A whole-grain-rich diet reduces urinary excretion of markers of protein catabolism and gut microbiota metabolism in healthy men after one week.

Epidemiological studies consistently find that diets rich in whole-grain (WG) cereals lead to decreased risk of disease compared with refined grain (RG)-based diets. Aside from a greater amount of fiber and micronutrients, possible mechanisms for why WGs may be beneficial for health remain speculative. In an exploratory, randomized, researcher-blinded, crossover trial, we measured metabolic profile differences between healthy participants eating a diet based on WGs compared with a diet based on RGs. Seventeen healthy adult participants (11 female, 6 male) consumed a controlled diet based on either WG-rich or RG-rich foods for 2 wk, followed by the other diet after a 5-wk washout period. Both diets were the same except for the use of WG (150 g/d) or RG foods. The metabolic profiles of plasma, urine, and fecal water were measured using (1)H-nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry (plasma only). After 1 wk of intervention, the WG diet led to decreases in urinary excretion of metabolites related to protein catabolism (urea, methylguanadine), lipid (carnitine and acylcarnitines) and gut microbial (4-hydroxyphenylacetate, trimethylacetate, dimethylacetate) metabolism in men compared with the same time point during the RG intervention. There were no differences between the interventions after 2 wk. Urinary urea, carnitine, and acylcarnitine were lower at wk 1 of the WG intervention relative to the RG intervention in all participants. Fecal water short-chain fatty acids acetate and butyrate were relatively greater after the WG diet compared to the RG diet. Although based on a small population and for a short time period, these observations suggest that a WG diet may affect protein metabolism.

A whole-grain cereal-rich diet increases plasma betaine, and tends to decrease total and LDL-cholesterol compared with a refined-grain diet in healthy subjects.

Epidemiological studies have repeatedly found that whole-grain (WG) cereal foods reduce the risk of several lifestyle-related diseases, though consistent clinical outcomes and mechanisms are elusive. To compare the effects of a WG-rich diet with a matched refined-grain (RG) diet on plasma biomarkers and bowel health parameters, seventeen healthy subjects (eleven females and six males) completed an exploratory cross-over study with a 2-week intervention diet based on either WG- or RG-based foods, separated by a washout of at least 5 weeks. Both diets were the same except for the use of WG (150 g/d) or RG foods. Subjects undertook a 4 h postprandial challenge on day 8 of each intervention diet. After 2 weeks, the WG diet tended to decrease plasma total and LDL-cholesterol (both P = 0·09), but did not change plasma HDL-cholesterol, fasting glucose, C-reactive protein or homocysteine compared with the RG diet. Plasma betaine and alkylresorcinol concentrations were elevated after 1 week of the WG diet (P = 0·01 and P < 0·0001, respectively). Clostridium leptum populations in faeces were increased after the WG diet, along with a trend for decreased faecal water pH (P = 0·096) and increased stool frequency (P < 0·0001) compared with the RG diet. A short controlled intervention trial with a variety of commercially available WG-based products tended to improve biomarkers of CVD compared with a RG diet. Changes in faecal microbiota related to increased fibre fermentation and increased plasma betaine concentrations point to both fibre and phytochemical components of WG being important in mediating any potential health effects.

Isotopomics: a top-down systems biology approach for understanding dynamic metabolism in rats using [1,2-(13)C(2)] acetate.

Isotope labeled tracers are commonly used to quantify the turnover rates of various metabolic intermediates and yield information regarding physiological regulation. Studies often only consider either one nutritional state (fasted or fed) and/or one question (e.g., measure of lipid or protein turnover). In this article, we consider a novel application combining the global approach of metabonomics with widespread stable isotope labeling as a way of being able to map metabolism in open mammalian systems, an approach we call "isotopomics". A total of 45 15-week-old male Zucker rats were administrated different amounts (from 0.5 to 8 mmol/kg) of sodium [1,2-(13)C(2)] acetate. Plasma samples taken at 1, 4, and 24 h were analyzed with (13)C nuclear magnetic resonance (NMR) and gas chromatography/mass spectrometry (GC/MS) to measure (13)C isotopic enrichment of 39 plasma metabolites across a wide range of compound classes (amino acids, short-chain fatty acids, lactate, glucose, and free fatty acids). Isotopic enrichment from 0.1-7.1 mole percent excess (MPE) for the highest dose could be reliably measured in 16 metabolites, and the kinetics of their (13)C isotopic enrichment are reported. Clustering metabolites based on (13)C kinetic curves enabled highlighting of time dependent patterns of (13)C distribution through the key metabolic pathways. These kinetic and quantitative data were reported into a biochemical map. This type of isotopomic approach for mapping dynamic metabolism in an open system has great potential for advancing our mechanistic knowledge of how different interventions and diseases can impact the metabolic response of animals and humans.

