High Field 1H NMR Spectroscopy Research Articles

Low vs high field 1h Nmr spectroscopy for the detection of adulteration of cold pressed rapeseed oil with refined oils

Cold pressed rapeseed oil (CPRO) is a relatively recent development in rapeseed processing, which produces a quality product with a high market value. High field NMR (400 MHz) is a well-established tool in food analysis, while low-field NMR (60 MHz) is much less studied. This study aims to establish the effectiveness of both techniques in identifying binary adulteration in CPRO. Three adulteration scenarios were investigated: a) CPRO and refined rapeseed oil (RRO); b) CPRO and refined sunflower oil (RSO); and c) CPRO and RRO or RSO. A range of classification techniques were trialled as well as partial least squares regression to gauge predictive quantification performance. The 400 MHz NMR achieved classification rates of 100% in the scenarios with a single adulterant, and 93% in the multiple adulterant scenario. The 60 MHz NMR produced lower but still encouraging classification rates (RSO 92%; RRO 85%; both RRO and RSO 87%).

Ovothiol A is the Main Antioxidant in Fish Lens.

Tissue protection from oxidative stress by antioxidants is of vital importance for cellular metabolism. The lens mostly consists of fiber cells lacking nuclei and organelles, having minimal metabolic activity; therefore, the defense of the lens tissue from the oxidative stress strongly relies on metabolites. Protein-free extracts from lenses and gills of freshwater fish, Sander lucioperca and Rutilus rutilus lacustris, were subjected to analysis using high-field 1H NMR spectroscopy and HPLC with optical and high-resolution mass spectrometric detection. It was found that the eye lenses of freshwater fish contain high concentrations of ovothiol A (OSH), i.e., one of the most powerful antioxidants exciting in nature. OSH was identified and quantified in millimolar concentrations. The concentration of OSH in the lens and gills depends on the fish genus and on the season. A possible mechanism of the reactive oxygen species deactivation in fish lenses is discussed. This work is the first to report on the presence of OSH in vertebrates. The presence of ovothiol in the fish tissue implies that it may be a significantly more common antioxidant in freshwater and marine animals than was previously thought.

Non-targeted detection of milk powder adulteration by 1H NMR spectroscopy and conformity index analysis

A non-targeted method for classifying authentic and adulterated skim and nonfat dry milk powders (MP) by solution-state, high-field 1H NMR spectroscopy and conformity index analysis has been developed. Authentic MP samples from the global market and synthetically-adulterated MP samples involving eight adulterants were used in a pre-validation study. Adulteration was detected at the lowest concentrations (≥ 0.005 – 0.05% w/w) for samples containing nitrogen-rich, small molecules (melamine and dicyandiamide). For urea, a milk metabolite, and for sucrose and maltodextrin, the detection thresholds were higher (≥ 0.5% w/w). An NMR peak associated with ammonium sulfate was not observed; however, adulteration was indirectly detected at 5% w/w via matrix effects on milk metabolites. Adulteration by soy protein isolate and whey protein concentrate was not detected even at 5% w/w of spiking, which was attributed in part to poor protein solubility. These results provide a successful proof of concept that 1H NMR combined with conformity index analysis can support MP authentication and detect adulteration.

Structure determination of a partially ordered layered silicate material with an NMR crystallography approach.

Structure determination of layered materials can present challenges for conventional diffraction methods due to the fact that such materials often lack full three-dimensional periodicity since adjacent layers may not stack in an orderly and regular fashion. In such cases, NMR crystallography strategies involving a combination of solid-state NMR spectroscopy, powder X-ray diffraction, and computational chemistry methods can often reveal structural details that cannot be acquired from diffraction alone. We present here the structure determination of a surfactant-templated layered silicate material that lacks full three-dimensional crystallinity using such an NMR crystallography approach. Through a combination of powder X-ray diffraction and advanced 29Si solid-state NMR spectroscopy, it is revealed that the structure of the silicate layer of this layered silicate material templated with cetyltrimethylammonium surfactant cations is isostructural with the silicate layer of a previously reported material referred to as ilerite, octosilicate, or RUB-18. High-field 1H NMR spectroscopy reveals differences between the materials in terms of the ordering of silanol groups on the surfaces of the layers, as well as the contents of the inter-layer space.

