Mass spectrometrically detected directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry for the identification of xenobiotic metabolites in maize plants

Reconstructed ion chromatograms have been used to identify relevant high performance liquid chromatography (HPLC) peaks in a directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry (HPLC/NMR/MS) experiment. This has been applied to a study of the metabolism of a model compound, 5-nitropyridone (2-hydroxy-5-nitropyridine), in maize plants grown hydroponically. By monitoring the on-flow reconstructed ion chromatogram corresponding to the 5-nitropyridone fragment at m/z 143, and additional molecular ions corresponding to metabolites identified as products from similar compounds, relevant peaks were identified rapidly for subsequent stopped-flow 1H NMR spectroscopic analysis. The combination of coupled HPLC/NMR/MS enabled the direct identification of three metabolites, namely the N-glucoside, N-malonylglucoside, and O-malonylglucoside. This work demonstrates the power of HPLC/NMR/MS for the structural elucidation of xenobiotic metabolites in complex biological matrices (such as plant material) with minimal sample preparation. In particular, using mass spectrometry for the initial identification of relevant HPLC peaks allows the analysis of complex samples without the necessity for other spectroscopic markers, such as 19F NMR signal for fluorinated compounds or UV spectroscopy for molecules with strong UV chromophores.     

Mass spectrometric analysis of cyclofenil and its metabolites in human urine

Using gas chromatography/electron impact-mass spectrometry (GC/EI-MS) and high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), the structures of cyclofenil metabolites in human urine have been assigned. The hydroxyl metabolites liberated from the glucuronide conjugates after acid hydrolysis were characterized as the trimethylsilyl (O-TMS) derivatives using GC/MS. The conjugate glucuronide forms were detected without hydrolysis by HPLC/MS. Cyclofenil was not observed in urine. Tentative structures for the two metabolites are proposed.     

Hair Analysis as Method for Determination of Level of Drugs and Pharmaceutical in Human Body: Review of Chromatographic Procedures

Abstract

Interest in hair analysis as an alternativ or complementary approach to urinalysis for drug abuse detection has grown in recent years. Hair analysis can be particularly advantageous for drugs and their enantiomers.

More than hundred pharmaceuticals, drugs of abuse agents are reported to be detectable in human and animal hair. This article reviews the aalysis of drugs and drug metabolites by chromatographic procedures, incuding the pretreatment steps, and the xtraction methods. Tihe eneral tendency in the last years, to highly sophisticated techiques gas chromatography–mass spectrometry (GC–MS–NCI), high pressure liquid chromatography–mass spectrometry (HPLC–MS), gas chromatography–mass spectrometry–mass spectrometry (GC–MS–MS) well illustrates this constant fight for sensitivity.     

Determination of Tripamide in human urine by high-performance liquid chromatography and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Tripamide is a drug widely used in clinical practice for the treatment of hypertension and edema. This work evaluated a screening method for Tripamide and its urinary metabolites in human urine, using high-performance liquid chromatography diode-array detection (HPLC/DAD). Identification of these metabolites was investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) after dosing with 15 mg Tripamide. Acid hydrolysis showed that Tripamide is conjugated in the body. Two suspected metabolites were detected by HPLC/DAD. HPLC/ESI-MS/MS analysis suggested that these metabolites were probably hydroxylated together with loss of the -NH(2) group and dehydrogenation. These results will be useful in confirmation methods for Tripamide in doping control.     

Liquid chromatography with mass spectrometry method based two‐step precursor ion scanning for the structural elucidation of flavonoids

Plant flavonoids are very important secondary metabolites for insect and virus control of their host plant and are potent nutrients for humans. To be able to understand the bioavailability and functions of plant flavonoids, it is necessary to reveal their exact chemical structures. Liquid chromatography with tandem mass spectrometry is a powerful approach for structural elucidation of metabolites. In this report, a two‐step precursor ion scanning based liquid chromatography with tandem mass spectrometry method was developed for the structural elucidation of plant flavonoids. The established method consists of the two‐step precursor ions scanning for possible flavonoids extraction, MS² fragment spectra acquisition and comparison with an online database, liquid chromatography retention rules correction, and commercial standards verification. The developed method was used for the structure elucidation of flavonoids in flowers and leaves of tobacco (Nicotiana tabacum), and 17 flavonoids were identified in the tobacco variety Yunyan 97. Nine of the 17 identified flavonoids were considered to be found in tobacco flowers or/and leaves for the first time based on the available references. This method was proved to be very effective and can be used for the identification of flavonoids in other plants.     

