Analysis of human urine metabolites using SPE and NMR spectroscopy

Nuclear magnetic resonance (NMR) spectroscopic analysis of metabonome/metabolome has widespread applications in biomedical science researches. However, most of NMR resonances for urinary metabolites remain to be fully assigned. In the present study, human urine samples from two healthy volunteers were pre-treated with C18 solid-phase extraction and the resultant 5 sub-fractions were subjected to one- and two-dimensional NMR studies, including 1H J-Resolved, 1H-1H COSY, 1H-1H TOCSY, 1H-13C HSQC, and HMBC 2D NMR. More than 70 low molecular weight metabolites were identified, and complete assignments of 1H and 13C resonances including many complex coupled spin systems were obtained.     

Simultaneous quantification and identification of individual chemicals in metabolite mixtures by two-dimensional extrapolated time-zero (1)H-(13)C HSQC (HSQC(0))

Quantitative one-dimensional (1D) (1)H NMR spectroscopy is a useful tool for determining metabolite concentrations because of the direct proportionality of signal intensity to the quantity of analyte. However, severe signal overlap in 1D (1)H NMR spectra of complex metabolite mixtures hinders accurate quantification. Extension of 1D (1)H to 2D (1)H-(13)C HSQC leads to the dispersion of peaks along the (13)C dimension and greatly alleviates peak overlapping. Although peaks are better resolved in 2D (1)H-(13)C HSQC than in 1D (1)H NMR spectra, the simple proportionality of cross peaks to the quantity of individual metabolites is lost by resonance-specific signal attenuation during the coherence transfer periods. As a result, peaks for individual metabolites usually are quantified by reference to calibration data collected from samples of known concentration. We show here that data from a series of HSQC spectra acquired with incremented repetition times (the time between the end of the first (1)H excitation pulse to the beginning of data acquisition) can be extrapolated back to zero time to yield a time-zero 2D (1)H-(13)C HSQC spectrum (HSQC(0)) in which signal intensities are proportional to concentrations of individual metabolites. Relative concentrations determined from cross peak intensities can be converted to absolute concentrations by reference to an internal standard of known concentration. Clustering of the HSQC(0) cross peaks by their normalized intensities identifies those corresponding to metabolites present at a given concentration, and this information can assist in assigning these peaks to specific compounds. The concentration measurement for an individual metabolite can be improved by averaging the intensities of multiple, nonoverlapping cross peaks assigned to that metabolite.     

Nuclear magnetic resonance studies of cytochalasin E and its decomposition product

The proton and carbon-13 nuclear magnetic resonance signals of cytochalasin E (1) were assigned with the aid of 1H-1H, 1H-13C and 1H-13C long-range chemical shift correlation spectroscopy spectra, and the structure of the decomposition product (2) generated under neutral conditions was determined.     

六氟磷酸二(2,2'-联吡啶)·(4,4'-二甲基-2,2'-联吡啶) 合钌(Ⅱ)的二维核磁共振谱分析

用一维NMR方法研究了电化学发光物质六氟磷酸二(2,2'-联吡啶)·(4,4'-二甲基-2,2'-联吡啶)合钌(Ⅱ)的立体结构,借助二维1H-1H COSY和1H-13C COSY实验技术对其氢谱和碳谱进行了完全的归属,并给出了其氢谱和碳谱的化学位移值.     

六氟磷酸二(4,4'-二甲基-2,2'-联吡啶)·(2,2'-联吡啶-4,4'-二羧酸)合钌(Ⅱ)的二维核磁共振

用1H NMR和13C NMR谱研究了新型电化学发光探针六氟磷酸二(4,4'-二甲基-2,2'-联吡啶)·(4,4'-二羧酸-2,2'-联吡啶)合钌(Ⅱ)的立体结构,通过1H-1H COSY、13C-1H HETCOR谱对其氢谱和碳谱中的各谱峰进行了归属,并给出了氢谱和碳谱峰的化学位移值.     

