Rapid peptide mapping by reversed-phase liquid chromatography on nonporous silica with on-line electrospray time-of-flight mass spectrometry

Reversed-phase liquid chromatography (LC) using a nonporous silica support has been combined with electrospray (ES) time-of-flight (TOF) mass spectrometry (MS) for the fast separation and mass detection of peptides. Using this LC method, the resolution of a peptide mixture can be completed is less than 35 s. The resulting chromatographic peak widths are less than 1 s wide. Because of the unique nature of a TOF mass analyzer, complete mass spectra can be acquired at a rate which is sufficient to sample these narrow peaks. When compared with conventional LC, the same separation takes nearly 20 min to complete, and the signal-to-noise ratio observed in the total ion chromatogram is dramatically lower due to the influence of increased background noise in the mass spectra. The limit of detection for a low molecular weight peptide, Val-Pro-Leu, was found to be 6 pmol with the total ion chromatogram and 500 fmol with the reconstructed ion chromatogram. A peptide map of horse heart myoglobin, completed in 3.5 min, is shown as an example of the results which can be obtained from combining this fast LC method with fast ES/TOF/MS detection capability.     

工业芴组份的测定

本文应用CBP^TM-5毛细管柱,对工业芴中各组份含量进行测定。结果表明：应用该色谱分析方法,色谱峰形较好,物质组份分离好,色谱柱的柱效高,分析结果的准确度和灵敏度高,能满足样品分析的要求。     

Haemoglobin O Padova and falsely low haemoglobin A1c in a patient with type I diabetes

Glycated haemoglobin (HbA1c) measured by high performance liquid chromatography (HPLC) in a 20 year old female with insulin dependent diabetes mellitus was consistently within the normal range although her daily blood glucose values were > 11.1 mmol/l. HbA1c measured by immunoagglutination and fructosamine was elevated and correlated with the patient's blood glucose values. The HPLC chromatogram showed an additional peak at HbA0. Electrophoresis of haemoglobin on citrate agar gel revealed an abnormal haemoglobin anodal of HbS. Cellulose acetate electrophoresis and isoelectric focusing demonstrated an additional haemoglobin migrating close to HbA2. Amino acid analysis and DNA sequencing revealed an alpha 30 (B11) Glu-->Lys replacement, that is, haemoglobin O Padova. Investigations of two family members without diabetes revealed the same rare haemoglobin variant. This case showed that this silent haemoglobin mutation caused an additional peak and falsely low HbA1c values when measured by HPLC, the gold standard for this evaluation.     

Reverse shear of shear bands and annealing embrittlement in amorphous alloy

Shear bands produced by bending before annealing could be reversed if isothermally annealed or continuously heated before the onset of embrittlement. The activation energy for the onset of embrittlement is 69–72 kcal/mol similar to that of crystallization, 67–68 kcal/mol, if the latter is evaluated at the same X-ray first peak height. The activation energy of crystallization based on Kissinger plots of DSC data is erroneous, since samples at the onset or peak of crystallization in the thermogram do not have similar X-ray first peak heights. It seems that whatever structural modification is inside the shear bands is stable during annealing until embrittlement and that the embrittlement is related to the incipience of crystallization.     

Analysis of human milk triacylglycerols by high-performance liquid chromatography with light-scattering detection

A high-performance liquid chromatography (HPLC) method for the separation of human milk triacylglycerols using a C18 Spherisorb ODS column and ternary gradient elution with dichloromethane, acetone and acetonitrile is described. The triacylglycerols are detected by light scattering. Several chromatographic conditions were assayed in order to optimize the method: sample solubility, mobile phase, column temperature and the mass detector oven temperature. The linearity, precision and relative response of the method were examined. A total of 34 peaks were separated and quantified based on the percentage peak area in the HPLC chromatogram. Mature human milk analyzed by this method contained six predominant triacylglyceride structures: POO, POL, LOO, POP, OOO and SOP, where P = palmitin, O = olein, L = linolein and S = stearin.     

Representing primary electrophoretic data in the 1/time domain: comparison to representations in the time domain

A plot of absorbance vs 1/time (the "1/time domain") is a more useful representation of the primary data in capillary electrophoresis than traditional plots of absorbance vs time (the "time domain") in a wide set of circumstances, especially when comparing electropherograms in which the rate of electroosmotic flow is not precisely the same. The quantity that is of fundamental interest in capillary electrophoresis (CE) is the electrophoretic mobility of an analyte. The electrophoretic mobility of a species is nonlinearly proportional to time and, therefore, not linearly represented in the time domain: that is, the distance between two peaks along the time axis is not linearly related to the difference in their electrophoretic mobilities. In contrast, the electrophoretic mobility is linearly proportional to 1/time, and the distance between two peaks along the 1/time axis is linearly related to the difference in electrophoretic mobilities. Plots in the 1/time domain are similar to the familiar plots in the time domain (each analyte is represented by a peak, and the order of peaks corresponds to the order in which these analytes reach the detector), but the spacing between the peaks corresponds linearly to differences in mobility. This article derives this useful, visually appealing, and broadly applicable plotting strategy and illustrates common situations in which these plots are more useful than plots in the time domain.     