反相高效液相色谱/基质辅助激光解吸电离飞行时间质谱 分离鉴定螺蛳血管紧张素转换酶抑制肽

运用反相高效液相色谱(RP-HPLC)对酶解螺蛳腹足肌得到的血管紧张素转换酶(ACE)抑制肽进行两步分离提纯,第一步主要得到8个组分;选取其中活性最高的组分进一步分离,得到2个组分,其中活性较高组分的ACE半抑制浓度为43.5 μmol/L,基本为单一肽组分。对提纯的组分分别使用高效液相色谱/电喷雾离子质谱法(HPLC/ESI-MS)和基质辅助激光解吸电离飞行时间质谱法(MALDI-TOF MS)进行分析,同时结合氨基酸组成分析结果,最终得到的肽链一级结构为Lys-Glu-Ile-Trp(KEIW),符合已知的高活性ACE抑制肽的结构规律。经过对两种方法分析过程的比较,认为ESI-MS可以得到多方面的信息,但无法确定肽的序列;MALDI-TOF MS可以得到精确的二级质谱图(m/z精确至0.0001),从而可以得到确定的肽的序列。     

Quantitative analysis of cytokinins in plants by liquid chromatography–single-quadrupole mass spectrometry

A sensitive method for the quantitative analysis of all natural isoprenoid cytokinins in plant material by electrospray single-quadrupole mass spectrometry is presented. A baseline chromatographic separation of 20 non-derivatised naturally occurring cytokinins has been developed. Precise analyses of O-glucoside and ribonucleotide fractions were also performed by the high-performance liquid chromatography–mass spectrometry (HPLC–MS) but run separately from the basic cytokinin metabolites. Using post-column splitting, the flux from narrow-bore (2.1 mm i.d.) reversed-phase liquid chromatography column was simultaneously introduced into the diode array and mass detector. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. When low cone voltage (20 V) was applied, all studied cytokinins were determined in aqueous methanol as dominant quasi-molecular ions of [M+H]⁺ with limits of detection ranging between 10 and 50 fmol. For routine analysis a linearity range between 25 (75) fmol and 100 pmol was obtained. Developed liquid chromatography–mass spectrometry (LC–MS) method in selective ion monitoring mode was employed to quantify cytokinin species in tobacco BY-2 suspension culture and poplar leaves (Populus×canadensis Moench, cv Robusta).

Purified plant cell (BY-2) and plant tissue (poplar leaves) extracts were obtained by using two different ion-exchange chromatography steps, in combination with immunoaffinity purification using a broad-spectrum monoclonal anti-cytokinin antibody. The antibody strongly recognises the presence of N⁶-substituent on purine skeleton and thus does not bind adenine and related compounds. The presence of authentic cytokinins in the extracts quantified by LC–MS was further verified by enzyme-linked immunosorbent assays (ELISAs) with prior LC preparation. The combination of liquid chromatography–single-quadrupole mass spectrometry with immunoaffinity chromatography offers an efficient and elegant method for detection and quantification of cytokinin metabolites.     

Effects of Pulsed Electric Field on Secondary Metabolism of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. Gamay Fréaux Suspension Culture and Exudates

Plant cell cultures provide a large potential for the production of secondary metabolites. Through the application of different physical and chemical cell stress factors, we investigated the production of the secondary metabolites in plant cell cultures. The effects of pulsed electric field (PEF) and ethephon on growth and secondary metabolism, particularly anthocyanins and phenolic acids synthesis, were investigated by using suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system. Anthocyanins were measured by spectrophotometer and extracellular phenolic acids were determined by high-performance liquid chromatography. The compounds were identified by liquid chromatography–mass spectrometry and nuclear magnetic resonance. After the treatments with PEF and ethephon, the concentrations of anthocyanins and phenolic acids in cell culture were higher than in the control, without loss of biomass. The combination of PEF treatment and ethephon improved secondary metabolites formation. Production levels of extracellular phenolic acids, 3-O-glucosyl-resveratrol were increased by PEF and ethephon treatments. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane’s dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolites biosynthesis in plant cell cultures.     