Simultaneous analysis of (13)C-glutathione as its dimeric form GSSG and its precursor [1-(13)C]glycine using liquid chromatography/isotope ratio mass spectrometry.

Determination of glutathione kinetics using stable isotopes requires accurate measurement of the tracers and tracees. Previously, the precursor and synthesized product were measured with two separate techniques, liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). In order to reduce sample volume and minimize analytical effort we developed a method to simultaneously determine (13)C-glutathione as its dimeric form (GSSG) and its precursor [1-(13)C]glycine in a small volume of erythrocytes in one single analysis. After having transformed (13)C-glutathione into its dimeric form GSSG, we determined both the intra-erythrocytic concentrations and the (13)C-isotopic enrichment of GSSG and glycine in 150 microL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of micromol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 micromol/mL. The (13)C-isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3 per thousand) and accuracy (measured between 0 and 5 APE). This novel method opens up a variety of kinetic studies with relatively low dose administration of tracers, reducing the total cost of the study design. In addition, only a minimal sample volume is required, enabling studies even in very small subjects, such as preterm infants.

Simultaneous measurement of 13C- and 15N-isotopic enrichments of threonine by mass spectrometry.

Under conditions of high isotopic dilution, e.g. in a tracer study, the ability to determine accurately and quantitatively small variations in isotopic enrichments of differently labelled chemical compounds (e.g. (13)C and (15)N in threonine) in a single run by gas chromatography/mass spectrometry (GC/MS) is desirable but remains a technological challenge. Here, we report a new, rapid and simple GC/MS method for simultaneously measuring the isotopic enrichments of doubly labelled threonine ([U(13)C] and (15)N) with isotopic enrichment lower than 1.5 Molar Percent Excess (MPE). The long-term reproducibility measured was around 0.09 MPE for both tracers (throughout a 6 week period). The intra-day repeatability was lower than 0.05 and 0.06 MPE for [U(13)C]-Thr and (15)N-Thr, respectively. To calculate both isotopic enrichments, two modes of calculations were used: one based on work by Rosenblatt et al. in 1992 and the other one using a matrix approach. Both methods gave similar results (ANOVA, P >0.05) with close precision for each mode of calculation. The GC/MS method was then used to investigate the differential utilization of threonine in different organs according to its route of administration in minipigs after administration of both tracers. In plasma samples, the lowest isotopic enrichment measured between two successive time points was at 0.01 and 0.02 MPE for [U(13)C]-Thr and (15)N-Thr, respectively. Moreover, the accuracy of GC/MS (13)C-isotopic enrichment measured was validated by analyzing the same plasma samples by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Statistical analysis showed that both techniques gave the same results (ANOVA, P >0.05). This new GC/MS method offers the possibility to measure (13)C- and (15)N-isotopic enrichments with higher throughput, and using a lower amount of sample, than using GC/C/IRMS.

Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of 13C-valine isotopic ratios in complex biological samples.

On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision (13)C isotopic enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure (13)C isotopic enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(delta(13)C) reported with this method was found to be below 1 per thousand . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (delta(13)C= -12.3 to 150.8 per thousand), the calculated root-mean-square (rms) of SD was 0.38 and 0.46 per thousand and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (delta(13)C) and absolute (atom%, APE and MPE) isotopic enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic enrichment of protein-bound (13)C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p>0.05). The results of this work indicate that the LC-IRMS was successful for high-precision (13)C isotopic measurements in tracer studies giving (13)C isotopic enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio.

Liquid chromatography combined with mass spectrometry for 13C isotopic analysis in life science research.