637 A NMR METABONOMIC APPROACH TO EXPLORE EARLY BIOMARKERS OF HEPATOCELLULAR CARCINOMA IN THE EUROPEAN PROSPECTIVE INVESTIGATION INTO CANCER AND NUTRITION (EPIC)

Primary liver cancer (PLC; ranked sixth in incidence and third in mortality worldwide) is a cancer grouping composed of hepatocellular (HCC) and intrahepatic bile duct carcinomas. HCC make up the bulk of PLC and are highly malignant, usually diagnosed at late stages and often have very poor prognosis with limited treatment options. Thus, there is a need to identify early diagnostic biomarkers of HCC risk. Also, it is plausible that metabolic imbalances, reflective of obesity, diabetes, and lifestyle habits play a central role in HCC etiology. Thus, a NMR metabonomic study was undertaken in a case-control study nested within EPIC, a large prospective cohort of over 520,000 subjects from 23 centers in 10 Western European countries. Detailed dietary/lifestyle data and biological samples (stored under liquid nitrogen) were collected at enrolment from the majority of participants. After an average of 7.6 years of follow-up, 112 first primary incident HCC cases were identified and matched to control subjects (1:2; by age, sex, study centre). Serum samples from matched cases and controls were analyzed by high-field 1H NMR spectroscopy with the following objectives: (i) to identify early predictive biomarkers of HCC by following a metabonomic approach and (ii) to identify metabolomic profiles representative of distinct dietary or lifestyle patterns. Although the protocols of the EPIC study were not specifically designed with metabolomic analyses in mind, extensive pilot testing has shown that EPIC serum samples are suitable for this type of analysis. High-resolution one-dimensional 1 H NMR (NOESY and CPMG) spectral profiles were recorded at 800MHz. Additional 2D 1H-1H (Jres, TOCSY) and 1H-13C (HSQC) data were recorded for metabolite assignments. We present here the first results of the metabonomic analysis of the EPIC HCC nested case-control study that enabled the identification of a metabolomic profile discriminating cases from controls based on serum collected prior to diagnosis. The potential of this study to identify both pre-diagnostic biomarkers as well as those related to exposures associated with disease risk, may have relevant public health impact and enhance understanding of HCC cancer aetiology, particularly in HCC cases arising in the absence of hepatitis B or C infections.

Synthesis of Planar Chiral Iridacycles by Cationic Metal π-Coordination: Facial Selectivity, and Conformational and Stereochemical Consequences

Facial selectivity during the π-coordination of pseudo-tetrahedral iridacycles by neutral (Cr(CO)(3)), monocationic (Cp*Ru(+)), and biscationic (Cp*Ir(2+)) metal centers was directly influenced by the coulombic imbalance in the coordination sphere of the chelated Ir center. We also showed by using theoretical calculations that the feasibility of the related metallacycles that displayed metallocenic planar chirality was dependent to the presence of an electron-donating group, such as NMe(2), which contributed to the overall stability of the complexes. When the π-bonded moiety was the strongly electron-withdrawing Cp*Ir(2+) group, the electron donation from NMe(2) resulted in major conformational changes, with a barrier to rotation of about 17 kcal mol(-1) for this group that became spectroscopically diastereotopic (high-field (1)H NMR spectroscopy). This peculiar property is proposed as a means to introduce a new type of constitutional chirality at the nitrogen center: planar chirality at tertiary aromatic amines.

19F NMR spectroscopy study of the metabolites of flucloxacillin in rat urine.

19F NMR spectroscopy was used to monitor the metabolites of flucloxacillin in the urine of a rat dosed with its sodium salt. 19F NMR signals were detected and quantified from flucloxacillin and three metabolites. The 19F NMR method involves minimal sample preparation and is free from interference by endogenous urine components. High-field spin-echo 1H NMR spectroscopy and HPLC were used to confirm the results.

ChemInform Abstract: Electrochemical Property and Formation of the Cation Radical and Dication of Dinaphtho(1,8-b,c)-1,5-dithiocin.

