Rapid screening assay of trimethylaminuria in urine with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Trimethyamine (TMA) and trimethylamine N-oxide (TMAO) are the most important urine parameters for diagnosing and monitoring trimethylaminuria. A rapid, simple, and specific method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was developed to determine the presence of TMA and TMAO in urine samples from patients with trimethylaminuria. Formation of the quaternary tetramethylamino iodide by derivatization of TMA with methyl iodide allows measurement of TMA by MALDI-TOFMS. The method is repeatable and reproducible, with coefficients of variance (CVs)<3%. This new method was used for direct determination of TMA and TMAO in urine specimens obtained from normal children and patients. The proposed method allows for rapid and reliable measurements of TMA and TMAO in urine specimens from patients affected by trimethylaminuria.     

离子色谱法测定饲料中氯化胆碱和三甲胺的含量

建立了离子色谱法测定饲料中氯化胆碱含量及鉴别饲料中氯化胆碱及掺假物三甲胺的方法。选用IonPac CS12阳离子交换色谱柱(250 mm×4 mm i.d.)和8.5 mmol/L H2SO4淋洗液,抑制型电导检测,在16 min内分离测定了包括胆碱和三甲胺在内的8种阳离子。胆碱和三甲胺的最小检出限分别为0.1 mg/L和0.05 mg/L。方法回收率为99.25%~102.5%。该方法具有灵敏度高、选择性强、操作简单等优点。     

Simultaneous determination of trimethylamine and trimethylamine N‐oxide in mouse plasma samples by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry

A method was developed that applies hydrophilic interaction liquid chromatography with tandem mass spectrometry in the multiple reaction monitoring mode to separate and accurately quantify trimethylamine and trimethylamine N‐oxide in a single chromatographic run. This was achieved by converting trimethylamine to ethyl betaine, which is less volatile and hence results in greatly improved quantitation. Ethyl betaine also gives a similar response to trimethylamine N‐oxide using positive‐ion electrospray ionization mass spectrometry. It is readily separated from trimethylamine N‐oxide by hydrophilic liquid chromatography in a 5 min run and with improved peak shape compared to underivatized trimethylamine. Validation of the method yielded a limit of detection (S/N ≥ 3) of 0.5 ng/mL for trimethylamine and 0.25 ng/mL for trimethylamine N‐oxide. Method accuracies of 91.4–105.3% with precisions of 0.4–5.5% were obtained for standard mixtures over the range of 2.5–500 ng/mL. Recoveries measured for the extraction of trimethylamine and trimethylamine N‐oxide spikes into mouse plasma were both >90%. The method, which simultaneously measures trimethylamine and trimethylamine N‐oxide, was successfully applied to mouse plasma samples and could be adapted for use with other biological fluids.     

Blue shifts of the C-H stretching vibrations in hydrogen-bonded and protonated trimethylamine. Effect of hyperconjugation on bond properties

The optimized geometry of isolated trimethylamine (TMA), its hydrogen bond complexes with phenol derivatives and protonated TMA is calculated at the B3LYP/6-31++G(d,p) level. A natural bond orbital (NBO) analysis on these systems is carried out at the same level of theory. In isolated TMA, one of the C-H bond in each of the three CH(3) groups is more elongated than the two other ones. As revealed by the NBO data, this results from a hyperconjugative interaction from the N lone pair to the sigma*(C-H) orbitals of the C-H bonds being in a transoid position with respect to the N lone pair. The formation of an intermolecular OH...N hydrogen bond with phenols results in a decrease of the lone pair effect. A linear correlation is found between the decrease in occupation of the sigma*(C-H) orbitals and the decrease in the hyperconjugative interaction energy in the complexes and isolated TMA. Complex formation with phenols results in a blue shift of 55-74 cm(-1) of the C-H stretching vibrations involved in the lone pair effect. Smaller blue shifts between 14 and 23 cm(-1) are predicted for the other C-H bonds. In these complexes, a linear correlation is found between the frequency shifts and the elongation of the C-H bonds. Protonation of TMA results in a nearly equalization of all the C-H distances and a blue shift of 180 cm(-1) of the C-H bonds involved in hyperconjugation with the N lone pair.     

