液相色谱串联质谱法测定烟草中的游离氨基酸

建立液相色谱串联质谱法测定烟草中20种游离氨基酸的方法。烟草样品经0.1%的盐酸溶液超声萃取并离心后,直接进样测定。色谱柱采用XTerra MS C18(50mm×2.1mm×2.5μm),0.1%甲酸溶液和乙腈为流动相。结果表明:20种氨基酸的检出限为0.001～0.011μg/mL,标准曲线的拟合度均大于0.999,回收率在86.4%～105.9%之间。该方法操作简单,灵敏度高,适用于烟草中游离氨基酸的检测。     

Analysis of underivatized amino acids and their D/L-enantiomers by sheathless capillary electrophoresis/electrospray ionization mass spectrometry

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of underivatized amino acids and the separation of their D/L-enantiomers. Under full-scan mode, all standard protein amino acids were separated and detected at low-femtomole levels using a 130-cm-long, 20-microm-i.d., 150-microm-o.d. underivatized fused-silica capillary with 1 M formic acid as the background electrolyte. The CE/ESI-MS technique was also applied to the separation of L-arginine from L-canavanine (a close analogue of arginine where the terminal methylene linked to the guanidine group of arginine is replaced by an oxygen atom) in a complex mixture containing all standard protein amino acids. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the identification of two metabolic diseases (PKU and tyrosinemia) through blood analysis with minimal sample preparation. In addition, the on-line separation of 11 underivatized L-amino acids from their D-enantiomers was achieved by using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the background electrolyte.     

Chiral analysis by electrospray ionization mass spectrometry/mass spectrometry. 2. Determination of enantiomeric excess of amino acids

The determination of enantiomeric excess (ee) of amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed by electrospray ionization of amino acids in the presence of one of the following chiral selectors: L- or D-N-tert-butoxycarbonylphenylalanine, L- or D-N-tert-butoxycarbonylproline, and L- or D-N-tert-butoxycarbonyl-O-benzylserine. The protonated trimers were dissociated to form protonated dimers, and the observed dissociation efficiency r (i.e., the intensity ratio of protonated dimers to protonated trimers) for an enantiomeric mixture was found to be related to its ee value by the following equation: r = a + b/(c + ee), where a, b, and c were constants. A linear calibration plot was obtained by plotting r versus 1/(c + ee), where c was calculated with the MATLAB software, or by plotting 1/(r - r0) versus 1/ee, where r0 was the r value for the racemic mixture. The latter "two-reciprocal" method was more convenient for application. Another practical method for ee determination was the "three-point" method, whereby the ee of an unknown sample with a measured r value could be derived from the equation ee = 100?1/(rL - r0) - 1/(rD - r0)?/?2/(r - r0) - 1/(rL - r0) - 1/(rD - r0)?, with rL and rD being the r values for the enantiomerically pure L- and D-forms of the sample, respectively. A calibration plot was not required. The ee determination was achieved with acceptable precision even for the worst case of acceptable chiral recognition with a particular chiral selector, suggesting that the ee determination of all 19 common amino acids could be achieved by the present method. The ee of a histidine sample was determined both by the two-reciprocal method, giving an error of 0.2% ee (1.1% relative error) and consuming only approximately 5.3 nmol of sample, and by the three-point method, giving an error of 0.4% ee and consuming only approximately 2.3 nmol of sample. In the latter case, it took 27 min for the mass spectrometric measurements of the three calibration standards and an additional 9 min for the unknown sample. The direct ee determination of more than one amino acid in a mixture was also demonstrated in the study.     