A new symmetrical dithio peri-bridged naphthalene, dinaphtho[l,8-b,c]-1,5-dithiocin (1) has been synthesized. The cyclic voltammogram of 1 showed one reversible oxidation peak. ESR spectrum of the coned H2SO4 solution of 1 showed the formation of the cation radical. The formation of the dication of 1 was observed in the reaction of the corresponding monosulfoxide with concd D2SO4 by both high-field 1H and 13C NMR spectroscopy.

ChemInform Abstract: Structures of α- and β-Turmerone

Procedures are described for isolating turmerones from Curcuma longa rhizomes and for chromatographically separating α- from β-turmerone. Using a combination of spectroscopic techniques (especially high field 1H NMR spectroscopy and chiroptical methods) these compounds are shown to be (1′R, 6S)-2-methyl-6-(4-methylcyclohexa-2,4-dienyl)hept-2-en-4-one 2(α-turmerone) and (1′R, 6S)-2-methyl-6-(4-methylenecyclohex-2-enyl)hept-2-en-4-one 3(β-turmerone).

Analysis of pharmaceutical heparins and potential contaminants using 1H-NMR and PAGE

In 2008, heparin (active pharmaceutical ingredient, API) lots were associated with anaphylactoid-type reactions. Oversulfated chondroitin sulfate (OSCS), a semi-synthetic glycosaminoglycan (GAG), was identified as a contaminant and dermatan sulfate (DS) as an impurity. While DS has no known toxicity, OSCS was toxic leading to patient deaths. Heparins, prepared before these adverse reactions, needed to be screened for impurities and contaminants. Heparins were analyzed using high-field 1H-NMR spectroscopy. Heparinoids were mixed with a pure heparin and analyzed by 1H-NMR to assess the utility of 1H-NMR for screening heparin adulterants. Sensitivity of heparinoids to deaminative cleavage, a method widely used to depolymerize heparin, was evaluated with polyacrylamide gel electrophoresis to detect impurities and contaminants, giving limits of detection (LOD) ranging from 0.1% to 5%. Most pharmaceutical heparins prepared between 1941 and 2008 showed no impurities or contaminants. Some contained DS, CS, and sodium acetate impurities. Heparin prepared in 2008 contained OSCS contaminant. Heparin adulterated with heparinoids showed additional peaks in their high-field 1H-NMR spectra, clearly supporting NMR for monitoring of heparin API with an LOD of 0.5–10%. Most of these heparinoids were stable to nitrous acid treatment suggesting its utility for evaluating impurities and contaminants in heparin API. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4017–4026, 2009

The Mechanism of Galactosamine Toxicity Revisited; A Metabonomic Study

1H NMR spectroscopy was used to investigate the metabolic effects of the hepatotoxin galactosamine (galN) and the mechanism by which glycine protects against such toxicity. Rats were acclimatized to a 0 or 5% glycine diet for 6 days and subsequently administered vehicle, galN (500 mg/kg), glycine (5% via the diet), or both galN and glycine. Urine was collected over 12 days prior to administration of galN and for 24 hours thereafter. Serum and liver tissue were sampled on termination, 24 hours post-dosing. The metabolic profiles of biofluids and tissues were determined using high-field 1H NMR spectroscopy. Orthogonal-projection to latent structures discriminant analysis (O-PLS-DA) was applied to model the spectral data and enabled the hepatic, urinary, and serum metabolites that discriminated between control and treated animals to be determined. Histopathological data and clinical chemistry measurements confirmed the protective effect of glycine. The level of N-acetylglucosamine (glcNAc) in the post-dose urine was found to correlate strongly with the degree of galN-induced liver damage, and the urinary level of glcNAc was not significantly elevated in rats treated with both galN and glycine. Treatment with glycine alone was found to significantly increase hepatic levels of uridine, UDP-glucose, and UDP-galactose, and in view of the known effects of galactosamine, this suggests that the protective role of glycine against galN toxicity might be mediated by changes in the uridine nucleotide pool rather than by preventing Kupffer cell activation. Thus, we present a novel hypothesis: that administration of glycine increases the hepatic uridine nucleotide pool which counteracts the galN-induced depletion of these pools and facilitates complete metabolism of galN. These novel data highlight the applicability of NMR-based metabonomics in elucidating multicompartmental metabolic consequences of toxicity and toxic salvage.