Selective determination of trimethylamine in air by liquid chromatography using solid phase extraction cartridges for sampling

The selective determination of trimethylamine (TMA) in air by liquid chromatography is reported. Sampling is effected by flushing air through C18-packed solid-phase extraction (SPE) cartridges at a flow rate of 15 mL/min for 15 min. Next, TMA is desorbed from the cartridges and injected into the chromatographic system. The analyte is then selectively retained on a precolumn (20 mm x 2.1 mm i.d., packed with 30 microm, Hypersil C18 phase), and derivatized on-line by injecting 9-fluorenylmethyl chloroformate (FMOC). Finally, the TMA-FMOC derivative is transferred to the analytical column (125 mm x 4 mm i.d., LiChrospher 100 RP18, 5 microm), and monitored at 262 nm. The method was applied to the measurement of TMA in air in the 0.25-2.5 microg interval (equivalent to concentrations of TMA of 1.1-11 mg/m3), providing good linearity, reproducibility and accuracy. The mean recovery of TMA was (96 +/- 7%) (n = 12), and the limit of detection was 0.05 microg. The proposed procedure allows the selective determination of TMA in the presence of other primary and secondary short-chain aliphatic amines.     

Simultaneous determination of dimethylamine,trimethylamine and trimethylamine-n-oxide in aquatic products extracts by ion chromatography with non-suppressed conductivity detection

An ion chromatography method with non-suppressed conductivity detection was developed for the simultaneous determination of dimethylamine (DMA), trimethylamine (TMA) and trimethylamine-n-oxide (TMAO) in aquatic products. They were separated by means of cation-exchange chromatography using a 3.0 mmol/L methanesulfonic acid solution as eluent and an IonPac CS17 column (250 mm × 4 mm i.d.) as the separation column. Detection limits of dimethylamine, trimethylamine and trimethylamine-n-oxide were 0.06, 0.08 and 0.10 mg/L, respectively. The relative standard deviations (RSDs) of peak area were less than 3.53%. The recoveries were between 93.7% and 104.1%. Unlike traditional methods, this validated method is inexpensive and stable.     

Determination of trimethylamine‐N‐oxide in combination with l‐carnitine and γ‐butyrobetaine in human plasma by UPLC/MS/MS

An ultra‐high‐performance liquid chromatography–mass spectrometry (UPLC/MS/MS) method was developed and validated for the quantification of trimethylamine‐N‐oxide (TMAO) simultaneously with TMAO‐related molecules l ‐carnitine and γ‐butyrobetaine (GBB) in human blood plasma. The separation of analytes was achieved using a Hydrophilic interaction liquid chromatography (HILIC)‐type column with ammonium acetate–acetonitrile as the mobile phase. TMAO determination was validated according to valid US Food and Drug Administration guidelines. The developed method was successfully applied to plasma samples from healthy volunteers. Copyright © 2015 John Wiley & Sons, Ltd.     

Liquid chromatographic determination of trimethylamine in water

A method for the selective determination of trimethylamine (TMA) in aqueous matrices by liquid chromatography is reported. The proposed procedure is based on the derivatization of the analyte with 9-fluorenylmethyl chloroformate (FMOC) in a precolumn (Hypersil C18, 30 microm, 20 mm x 2.1 mm i.d.) connected on-line to the analytical column (LiChrosphere 100 RP18, 5 microm, 125 mm x 4 mm i.d.). Gradient elution was performed with a mixture of acetonitrile-water-0.05 M borate buffer (pH 9.0). The method has been applied to the direct determination of TMA in water within the 0.25-10.0 microg/ml concentration interval, and can also be adapted to the determination of TMA over the range 0.05-1.0 microg/ml by incorporating a preconcentration stage with C18 solid-phase extraction (SPE) cartridges. Good linearity, reproducibility and accuracy was achieved within the tested concentration intervals. The limits of detection at 262 nm were 50 and 5 ng/ml for the direct method and for the method involving preconcentration, respectively. The proposed conditions allowed the selective determination of TMA in the presence of other primary and secondary short-chain aliphatic amines. The utility of the described procedure has been tested by determining TMA in different water samples.     