Analysis of hydrophilic metabolites by high-performance liquid chromatography-mass spectrometry using a silica hydride-based stationary phase

A novel silica hydride-based stationary phase was used to evaluate the retention behavior in the aqueous normal-phase (ANP) mode of standards representing three classes of metabolites. The effects on retention behavior of amino acids, carbohydrates and small organic acids were examined by altering the column temperature, and by adding different additives to both the mobile phase and sample solvent. Gradient mode results revealed the repeatability of retention times to be very stable for these compound classes. At both 15 and 30 degrees C, excellent RSD values were obtained with less than 1% variation for over 50 injections of an amino acid mixture. The ability to separate the 19 nonderivatized amino acid standards, organic acids and carbohydrates was demonstrated as well as the potential for this material to separate polar metabolites in complex fluids such as urine.     

苯并咪唑萘酰亚胺类化合物的合成与结构确证

本文合成并分离提纯了1位和4位单取代的苯并咪唑萘酰亚胺(NBI)类化合物1～4,对化合物1的紫外-可见光谱、红外光谱、质谱、核磁共振谱(包括1H NMR、13C NMR、DEPT、1H-13C HSQC、1H-13C HMBC、1H-1HCOSY多种NMR技术)进行了详细解析,对其所有的1H和13C NMR信号进行了归属,并使用了GIAO和CSGT两种方法量化计算核磁位移,通过多种波谱学技术确证了化合物的结构。     

Assignments of 1H and 13C NMR spectral data for ondansetron and its two novel metabolites, 1-hydroxy-ondansetron diastereoisomers

Assignments of 1H and 13C NMR chemical shifts were made by means of heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments for ondansetron, and by means of 1H-1H correlation spectroscopy (1H-1H COSY) and two-dimensional nuclear Overhauser effect spectroscopy (NOESY) experiments for two novel metabolites (M1 and M2) of ondansetron. These two metabolites were isolated for the first time from Mucor circinelloides.     

Characterisation of uniformly 13C, 15N labelled bacteriochlorophyll a and bacteriopheophytin a in solution and in solid state: complete assignment of the 13C, 1H and 15N chemical shifts

In this investigation we report a complete assignment of (13)C, (1)H and (15)N solution and solid state chemical shifts of two bacterial photosynthetic pigments, bacteriochlorophyll (BChl) a and bacteriopheophytin (BPheo) a. Uniform stable-isotope labelling strategies were developed and applied to biosynthetic preparation of photosynthetic pigments, namely uniformly (13)C, (15)N labelled BChl a and BPheo a. Uniform stable-isotope labelling with (13)C, (15)N allowed performing the assignment of the (13)C, (15)N and (1)H resonances. The photosynthetic pigments were isolated from the biomass of photosynthetic bacteria Rhodopseudomonas palustris 17001 grown in uniformly (13)C (99%) and (15)N (98%) enriched medium. Both pigments were characterised by NMR in solution (acetone-d(6)) and by MAS NMR in solid state and their NMR resonances were recorded and assigned through standard liquid 2D (13)C-(13)C COSY, (1)H-(13)C HMQC, (1)H-(15)N HMBC and solid 2D (13)C-(13)C RFDR, (1)H-(13)C FSLG HETCOR and (1)H-(15)N HETCOR correlation techniques at 600 MHz and 750 MHz. The characterisation of pigments is of interest from biochemical to pharmaceutical industries, photosynthesis and food research.     

2,2'-二(对胺苯甲酯)-1,1'联萘的核磁共振谱解析

利用~1HNMR,~(13)CNMR谱研究了2,2’-二(对胺苯甲酯)-1,1’联萘的结构,并通过~1H-~1HCOSY,~(13)C-~1H异核相关及~(13)C-~1H异核远程相关谱进一步地确定了~1H谱和~(13)C谱中各谱峰的归属,为同类化合物的表征提供了一个依据.     