Chiral analysis by electrospray ionization mass spectrometry/mass spectrometry. 1. Chiral recognition of 19 common amino acids

Chiral recognition of 19 common amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed from the electrospray ionization of amino acids in the presence of one of the following chiral selectors: L- or D-N-tert-butoxycarbonylphenylalanine, L- or D-N-tert-butoxycarbonylproline, and L- or D-N-tert-butoxycarbonyl-O-benzylserine. The protonated trimers were dissociated to protonated dimers, and the intensity ratios of the protonated dimer (product ion) to the protonated trimer (precursor ion), i.e., the observed dissociation efficiency, was found to be strongly dependent on the chirality of the amino acids with respect to that of the chiral selectors. The results showed that the chirality of all 19 common amino acids can be definitely differentiated. The method was demonstrated as rapid, sensitive, precise, robust, and requiring no reference standards and only minimal sample preparation. The chirality of all three amino acids in a mixture was determined without prior separation of the amino acids, consuming only 70 pmol of sample and requiring only approximately 14 min of mass spectrometric measurements. A cyclodipeptide with unknown chirality was determined to be cyclo-(L-Pro-L-Leu) by acid hydrolysis followed by the present method, and the results were consistent with the physiochemical properties and NMR data of the compound. This study suggested that ESI-MS/MS can be a promising approach for the chiral recognition of other compounds.     

Electrospray mass spectrometry: application of ion evaporation theory to amino acids

We describe the result of applying the ion evaporation theory to a series of amino acids. The very good correlation (r = 0.98) of the natural logarithms of protonated molecule intensities observed by electrospray with the difference between the hydration free energies of molecules and the gas-phase binding free energies of molecules and protons in amino acids is consistent with the ion evaporation model. It seems that the difference in the protonated molecule intensities of amino acids obtained by electrospray can be explained by a scheme in which protonated molecules in the liquid phase are extracted into the gas phase after a charged droplet is formed.     

Sizing nanomatter in biological fluids by fluorescence single particle tracking

Accurate sizing of nanoparticles in biological media is important for drug delivery and biomedical imaging applications since size directly influences the nanoparticle processing and nanotoxicity in vivo. Using fluorescence single particle tracking we have succeeded for the first time in following the aggregation of drug delivery nanoparticles in real time in undiluted whole blood. We demonstrate that, by using a suitable surface functionalization, nanoparticle aggregation in the blood circulation is prevented to a large extent.     

Analysis of chiral amino acids in conventional and transgenic maize

In this work, a new chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC-LIF) method is proposed to identify and quantify D- and L-amino acids in three lines of transgenic maize and their corresponding nontransgenic parental lines grown under identical conditions. The optimized procedure includes amino acids extraction, derivatization with FITC and chiral-MEKC-LIF separation in a background electrolyte composed of 100 mM sodium tetraborate, 80 mM SDS, and 20 mM beta-CD at pH 10.0. The D- and L-forms of Arg, Ser, Ala, Glu, and Asp, corresponding to the majority amino acids usually found in maize, are separated in less than 25 min with efficiencies up to 890,000 plates/m and high sensitivity (i.e., LODs as low as 160 nM were obtained for D-Arg for a signal-to-noise ratio of three), allowing the detection of 1% D-Arg in the presence of 99% of its opposite enantiomer. Using this method, different D-amino acids are detected in all investigated maize samples providing the reproducible quantification of the D-enantiomeric excess (% d-aa) for each amino acid calculated as % D-aa = 100D-aa/(D-aa + L-aa). Thus, significant differences were observed among the % d-aa values for the different conventional varieties (Aristis, Tietar, and PR33P66 maize) as could be expected from their natural variability. More interestingly, comparing each conventional maize with its corresponding transgenic line, very similar % D-aa values were obtained for one of the studied maize couples (Tietar vs Tietar-Bt) what could be presented as a new proof of their substantial equivalence. However, significant differences in the % d-aa values were observed for the other lines of maize studied. It is concluded that enantioselective procedures can open new perspectives in the study of transgenic organisms in order to corroborate (or not) the equivalence with their conventional counterparts.     

