Progress towards the characterisation of faecal compounds.

Intestinal nitrosation produces ATNCs (Apparent Total N-nitroso Compounds) and these have been linked with an increased risk of colon cancer from eating red meat. Modern LC-MS instrumentation makes direct detection of ATNC components in faecal water a possibility. The difficulty is in determining which of the many compounds present are N-nitrosamines before embarking on efforts to characterise them. We have assumed that any in vivo nitrosation of alimentary tract contents will be non-specific and depend on the amount and basicity of amine present, with concentration of nitrosating agent being the limiting factor. By further nitrosating faecal waters (and ileostomy fluids) we can increase the amount of ATNC and readily access these compounds. The amount and the number of nitrosamines generated depend on the concentration of individual amines present. By derivatisation separately using both 14N and 15N labelled nitrite, we demonstrated that inspecting chromatograms in parallel for a unit mass difference provides a novel and practicable means for identifying unknown ATNC in faecal and ileostomy samples. MS procedures were linked with the traditional approaches of thermal energy analyser (TEA), preparative HPLC, visualisation of nitrosamines with Griess reagent and degradation of N-nitroso compounds by UV irradiation. We have demonstrated that this approach is repeatable and have used it to identify 30 putative N-nitroso compounds (as protonated parent masses [M + H]+ at: 242, 258, 312, 313, 333, 348, 365, 377, 382, 386, 392, 412, 414, 421, 434, 442, 466, 467, 483, 493, 572, 582, 625, 636, 637, 656, 662, 752, 808, and 870.

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%.

Determination of chlormequat and mepiquat in foods by liquid chromatography/mass spectrometry or liquid chromatography/tandem mass spectrometry: interlaboratory study.

Interlaboratory validation studies have been performed on 2 methods for the determination of chlormequat (CLQ) and mepiquat (MPQ). Both methods used identical extraction procedures and stable isotope internal standardization but differed in the use of liquid chromatography/mass spectrometry (LC/MS) or LC/tandem mass spectrometry (LC/MS/MS) for the determination, the amount of internal standard used, and the expected limit of detection. After addition of deuterated internal standards, CLQ and MPQ were extracted with methanol-water and determined by LC//MS or LC/MS/MS with positive electrospray ionization. Eight European laboratories participated in the LC/MS method study, analyzing mushroom, pear, wheat flour, and fruit puree with residues of CLQ in the range 0.040-1.19 mg/kg and of MPQ in the range 0.041-0.39 mg/kg. For CLQ, the Horwitz ratio (HoRat) values for individual test materials/levels were in the range 0.85-1.13 with a mean of 1.00, showing good method performance. For MPQ, the Ho values for mushroom, pear (both levels), and wheat flour were in the range 0.83-0.94, again indicating good method performance. For the determination of MPQ in infant food (fruit puree) at 0.041 mg/kg, the Ho was 1.7 when a value of 0 reported by one participant was excluded. In the LC/MS/MS study, in which 11 laboratories participated, a separate sample set was analyzed with residues of CLQ in the range 0.007-1.03 mg/kg and of MPQ in the range 0.008-0.72 mg/kg. Ho values for CLQ were in the range 0.27-1.36 and for MPQ in the range 0.51-2.10, all corresponding to acceptable method performance.

Negative ion electrospray mass spectrometry of aminomethylphosphonic acid and glyphosate: elucidation of fragmentation mechanisms by multistage mass spectrometry incorporating in-source deuterium labelling.

Glyphosate and its main metabolite, aminomethylphosphonic acid, introduced by direct infusion in (2)H(2)O, appear in negative ion electrospray mass spectrometry (ES-MS) as triply deuteriated [M[bond]H](-) ions. Sites of deuterium residence and loss were established using the multistage (MS(n)) capabilities of an ion trap mass spectrometer to assist in the determination of fragmentation mechanisms. The study reveals specific mechanisms, common to each analyte, such as those involving a five-membered transition state between the amine and phosphonate group, as well as analyte specific transitions.

Analysis of glyphosate and glufosinate by capillary electrophoresis-mass spectrometry utilising a sheathless microelectrospray interface.

The potential of capillary electrophoresis combined with mass spectrometry for the simultaneous determination of two herbicides (glyphosate and glufosinate) and their metabolites (aminomethylphosphonic acid and methylphosphinicopropionic acid) as the native species is demonstrated utilising a simple microelectrospray interface. The interface uses the voltage applied to the CE capillary to drive separation and generate the electrospray, avoiding sample dilution associated with the use of a sheath liquid interface. The chemistry of the internal walls of the capillary has a marked influence on peak shape, and appropriate choice is essential to successful operation of the interface. A linear polyacrylamide coated capillary, which has no electroosmotic flow, gave best reproducibility, with precision of migration time and peak area in the range 1-2 and 7-12% RSD, respectively, for the four analytes. Limits of detection, low-pg on-column, are substantially better than for previous methods and calibration curves over the range 1-100 microM have R2 values greater than 0.97. The observed concentration limit of detection for glyphosate in water is 1 microM and for a water-acetone extract of wheat is 2.5 microM, allowing the underivatised herbicide to be detected at 10% of the maximum residue limit in wheat.

Tandem mass spectrometric analysis of glyphosate, glufosinate, aminomethylphosphonic acid and methylphosphinicopropionic acid.

A detailed MS(n) study of glyphosate, glufosinate and their main metabolites, aminomethylphosphonic acid and methylphosphinicopropionic acid, using an ion trap mass spectrometer, was performed. The analytes show good response in negative ion electrospray mass spectrometry (ES-MS) as [M-H](-) ions. Tandem-MS spectra reveal a wealth of structurally specific ions, allowing characterisation of the fragmentation pathways of the four analytes in their native form for the first time. The ions formed at each stage of fragmentation reveal ions common to each analyte, such as phosphinate, as well as analyte specific transitions. Simplex optimisation allows optimum trapping and fragmentation parameters to be determined leading to improved response for particular transitions and transition sequences, and revealing previously unseen ions.