Simultaneous quantification of phytohormones in fermentation extracts of Botryodiplodia theobromae by liquid chromatography–electrospray tandem mass spectrometry

Fermentation broth and biomass from three strains of Botryodiplodia theobromae were characterized by high performance liquid chromatography–electrospray tandem mass spectrometry (HPLC–ESI–MS/MS) method, in order to quantify different phytohormones and to identify amino acid conjugates of jasmonic acid (JA) present in fermentation broths. A liquid–liquid extraction with ethyl acetate was used as sample preparation. The separation was carried out on a C18 reversed-phase HPLC column followed by analysis via ESI–MS/MS. The multiple reaction monitoring mode was used for quantitative measurement. For the first time, indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and JA were identified and quantified in the ethyl acetate extracts from the biomass, after the separation of mycelium from supernatant. The fermentation broths showed significantly higher levels of JA in relation to the other phytohormones. This is the first report of the presence of gibberellic acid, abscisic acid, salicylic acid and the cytokinins zeatin, and zeatin riboside in fermentation broths of Botryodiplodia sp. The presence of JA-serine and JA-threonine conjugates in fermentation broth was confirmed using HPLC-ESI tandem mass spectrometry in negative ionization mode, while the occurrence of JA-glycine and JA-isoleucine conjugates was evidenced with the same technique but with positive ionization. The results demonstrated that the used HPLC–ESI–MS/MS method was effective for analysing phytohormones in fermentation samples.     

Stationary phase thickness determines the quality of thin-layer chromatography/matrix-assisted laser desorption and ionization mass spectra of lipids

Normal phase thin-layer chromatography (NP TLC) is an established method of (phospho)lipid analysis. The determination of the fatty acyl composition is, however, a more challenging task by NP TLC. The direct coupling of TLC separation with mass spectrometric detection (e.g., matrix-assisted laser desorption/ionization mass spectrometry, MALDI MS), however, enables a detailed characterization of complex lipid mixtures. Here we show that the thickness of the silica gel layer has a considerable effect on the quality of the mass spectra recorded directly from the TLC plate. In particular, the intensity of the matrix background signals can be reduced if “thinner” TLC layers are used.     

Identification of tryptophan in leachate of oat hulls (Avena sativa) as a mediator of root growth regulation

Leachate of oat hulls ( Avena sativa L. cv. Sang) inhibits root elongation and causes swollen roots and abundant root hair formation. The active substance was isolated by column chromatography and thin layer chromatography (TLC) systems. High performance liquid chromatography (HPLC) revealed 3 peaks, one of which corresponded to the substance responsible. The latter was identified as tryptophan by means of its UV spectrum, amino acid analysis, nuclear magnetic resonance spectrometry (NMR) and mass spectrometry (MS).     

Identification of delta-guanidinovaleric acid in human urine

delta-Guanidinovaleric acid (DGVA) was identified in human urine using thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). In the TLC, all Rfs of sample from urine developed by 6 solvent systems were identical to that of authentic DGVA. In the GC/MS, the mass spectrum of the sample was identical to the trifluoroacetylated dimethylpyrimidyl derivative of DGVA butylester (M+ = 375). In the HPLC analysis, the DGVA peak was observed just before 15 min in either chromatogram obtained by analysis of human urine or authentic DGVA, and the content of DGVA in pooled human urine was calculated at 2.4 nmol/ml.     

Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography.

Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1-2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1-2 nmol/ml, but the response was non-linear.     

Analysis of oligosaccharides by on-line high-performance liquid chromatography and ion-spray mass spectrometry.

Oligosaccharides were analyzed by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). First, oligosaccharides labeled with 2-aminopyridine were studied to see if they could be analyzed by MS under the conditions used for separation by HPLC. Pyridylamino (PA)-oligosaccharides could be analyzed under these conditions, although the mass spectra were affected. Then, liquid chromatography-mass spectrometry was used to analyze a PA-oligosaccharide mixture derived from human immunoglobulin G. The PA-oligosaccharides were separated on a reversed-phase column and mass-analyzed directly. The observed molecular weights were close to or identical to those expected from the structures, which were estimated from the elution position on HPLC. This method is rapid and simple, as the mass spectrometer can give the accurate molecular weight of each PA-oligosaccharide in one chromatography run, even if the HPLC separation is incomplete. This method can be used to extend the so-called two-dimensional mapping of PA-oligosaccharides. The structure can be studied in greater detail by tandem MS.     