Among the different disciplines covered by mass spectrometry, measurement of (13)C/(12)C isotopic ratio crosses a large section of disciplines from a tool revealing the origin of compounds to more recent approaches such as metabolomics and proteomics. Isotope ratio mass spectrometry (IRMS) and molecular mass spectrometry (MS) are the two most mature techniques for (13)C isotopic analysis of compounds, respectively, for high and low-isotopic precision. For the sample introduction, the coupling of gas chromatography (GC) to either IRMS or MS is state of the art technique for targeted isotopic analysis of volatile analytes. However, liquid chromatography (LC) also needs to be considered as a tool for the sample introduction into IRMS or MS for (13)C isotopic analyses of non-volatile analytes at natural abundance as well as for (13)C-labeled compounds. This review presents the past and the current processes used to perform (13)C isotopic analysis in combination with LC. It gives particular attention to the combination of LC with IRMS which started in the 1990's with the moving wire transport, then subsequently moved to the chemical reaction interface (CRI) and was made commercially available in 2004 with the wet chemical oxidation interface (LC-IRMS). The LC-IRMS method development is also discussed in this review, including the possible approaches for increasing selectivity and efficiency, for example, using a 100% aqueous mobile phase for the LC separation. In addition, applications for measuring (13)C isotopic enrichments using atmospheric pressure LC-MS instruments with a quadrupole, a time-of-flight, and an ion trap analyzer are also discussed as well as a LC-ICPMS using a prototype instrument with two quadrupoles.

Novel method for measurement of glutathione kinetics in neonates using liquid chromatography coupled to isotope ratio mass spectrometry.

A novel analytical method using liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) was developed for measuring the fractional synthesis rate (FSR) of glutathione (GSH) in neonates after infusion of [1-(13)C]-glycine as a tracer. After transformation of GSH into GSSG, its dimeric form, the intra-erythrocytic concentration and (13)C-isotopic enrichment of GSH were determined using 200 microL of blood. The results showed that, using LC/IRMS, the concentration (range of micromol/mL) was reliably measured using norvaline as internal standard with precision better than 0.1 micromol/mL. In addition, the (13)C-isotopic enrichment measured in the same run gave reliable values with excellent precision (with standard deviation (sd) lower than 0.3 per thousand) and accuracy (measured between 0 and 2 Atom % Excess (APE)). The inter-assay repeatability of delta(13)C of norvaline used as internal standard with in vivo samples was assessed at -26.07 +/- 0.28 per thousand with coefficient of variance (CV) at 1.1%. The FSR calculated either with GSH or GSSG showed similar results with slightly higher values for GSSG (41.6 +/- 4.7 and 46.5 +/- 4.4, respectively). The slightly lower FSR of GSH is probably due to interfering compounds in the biological matrix. Successfully used in a clinical study, this rapid and reliable method opens up a variety of kinetic studies with relatively low administration of tracer infusates, reducing the total cost of the study design. The small volume of blood needed enables studies even in extremely small subjects, such as premature infants, as reported in this study.

Determination of 13C isotopic enrichment of valine and threonine by GC-C-IRMS after formation of the N(O,S)-ethoxycarbonyl ethyl ester derivatives of the amino acids.

We describe a new method of assessing, in a single run, (13)C isotopic enrichment of both Val and Thr by gas chromatography-combustion-isotope-ratio mass spectrometry (GC-C-IRMS). This method characterised by a rapid one-step derivatisation procedure performed at room temperature to form the N(O,S)-ethoxycarbonyl ethyl ester derivatives, and a polar column for GC. The suitability of this method for Val and Thr in in-vivo samples (mucosal hydrolysate) was demonstrated by studying protein metabolism with two tracers ((13)C-valine or (13)C-threonine). The intra-day and inter-day repeatability were both assessed either with standards or with in-vivo samples at natural abundance and at low (13)C isotopic enrichment. For inter-day repeatability CVs were between 0.8 and 1.5% at natural abundance and lower than 5.5% at 0.112 and 0.190 atom% enrichment for Val and Thr, respectively. Overall isotopic precision was studied for eleven standard amino acid derivatives (those of Val, Ala, Leu, Iso, Gly, Pro, Asp, Thr, Ser, Met, and Phe) and was assessed at 0.32 per thousand. The (13)C isotopic measurement was then extended to the other amino acids (Ala, Val, Leu, Iso, Gly, Pro, Thr, and Phe) at natural abundance for in-vivo samples. The isotopic precision was better than 0.002 atom% per amino acid (for n = 4 rats). This analytical method was finally applied to an animal study to measure Thr utilization in protein synthesis.

Metabolomics in practice: emerging knowledge to guide future dietetic advice toward individualized health.