Adenosine 5'-phosphosulfate (APS) kinase: diagnosing the mechanism of substrate inhibition

Adenosine 5'-phosphosulfate (APS) kinase is subject to strong substrate inhibition by APS. The inhibition has been variously reported to be uncompetitive with respect to MgATP (resulting from the formation of a dead-end E. APS. MgADP complex) or competitive with MgATP (resulting from the formation of a dead-end E. APS complex). It is shown that these two types of substrate inhibition can be differentiated for ordered kinetic mechanisms by simple inspection of the v versus [APS] plots at different fixed concentrations of MgATP. Linear diagnostic plots are unnecessary. One diagnostic feature is the changing position of [APS]opt, the concentration of APS that yields the peak velocity. In the uncompetitive system, [APS]opt decreases asymptotically to a limit as the fixed [MgATP] is increased, while in the competitive system, [APS]opt increases continuously as the fixed [MgATP] is increased. A second (and more easily discerned) diagnostic feature is that, at any given inhibitory level of APS, enzyme activity relative to the velocity at [APS]opt (v/vopt) decreases as the fixed [MgATP] is increased in the uncompetitive system, while in the competitive system the relative activity increases as the fixed [MgATP] is increased. Normalized plots of v/vopt versus [APS] clearly display these distinguishing characteristics. The method confirmed that Penicillium chrysogenum APS kinase exhibits uncompetitive inhibition by APS.     

Reconstruction of the extremely resorbed mandible with interposed bone grafts and placement of endosseous implants. A preliminary report on outcome of treatment and patients'' satisfaction

An analysis method for volatile organic compounds in blood based on purge-and-trap extraction coupled with gas chromatography-Fourier transform infrared spectroscopy (GC-FTIR) was developed. The sample volume was 5 mL, and the internal standard was diethyl ketone. The chromatographic separation was carried out on a PoraPLOT Q capillary column, and the effluent was first directed to the FTIR and then to a flame ionization detector (FID). FTIR identification limits were measured for 27 volatile organic compounds; the criteria for the limit were that the first hit-list position should be obtained against the Sadtler library, which contains 3240 spectra, and that the correlation value should exceed 0.5. It was required that the peak be seen by FID but not necessarily by a Gram-Schmidt chromatogram. The FTIR identification limits, ranging from 0.01 mg/L for ethyl acetate, methylethyl ketone, and sevoflurane to 24 mg/L for methanol, generally allowed the detection of volatile-substance exposure at a lower level than is acutely toxic. Quantitative calibration data were presented for selected substances, based on the FID response, which shows that the method is also amenable to quantitative analysis. The throughput of the method without additional automation is five samples per day, the purge-and-trap stage being the limiting factor.     

Polymorphism and identification of metallothionein isoforms by reversed-phase HPLC with on-line ion-spray mass spectrometric detection

Microbore reversed-phase HPLC with on-line ion-spray mass spectrometric detection is proposed for a study of polymorphism of rabbit liver metallothionein (MTRL) and its major isoforms MT-1 and MT-2. Separation conditions had been optimized until each chromatographic peak could be attributed to a single metalloprotein species, of which the molecular mass could be determined by ionspray MS. At the optimized conditions (elution with the gradient 5-8% of acetonitrile within 50 min using a 5 mM acetate buffer at pH 6), the chromatogram of MTRL showed five peaks, whereas those of MT-1 and MT-2 showed nine and seven different peaks, respectively. The on-line determination of the molecular masses (+/- 1 Da) of the compounds eluted permits the unambiguous cataloguing and further referencing of putative and true MT subisoforms. The results obtained are compared with those obtained by direct ion-spray MS of the MT preparations at different pHs, with a goal to identify possible chromatographic artifacts. Metals (Cd, Cu) in the eluted complexes were studied by HPLC with on-line ICP MS detection.     

Autologous hematopoietic stem cell transplantation in chronic myeloid leukemia

A circuit that simulates T-type calcium-channel current characteristics of the sinoatrial (SA) node was developed from discrete electronic components and tested at physiologic membrane voltage ranges. The circuit design was based on the T-type calcium-channel current dynamics obtained from a mathematical model of the SA node membrane, which, in turn, is based on physiologic data. The design was held at a resting membrane potential and then stepped to new voltages over the entire operating range of the T-type calcium channel. The circuit was validated by comparing its transient response current with the predicted current from the mathematical model. In addition, the peak currents of the circuit were compared with plots of peak current obtained from the mathematical model and physiologic data. By showing that the electronic circuit mimics the T-type calcium-channel current dynamics found within the SA node, the results may provide a foundation for developing a novel cardiac pacemaker that is based on the ion-channel characteristics of excitable tissue.     