Proteome analysis of Escherichia coli using high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry

The basic problem of complexity poses a significant challenge for proteomic studies. To date two-dimensional gel electrophoresis (2-DE) followed by enzymatic in-gel digestion of the peptides, and subsequent identification by mass spectrometry (MS) is the most commonly used method to analyze complex protein mixtures. However, 2-DE is a slow and labor-intensive technique, which is not able to resolve all proteins of a proteome. To overcome these limitations gel-free approaches are developed based on high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The high resolution and excellent mass accuracy of FT-ICR MS provides a basis for simultaneous analysis of numerous compounds. In the present study, a small protein subfraction of an Escherichia coli cell lysate was prepared by size-exclusion chromatography and proteins were analyzed using C4 reversed phase (RP)-HPLC for pre-separation followed by C18 RP nanoHPLC/nanoESI FT-ICR MS for analysis of the peptide mixtures after tryptic digestion of the protein fractions. We identified 231 proteins and thus demonstrated that a combination of two RP separation steps - one on the protein and one on the peptide level - in combination with high-resolution FT-ICR MS has the potential to become a powerful method for global proteomics studies.     

Comparative quantitative analysis of artemisinin by chromatography and qNMR

Introduction – Since the discovery of artemisinin in the 1970s, many techniques based on diverse chromatography techniques have been developed to detect and quantify this important antiplasmodial compound. The accurate quantification of this compound in the Artemisia annua plant material is mainly needed for breeding purposes in order to cultivate higher yielding varieties. It is also important for the quality control of herbal preparations containing A. annua plant material. Objective – To evaluate the most common validated quantification techniques (LC‐MS, HPLC‐ELSD and TLC) and compare the results to quantitative nuclear magnetic resonance spectroscopy (qNMR) in eight different A. annua samples collected from around the world. Methodology – The leaf material were extracted according to standard procedures and analysed with the validated quantification techniques. For the qNMR analysis we did not employ a standard curve but instead used an internal standard (maleid acid) which is not chemically related to artemisinin. Results – We found a significant difference between the results in this study. Compared with the qNMR results the HPLC‐ELSD corresponded closely, followed by LC‐MS. Quantitation with TLC led to an estimation range of ?0.5 to +3.2 mg artemisinin/g of A. annua. Conclusion – These results imply that qNMR, with the addition of an internal standard, can be used to quantify artemisinin in A. annua samples in a rapid and reproducible manner. Copyright © 2010 John Wiley & Sons, Ltd.     

Affinity chromatography as a method for sample preparation in gas chromatography/mass spectrometry.

Analytical chemistry aims at developing analytical methods and techniques for unequivocal identification and accurate quantitation of natural and synthetic compounds in a given matrix. Analytical methods based on the mass spectrometry (MS) technology, e.g., GC/MS and LC/MS and their variants, GC/tandem MS and LC/tandem MS, are best suited both for qualitative and quantitative analyses. GC/MS methods not only serve as reference methods, e.g., in clinical chemistry, but they are now widely and routinely used for quantitative determination of numerous analytes. However, despite inherent accuracy, analytical methods based on GC/MS commonly consist of several analytical steps, including extraction and derivatization of the analyte. In general, unequivocal identification and accurate quantification of an analyte in very low concentrations in complex matrices require further chromatographic techniques, such as high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) for sample purification. In recent years, affinity chromatography (e.g., boronate and immunoaffinity chromatography) has been developed to a superior technique for sample preparation of numerous classes of compounds in GC/MS. In this article, the application and importance of affinity chromatography as a method for sample preparation in modern quantitative GC/MS method is described and discussed, using as examples various natural and synthetic compounds, such as arachidonic acid derivates, nitrosylated and nitrated proteins, steroids, drugs, and toxins.     