Effects of p‐hydroxybenzoic acid monomer units on physical properties of thermal liquid crystalline poly(ester imide ketone)s

AbstractA series of poly(ester imide ketone)s (PEIKs) with varied p‐hydroxybenzoic acid (HBA) molar fraction derived from N,N′‐hexane‐1,6‐diylbis(trimellitimide), 4,4′‐dihydroxybenzophenone, and p‐hydroxybenzoic acid were synthesized by a “step‐feeding” polycondensation method in benzene sulfonyl chloride, dimethylformamide, and pyridine. High field 1H, 13C, and 2D NMR spectroscopy measurements were combined to determine the assignments of hydrogen and carbon atoms in the copolymers. 13C inverse gated decoupling NMR spectra were taken and used as a quantitative method to analyze the chain sequence structures of these copolymers. The liquid crystalline behaviors and thermal properties of the PEIKs were characterized by polarized light microscopy (PLM), wide‐angle X‐ray diffraction (WAXD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The NMR studies show that the monomeric unit ratios of the PEIKs are very close to the ratios of the monomers added into the polycondensation process. The analytical results of sequence distribution indicate that the sequence ratios of I‐H, H‐D, and H‐H dias rise with the increase of HBA molar fraction, while that of I‐D decrease. It is worth noting that the sequence ratios of H‐H are always very small among the four sequence ratios although the HBA molar fraction varied from 0 to 50%. It was shown that the copolymers possess a typical nematic thermotropic liquid crystalline character and high thermal stability, which is strongly related with the changes in sequence structure of the molecular chains. This type of liquid crystalline polymers also exhibits excellent fiber‐forming character in the melting state, which would find its potential usages in high performance fiber and fiber modification materials. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 3183–3193, 2007

Flame-Retardancy Improvement of Novel Styrene–Maleic Anhydride Based Copolymers

Brominated flame-retardants are one of the common and efficient compounds in producing flame retardant plastics and antimony trioxide is often used as a synergistic agent for halogenated flame-retardants. In this research, a new reactive flame retardant, maleated tetrabromobiphenol-A (MTBBA) crystals, has been introduced for the first time and its structure was fully characterized by high-field 1H NMR and 13C NMR spectroscopy. The improvement in flame-retardancy effect of styrene–maleic anhydride copolymer by MTBBA and also their compounded samples with Sb2O3 was investigated as the main purpose in this work. Limiting oxygen indexes (LOI) for the neat terpolymers and the compounded samples were measured by flammability tests. Also thermal gravimetry analysis (TGA) and differential scanning calorimetry (DSC) were applied in order to study thermal behavior of the samples. The results of thermal analysis were used to observe the effect of insertion of this novel monomer into the copolymer chain in comparison with styrene–maleic anhydride copolymer.

Synthesis and NMR Characterization of New Hyaluronan-Based NO Donors

Nitric oxide (NO) and hyaluronic acid (HA), two species widely different in terms of molecular complexity and biological competence, are both known to play an important role in the wound healing process. To combine the properties of HA and NO, we synthesized new NO-donors based on hyaluronic acid derivatives exhibiting a controlled NO-release under physiological conditions (in vitro tests). Since two molecules of NO can form a covalent bond with secondary amines to yield structures, named NONO-ates, able to release NO in solution, we used spermidine bound to HA as the NO-linker. The HA-spermidine derivative was obtained by controlled HA amidation in aqueous media, activating the biopolymer carboxylate groups with a water soluble carbodiimide. The resulting derivative, soluble in water, was fully characterized by high field 1H and 13C NMR spectroscopy. The amount of grafting of spermidine on HA was determined by integration of suitable 1H NMR signals. In addition, cross-linked derivatives of HA were synthesized by the Ugi's four-component reaction using formaldehyde, cyclohexylisocyanide, and spermidine. The HA-spermidine networks were characterized by 13C CP-MAS NMR spectroscopy. The degree of cross-linking of the networks was also determined. Finally, the release of NO from the swollen hydrogels freshly saturated with NO, in contact with aqueous media, was monitored by means of UV spectrophotometric measurements.