直接萃取-GC/MS法测定卷烟主流烟气中四种芳香胺

采用气相色谱/质谱法直接测定了卷烟主流烟气中的1-氨基萘、 2-氨基萘、 3-氨基联苯和4-氨基联苯等4种芳香胺.该方法直接用CH2Cl2溶剂超声振荡萃取滤片中的芳香胺,超声萃取的同时加入衍生试剂,使萃取和衍生化同步进行,萃取液过硅酸镁柱进行进一步纯化,然后旋转浓缩后进行GC/MS分析.方法的RSD在1.0%～3.7%之间,加标回收率在92.9%～101.1%之间.     

An X-ray and neutron scattering study of the equilibrium between trimethylamine N-oxide and urea in aqueous solution

The interaction of the osmolytes trimethylamine N-oxide (TMAO) and urea in aqueous solutions at 40 °C was investigated by isotopic substitution neutron scattering at a TMAO mole fraction of 0.05 and TMAO/urea concentration ratios of 1?:?2 and 1?:?4. The partial pair distribution functions obtained by the empirical potential structure refinement method are consistent with those obtained previously for similar pure TMAO and 1?:?1 TMAO-urea solutions and indicate that urea progressively replaces the water molecules in the first coordination shell of the TMAO oxygen atom. The apparent association constant for the TMAO?:?urea complex (K(1)) was calculated to be 0.14 M(-1), which is of the same order as the experimental urea-protein binding constants per site reported in the literature. This confirms that the two osmolytes act independently at least in the physiological range.     

Gas chromatographic determination of 2-ethylhexanoic acid in urine as its pentafluorobenzyl ester

A gas chromatographic (GC) method was developed for the determination of 2-ethylhexanoic acid (2-EHA), initially in the urine of animals, but subsequently in samples of urine from sawmill workers in order to evaluate their exposure to 2-EHA which is used as a wood preservative. The 2-EHA was derivatised to the pentafluorobenzyl ester, which was then analysed by means of a cross-linked methyl silicone GC column with electron capture detection. Gas chromatography-mass spectrometry was used to confirm the identity of the GC peaks. The analytical range of the method was 0.03-2.70 mmol of 2-EHA per mol of creatinine in urine and the limit of detection was 0.01 mmol per mol of creatinine. The recovery of 2-EHA was 81-90% with a coefficient of variation of 9.8%. The amount of 2-EHA excreted in urine was corrected for the excretion of creatinine. The concentration of 2-EHA in the urine of the workers studied varied from 0.01 to 5.40 mmol per mol of creatinine; the median was 0.1 mmol per mol of creatinine.     

Interactions of trimethylamine N-oxide and water with cyclo-alanylglycine

The osmolyte trimethylamine N-oxide (TMAO) is one of a family of compounds found in living systems that can stabilize biomolecular tertiary structures. As a step in exploring the interactions between this material and polyamino acids, we have determined intermolecular 1H{1H} nuclear Overhauser effects (NOEs) between the protons of cyclo-alanylglycine and protons of solvent components in TMAO-water solutions. Comparison of the results to effects predicted on the basis of the molecular shape of the dipeptide and experimental translational diffusion coefficients suggests that both water and TMAO molecules have properties in the vicinity of the dipeptide that are different from those in the bulk solution. Changes of local concentrations of water and TMAO and changes in the diffusive behavior of these components near the dipeptide are rejected as possible explanations of the discrepancies between observed and calculated Overhauser effects. Rather, it is concluded that TMAO molecules, and the water molecules associated with them, participate to some extent in the formation of long-lived solute-solvent complexes. The aliphatic alcohol tert-butyl alcohol is structurally similar to TMAO. Overhauser effect studies of its interaction with cyclo-alanylglycine in tert-butyl alcohol-water suggest similar kinds of interactions are present in this system but that they are significantly weaker, presumably because of the lower polarity of this alcohol compared to TMAO.     