Multiplexed two-dimensional liquid chromatography for MALDI and nanoelectrospray ionization mass spectrometry in proteomics

We developed a multiplexed two-dimensional separation system based on reversed phase (RP)--strong cation exchange (SCX) chromatography as a front-end device for matrix-assisted laser desorption ionization (MALDI) or nanoelectrospray ionization (nanoESI) mass spectrometry. Tryptic peptide mixtures were fractionated on a reversed-phase HPLC column, and each fraction was loaded onto multiplexed SCX microcolumns. Because this second chromatography was carried out in parallel, the analysis time is independent of the fraction number in the first RP-HPLC separation. The resultant samples were desalted/concentrated and eluted onto a MALDI plate with matrix-containing elution solutions in parallel, or eluted with optimized solutions for nanoESI and loaded onto nanoESI sprayers by an automated instrument. The soluble portion of HCT116 lysate was digested and fractionated using a 48-plexed chromatography system. Approximately 1000 unique peaks were detected in MALDI-MS with 3000 MS/MS spectra, while 724 peptides with ultrahigh peptide mass accuracy (sub-ppm error) were identified in nanoESI-FTICR mass spectrometry with five integrated selected ion monitoring scans. Since MS measurement with this off-line LC-LC approach is not restricted by continuous LC elution, it is expected to be useful especially in cases where repeated analysis with different scan modes or long-term data acquisition is required.     

Chemistry and liquid chromatography methods for the analyses of primary oxidation products of triacylglycerols

Abstract

Triacylglycerols (TAGs) are one of the major components of the cells in higher biological systems, which can act as an energy reservoir in the living cells. The unsaturated fatty acid moiety is the key site of oxidation and formation of oxidation compounds. The TAG free radical generates several primary oxidation compounds. These include hydroperoxides, hydroxides, epidioxides, hydroperoxy epidioxides, hydroxyl epidioxides, and epoxides. The presence of these oxidized TAGs in the cell increases the chances of several detrimental processes. For this purpose, several liquid chromatography (LC) methods were reported in their analyses. This review is therefore focused on the chemistry, oxidation, extraction, and the LC methods reported in the analyses of oxidized TAGs. The studies on thin-layer chromatography were mostly focused on the total oxidized TAGs separation and employ hexane as major solvent. High-performance LC (HPLC) methods were discussed in details along with their merits and demerits. It was found that most of the HPLC methods employed isocratic elution with methanol and acetonitrile as major solvents with an ultraviolet detector. The coupling of HPLC with mass spectrometry (MS) highly increases the efficiency of analysis as well as enables reliable structural elucidation. The use of MS was found to be helpful in studying the oxidation chemistry of TAGs and needs to be extended to the complex biological systems.     

紫细菌光合色素指纹图谱的建立与色素分析

【目的】探索快速高效的色素样品制备方法;为建立紫细菌全色素TLC和HPLC标准指纹图谱和数据库提供研究方法和思路;为色素代谢与调控等研究提供快速的色素分析方法。【方法】选择沼泽红假单胞菌(Rhodopesudomonas palustris CQV97)为材料,采用改良丙酮甲醇提取法、TLC重复展层法、图像灰度分析法、吸收光谱法、HPLC和MS法进行色素分析。【结果】甲醇或丙酮可选择性地提取样品的细菌叶绿素和类胡萝卜素,通过对丙酮甲醇法的改良,使色素提取总量提高了13.5%。建立了CQV97菌株全色素的TLC和HPLC指纹图谱,二者均含有11种色素组分。图像灰度分析法估算了TLC指纹图谱各色素组分的表观相对含量。以TLC图谱的各色素组分为外标物,明确了HPLC图谱中各色素组分的保留时间(Rt)与TLC图谱中各色素组分的迁移率(Rf)之间的对应关系。结合色素代谢途径、光谱分析和MS,定性分析了指纹图谱中11种色素组分。TLC或HPLC分析结果表明,理论样品与对照样品色素组分和含量一致,而实际样品与对照样品色素组分一致,但含量不同,重复测定3次,样品中色素相对含量的RSD均小于5%。【结论】改良的丙酮甲醇法可以快速高效地提取光合细菌的色素。采用TLC重复展层法和HPLC法建立的全色素指纹图谱色素组成一致,重复稳定性好,各具特色。TLC和HPLC两种指纹图谱分析方法均能进行光合色素的快速分析,适合于紫细菌色素合成途径中主要积累色素的组分和含量变化规律研究。     

Near infrared spectroscopy compared to liquid chromatography coupled to mass spectrometry and capillary electrophoresis as a detection tool for peptide reaction monitoring

