Analysis of biological fluids by high-field nuclear magnetic resonance spectroscopy

The application of high-field Fourier transform nuclear magnetic resonance (NMR) spectroscopy to the analysis of biological fluids such as urine, plasma and bile is described. Applications include areas such as clinical chemistry, experimental and clinical toxicology and drug metabolism studies. In the case of proton NMR some means of attenuating or eliminating the interference due to water protons is required and suitable strategies for achieving this are discussed. The use of 2-dimensional NMR or solid-phase extraction/chromatography to enable the identification of unknowns is discussed and the potential usefulness of ¹⁹F NMR for studying the metabolism of fluorinated xenobiotics is highlighted.     

Transmission of polar substituent effects in bicycloalkane systems as probed by fluorine-19 nuclear magnetic resonance

The results of a correlative analysis indicate that the ¹⁹F substituent chemical shifts (SCS) of 4-substituted bicyclo[2.2.1]hept-1-y1 fluorides are essentially a manifestation of electronegativity effects which are $\text{opposite}$ in sign to those previously disclosed for the corresponding 4-substituted bicyclo[2.2.2]oct-1-y1 fluorides.     

Determination of metformin hydrochloride in tablets by nuclear magnetic resonance spectrometry

The ¹H-n.m.r. signal of the drug (10–35 mg ml^?1) in deuterium oxide, with maleic acid as internal reference standard, is used. The integral of the peak at 3.06 ppm with respect to 3-trimethylsilylpropionic acid is compared with that at 6.3 ppm for the internal standard. The method is quantitative and free from interference by tablet excipients.     

Identification of a probable new adrenergic agonist by nuclear magnetic resonance and mass spectrometry

In animal production, it is consolidated the synthesis and the illegal use of growth promoters of new generation, able to skip routine screening and confirmatory analysis. In this work it is reported the nuclear magnetic resonance (NMR) and the mass spectrometry identification of a probable new adrenergic drug found in a feed premix. The substance was selectively purified on alpha 1 acid glycoprotein affinity columns; then its structure was first achieved by recording the 13C NMR spectrum that gave the total number of carbons of the molecule, successively sorted by DEPT experiments into quaternary, CH, CH2, and CH3 groups. However, the complete assignments of all resonances were derived from the bi-dimensional analysis and the crucial indications from the 1H-13C reverse experiments. Further characterisation was performed by atmospheric pressure chemical ionisation both in positive and negative ion mode, matching the molecular ion and the fragmentation pattern with those of most recently described new adrenergic agonists. After the loss of a ter-butylic group, the structure shows an internal symmetry along with the presence of Chlorine clusters. The proposed formula of the compound, the 8,8'-diamino-9,9'-dichloro-1-terbutyl-1,1',4,4-tetrahydro-5H,5'H-2,2'-bi-1-benzazepine-5,5'-dione, partially resembles that of Zilpaterol for the presence of a heterocyclic ring; Further work is in progress to characterise the structure-activity relationship.     

Conditions to obtain precise and true measurements of the intramolecular C distribution in organic molecules by isotopic C nuclear magnetic resonance spectrometry

Intramolecular ¹³C composition gives access to new information on the (bio) synthetic history of a given molecule. Isotopic ¹³C NMR spectrometry provides a general tool for measuring the position-specific ¹³C content. As an emerging technique, some aspects of its performance are not yet fully delineated. This paper reports on (i) the conditions required to obtain satisfactory trueness and precision for the determination of the internal ¹³C distribution, and (ii) an approach to determining the “absolute” position-specific ¹³C content. In relation to (i), a precision of <1% can be obtained whatever the molecule on any spectrometer, once quantitative conditions are met, in particular appropriate proton decoupling efficiency. This performance is a prerequisite to the measurement of isotope fractionation either on the transformed or residual compound when a chemical reaction or process is being studied. The study of the trueness has revealed that the response of the spectrometer depends on the ¹³C frequency range of the studied molecule, i.e. the chemical shift range. The “absolute value” and, therefore, the trueness of the ¹³C NMR measurements has been assessed on acetic acid and by comparison to the results obtained on the fragments from COOH and CH₃ by isotopic mass spectrometry coupled to a pyrolysis device (GC-Py–irm-MS), this technique being the reference method for acetic acid. Of the two NMR spectrometers used in this work, one gave values that corresponded to those obtained by GC-Py–irm-MS (thus, the “true” value) while the other showed a bias, which was dependent to the range covered by the resonance frequencies of the molecule. Therefore, the former can be used directly for studying isotope affiliations, while the latter can only be used directly for comparative data, for example in authenticity studies, but can also be used to obtain the true values by applying appropriate correction factors. The present study assesses several key protocol steps required to enable the determination of position-specific ¹³C content by isotopic ¹³C NMR, irrespective of the NMR spectrometer: parameters to be adjusted, performance test using [1,2-¹³C₂]acetic acid, generation of correction factors.     

A carbon-13 nuclear magnetic resonance study of compounds substituted by a perfluoroalkyl chain

The ¹³C NMR spectra of alkanes, alkanones and cyclohexanones substituted by perfluoroalkyl groups, R_F, have been studied. The influence of the perfluoroalkyl group on the chemical shifts of other carbons of the molecules is the same regardless of the R_F chain length.     