IsoQuant: a software tool for stable isotope labeling by amino acids in cell culture-based mass spectrometry quantitation

Accurate protein identification and quantitation are critical when interpreting the biological relevance of large-scale shotgun proteomics data sets. Although significant technical advances in peptide and protein identification have been made, accurate quantitation of high-throughput data sets remains a key challenge in mass spectrometry data analysis and is a labor intensive process for many proteomics laboratories. Here, we report a new SILAC-based proteomics quantitation software tool, named IsoQuant, which is used to process high mass accuracy mass spectrometry data. IsoQuant offers a convenient quantitation framework to calculate peptide/protein relative abundance ratios. At the same time, it also includes a visualization platform that permits users to validate the quality of SILAC peptide and protein ratios. The program is written in the C# programming language under the Microsoft .NET framework version 4.0 and has been tested to be compatible with both 32-bit and 64-bit Windows 7. It is freely available to noncommercial users at http://www.proteomeumb.org/MZw.html .     

Design of a room-temperature phosphorescence-based molecular beacon for highly sensitive detection of nucleic acids in biological fluids

Ultrasensitive fluorescent analysis or monitoring of significant molecules in complex samples is important for many biological studies, clinical diagnosis, and forensic investigations, the major obstacle for which is the background signals from ubiquitous endogenous fluorescent components of the environments. Herein, a room-temperature phosphorescence (RTP)-based molecular beacon (MB), employing a Eu(3+) complex of chlorosulfonylated tetradentate β-diketone (L) and the quencher BHQ-2, was engineered for highly sensitive detection of DNA sequences in biological fluids. Complexation of Eu(3+) with the ligand L formed a strongly luminescent complex EuL(2). But when EuL(2) and BHQ-2 were labeled to two ends of a DNA molecule with hairpin structure, the luminescence of EuL(2) was quenched by BHQ-2 due to the stem-closed conformation of the beacon. Due to very low background luminescence from the probe molecule, >200-fold signal enhancement was achieved when nanomolar target sequence was introduced. This sensitivity is about 20-fold higher than the level achieved with conventional fluorescence-based molecular beacons. Furthermore, because the Eu(3+) complex has a much longer luminescence lifetime (≈0.8 ms) than that of the background (<10 ns), RTP measurements were used to directly detect as low as 500 pM DNA in cell media quantitatively without any sample pretreatment.     

Direct determination of benzodiazepines in biological fluids by restricted-access solid-phase microextraction

A biocompatible solid-phase microextraction (SPME) fiber was prepared using an alkyl-diol-silica (ADS) restricted-access material as the SPME coating. The ADS-SPME fiber was able to simultaneously fractionate the protein component from a biological sample, while directly extracting several benzodiazepines, overcoming the present disadvantages of direct sampling in biological matrixes by SPME. The fiber was interfaced with an HPLC-UV system, and an isocratic mobile phase was used to desorb, separate, and quantify the extracted compounds. The calculated clonazepam, oxazepam, temazepam, nordazepam, and diazepam detection limits were 600, 750, 333, 100, and 46 ng/mL in urine, respectively. The method was confirmed to be linear over the range of 500-50000 ng/mL with an average linear coefficient (R2) value of 0.9918. The injection repeatability and intraassay precision of the method were evaluated over 10 injections, resulting in a RSD of approximately 6%. The ADS-SPME fiber was robust and simple to use, providing many direct extractions and subsequent determination of benzodiazepines in biological fluids.     

乳状液膜法分离氨基酸的动力学

探讨了含载体P2 0 4 (磷酸二异辛酯 )的液膜体系用于苯丙氨酸促进运输的动力学行为 .采用液滴法装置 ,控制实验条件 ,测定了内相氢离子浓度随时间的变化 ,适用于有机相界面的化学反应对溶质传递的影响 ,并将计算的结果同实验相比较 ,建立了水相 -有机相界面化学反应对溶质传质具有决定作用时的传质模型     

Simultaneous derivatization and quantification of the nitric oxide metabolites nitrite and nitrate in biological fluids by gas chromatography/mass spectrometry