NMR metabolic profiling of organic and aqueous sea bass extracts: implications in the discrimination of wild and cultured sea bass

The nuclear magnetic resonance (NMR) technique was used as analytical tool to determine the complete metabolic profiling of sea bass extracts: water-soluble metabolites belonging to different classes such as sugars, amino acids, dipeptides and organic acids as well as metabolites soluble in organic solvent such as lipids, sterols and fatty acids were identified. The metabolite profiling together with a suitable statistical analysis were used to discriminate between wild and cultured sea bass samples. Preliminary results show that discrimination between wild and cultured sea bass was obtained not only using fatty acid composition but also cholesterol and phosphatidylethanolamine and some water-soluble metabolites such as choline, trimethylamine oxide, glutamine, fumaric and malic acids.     

去羧氯雷他定的波谱学数据与结构确证

对去羧氯雷他定的红外（IR）、质谱（MS）、氢-氢相关谱(^1H-HCOSY)、碳谱（BB、DEPT-90、DEPT-135）、碳氢相关谱（HMQC）、碳氢远程相关谱（HMBC）解析并进行了报道。对所有的^1HNMR、^13CNMR谱的信号进行了归属，同时讨论了质谱的主要碎片离子的可能的裂解方式和红外特征吸收峰所对应的官能团的振动形式。     

咪喹莫特的波谱学数据与结构表征

对咪喹莫特的红外(IR)、紫外(UV)、质谱(MS)、氢-氢相关谱(^1H--^1H COSY)、碳氢相关谱(HMQC)、碳氢远程相关谱(HMBC)予以解析并进行了报道。对所有的^1H NMR、^13C NMR谱的信号进行了归属；讨论了质谱的主要碎片离子的可能的裂解方式和红外特征吸收峰所对应的官能团的振动形式。     

含穴醚N8O3配体的金属大环化合物的NMR研究

在溶液中,室温下对含有穴醚Ｎ８Ｏ３配体（Ｈ３Ｌ）的金属大环化合物［Ｎａ（Ｈ３Ｌ）］（ＣｌＯ４）３Ｈ２Ｏ（２）和［Ｌａ（Ｈ３Ｌ）（ＯＨ）２］（ＣｌＯ４）３（３）,作了１ＨＮＭＲ谱的测定,在化合物（２）－乙腈溶液中加入过量Ｌａ（ＣｌＯ４）３后的（２＋Ｌａ）和化合物（３）还作了１３Ｃ和１Ｈ－１ＨＣＯＳＹ（氢－氢相关二维谱）、１Ｈ－１３ＣＨＭＱＣ（氢核检测的异核多量子相干相关谱）及１Ｈ－１３ＣＨＭＢＣ（氢核检测的异核多键相关谱）的ＮＭＲ谱测定,归属了所有１Ｈ,１３Ｃ的谱线。对其配位行为通过１ＨＮＭＲ试验作了简单的讨论。     

核磁共振方法表征2,2''''-二(对羧酸苯氧基)-1,1''''联萘

利用１Ｈ ＮＭＲ，１３Ｃ ＮＭＲ研究了 ２，２’－二（对羧酸苯氧基）－１，１’－联萘结构，并通过１Ｈ－１Ｈ ＣＯＳＹ及１３Ｃ－１Ｈ异核相关谱进一步确定了其１Ｈ谱和１３Ｃ谱中各谱峰的归属，为同类化合物的表征提供了一个依据。     

基于~1H-NMR、~(31)P-NMR的三苯基膦三间磺酸钠定量分析研究

该文建立了三苯基膦三间磺酸钠(TPPTS)的核磁氢谱(~1H-NMR)、核磁磷谱(~(31)P-NMR)、定量分析方法。通过~1H-NMR、~(13)C-NMR、~(31)P-NMR并结合~1H-~1H COSY、~1H-~(13)C、~1H-~(13)C HMBC对TPPTS的氢原子、碳原子以及磷原子的化学位移进行归属;氢谱定量选用三■烷作内标、氘代N,N-二甲基甲酰胺(DMF-D_7)作溶剂,磷谱定量选用KH_2PO_4作内标、氘代水(D_2O-D_2)作溶剂,通过对混合体系中各自旋核纵向弛豫时间(T1)的测定,为弛豫延迟时间(D1)的合理设置提供依据。以上两种方法对体系中TPPTS的定量分析结果分别为(58.72±0.21)%、(58.51±0.21)%,测定结果一致性高、平行性好,且检测过程无需待测组分的标准品,能实现TPPTS含量的快速准确测定。     