Characterization of oligosaccharides from the chondroitin/dermatan sulfates

Chondroitin and dermatan sulfate (CS and DS) chains were isolated from bovine tracheal cartilage and pig intestinal mucosal preparations and fragmented by enzymatic methods. The oligosaccharides studied include a disaccharide and hexasaccharides from chondroitin ABC lyase digestion as well as trisaccharides already present in some commercial preparations. In addition, other trisaccharides were generated from tetrasaccharides by chemical removal of nonreducing terminal residues. Their structures were examined by high-field 1H and 13C NMR spectroscopy, after reduction using sodium borohydride. The main hexasaccharide isolated from pig intestinal mucosal DS was found to be fully 4-O-sulfated and have the structure: DeltaUA(beta1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S-ol, whereas one from bovine tracheal cartilage CS comprised only 6-O-sulfated residues and had the structure: DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. No oligosaccharide showed any uronic acid 2-sulfation. One novel disaccharide was examined and found to have the structure: GalNAc6S(beta1-4)GlcA-ol. The trisaccharides isolated from the CS/DS chains were found to have the structures: DeltaUA(beta1-3)GalNAc4S(beta1-4)GlcA-ol and DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA-ol. Such oligosaccharides were found in commercial CS/DS preparations and may derive from endogenous glucuronidase and other enzymatic activity. Chemically generated trisaccharides were confirmed as models of the CS/DS chain caps and included: GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc4S-ol and GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. The full assignment of all signals in the NMR spectra are given, and these data permit the further characterization of CS/DS chains and their nonreducing capping structures.

Evaluation of the speciation status of aluminium(III) ions in isolated osteoarthritic knee-joint synovial fluid

High field 1H NMR spectroscopy demonstrated that the equilibration of added Al(III) ions in osteoarthritic (OA) knee-joint synovial fluid (SF) resulted in its complexation by citrate and, to a much lesser extent, tyrosine and histidine. The ability of these ligands, together with inorganic phosphate, to compete for the available Al(III) in terms of (1) thermodynamic equilibrium constants for the formation of their complexes and (2) their SF concentrations was probed through the use of computer speciation calculations, which considered low-molecular-mass binary and ternary Al(III) species, the predominant Al(III) plasma transport protein transferrin, and also relevant hydrolysis and precipitation processes. It was found that, at relatively low added Al(III) concentrations, citrate species were more favoured, whilst phosphate species became dominant at higher levels. The significance of these findings with regard to the in vivo corrosion of aluminium-containing metal alloy joint prostheses (e.g., TiAlV alloys) is discussed.

Examination of the molecular nature of low-molecular-mass chromium(III) ions in isolated osteoarthritic knee-joint synovial fluid

High field 1H NMR spectroscopy demonstrated that equilibration of added Cr(III) ions in osteoarthritic knee-joint synovial fluid (SF) resulted in its complexation by a range of biomolecules, the relative efficacies of these complexants/chelators being threonine ∼ alanine > glycine > glutamine > citrate > histidine ∼ phenylalanine ∼ tyrosine > valine ∼ isoleucine ∼ leucine > glutamate > lactate ∼ acetate ∼ formate ∼ pyruvate, this order reflecting the ability of these ligands to compete for the available Cr(III) in terms of (1) thermodynamic equilibrium constants for the formation of their complexes and (2) their SF concentrations. The significance of these observations with regard to the in vivo corrosion of chromium-containing metal alloy joint prostheses (e.g., CoCr alloys) is discussed.

Stereochemistry of the Macrocyclization and Elimination Steps in Taxadiene Biosynthesis through Deuterium Labeling