气相色谱法测定室内空气中TVOC的影响因素

研究了气相色谱法测定室内空气中总有机挥发物(TVOC)的几个主要影响因素.结果表明:采样体积的准确性对测定结果的真实性有很大影响;采样现场在封闭前由于至少通风24 h;在气相色谱仪、热解吸仪最佳条件参数下可得到理想的色谱图;解吸时间过长,会导致组分解吸率下降;采用极性毛细管色谱柱(PEG-20M型)可将对、间二甲苯组分峰完全分开;Tenax-TA采样管中的吸附剂粒度和吸附剂装管时的密实程度对组分在采样管上的吸附和解吸有影响;各组分的解吸率不同使得苯乙烯和苯的解吸率偏低;组分解吸率越低,结果偏差越大.     

Passivation process of X80 pipeline steel in bicarbonate solutions

The passivation process of X80 pipeline steel in bicarbonate solutions was investigated using potentiodynamic, dynamic electrochemical impedance spectroscopy (DEIS), and Mott-Schottky measurements. The results show that the shape of polarization curves tions. No anodic current peak exists in HCO3- solutions when the concentration is lower than 0.009 mol/L, whereas there are one and two anodic current peaks when the HCO3- concentration ranges from 0.009 to 0.05 mol/L and is higher than 0.1 mol/L, respectively. DEIS measurements show that there exist active dissolution range, transition range, pre-passive range, passive layer formation range, passive range,and trans-passive range for X80 pipeline steel in the 0.1 mol/L HCO3- solutions. The results of DEIS measurements are in complete agreement with the potentiodynamic diagram. An equivalent circuit containing three sub-layers is used to explain the Nyquist plots in the passive range. Analyses are well made for explaining the corresponding fitted capacitance and impedance. The Mott-Schottky plots show that the passive film of X80 pipeline steel is an n-type semiconductor, and capacitance measurements are in good accordance with the results of DEIS experiment.     

Bypass of abnormal MDM2 inhibition of p53-dependent growth suppression

Fourier transform infrared (FT-IR) microspectroscopy and chemometric methods have been applied to the study of fresh and simulated post-mortem human arterial tissue. The results have shown that although physical differences were observed using light microscopy, no spectroscopic distinction could be made between these groups. The presence of collagen throughout the artery wall results in characteristic absorptions which may mask any biochemical changes that could otherwise have been detected by the FT-IR technique. Because the structure of the artery is unique, these findings should be regarded as tissue specific.     

Octoxynol presence in bear-bile

The presence of octoxynol from dried bear-bile was examined. Octoxynol was coextracted when glycolipids by Folch-Suzuki partition method. Octoxynol formed mixed-micelles with glycosphingolipids. The glycolipids were purified by DEAE-Sephadex A-25 column chromatography. The fractions containing mixed micelles were obtained from linear gradient solvent of 0.05M-0.5M ammonium acetate in methanol. HPLC ( Bondapak-NH(2) - linked to a Bondapak-C(18) column) chromatogram showed five peaks. Two possible structures for the fourth peak fraction were proposed as (CH(3))(3)C-CH(2)-C(CH(3))(2)-C(6)H(4)-OR and (CH(3))(3)C-C(CH(3))(2)-CH(2)-C(6)H(4)-OR by NMR spectroscopy. The structure was further confirmed by electrospray tandem mass spectrometry (ESI MS/MS). The spectrum showed a protonated molecule at m/z 559 and three different series of ions with mass difference of 44 were detected in the MS/MS spectrum. Therefore, the structure of the fourth peak fraction from HPLC was confirmed as octoxynol, (CH(3))(3)C-CH(2)-C(CH(3))(2)-C(6)H(4)-(OCH(2)-CH(2))n-OH, based on mass spectrometry and NMR spectroscopy.     

Development of a three-dimensional topographic map display for capillary electrophoresis/mass spectrometry with an ion trap/reflectron time-of-flight mass spectrometer detector: applications to tryptic digests of isoforms of myelin basic protein

A three-dimensional (3-D) contour map format has been developed to display the large amount of data continuously collected throughout an on-line capillary separation using an ion trap storage/reflectron time-of-flight detector (IT/reTOF). The resulting data are displayed on a single computer screen with a mass-to-charge ratio value-elution time-intensity representation. The intensity of various components is represented by 16 different colors so that the mass-to-charge ratio value, the elution time, and the intensity can be conveniently determined for each component. In addition, the mass spectrum and total ion chromatogram or total ion electropherogram (TIE) are shown on the same screen as the 3-D map that enables the correlation of a single spot in the 3-D map to the peaks in the TIE and the corresponding mass spectrum. The 3-D map has been used to identify various posttranslational modification sites of bovine myelin basic protein charge isomers, where the datafiles of tryptic digests of proteins analyzed by capillary electrophoresis/mass spectrometry were processed by this software and a comparison could be performed among the isoforms. The feature of in-screen integration over both the separation domain and the mass domain makes the acquisition of the selected ion chromatogram very convenient and greatly improves the ability to detect modified components present in low amounts.     