Simultaneous quantification of nitrite and nitrate, the major oxidative metabolites of L-arginine-derived nitric oxide (NO), in biological fluids by GC or GC/MS methods is currently impossible. The separate analysis of these anions is associated with severe methodological problems. Therefore, a GC/MS method was developed which allows, for the first time, simultaneous quantification of nitrite and nitrate in various biological fluids. The method involves a single derivatization procedure, by which endogenous nitrite and nitrate and their externally added 15N-labeled analogues are simultaneously converted in aqueous acetone by pentafluorobenzyl bromide to the nitro and nitric acid ester pentafluorobenzyl derivatives, respectively, and a single GC/MS analysis. Nitrite and nitrate concentrations measured in plasma and urine of humans by this method correlated excellently with those from quantification of nitrite and nitrate in these matrixes using a previously reported GC/MS method that, however, requires reduction of nitrate to nitrite. Also, the present method enables discrimination between S-nitro- and S-nitroso-glutathione, which have identical chromatographic and spectrophotometric properties. The method is very useful to routinely study metabolism and reactions of NO and its metabolites in vitro and in vivo. It is accurate, interference-free, sensitive-50 fmol of [15N]-nitrite and [15N]nitrate were detected at signal-to-noise ratios of 870:1 and 95:1, respectively-and should be a reference method for nitrite and nitrate measurements.     

Isotopic determination of organic keto acid pentafluorobenzyl esters in biological fluids by negative chemical ionization gas chromatography/mass spectrometry.

A rapid, single-step procedure for the extraction and derivatization of organic alpha-keto acids from microliter quantities of human plasma has been developed. The keto acids were analyzed as the pentafluorobenzyl (PFB) ester by methane negative chemical ionization gas chromatography/mass spectrometry. The PFB esters possess excellent chromatographic properties and required no further derivatization to block the keto group. They fragment to produce intense carboxylate anions, often as the sole ion in the spectrum, and offer detection limits below 1 pmol. This derivative is suitable for isotopic analysis of organic keto acids because it does not introduce any additional isotopic complexity into the target molecule. Normal human plasma 4-methyl-2-oxopentanoic acid levels were 34.9 +/- 5.3 mumol.L-1 and could be determined with 1.1% precision by isotope dilution GC/MS. We have used this procedure to study leucine and 4-methyl-2-oxopentanoic acid metabolism by using stable isotopically labeled tracers in a variety of normal and abnormal conditions.     

Accurate molecular mass determination of mycolic acids by MALDI-TOF mass spectrometry

Mycolic acids, major and specific long-chain fatty (C70-C90) acid components of the mycobacterial cell envelope, were analyzed for the first time using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry operating in a reflectron mode. The various types of purified mycolates from representative mycobacterial species were analyzed using 2,5-DHB as matrix, because less than 10 pmol of mycolates was sufficient to obtain well-resolved mass spectra composed exclusively of pseudomolecular [M + Na]+ ions consistent with the structures deduced from the chemical analytical techniques applied to these molecules. Examination of the MALDI mass spectra demonstrated that the chain lengths of the various mycolates correlated with the growth rate of mycobacterial strains. Although slow growers, such as Mycobacterium tuberculosis and Mycobacterium ulcerans, produced a series of odd carbon numbers (C74-C82) of alpha-mycolic acids, rapid growers synthesized both odd and even carbon numbers. In addition, the main chain of oxygenated mycolic acids from slow growers were four to six carbon atoms longer than the corresponding alpha-mycolic acids, whereas rapid growers elaborated oxygenated homologues possessing the same chain lengths as their alpha-mycolic acids. Furthermore, a comparative analysis of the crude fatty acid mixtures from a wild-type strain of M. tuberculosis and its isogenic mutant effected in the synthesis of oxygenated mycolates by MALDI mass spectrometry revealed structural differences between the alpha-mycolates from the two strains. Thus, this technique appeared to be a rapid and highly sensitive technique for the analysis of mycolic acids, not only by providing accurate molecular masses and new structural information, but also by both reducing sample consumption and saving time.