2D-NMR归属1,1,1-Kestopentaose的^1H和^1^3C共振谱线

１,１,１－Ｋｅｓｔｏｐｅｎｔａｏｓｅ是菊粉（Ｉｎｕｌｉｎ）类中的一个葡果糖化合物（β－Ｄ－果糖－（２→１）－β－Ｄ－果糖（２→１）－β－Ｄ－果糖－（２→１）－α→Ｄ－葡萄糖）本文采用各种２Ｄ－ＮＭＲ技术,包括Ｈ－ＨＣＯＳＹＦ１－去偶Ｈ－ＨＣＯＳＹ ＨＭＱＣ和ＨＭＢＣ等,归属了它的Ｈ和Ｃ的谱线,并得到了有关质子间的偶合常数,为进一步分析其在溶液中的三维空间结构打下了基础。     

Ultrahigh-resolution (1)H-(13)C HSQC spectra of metabolite mixtures using nonlinear sampling and forward maximum entropy reconstruction

To obtain a comprehensive assessment of metabolite levels from extracts of leukocytes, we have recorded ultrahigh-resolution 1H-13C HSQC NMR spectra of cell extracts, which exhibit spectral signatures of numerous small molecules. However, conventional acquisition of such spectra is time-consuming and hampers measurements on multiple samples, which would be needed for statistical analysis of metabolite concentrations. Here we show that the measurement time can be dramatically reduced without loss of spectral quality when using nonlinear sampling (NLS) and a new high-fidelity forward maximum-entropy (FM) reconstruction algorithm. This FM reconstruction conserves all measured time-domain data points and guesses the missing data points by an iterative process. This consists of discrete Fourier transformation of the sparse time-domain data set, computation of the spectral entropy, determination of a multidimensional entropy gradient, and calculation of new values for the missing time-domain data points with a conjugate gradient approach. Since this procedure does not alter measured data points, it reproduces signal intensities with high fidelity and does not suffer from a dynamic range problem. As an example we measured a natural abundance 1H-13C HSQC spectrum of metabolites from granulocyte cell extracts. We show that a high-resolution 1H-13C HSQC spectrum with 4k complex increments recorded linearly within 3.7 days can be reconstructed from one-seventh of the increments with nearly identical spectral appearance, indistinguishable signal intensities, and comparable or even lower root-mean-square (rms) and peak noise patterns measured in signal-free areas. Thus, this approach allows recording of ultrahigh resolution 1H-13C HSQC spectra in a fraction of the time needed for recording linearly sampled spectra.     

1H and 13C spectral assignment of substituted 5H-[1,3]thiazolo[2,3-b]quinazolin-5-one and 12H-[1,3]benzothiazolo[2,3-b] quinazolin-12-one

(1)H and (13)C spectroscopic data for 5H-[1,3]thiazolo[2,3-b]quinazolin-5-one and 12H-[1,3]benzothiazolo[2,3-b]quinazolin-12-one derivatives were fully assigned by combination of one- and two-dimensional experiments (DEPT, HMBC and HMQC). Both heterocyclic systems show similar spectroscopic properties with some remarkable differences.     

Novel steroids from the seeds of Centratherum anthelminticum

Two novel and two known steroids were isolated respectively from benzene: acetone and ethanolic extracts of the seeds of C. anthelminticum and, identified as (24alpha/R)-Stigmasta-7-en-3-one (1), (24alpha/R)-Stigmasta-7, 9(11)-dien-3-one (2), (24alpha/S)-Stigmasta-5, 22-dien-3beta-ol (3) and (24alpha/S)-Stigmasta-7, 22-dien-3beta-ol (4). The structures were assigned on the basis of their IR, 1H NMR, 13C NMR (DEPT), 2D NMR (1H-1H COSY and 1H-13C COSY), mass spectra, chemical analyses and on comparison with literature values. The novel steroids (1) and (2) were also tested against several human pathogenic bacteria and fungi.