Simultaneous Determination of Nine Phthalates in Vegetable Oil by Atmospheric Pressure Gas Chromatography with Tandem Mass Spectrometry (APGC-MS/MS).

Although the use of phthalates has been restricted worldwide, they remain an issue due to health concerns. Diet is one of the most important exposure pathways for humans and due to their solubility in oil, phthalates are commonly found in edible oil and food high in fat. Gas chromatography-mass spectrometry (GC-MS) using electron ionization (EI) has been commonly used for the analysis of the phthalates in foodstuffs, including edible oil. However, this method suffers from issues with sensitivity and selectivity, as most phthalates are fragmented to generate a common phthalic anhydride fragment ion at m/z 149. The molecular ion cannot be observed due to strong fragmentation in EI. In contrast, atmospheric pressure gas chromatography (APGC) is a soft ionization technique with less fragmentation, whereby the molecular ion can be used as the precursor ion for multiple reaction monitoring (MRM). In this study, a simple and quick method for the determination of phthalates in vegetable oil using APGC-MS/MS was developed, and performance was assessed. The method was based on dilution of the oil in solvent and direct injection without the need for further cleanup. The established method was evaluated for linearity, recovery, precision, method detection limit (MDL), and method quantitation limit (MQL). The obtained MQL in vegetable oil was in the range of 0.015-0.058 mg/kg, despite limiting the injection volume to 1 µL, which is suitable for investigating dietary exposure and future proof against decreases to the regulatory limit. Finally, the developed method was successfully applied to analyze nine phthalates in eight commercially available vegetable oil.

A novel screening method for free non-standard amino acids in human plasma samples using AccQ·Tag reagents and LC-MS/MS.

There are at least 500 naturally occurring amino acids, of which only 20 standard proteinogenic amino acids are used universally across all organisms in the synthesis of peptides and proteins. Non-standard amino acids can be incorporated into proteins or are intermediates and products of metabolic pathways. While the analysis of standard amino acids is well-defined, the analysis of non-standard amino acids can be challenging due to the wide range of physicochemical properties, and the lack of both reference standards and information in curated databases to aid compound identification. It has been shown that the use of an AccQ·Tag™ derivatization kit along with LC-MS/MS is an attractive option for the analysis of free standard amino acids in complex samples because it is fast, sensitive, reproducible, and selective. It has been demonstrated that the most abundant quantitative transition for MS/MS analysis of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids corresponds to the fragmentation of the molecule at the 6-aminoquinoline carbonyl group producing a common m/z 171 fragment ion and occurs at similar mass spectrometry collision energy and cone voltages. In this study, the unique properties of AQC derivatized amino acids producing high intensity common fragment ions, along with chromatographic separation of amino acids under generic chromatography conditions, were used to develop a novel screening method for the detection of trace levels of non-standard amino acids in complex matrices. Structural elucidation was carried out by comparing the MS/MS fragment ion mass spectra generated with in silico predicted fragmentation spectra to enable a putative identification, which was confirmed using an appropriate analytical standard. This workflow was applied to screen human plasma samples for bioactive thiol-group modified cysteine amino acids and S-allylmercaptocysteine (SAMC), S-allylcysteine sulfoxide (SACS or alliin) and S-propenylcysteine (S1PC) are reported for the first time to be present in human plasma samples after the administration of garlic supplements.

A twenty-volunteer study using deuterium labelling to determine the kinetics and fractional excretion of primary and secondary urinary metabolites of di-2-ethylhexylphthalate and di-iso-nonylphthalate.

This study has obtained estimates of the kinetics and fractional excretion factors of metabolism of DEHP and DINP to their main primary and secondary metabolites. Samples were obtained from an open-label, fixed sequence, single oral dose study in 10 male and 10 female subjects. The dosed substances were deuterated di-2-ethylhexylphthalate (D(4)-DEHP) and di-isononylphthalate (D(4)-DINP) at two dose levels. Urine samples were collected at intervals up to 48 h post-dose. LC-MS/MS was used to measure metabolite concentrations. Excreted amounts were then calculated using urine volumes. Metabolite half-lives were estimated to be 4-8h with more than 90% of metabolites in the first 24h of urine collections and the remainder in the 24-48 h period. The four metabolites of DEHP amounted to 47.1 ± 8.5% fractional excretion on a molar basis. For DINP the identified metabolites totalled 32.9 ± 6.4%. For both DEHP and DINP the metabolites were in the abundance order -monoester<-oxo<-carboxy<-hydroxy. These robust fractional excretion values for the main primary and secondary phthalate metabolites along with estimates of their uncertainty can be used in future surveys of human exposure to DEHP and DINP.

Mass spectral studies towards more reliable measurement of perfluorooctanesulfonic acid and other perfluorinated chemicals (PFCs) in food matrices using liquid chromatography/tandem mass spectrometry.

