit pulp (Adansonia digitata).

The baobab (Adansonia digitata L.) is a magnificent tree revered throughout Africa and is becoming recognized for its high nutritional and medicinal values. Despite numerous reports on the pharmacological potential, little is known about its chemical compositions. In this study, four hydroxycinnamic acid glycosides (1-4), six iridoid glycosides (5-10), and three phenylethanoid glycosides (11-13) were isolated from the dried baobab fruit pulp. Their structures were determined by means of spectroscopic analyses, including HRMS, 1H and 13C NMR and 2D experiments (COSY, HSQC, HMBC, and ROESY). All 13 compounds isolated were reported for the first time in the genus of Adansonia. An ultra high-performance liquid chromatography high-resolution accurate-mass mass spectrometry (UHPLC HRAM MS) method was used to conduct further investigation of the chemical compositions of the hydro-alcohol baobab fruit pulp extract. Hydroxycinnamic acid glycosides, iridoid glycosides and phenylethanoid glycosides were found to be the main components in baobab fruit pulp.

Determination of Sudan I and a newly synthesized Sudan III positional isomer in the color additive D&C Red No. 17 using high-performance liquid chromatography.

Specifications in the Code of Federal Regulations for the color additive D&C Red No. 17 (Colour Index 26100) limit the levels of two subsidiary colors, 1-(phenylazo)-2-naphthol (Sudan I) and 1-[[2-(phenylazo)phenyl]azo]-2-naphthalenol (Sudan III o-isomer), to 3% and 2%, respectively. The present work reports the development of a high-performance liquid chromatography (HPLC) method for the quantitative determination of these subsidiary colors. Since Sudan III o-isomer needed to be synthesized for use as a reference material, a two-step procedure was devised: (i) preparative-scale synthesis of the intermediate 2-aminoazobenzene (2AAB) and its purification by counter-current chromatography and (ii) diazotization of 2AAB and coupling with 2-naphthol. Characterization of the newly synthesized Sudan III o-isomer is also reported. Sudan I and Sudan III o-isomer were quantified by using five-point calibration curves with data points ranging from 0.108 to 3.240% and 0.077 to 2.227% by weight, respectively. The HPLC method is rapid (14 min for the total analysis cycle) and simple to implement. It was applied to the analysis of test portions from 25 batches of D&C Red No. 17 submitted to the U.S. Food and Drug Administration (USFDA) for certification, and it has recently been implemented by USFDA for routine batch certification of that color additive.

Application of a computer-assisted structure elucidation program for the structural determination of a new terpenoid aldehyde with an unusual skeleton.

The structure of a novel compound from Adansonia digitata has been elucidated, and its 1 H and 13 C NMR spectra have been assigned employing a variety of one-dimensional and two-dimensional NMR techniques without degradative chemistry. The Advanced Chemistry Development ACD/Structure Elucidator software was important for determining part of this structure that contained a fused bicyclic system with very few hydrogen atoms, which in turn, exhibited essentially no discriminating HMBC connectivities throughout that portion of the molecule. Copyright © 2016 John Wiley & Sons, Ltd.

Isolation and characterization of roridin E.

The commonly occurring, high-cytotoxicity macrolide roridin E has been re-isolated from Stachybotrys chartarum and characterized by 1-D and 2-D NMR spectroscopy. Assignment of the spectral data for roridin E revealed differences from the accepted literature, and spectra are reported herein to aid in future identification. For the first time confirmation of structure was provided by a crystallographic solution for roridin E. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of unknown compounds from polyester cans coatings that may potentially migrate into food or food simulants.

Cross-linked polyester resins are being introduced into the market as alternatives to epoxy resins as coatings for metal food cans. Identification of potential migrants, from these coatings into food, is a significant analytical challenge due to the diversity of substances employed in the manufacture of the coatings. However, such identification is required to assess migration from the can coating into the food and quantify dietary exposure. Polyester can coatings were extracted with acetonitrile at 40°C for 24h and the extracts were analyzed by a variety of analytical techniques, including GC-MS, HPLC-DAD/MS, HPLC-DAD/CAD and UHPL C-HRMS. Twenty nine non-volatile oligomers were tentatively identified using retention times, UV spectra, and accurate mass measurements. Identified oligomers suggest the coating in use for food cans is a polyester coating and is mainly based on the monomers isophthalic acid, terephthalic acid and nadic acid. To give confidence in the identification, one of the tentatively identified oligomer was synthetized and analyzed by (13)C and (1)H NMR and UHPL C-HRMS. The NMR and HRMS results, confirmed the presence of this compound in the can extracts. Finally, to determine if rapid, direct detection of the oligomers was practical, the coatings were analyzed by DART-HRMS. Twenty three out of the 29 oligomers were identified in the coating by direct measurement with DART-HRMS in few minutes.

Sesquiterpenoid tropolone glycosides from Liriosma ovata.

Two new sesquiterpenoid tropolone glycosides, liriosmasides A (1) and B (2), along with two known compounds, secoxyloganin and oplopanpheside C, were isolated from a methanol extract of the roots of Liriosma ovata. The structures of 1 and 2 were elucidated by spectroscopic methods including 1D and 2D NMR and by high-resolution mass spectrometry involving an ultra-high-performance liquid chromatography-quadrupole-orbital ion trap mass spectrometric (UHPLC-Q-Orbitrap MS) method. Compound 1 showed weak inhibitory activity against HIV RNase H.

Preparative separation and identification of novel subsidiary colors of the color additive D&C Red No. 33 (Acid Red 33) using spiral high-speed counter-current chromatography.