Peptide interaction is normally monitored by liquid chromatography (LC), liquid chromatography coupled to mass spectrometry (LC-MS), mass spectrometric (MS) methods such as MALDI-TOF/MS or capillary electrophoresis (CE). These analytical techniques need to apply either high pressure or high voltages, which can cause cleavage of newly formed bondages. Therefore, near infrared reflectance spectroscopy (NIRS) is presented as a rapid alternative to monitor the interaction of glutathione and oxytocin, simulating physiological conditions. Thereby, glutathione can act as a nucleophile with oxytocin forming four new conjugates via a disulphide bondage. Liquid chromatography coupled to UV (LC-UV) and mass spectrometry via an electrospray ionisation interface (LC-ESI-MS) resulted in a 82% and a 78% degradation of oxytocin at pH 3 and a 5% and a 7% degradation at pH 6.5. Capillary electrophoresis employing UV-detection (CE-UV) showed a 44% degradation of oxytocin. LC and CE in addition to the NIRS are found to be authentic tools for quantitative analysis. Nevertheless, NIRS proved to be highly suitable for the detection of newly formed conjugates after separating them on a thin layer chromatography (TLC) plate. The recorded fingerprint in the near infrared region allows for a selective distinct qualitative identification of conjugates without the need for expensive instrumentation such as quadrupole or MALDI-TOF mass spectrometers. The performance of the established NIRS method is compared to LC and CE; its advantages are discussed in detail.     

Automated in-solution protein digestion using a commonly available high-performance liquid chromatography autosampler

A completely automated peptide mapping liquid chromatography/mass spectrometry (LC/MS) system for characterization of therapeutic proteins in which a common high-performance liquid chromatography (HPLC) autosampler is used for automated sample preparation, including protein denaturation, reduction, alkylation, and enzymatic digestion, is described. The digested protein samples are then automatically subjected to LC/MS analysis using the same HPLC system. The system was used for peptide mapping of monoclonal antibodies (mAbs), known as a challenging group of therapeutic proteins for achieving complete coverage and quantitative representation of all peptides. Detailed sample preparation protocols, using an Agilent HPLC system, are described for Lys-C digestion of mAbs with intact disulfide bonds and tryptic digestion of mAbs after reduction and alkylation. The automated procedure of Lys-C digestion of nonreduced antibody, followed by postdigestion disulfide reduction, produces both the nonreduced and reduced digests that facilitate disulfide linkage analysis. The automated peptide mapping LC/MS system has great utility in preparing and analyzing multiple samples for protein characterization, identification, and quantification of posttranslational modifications during process and formulation development as well as for protein identity and quality control.     

Sensitive and selective liquid chromatography/tandem mass spectrometry methods for quantitative analysis of 1-methyl-4-phenyl pyridinium (MPP+) in mouse striatal tissue

The systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice produces a reliable and selective degeneration of the nigrostriatal pathway, a hallmark feature of Parkinson's disease (PD). Determining the brain concentrations of 1-methyl-4-phenyl pyridium (MPP+), the neurotoxic metabolite of MPTP, is critical for evaluating drugs designed to potentially treat PD. We have developed sensitive and specific quantitative methods for the determination of MPP+ in mouse striatal tissue by liquid chromatography/tandem mass spectrometry. The separations were carried out based on reversed phase chromatography or cation exchange chromatography with volatile elution buffer. Neutralizing the brain sample with 0.2M phosphate buffer successfully solved a high-performance liquid chromatography (HPLC) peak tailing of MPP+ in brain extracts with 0.4M perchloric acid (HClO4) under the reversed phase HPLC conditions, which significantly improved the sensitivity of the method. The HPLC peak shape of MPP+ using cation exchange chromatography was not affected by the pH of the samples. Optimization of electrospray ionization (ESI) conditions for the quaternary ammonium compound MPP+ established the limits of detection (LOD) (S/N=3) at 0.34pg/mg tissue and 0.007pg/mg tissue (5microl of injection) using the reversed phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the cation exchange LC/MS/MS, respectively. Both methods were selective, precise (%R.S.D.<6%), and sensitive over a range of 0.001-1ng/mg tissue. The cation exchange method showed greater sensitivity and tolerance to low pH samples than the reversed phase method. The developed methods were applied to monitoring changes in MPP+ concentrations in vivo. Two reference agents, R-(-) Deprenyl and MK-801, known to alter the concentration of MPP+ in MPTP treated mice were evaluated.     