Liquid chromatography/mass spectrometry (LC/MS) experiments are described, leading to a reliable method for the measurement of perfluorooctanesulfonic acid (PFOS) and other perfluorinated chemicals (PFCs) in foods. Separations were performed on new fluorinated stationary phases, RP Octyl (-C(8)F(17)) or propyl-perfluorobenzene (-C(3)H(6)-C(6)F(5)), to ensure resolution of PFOS and interfering taurohydroxycholate isomers. Aqueous ammonium formate (5 mM) and methanol were used as the mobile phases. The mass spectrometer was operated in negative electrospray ionisation mode, recording two transitions for each analyte and one for each internal standard. The purities of the analytical standards for the eleven target perfluoro analytes (C(7) to C(12) carboxylic acids, C(4), C(6) and C(8) sulfonic acids, and octanesulfonamide (PFOSA)) were found to be in close agreement with the supplied values; the lowest purity was 91%. Five candidate internal standards were investigated, (13)C(4)-PFOS, (13)C(4)-perfluorooctanoic acid, (13)C(2)-perfluorodecanoic acid, D(9)-n-ethylperfluorooctanesulfonamidoethanol (D(9)-n-Et-FOSE) and D(3)-n-methylperfluorooctanesulfonamide (D(3)-n-Me-FOSA); the purities were all >98%. The use of tetrahydro-PFOS generated backgrounds (>1 microg/kg) for perfluoroheptanoic acid and perfluorobutanesulfonic acid. Similarly D(9)-n-Et-FOSE was unacceptable and D(3)-n-Me-FOSA was volatile, leaving no clear candidate for normalisation of the measurement of PFOSA. Severe matrix-induced suppression and enhancement effects influenced ionisation, making external calibration and quantification problematic. This was addressed by a parallel standard addition and matrix-matching approach, comparing ionisation in methanol, in procedural blanks and in food-based extracts. The limits of detection (LODs) of 0.001-0.01 microg/kg in solvent and 0.01-1 microg/kg in foods demonstrate that this method is suitable for the determination of PFCs in all food to the required 1 microg/kg reporting level.

Application of stable carbon isotope analysis to the detection of testosterone administration to cattle.

The use of anabolic substances is prohibited in food-producing animals throughout the European Union. No method is available to reliably detect the misuse of natural hormones in cattle. A method was developed to detect the abuse of testosterone in cattle fattening. Synthesized testosterone is rather depleted in the (13)C/(12)C ratio. Hence, the method is based on gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis of urine. To select testosterone metabolites and endogenous reference compounds (ERC), the concentration of urinary steroids of cattle was investigated. Dehydroepiandrosterone and androst-5ene-3beta,17alpha-diol were chosen as ERCs to show endogenous (13)C/(12)C ratios. Etiocholanolone and 5alpha-androstane-3beta,17alpha-diol were chosen as the most important testosterone metabolites. Other metabolites known from literature like epitestosterone were less promising. In principle, GC/C/IRMS is a nonspecific method because finally carbon dioxide is analyzed. Therefore, a dedicated cleanup procedure for the selected steroids was developed. By means of proposed confidence intervals in the isotopic composition of ERCs and metabolites, the administration of testosterone to cattle could be detected reliably. Differences of up to 11 per thousand on the delta-scale between ERC and testosterone metabolites were found after testosterone administration, whereas endogenous differences did not exceed 2 per thousand.

Determination of ethylenethiourea (ETU) and propylenethiourea (PTU) in foods by high performance liquid chromatography-atmospheric pressure chemical ionisation-medium-resolution mass spectrometry.

A robust and sensitive method for the determination of ethylenethiourea (ETU) and iso-propylenethiourea (i-PTU) in foods is reported. ETU and i-PTU were extracted by blending with dichloromethane (DCM) in the presence of sodium sulphate, sodium carbonate, thiourea and ascorbic acid. 2H4-ETU and n-PTU were used as internal standards. After filtration the DCM was removed by rotary evaporation and the extract re-dissolved in water before analysis by reversed-phase liquid chromatography with detection by atmospheric pressure chemical ionization-mass spectrometry using a double focusing mass spectrometer at a resolution of 5000. Mean recoveries of ETU and i-PTU from fruit-based, cereal-based and meat-based infant foods, potato chips and tinned potatos at 0.01 mg kg(-1) and from pizza and yoghurt at 0.02-0.1 mg kg(-1) were 95% and 97% respectively. Precision, including both repeatability and internal reproducibility, was in the range of 3.1-13.1%.

Gas chromatographic/mass spectrometric determination of 3-methoxy-1,2-propanediol and cyclic diglycerols, by-products of technical glycerol, in wine: interlaboratory study.

The aim of the present study was to provide the official wine control authorities with an internationally validated method for the determination of 3-methoxy-1,2-propanediol (3-MPD) and cyclic diglycerols (CycDs)-both of which are recognized as impurities of technical glycerol-in different types of wine. Because glycerol gives a sweet flavor to wine and contributes to its full-body taste, an economic incentive is to add glycerol to a wine to mask its poor quality. Furthermore, it is known that glycerol, depending on whether it is produced from triglycerides or petrochemicals, may contain considerable amounts of 3-MPD in the first case or CycDs in the second. However, because these compounds are not natural wine components, it is possible to detect glycerol added to wine illegally by determining the above-mentioned by-products. To this end, one of the published methods was adopted, modified, and tested in a collaborative study. The method is based on gas chromatographic/mass spectrometric analysis of diethyl ether extracts after salting out with potassium carbonate. The interlaboratory study for the determination of 3-MPD and CycDs in wine was performed in 11 laboratories in 4 countries. Wine samples were prepared and sent to participants as 5 blind duplicate test materials and 1 single test material. The concentrations covered ranges of 0.1-0.8 mg/L for 3-MPD and 0.5-1.5 mg/L for CycDs. The precision of the method was within the range predicted by the Horwitz equation. HORRAT values obtained for 3-MPD ranged from 0.8 to 1.7, and those obtained for CycDs ranged from 0.9 to 1.3. Average recoveries were 104 and 109%, respectively.