Three low-level subsidiary color impurities (A, B, and C) often present in batches of the color additive D&C Red No. 33 (R33, Acid Red 33, Colour Index No. 17200) were separated from a portion of R33 by spiral high-speed counter-current chromatography (HSCCC). The separation involved use of a very polar solvent system, 1-BuOH/5mM aq. (NH4)2SO4. Addition of ammonium sulfate to the lower phase forced partition of the components into the upper phase, thereby eliminating the need to add a hydrophobic counterion as was previously required for separations of components from sulfonated dyes. The very polar solvent system used would not have been retained in a conventional multi-layer coil HSCCC instrument, but the spiral configuration enabled retention of the stationary phase, and thus, the separation was possible. A 1g portion of R33 enriched in A, B, and C was separated using the upper phase of the solvent system as the mobile phase. The retention of the stationary phase was 38.1%, and the separation resulted in 4.8 mg of A of >90% purity, 18.3mg of B of >85% purity, and 91 mg of C of 65-72% purity. A second separation of a portion of the C mixture resulted in 7 mg of C of >94% purity. The separated impurities were identified by high-resolution mass spectrometry and NMR spectroscopic techniques as follows: 5-amino-3-biphenyl-3-ylazo-4-hydroxy-naphthalene-2,7-disulfonic acid, A; 5-amino-4-hydroxy-6-phenyl-3-phenylazo-naphthalene-2,7-disulfonic acid, B; and 5-amino-4-hydroxy-3,6-bis-phenylazo-naphthalene-2,7-disulfonic acid, C. The isomers A and B are compounds reported for the first time. Application of the spiral HSCCC method resulted in the additional benefit of yielding 930 mg of the main component of R33, 5-amino-4-hydroxy-3-phenylazo-naphthalene-2,7-disulfonic acid, of >97% purity.

Conditional rotations of heteronuclear coupled spins.

We present a new pulse sequence that conditionally excites I spin magnetization only in the presence of a nonzero heteronuclear coupling to an S spin. The pulse sequence, referred to as the reverse INEPT pathway selective pulse or RIPSP, generates a pure I spin rotation by an angle that depends upon the heteronuclear coupling constant in InS spin systems. Experimental demonstrations are shown in (13)C labeled chloroform, dichloromethane, and toluene samples and in unlabeled 2,3-dibromopropionic acid and brucine samples.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct detection of 2-monochloropropanediol (2-MCPD) esters in edible oils.

A new analytical method has been developed and validated for the detection and quantification of 2-monochloropropanediol (2-MCPD) esters in edible oils. The target compounds are potentially carcinogenic contaminants formed during the processing of edible oils. As the 2-MCPD esters that occur most frequently in refined edible oils were not commercially available, standards were synthesized with identity and purity (95+%) confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (1)H NMR. Target analytes are separated from edible oil matrices using a two-step solid-phase extraction (SPE) procedure. The extracts are then analyzed using LC-MS/MS with electrospray ionization (ESI). The method has been validated for 11 2-MCPD diesters and 3 2-MCPD monoesters in soybean oil, olive oil, and palm oil using an external calibration curve. The ranges of average recoveries and relative standard deviations (RSD) across the three oil matrices at three spiking concentrations are 79-106% (3-13% RSD) for the 2-MCPD diesters and 72-108% (4-17% RSD) for the 2-MCPD monoesters, with limits of quantitation at or below 30 ng/g for the diesters and 90 ng/g for the monoesters.

Preparative separation of two subsidiary colors of FD&C Yellow No. 5 (Tartrazine) using spiral high-speed counter-current chromatography.

Specifications in the U.S. Code of Federal Regulations for the color additive FD&C Yellow No. 5 (Color Index No. 19140) limit the level of the tetrasodium salt of 4-[(4',5-disulfo[1,1'-biphenyl]-2-yl)hydrazono]-4,5-dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylic acid and that of the trisodium salt of 4,4'-[4,5-dihydro-5-oxo-4-[(4-sulfophenyl)hydrazono]-1H-pyrazol-1,3-diyl]bis[benzenesulfonic acid], which are subsidiary colors abbreviated as Pk5 and Pk7, respectively. Small amounts of Pk5 and Pk7 are needed by the U.S. Food and Drug Administration for confirmatory analyses and for development of analytical methods. The present study describes the use of spiral high-speed counter-current chromatography (HSCCC) to separate the closely related minor components Pk5 and Pk7 from a sample of FD&C Yellow No. 5 containing ∼3.5% Pk5 and ∼0.7% Pk7. The separations were performed with highly polar organic/high-ionic strength aqueous two-phase solvent systems that were chosen by applying the recently introduced method known as graphic optimization of partition coefficients (Zeng et al., 2013). Multiple ∼1.0g portions of FD&C Yellow No. 5 (totaling 6.4g dye) were separated, using the upper phase of the solvent system 1-butanol/abs. ethanol/saturated ammonium sulfate/water, 1.7:0.3:1:1, v/v/v/v, as the mobile phase. After removing the ammonium sulfate from the HSCCC-collected fractions, these separations resulted in an enriched dye mixture (∼160mg) of which Pk5 represented ∼46% and Pk7, ∼21%. Separation of the enriched mixture, this time using the lower phase of that solvent system as the mobile phase, resulted in ∼61mg of Pk5 collected in fractions whose purity ranged from 88.0% to 92.7%. Pk7 (20.7mg, ∼83% purity) was recovered from the upper phase of the column contents. Application of this procedure also resulted in purifying the major component of FD&C Yellow No. 5 to >99% purity. The separated compounds were characterized by high-resolution mass spectrometry and several (1)H and (13)C nuclear magnetic resonance spectroscopic techniques.