一株高抗氧化活性内生真菌的分子鉴定及产酚酸类物质研究

【目的】以疏花水柏枝(Myricaria laxiflora)淹水前根部分离到的内生真菌SG17为研究对象,为明确其抗氧化能力及可能的物质基础,对其进行生物学鉴定、抗氧化能力分析及产酚酸类物质研究。【方法】对该菌nr DNA的内转录间隔区进行测序;应用总抗氧化试剂盒,以维生素C为阳性对照,测定其抗氧化能力;采用高效液相色谱(HPLC)和薄层色谱(TLC)探讨其发酵液中的酚酸类化合物。【结果】SG17具有很高的抗氧化活性,其发酵液粗提物的抗氧化活性达到同浓度抗坏血酸的31.86%,分子和形态鉴定表明SG17是一株烟曲霉(Aspergillus fumigatus)。其发酵产物中酚酸类物质含量丰富,HPLC能检测到含有没食子酸,且通过HPLC和TLC分析还发现一种光学性质与绿原酸极为相似的类似物。【结论】内生真菌烟曲霉SG17发酵液的抗氧化能力远高于国际报道水平,是一种具有潜在开发价值的抗氧化剂资源。     

Use of direct-probe mass spectrometry as a toxicology confirmation method for demoxepam in urine following high-performance liquid chromatography

The identification of the metabolite demoxepam in human urine establishes that chlordiazepoxide, a common benzodiazepine, has been administered. Like N-oxide metabolites of other drugs, demoxepam cannot be detected by gas chromatography—mass spectrometry (GC-MS), due to thermal decomposition, and the product, nordiazepam, is a metabolite common to many benzodiazepines. Demoxepam can be readily screened using a high-performance liquid chromatography (HPLC) system such as REMEDi HS; at 35°C, no thermal decomposition will occur. Currently, there is no confirmation method available for the detection of demoxepam in urine samples. In this study, we demonstrated that following collection of the HPLC fraction, demoxepam can be confirmed using the technique of direct-probe MS. The mass spectra of demoxepam and nordiazepam differ and are easily distinguishable from each other. Ten urine samples that were analyzed by HPLC and determined to contain demoxepam were evaluated; demoxepam was confirmed in each case by direct-probe MS.     

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 μg protein) or preparative (>250 μg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.     

Narrow-bore sample trapping and chromatography combined with quadrupole/time-of-flight mass spectrometry for ultra-sensitive identification of in vivo and in vitro metabolites

The identification of in vitro and in vivo metabolites is vital to the discovery and development of new pharmaceutical therapies. Analytical strategies to identify metabolites at different stages of this process vary, but all involve the use of liquid chromatography separations combined with detection via mass spectrometry (HPLC/MS). Reported here is the use of narrow-bore column (0.5-1.0 mm i.d.) trapping of metabolites, followed by back-flushing onto a matching analytical column. Separated metabolites were then identified using quadrupole time-of-flight mass spectrometry (MS) and tandem MS. Metabolites in human plasma and from low-level in vitro incubations, that were not identified using standard HPLC/MS approaches, were characterized using the instrumental configuration described here.     

Comparative studies of HPLC-fluorometry and LC/MS method for the determination of N-acetylneuraminic acid as a marker of deteriorated ophthalmic solutions

Methods for determining the deterioration of ophthalmic solutions using both high-performance liquid chromatography (HPLC) with fluorescence detection and liquid chromatography coupled with selected ion monitoring mass spectrometry (LC/MS) are described. The methods are based on the determination of N-acetylneuraminic acid (NeuAc) released by the hydrolysis of foreign bodies that contaminate eye drops during use. The released NeuAc was either labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) for fluorometric detection or detected without derivatization by mass spectrometry. The calibration curves for NeuAc showed good linearity between 1.2 ng/mL and 39 ng/mL for fluorometric HPLC and 5.0 ng/mL and 100 ng/mL for LC/MS, respectively. Detection limits for fluorometric HPLC and LC/MS were 0.20 ng/mL and 0.88 ng/mL, respectively. The NeuAc content of some model glycoproteins determined by LC/MS method were 62-78% of those determined by fluorometry. The differences are attributed to matrix effects but the LC/MS method afforded sufficiently high sensitivity that NeuAc in the foreign bodies could be determined in eight of nine test samples.