Quantification of glycine betaine,choline and trimethylamine N-oxide in seawater particulates: Minimisation of seawater associated ion suppression

A liquid chromatography/mass spectrometry (LC/MS, electrospray ionisation) method has been developed for the quantification of nitrogenous osmolytes (N-osmolytes) in the particulate fraction of natural water samples. Full method validation demonstrates the validity of the method for measuring glycine betaine (GBT), choline and trimethylamine N-oxide (TMAO) in particulates from seawater. Limits of detection were calculated as 3.5, 1.2 and 5.9 pg injected onto column (equivalent to 1.5, 0.6 and 3.9 nmol per litre) for GBT, choline and TMAO respectively. Precision of the method was typically 3% for both GBT and choline and 6% for TMAO. Collection of the particulate fraction of natural samples was achieved via in-line filtration. Resulting chromatography and method sensitivity was assessed and compared for the use of both glass fibre and polycarbonate filters during sample collection. Ion suppression was shown to be a significant cause of reduced instrument response to N-osmolytes and was associated with the presence of seawater in the sample matrix.     

H-bonded clusters in the trimethylamine/water system: a matrix isolation and computational study

The environmentally important interaction products of trimethylamine (TMA) and water molecules have been observed by Matrix Isolation Fourier Transform Infrared Spectroscopy (MIS-FTIR). Infrared spectra of solid argon matrix layers, in which both TMA and H(2)O molecules were entrapped as impurities, were analyzed for bands in the ν(O-H) region, not seen in matrix layers containing either of the parent molecules alone. Results were interpreted on the basis of the emergence of several spectral band pairs and their red shifts from the position of the matrix isolated H(2)O monomers as compared to semiempirically scaled frequencies from the B3LYP/aug-cc-pVTZ calculations and empirical correlations with a large body of data on H-bonded complexes. Bands were assigned to a complex cluster of two TMA molecules flanking a closed ring of four H-bonded H(2)O molecules. The formation of this cluster is argued to be formed in the vapor phase (as opposed to being a result of diffusion of the trapped species) and is related to its large stabilization energy (enthalpy) because of strong cooperative effects in its H-bond system.     

G protein-coupled receptor mediated sensing of TMA

A new approach for the detection of trimethylamine (TMA) using recombinant Xenopus laevis melanophores was developed. The cells were genetically modified to express the mouse trace amine-associated receptor 5 (mTAAR5), a G protein-coupled receptor from the olfactory epithelium, which conferred high sensitivity to TMA. A focused chemical screen allowed the discovery of additional, previously unknown stimuli of mTAAR5. The cell-based sensor demonstrated no sensitivity to trimethylamine N-oxide (TMAO), making it suitable for a convenient evaluation of TMA levels in fish tissue extracts. The developed gas measurement platform was able to detect TMA from 1 to 100 ppm within thirty-five minutes.     

Adsorption of trimethylamine on zirconium silicate and polyethylene powder surfaces

Sorptive interactions of volatile organic compounds (VOCs) with indoor surfaces play a major role in inhalation exposure to these species. Using ZrSiO₄ and polyethylene (PE) to model mineral surfaces and carpeting, respectively, the adsorption behavior of gaseous trimethylamine (TMA) was examined under conditions of 80% relative humidity (RH) in N₂ and in the presence of 1000 ppm CO₂ or NH₃. TMA adsorption and desorption behavior were studied using attenuated total reflection infrared (ATR-IR) and X-ray photoelectron (XPS) spectroscopies. Spectral data revealed that TMA adsorbed on both surfaces in a protonated state. Stronger adsorption was observed to occur on ZrSiO₄. XPS scans indicate that the “dry” ZrSiO₄ surface maintains OH groups available for bonding, supporting earlier research showing that partition coefficients increase as RH decreases.     

The effect of trimethylamine N-oxide on RNase a stability

The thermal stability of bovine pancreatic ribonuclease (RNase A) has been investigated in the presence of trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, by means of differential scanning calorimetry (DSC) and circular dichroism (CD) measurements at neutral and acid pH conditions. It is well known that compatible osmolytes such as TMAO are effective in stabilizing protein structure and counteracting the denaturing the effect of urea and guanidinium hydrochloride (GuHCl). Calorimetric results show that TMAO stabilizes RNase A at pH 7.0 and does not stabilize the protein at pH 4.0. RNase A thermal denaturation in the presence of TMAO is a reversible two-state N ⇆ D process. We also show that TMAO counteracts the urea and GuHCl denaturing effect at neutral pH, whereas the counteracting ability is lost at acid pH.     