Taxadiene synthase catalyzes the cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP) to taxa-4(5),11(12)-diene (Scheme 1, 5 --> 2) as the first committed step of Taxol biosynthesis. Deuterated GGPPs labeled stereospecifically at C-1, C-4, and C-16 were synthesized and incubated with recombinant taxadiene synthase from Taxus brevifolia to elucidate the stereochemistry of the cyclization reaction at these positions. The deuterium-labeled taxadienes obtained from (R)-[1-(2H1)]-, (S)-[1-(2H1)]-, and [16,16,16-(2H3)]GGPPs (9, 10, and 23b) were established to have deuterium in the 2alpha and 2beta CH2 and 16CH3 positions, respectively, by high-field 1H NMR spectroscopy (eqs 1-3). Incubation of (R)-[4-(2H1)]GGPP (17) with the recombinant enzyme gave a 10:10:80 mixture of [5beta-(2H1)]taxa-3(4),11(12)-diene, [5beta-(2H1)]taxa-4(20),11(12)-diene, and unlabeled taxa-4(5),11(12)-diene according to GC/MS analyses of the products (eq 4). It follows that C-1 of GGPP underwent inversion of configuration, that the A ring cyclization occurs on the si face of C15, and that the terminating proton abstraction removes H5beta from the final taxenyl carbocation intermediate. Thus, the C1-C14 and C15-C10 bonds are formed on the opposite faces of the 14,15 double bond of the substrate, i.e., overall anti electrophilic addition. The implications of these findings for the mechanism of the cyclization and rearrangement are discussed.

Qualitative high field 1H-NMR spectroscopy for the characterization of endogenous metabolites in earthworms with biochemical biomarker potential

This study was designed to provide a database of the endogenous metabolites in earthworm extracts of the species, Eisenia veneta and Lumbricus terrestris by high resolution 1H-NMR spectroscopy in view of identifying biomarkers of toxicity or stress in environmental metabolomics studies. 1D and 2D NMR spectroscopic techniques enabled the identification and confirmation of the organic components in the tissue extracts of whole and segmented earthworms, dissected organs, and coelomic fluid. The extracts gave rise to characteristic 1H-NMR spectral fingerprints of the low MW metabolites contained, specific to the species of earthworm, and to the specific regions or dissected organs of the earthworms under investigation. Distinct changes in the normal biochemistry were observed upon starvation and cooling, such as markedly decreased glucose and maltose, but increased lactate, acetate, succinate, formate and acetone. Additionally, slightly decreased threonine, arginine, lysine, leucine, citrate, asparagine and glycine were observed. Furthermore, lactate could be identified as a biomarker of acute toxic stress in expressed coelomic fluid following exposure to a model ecotoxin (3-trifluoromethylaniline). This work supports the application of 1H-NMR spectroscopy for the study of changes in the normal invertebrate biochemistry in order to allow for the reliable assessment of biomarker responses following toxicity testing.

High-field localized 1H NMR spectroscopy in the anesthetized and in the awake monkey

Localized cerebral in vivo 1H NMR spectroscopy (MRS) was performed in the anesthetized as well as the awake monkey using a novel vertical 7 T/60 cm MR system. The increased sensitivity and spectral dispersion gained at high field enabled the quantification of up to 16 metabolites in 0.1- to 1-ml volumes. Quantification was accomplished by using simulations of 18 metabolite spectra and a macromolecule (MM) background spectrum consisting of 12 components. Major cerebral metabolites (concentrations >3 mM) such as glutamate (Glu), N-acetylaspartate (NAA), creatine (Cr)/phosphocreatine (PCr) and myo-inositol (Ins) were identified with an error below 3%; most other metabolites were quantified with errors in the order of 10%. Metabolite ratios were 1.39:1 for total NAA, 1.38:1 for glutamate (Glu)/glutamine (Gln) and 0.09:1 for cholines (Cho) relative to total Cr. Taurine (Tau) was detectable at concentrations lower than 1 mM, while lactate (Lac) remained below the detection limit. The spectral dispersion was sufficient to separate metabolites of similar spectral patterns, such as Gln and Glu, N-acetylaspartylglutamate (NAAG) and NAA, and PCr-Cr. MRS in the awake monkey required the development and refinement of acquisition and correction strategies to minimize magnetic susceptibility artifacts induced by respiration and movement of the mouth or body. Periods with major motion artifacts were rejected, while a frequency/phase correction was performed on the remaining single spectra before averaging. In resting periods, both spectral amplitude and line width, that is, the voxel shim, were unaffected permitting reliable measurements. The corrected spectra obtained from the awake monkey afforded the reliable detection of 6-10 cerebral metabolites of 1-ml volumes.