The profession of dietetics can take an increasingly prominent role in managing health and patient care as clinicians gain access to three new resources: detailed information about the metabolic status of healthy individual clients, metabolic knowledge about the relationships between metabolite abundances and health, and bioinformatics tools that link clients' metabolism to their present and future health status. The current use of single biomarkers as indicators of disease will be replaced by comprehensive profiling of individual metabolites linked to an understanding of health and human metabolism--the emerging science now known as metabolomics. Industrial and academic initiatives are currently developing the analytical and bioinformatic technologies needed to assemble the quantitative reference databases of metabolites as the metabolic analog of the human genome. With these in place, dietetics professionals will be able to assess both the current health status of individuals and predict their health trajectories. Another important role for dietetics professionals will be to assist in the development of the tools and their application in predicting how an individual's specific metabolic pattern can be changed by diet, drugs, and lifestyle, with the goal of improving health and preventing the development of chronic diseases.

Isotope ratio monitoring of small molecules and macromolecules by liquid chromatography coupled to isotope ratio mass spectrometry.

In the field of isotope ratio mass spectrometry, the introduction of an interface allowing the connection of liquid chromatography (LC) and isotope ratio mass spectrometry (IRMS) has opened a range of new perspectives. The LC interface is based on a chemical oxidation, producing CO2 from organic molecules. While first results were obtained from the analysis of low molecular weight compounds, the application of compound-specific isotope analysis by irm-LC/MS to other molecules, in particular biomolecules, is presented here. The influence of the LC flow rate on the CO2 signal and on the observed delta13C values is demonstrated. The limits of quantification for angiotensin III and for leucine were 100 and 38 pmol, respectively, with a standard deviation of the delta13C values better than 0.4 per thousand. Also, accuracy and precision of delta13C values for elemental analyser-IRMS and flow injection analysis-IRMS (FIA-LC/MS) were compared. For compounds with molecular weights ranging from 131 to 66,390 Da, precision was better than 0.3 per thousand, and accuracy varied from 0.1 to 0.7 per thousand. In a second part of the work, a two-dimensional (2D)-LC method for the separation of 15 underivatised amino acids is demonstrated; the precision of delta13C values for several amino acids by irm-LC/MS was better than 0.3 per thousand at natural abundance. For labelled mixtures, the coefficient of variation was between 1% at 0.07 atom % excess (APE) for threonine and alanine, and around 10% at 0.03 APE for valine and phenylalanine. The application of irm-LC/MS to the determination of the isotopic enrichment of 13C-threonine in an extract of rat colon mucosa demonstrated a precision of 0.5 per thousand, or 0.001 atom %.

Both free and esterified plant sterols reduce cholesterol absorption and the bioavailability of beta-carotene and alpha-tocopherol in normocholesterolemic humans.

BACKGROUND: Plant sterols reduce cholesterol absorption, which leads to a decrease in plasma and LDL-cholesterol concentrations. Plant sterols also lower plasma concentrations of carotenoids and alpha-tocopherol, but the mechanism of action is not yet understood. OBJECTIVES: The aims of this clinical study were to determine whether plant sterols affect the bioavailability of beta-carotene and alpha-tocopherol in normocholesterolemic men and to compare the effects of plant sterol esters and plant free sterols on cholesterol absorption. DESIGN: Twenty-six normocholesterolemic men completed the double-blind, randomized, crossover study. Subjects consumed daily, for 1 wk, each of the following 3 supplements: a low-fat milk-based beverage alone (control) or the same beverage supplemented with 2.2 g plant sterol equivalents provided as either free sterols or sterol esters. During this 1-wk supplementation period, subjects consumed a standardized diet. RESULTS: Both of the milks enriched with plant sterols induced a similar (60%) decrease in cholesterol absorption. Plant free sterols and plant sterol esters reduced the bioavailability of beta-carotene by approximately 50% and that of alpha-tocopherol by approximately 20%. The reduction in beta-carotene bioavailability was significantly less with plant free sterols than with plant sterol esters. At the limit of significance (P = 0.054) in the area under the curve, the reduction in alpha-tocopherol bioavailability was also less with plant free sterols than with plant sterol esters. CONCLUSIONS: Both plant sterols reduced beta-carotene and alpha-tocopherol bioavailability and cholesterol absorption in normocholesterolemic men. However, plant sterol esters reduced the bioavailability of beta-carotene and alpha-tocopherol more than did plant free sterols.