Structure of the molybdenum site of Escherichia coli trimethylamine N-oxide reductase

We report a structural characterization of the molybdenum site of recombinant Escherichia coli trimethylamine N-oxide (TMAO) reductase using X-ray absorption spectroscopy. The enzyme active site shows considerable similarity to that of dimethyl sulfoxide (DMSO) reductase, in that, like DMSO reductase, the TMAO reductase active site can exist in multiple forms. Examination of the published crystal structure of TMAO oxidase from Shewanella massilia indicates that the postulated Mo coordination structure is chemically impossible. The presence of multiple active site structures provides a potential explanation for the anomalous features reported from the crystal structure.     

Dissociative photoionization of mono-, di- and trimethylamine studied by a combined threshold photoelectron photoion coincidence spectroscopy and computational approach

Energy selected mono-, di- and trimethylamine ions were prepared by threshold photoelectron photoion coincidence spectroscopy (TPEPICO). Below 13 eV, the main dissociative photoionization path of these molecules is hydrogen atom loss. The ion time-of-flight (TOF) distributions and breakdown diagrams for H loss are analyzed in terms of the statistical RRKM theory, which includes tunneling. Experimental evidence, supported by quantum chemical calculations, indicates that the reverse barrier along the H loss potential energy curve for monomethylamine is 1.8 +/- 0.6 kJ mol(-1). Accurate dissociation onset energies are derived from the TOF simulation, and from this analysis we conclude that Delta(f)H degrees (298K)[CH(2)NH(2)(+)] = 750.4 +/- 1.3 kJ mol(-1) and Delta(f)H degrees (298K)[CH(2)NH(CH(3))(+)] = 710.9 +/- 2.8 kJ mol(-1). Quantum chemical calculations at the G3, G3B3, CBS-APNO and W1U levels are extensively used to support the experimental data. The comparison between experimental and ab initio isodesmic reaction heats also suggests that Delta(f)H degrees (298K)[N(CH(3))(3)] = -27.2 +/- 2 kJ mol(-1), and that the dimethylamine ionization energy is 8.32 +/- 0.03 eV, both of which are in slight disagreement with previous experimental values. Above 13 eV photon energy, additional dissociation channels appear besides the H atom loss, such as a sequential C(2)H(4) loss from trimethylamine for which a dissociation mechanism is proposed.     

The chemistry of trimethylamine on Ru(001) and O/Ru(001)

The interaction and reactivity of trimethylamine (TMA) has been studied over clean and oxygen-covered Ru(001) under UHV conditions, as a model for the chemistry of high-density hydrocarbons on a catalytic surface. The molecule adsorbs intact at surface temperature below 100 K with the nitrogen end directed toward the surface, as indicated from work function change measurements. At coverage less than 0.05 ML (relative to the Ru substrate atoms), TMA fully dissociates upon surface heating, with hydrogen as the only evolving molecule following temperature-programmed reaction/desorption (TPR/TPD). At higher coverage, the parent molecule desorbs, and its desorption peak shifts down from 270 K to 115 K upon completion of the first monolayer, indicating a strong repulsion among neighbor molecules. The dipole moment of an adsorbed TMA molecule has been estimated from work function study to be 1.4 D. Oxygen precoverage on the ruthenium surface has shown efficient reactivity with TMA. It shifts the surface chemistry toward the production of various oxygen-containing stable molecules such as H2CO, CO2, and CO that desorb between 200 and 600 K, respectively. TMA at a coverage of 0.5 ML practically cleans off the surface from its oxygen atoms as a result of TPR up to 1650 K, in contrast to CO oxidation on the O/Ru(001) surface. The overall reactivity of TMA on the oxidized ruthenium surface has been described as a multistep reaction mechanism.