液液萃取-气相色谱-质谱法同时测定水中46种半挥发性有机物

半挥发性有机物主要包括多环芳烃类(PAHs)、邻苯二甲酸酯类(PAEs)、有机氯农药类(OCPs)和硝基苯类(NBs)等化合物,这些物质多具有致癌、致畸、致突变作用,以及内分泌干扰效应.因此,快速准确测定水中半挥发性有机物非常重要,目前国内尚无水中半挥发性有机物的检测标准.该研究从氮吹温度、水样pH值和萃取时间3个方面...     

Oxygen determination in organic fluorine compounds by means of a glassy-carbon pyrolysis tube

A conventional apparatus for determination of oxygen in organic compounds has been improved for application to organic fluorine compounds. A feature of the apparatus is the use of a pyrolysis tube made of glassy carbon instead of quartz, which eliminates effects due to hydrogen fluoride produced in pyrolysis of the sample. Ten analyses of dexamethasone with the apparatus gave a mean value of 20.44% for oxygen (theory, 20.38%), with a standard deviation of 0.16%. Oxygen in 9 organic fluorine compounds was accurately determined by using the apparatus, with an average error of +0.1%. One analysis by a gravimetric or a coulometric method took about 40 or 25 min, respectively.     

Determination of isotope composition in protic solvents by cobal-59 nuclear magnetic resonance spectrometry

The very large chemical shift range provided by cobalt-59 yields well resolved lines for each of the nineteen isotopic species in the series of complexes ranging from Co(NH₃)³⁺₆ to Co(ND₃)³⁺₆ and each of the thirteen isotopic species in the similar series of tris(ethylenediamine)cobalt(III) complexes. The relative intensily distribution of these n.m.r. signals at equilibrium is related directly to the proton isotope composition of the solvent. This n.m.r. method of quantifying the isotope composition is examined with respect to the equilibrium isotope and the application to determination in aquerous and non-aqueous solvents.     

Photoelectron resonance capture ionization mass spectrometry: a soft ionization source for mass spectrometry of particle-phase organic compounds

Photoelectron resonance capture ionization (PERCI) is a soft and sensitive ionization method, based on the attachment of low-energy (<1 eV) photoelectrons to organic analyte molecules. PERCI has been developed in our laboratory for the real-time analysis of organic particles by mass spectrometry, and is employed here to monitor the heterogeneous reaction of ozone with oleic acid. Simplified identification of the reaction products is possible as a result of the soft nature of PERCI, giving predominantly the [M--H](-) ions. The major particle-phase products are identified as: 1-nonanal, nonanoic acid, 9-oxononanoic acid, and azelaic acid, consistent with proposed mechanisms. New insight into this well-studied heterogeneous reaction is gained as additional minor particle-phase products, consistent with the Criegee mechanism, are readily detected.     

Application of nuclear quadrupole resonance to organic chemistry : Probing delocalization effects in tetrachlopentafulvenes

The ³⁵Cl quadrupole resonance frequencies of a series of 6 - aryl - 1,2,3,4 - tetrachloropentafulvenes (2, R₁ = H, R₂ = Aryl) and related substances were determined at 77°K The results indicate the negligible contnbution of the delocalized ‘aromatic’ cyclopentadienide structures in the ground-state. In the relatively dipolar p-dimethylaminophenyl and triapentafulvalene derivatives (9 and 11, respectively), the partial negative-charge is not distributed evenly in the five-membered ring but is localized mostly in the vicinity of the fulvenic double bonds. In most of the 6 - aryl -1,2,3,4 - tetrachloropentafulvenes under study, the NQR absorptions of the chlorine nuclei Z to the aryl groups are considerably shifted to higher frequencies. The effect, which is most pronounced in the 6,6-diphenyl derivative (10), is explained in terms of the spatial structures of the compounds and their substantial deviations from planarity.     

Detection of 28 neurotransmitters and related compounds in biological fluids by liquid chromatography/tandem mass spectrometry

This work presents two liquid chromatography/tandem mass spectrometry (LC/MS/MS) acquisition modes: multiple reaction monitoring (MRM) and neutral loss scan (NL), for the analysis of 28 compounds in a mixture. This mixture includes 21 compounds related to the metabolism of three amino acids: tyrosine, tryptophan and glutamic acid, two pterins and five deuterated compounds used as internal standards. The identification of compounds is achieved using the retention times (RT) and the characteristic fragmentations of ionized compounds. The acquisition modes used for the detection of characteristic ions turned out to be complementary: the identification of expected compounds only is feasible by MRM while expected and unexpected compounds are detected by NL. In the first part of this work, the fragmentations characterizing each molecule of interest are described. These fragmentations are used in the second part for the detection by MRM and NL of selected compounds in mixture with and without biological fluids. Any preliminary extraction precedes the analysis of compounds in biological fluids.     