Immunoaffinity extraction of 4-hydroxy-2-(4-methylphenyl)benzothiazole and its metabolites for determination by gas chromatography-mass spectrometry

Immunoaffinity extraction of 4-hydroxy-2-(4-methylphenyl)benzothiazole and its metabolites, together with the corresponding meta-isomers has been achieved by the use of an antibody raised against an immunogen, an O-carboxymethyloxime-bovine serum albumin conjugate of 4-hydroxy-2-(4-formylphenyl)benzothiazole. The antibody produced exhibited a broad spectrum of affinity, not only for metabolites oxidized at the 4-methyl group of the benzene moiety but also for the corresponding meta-isomers. Up to 4 micrograms in total of these benzothiazoles could be extracted on the immunoaffinity adsorbent and recovered almost quantitatively by elution with 90% methanol. The resulting chromatogram was free from any interference. The eluted compounds were derivatized by conversion to their methyl esters and/or trimethylsilyl ethers, and subsequently separated into individual benzothiazoles by means of gas chromatography-mass spectrometry. The derivatized compounds were monitored using a characteristic ion, [M-CH3]+., and the limit of detection was 10 fmole. The peak height ratio of each metabolite to its corresponding meta-isomer internal standard was plotted against the concentration of the former and good linearity was observed over the range 0.2-5 ng/ml.     

高纯度、高收率L-丙交酯的制备

以L-乳酸为单体,在催化剂作用下用乳酸先缩聚再解聚制备L-丙爻酯(LLA),讨论了温度、压强对制备丙交酯的影响。采用水洗加重结晶的方法制得高纯度的LLA,LLA的收率达到40.6％。用旋光度、红外光谱、色谱质谱对所制得的LLA进行了检测,结果表明,提纯后LLA的纯度较高。     

Pyrolysis gas-liquid chromatography of the genus Bacillus: effect of growth media on pyrochromatogram reproducibility

Pyrolysis gas-liquid chromatography was performed on dried Bacillus microorganisms to evaluate the effects of growth media. Six cultures of Bacillus and six lot numbers of Trypticase soy agar (BBL) were used to test the hypothesis that a microorganism grown on various lot numbers of the same chromatogram. Also tested was the effect of three different media on chromatogram reproduction using the same six cultures. Results show little or no differences observed between the chromatograms of the individual Bacillus spp. grown on the six lot numbers of Trypticase soy agar. When chromatograms of the three different media were compared, several differences were observed, particularly in the areas most characteristic of individual species. Pryolysis gas-liquid chromatography can be a useful tool for the characterization or identification of the genus Bacillus if the chromatographic and cultural conditions are maintained.     

Diagnosis and surgical treatment of traumatic heart defects

The authors suggest a method of aldosterone determination in the urine. Aldosterone is extracted from the sample and purified with the aid of columnar and thin-layer chromatography on silicagel. Aldosterone is distinctly identified in ultraviolet light on the chromatogram by its green fluorescence developing after sprinkling the plate with phosphoric acid and its subsequent heating. Quantitative determination is carried out by the measurement of the intensity of aldosterone fluorescence in sulfuric acid.     

Full second-order chromatographic/spectrometric data matrices for automated sample identification and component analysis by non-data-reducing image analysis

A data analysis method is proposed for identification and for confirmation of classification schemes, based on single- or multiple-wavelength chromatographic profiles. The proposed method works directly on the chromatographic data without data reduction procedures such as peak area or retention index calculation. Chromatographic matrices from analysis of previously identified samples are used for generating a reference chromatogram for each class, and unidentified samples are compared with all reference chromatograms by calculating a resemblance measure for each reference. Once the method is configured, subsequent sample identification is automatic. As an example of a further development, it is shown how the method allows identification of characteristic sample components by local similarity calculations thus finding common components within a given class as well as component differences between classes from the reference chromatograms. This feature is a valuable aid in selecting components for further analysis. The identification method is demonstrated on two data sets: 212 isolates from 41 food-borne Penicillium species and 61 isolates from 6 soil-borne Penicillium species. Both data sets yielded over 90% agreement with accepted classifications. The method is highly accurate and may be used on all sorts of chromatographic profiles. Characteristic component analysis yielded results in good agreement with existing knowledge of characteristic components, but also succeeded in identifying new components as being characteristic.