[2H/H] Isotope ratio analyses of [2H5]cholesterol using high-temperature conversion elemental analyser isotope-ratio mass spectrometry: determination of cholesterol absorption in normocholesterolemic volunteers.

This paper validates the use of high-temperature conversion elemental analyser isotope-ratio mass spectrometry (TC-EA/IRMS) for measuring the [(2)H/H] enrichment of plasma [(2)H(5)]cholesterol. From a molecular point of view, the free cholesterol is initially separated from plasma by thin-layer chromatography (TLC) and then injected onto the TC-EA reactor which converts cholesterol molecules into CO and H(2) gases. The slope of the curve of the experimental mole percent excess (MPE((exp.))) versus MPE((theor.)) was very close to 1, demonstrating that no significant isotopic fractionation was observed during all processing of the samples (i.e., isolation of plasma free cholesterol by TLC and pyrolysis in the TC-EA reactor). Excellent linearity (r(2) = 0.9994, n = 4) of delta ( per thousand ) of [(2)H/H] isotopic measurements versus mole percent (MP) was assessed over the range 0 to 0.1 MP. The precision of the [(2)H/H] measurement, evaluated with two calibration points processed with TLC, was delta(2)H(V-SMOW) = -192.5 +/- 3.4 per thousand and delta(2)H(V-SMOW) = -136.9 +/- 2.9 per thousand. The standard deviations of the within-assay and between-assay repeatabilities of the analytical process, evaluated using the quality control (QC) of plasma samples, were 4.6 and 6.1 per thousand, respectively. Plant sterols are known to reduce cholesterol absorption and therefore were used as a positive control in a clinical study performed with normocholesterolemic volunteers. This present method produces biological results consistent with those already reported in the literature.

Quantitative analysis of triacylglycerol regioisomers in fats and oils using reversed-phase high-performance liquid chromatography and atmospheric pressure chemical ionization mass spectrometry.

Positional distribution of fatty acyl chains of triacylglycerols (TGs) in vegetable oils and fats (palm oil, cocoa butter) and animal fats (beef, pork and chicken fats) was examined by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to atmospheric pressure chemical ionization using a quadrupole mass spectrometer. Quantification of regioisomers was achieved for TGs containing two different fatty acyl chains (palmitic (P), stearic (S), oleic (O), and/or linoleic (L)). For seven pairs of 'AAB/ABA'-type TGs, namely PPS/PSP, PPO/POP, SSO/SOS, POO/OPO, SOO/OSO, PPL/PLP and LLS/LSL, calibration curves were established on the basis of the difference in relative abundances of the fragment ions produced by preferred losses of the fatty acid from the 1/3-position compared to the 2-position. In practice the positional isomers AAB and ABA yield mass spectra showing a significant difference in relative abundance ratios of the ions AA(+) to AB(+). Statistical analysis of the validation data obtained from analysis of TG standards and spiked oils showed that, under repeatability conditions, least-squares regression can be used to establish calibration curves for all pairs. The regression models show linear behavior that allow the determination of the proportion of each regioisomer in an AAB/ABA pair, within a working range from 10 to 1000 microg/mL and a 95% confidence interval of +/-3% for three replicates.

Synthesis and sensory characterization of novel umami-tasting glutamate glycoconjugates.

Two glycoconjugates of glutamic acid, namely, the N-glycoside dipotassium N-(d-glucos-1-yl)-l-glutamate (1) and the corresponding Amadori compound N-(1-deoxy-d-fructos-1-yl)-l-glutamic acid (2), have been synthesized in yields of 35 and 52%, respectively, using new Maillard-mimetic approaches, and their chemical structures have unequivocally been elucidated by 1D- and 2D-NMR and MS experiments. Systematic sensory studies revealed that both glycoconjugates exhibit pronounced umami-like taste with recognition taste thresholds of 1-2 mmol/L, close to that of monosodium glutamate (MSG). Contrary to an aqueous solution of MSG, 1 does not show the sweetish and slightly soapy by-note, but evokes an intense umami taste. Aqueous solutions of 2 were described by the descriptors umami, seasoning, and bouillon-like. Added to a bouillon base, which did not contain any taste enhancers, both glycoconjugates imparted a distinct umami character similar to the control sample containing the same amount of MSG on a molar basis. To the best of our knowledge, these types of glycoconjugates in general and, in particular, N-glucosyl glutamate and N-deoxyfructosyl glutamate have never been reported as taste active compounds having umami-like properties. Therefore, 1 and 2 represent a new class of umami-type taste compounds showing properties similar to the umami reference compound MSG. Systematic (13)C NMR measurements revealed that 1 was fairly stable in aqueous solutions under alkaline conditions (pH 8-10) as well as in dry form. However, it rapidly hydrolyzes in neutral and acidic solutions, giving rise to glucose and glutamate. In contrast, glycoconjugate 2 was observed to be rather stable in aqueous solution as well as in the presence of human saliva.