Arylthallium trifluoroacetates carbon-13 and fluorine-19 nuclear magnetic resonance: The substituent effect of the bis(trifluoroacetato)- thallium(III) group

¹³C NMR data for a series of arylthallium trifluoroacetates (ArTlX₂, X = OCOCF₃) are reported and assigned. The range of carbon—thallium couplings to be expected, the dependence on the disposition of coupled nuclei, and chemical shift effects are discussed. The Tl(OCOCF₃)₂ group is shown to be a powerful electron withdrawing group, from both the ¹³C data and ¹⁹F substituent chemical shifts of the p-fluorophenyl derivative.     

Simultaneous determination of semivolatile organic compounds in indoor air by gas chromatography-mass spectrometry after solid-phase extraction

A method is described for simultaneous determination of semivolatile organic compounds (SVOCs) in indoor air by gas chromatography-mass spectrometry (GC-MS). The selected 73 SVOCs were collected using combined adsorbents (quartz fiber filter disk and Empore disk) for 24 h at a 5.0 l/min flow rate. The SVOCs collected were extracted with acetone, concentrated, then analyzed by an internal standard method. Forty compounds (19 plasticizers and flame retardants; 19 insecticides; 1 synergist; and 1 fungicide) among the target SVOCs were determined accurately and precisely. The method of detection limits for these compounds were approximately 0.5 ng/m3 for most of the SVOCs. The collected SVOC samples could be stored for up to 1 month at 4 C in the refrigerator.     

Simultaneous quantitation of phenobarbitone and p-hydroxyphenobarbitone and their perdeuteroethyl analogues in biological fluids by combined gas chromatography-mass spectrometry

In order to develop 5-pentadeuteroethyl-5-phenyl barbituric acid as an alternative tracer in pharmacokinetic and metabolic studies of phenobarbitone, and to search for possible isotope effects associated with such labelling, we propose a gas chromatographic-mass spectrometric assay for simultaneous measurement of phenobarbitone, p-hydroxyphenobarbitone and their perdeuteroethyl analogues, using [1,3-15N2,2-13C] phenobarbitone as internal standard. These compounds were extracted from plasma (50 microliter) or urine (500 microliter) and pentylated according to Greeley's method. Linear calibration curves were obtained in the concentration range from 0.5 to 3 micrograms/ml. The interday precision, mean accuracy and detection limit were 0.77-5.28%, 99.99-100.80% and 0.03-0.05 microgram/ml, respectively. Results for plasma and urine concentrations, and pharmacokinetic parameters in humans, are presented to illustrate this method.     

Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods

The organic acids present in beer provide important information on the product's quality and history, determining organoleptic properties and being useful indicators of fermentation performance. NMR spectroscopy may be used for rapid quantification of organic acids in beer and different NMR-based methodologies are hereby compared for the six main acids found in beer (acetic, citric, lactic, malic, pyruvic and succinic). The use of partial least squares (PLS) regression enables faster quantification, compared to traditional integration methods, and the performance of PLS models built using different reference methods (capillary electrophoresis (CE), both with direct and indirect UV detection, and enzymatic essays) was investigated. The best multivariate models were obtained using CE/indirect detection and enzymatic essays as reference and their response was compared with NMR integration, either using an internal reference or an electrical reference signal (Electronic REference To access In vivo Concentrations, ERETIC). NMR integration results generally agree with those obtained by PLS, with some overestimation for malic and pyruvic acids, probably due to peak overlap and subsequent integral errors, and an apparent relative underestimation for citric acid. Overall, these results make the PLS-NMR method an interesting choice for organic acid quantification in beer.     

Identification of free phosphopeptides in different biological fluids by a mass spectrometry approach

Human body fluids have been rediscovered in the post-genomic era as a great source of biological markers and perhaps as source of potential biomarkers of disease. Recently, it has been found that not only proteins but also peptides and their modifications can be indicators of early pathogenic processes. This paper reports the identification of free phosphopeptides in human fluids using an improved IMAC strategy coupled to iterative mass spectrometry-based scanning techniques (neutral loss, precursor ion, multiple reaction monitoring). Many peptides were detected in the enriched extract samples when submitted to the MS-integrated strategy, whereas they were not detected in the initial extract samples. The combination of the IMAC-modified protocol with selective "precursor ion" and constant "neutral loss" triple quadrupole scan modes confers a high sensitivity on the analysis, allowing rapid phosphopeptide identification and characterization, even at low concentrations. To the best of our knowledge this work represents the first report exclusively focused on the detection of free phosphorylated peptides in biological fluids.     

Solid-State nuclear magnetic resonance: performance of point-charge distributions to model intermolecular effects in 19F chemical shifts

This contribution presents results from applying two different charge models to take into account intermolecular interactions to model the solid-state effects on the ¹⁹F NMR chemical-shift tensors. The density functional theory approach with the B3LYP gradient-corrected exchange correlation functional has been used because it includes electron correlation effects at a reasonable cost and is able to reproduce chemical shifts for a great variety of nuclei with reasonable accuracy. The results obtained with the charge models are compared with experimental data and with results obtained from employing the cluster model, which explicitly includes neighboring molecular fragments. The results show that the point-charge models offer similar accuracy to the cluster model with a lower cost. Received: 3 October 1999 / Accepted: 3 February 2000 / Published online: 5 June 2000