Simultaneous determination of deuterated and non-deuterated alpha-tocopherol in human plasma by high-performance liquid chromatography.

Labelled tocopherol is used to evaluate its absorption by biodiscriminating the dietary intake from the endogenous tocopherol pool of subject. A normal-phase high-performance liquid chromatographic method is described for the easy separation and quantification of deuterated (d(6)) and non-deuterated alpha-tocopherol. The alpha-tocopherol isotopomers were extracted from plasma triacylglycerol-rich lipoproteins in hexane, separated by two EC Nucleosil columns in series with a mobile phase of hexane-isopropanol (659.34:0.786, w/w) running isocratically. The detection of d(6)-alpha-tocopherol was performed by its UV absorbance at 297 nm with a limit of detection of 34 pmol/ml, a limit of quantification of 83 pmol/ml and a range of determination of 34-9905 pmol/ml. Between- and within-assay RSDs were 2.4% (n=10) and 2.7% (n=5), respectively.

Chemoenzymatic synthesis of aroma active 5,6-dihydro- and tetrahydropyrazines from aliphatic acyloins produced by baker's yeast.

Twenty-five acyloins were generated by biotransformation of aliphatic aldehydes and 2-ketocarboxylic acids using whole cells of baker's yeast as catalyst. Six of these acyloins were synthesized and tentatively characterized for the first time. Subsequent chemical reaction with 1,2-propanediamine under mild conditions resulted in the formation of thirteen 5,6-dihydropyrazines and six tetrahydropyrazines. Their odor qualities were evaluated, and their odor thresholds were estimated. Among these pyrazine derivatives, 2-ethyl-3,5-dimethyl-5,6-dihydropyrazine (roasted, nutty, 0.002 ng/L air), 2,3-diethyl-5-methyl-5,6-dihydropyrazine (roasted, 0.004 ng/L air), and 2-ethyl-3,5-dimethyltetrahydropyrazine (bread crustlike, 1.9 ng/L air) were the most intensive-smelling aroma active compounds.

Quantification of o,o'-dityrosine, o-nitrotyrosine, and o-tyrosine in cat urine samples by LC/ electrospray ionization-MS/MS using isotope dilution.

Quantification of o-tyrosine, o-nitrotyrosine, and o,o'dityrosine from cat urine samples was achieved by LC/ electrospray ionization-MS/MS (LC/ESI-MS/MS) using an isotope dilution technique in multiple reaction monitoring mode before butylation of o,o'-dityrosine and after butylation of o-tyrosine and o-nitrotyrosine. This novel approach of amino acids butylation enhanced the MS response by a factor of 7-fold for o-tyrosine and 6-fold for o-nitrotyrosine and decreased the overall chemical background noise. Butylation of o,o'-dityrosine resulted in a lower MS response as a result of the formation of both mono- and doubly butylated species. The mean recovery for the oxidized amino acids was estimated at 73 +/- 2%. The limits of quantitation of NO2-Tyr butyl ester, o-Tyr butyl ester, and di-Tyr in cat urine samples were calculated at 14.5, 28.2 and 140.9 nM, respectively. The oxidized amino acids levels in cat urine extracts ranged from 157 to 250 ng/day for o-Tyr and from 3,289 to 11,803 ng/day for di-Tyr. NO2-Tyr was found in only two urine extracts at levels below 58 ng/day. A certain trend of correlation was observed between o,o'-dityrosine and o-tyrosine when comparing these values against their respective creatinine amounts. A comparison of the data gathered from the ThermoFinnigan TSQ 7000 and Micromass Q-TOF instruments revealed several advantages of using the Q-TOF regarding the exact mass measurement, a lower ion suppression effect and the possibility to perform analyses in full scan product ion mode. These results demonstrate that a Q-TOF instrument can be a good alternative to classical triple quadrupole for quantitative purposes on a relatively small linear dynamic range (4 orders of magnitude for the Q-TOF, as compared to 6 for the triple quadrupole).