Screening for anthocyanins using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with precursor-ion analysis, product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring

A systematic method for anthocyanin identification using tandems mass spectrometry (MS/MS) coupled to high-performance liquid chromatography (HPLC) with photo-diode array detection (PDA) was developed. Scan for the precursor ions of commonly found anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, petunidin, and peonidin) using LC/MS/MS on a triple quadrupole instrument allows for the specific determination of each category of anthocyanins. Further characterization of each anthocyanin was performed using MS/MS product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring (SRM). The method was demonstrated for analysis of anthocyanins in black raspberries, red raspberries, highbush blueberries, and grapes (Vitis vinifera). Previous reported anthocyanins in black raspberries and red raspberries are confirmed and characterized. Common-neutral-loss analysis allows for the distinction of anthocyanin glucosides or galactoside and arabinosides in highbush blueberries. Separation and identification of anthocyanin glucosides and galactosides were achieved by LC/MS/MS using SRM. Anthocyanin isomers such as cyanidin sophoroside and 3,5-diglucoside were differentiated by their fragmentation pattern during product-ion analysis. Fifteen anthocyanins (all possible combinations of five anthocyanidins and three sugars) were characterized in highbush blueberries. Pelargonidin 3-glucoside and pelargonidin 3,5-diglucoside were detected and characterized for the first time in grapes. The present approach allows mass spectrometry to be used as a highly selective detector for rapid identification and characterization of anthocyanins and can be used as a sensitive procedure for screening anthocyanins in fruits and vegetables.     

固相微萃取-气相色谱/质谱测定植物叶片中的挥发性物质

采用固相微萃取（SPME)方法吸附植物叶片中的挥发性物质,然后采用气相色谱/质谱法（GC/MS）分析了挥发性物 质的成分。在45 ℃水浴温度下,采用Polyacrylate(85 μm)固相微萃取头,在广口瓶中植物叶片的上方顶空吸附60 min,然后进行GC/MS分析。结果表明,植物叶片中的挥发性物质得到了很好的分离,受山楂叶螨(Tetraychus vienneis) 危害严重的植物的完好叶片中的挥发性物质均含有顺-3-己烯-1-醇乙酸酯、顺-3-己烯-1-醇丁酸酯和α-法呢烯,且含量 较大。初步确定这些物质是对山楂叶螨具有引诱作用的主要物质,从而为利用天然生物活性物质防治山楂叶螨提供了理论 依据。     

Analysis of Flavonoid Metabolites in Watercress (Nasturtium officinale R. Br.) and the Non-Heading Chinese Cabbage (Brassica rapa ssp. chinensis cv. Aijiaohuang) Using UHPLC-ESI-MS/MS

Flavonoids from plants play an important role in our diet. Watercress is a special plant that is rich in flavonoids. In this study, four important watercress varieties were compared with non-heading Chinese cabbage by ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS). A total of 132 flavonoid metabolites (including 8 anthocyanins, 2 dihydroflavone, 3 dihydroflavonol, 1 flavanols, 22 flavones, 11 flavonoid carbonosides, 82 flavonols, and 3 isoflavones) were detected. Flavonoid metabolites varied widely in different samples. Both the non-heading Chinese cabbage and the variety of watercress from Guangdong, China, had their own unique metabolites. This work is helpful to better understand flavonoid metabolites between the non-heading Chinese cabbage and the other four watercress varieties, and to provide a reliable reference value for further research.     

Use of liquid chromatography/electrospray ionization tandem mass spectrometry to study the degradation pathways of terbuthylazine (TER) by Typha latifolia in constructed wetlands: identification of a new TER metabolite

S-Triazines are used worldwide as herbicides for agricultural and non-agricultural purposes. Although terbuthylazine (TER) is the second most frequently used S-triazine, there is limited information on its metabolism. For this reason, an analytical method based on liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) has been developed aiming at the identification of TER and its five major metabolites (desisopropyl-hydroxy-atrazine, desethyl-hydroxy-terbuthylazine, desisopropyl-atrazine, hydroxy-terbuthylazine and desethyl-terbuthylazine) in constructed wetland water samples. The separation of TER and its major metabolites was performed by reversed-phase high-performance liquid chromatography (HPLC) on a C(8) column using a gradient elution of aqueous acetic acid 1% (solvent A) and acetonitrile (solvent B), followed by MS/MS analysis on a triple quadrupole mass spectrometer. The data-depended analysis (DDA) scan approach has been employed and the main degradation pathways of both hydroxyl and chloro (dealkylated and alkylated) metabolites are elucidated through the tandem mass spectral (MS/MS) interpretation of triazine fragments under CID conditions. In addition, another major metabolite of TER, namely N2-tert-butyl-N4-ethyl-6-methoxy-1,3,5-triazine-2,4-diamine, has been identified. This methodology can be further employed in biodegradation studies of TER, thus assisting the assessment of its environmental impact.     

Proteomic profiling of a snake venom using high mass detection MALDI-TOF mass spectrometry

Proteomic profiling involves identification and quantification of protein components in complex biological systems. Most of the mass profiling studies performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been restricted to peptides and small proteins (<20 kDa) because the sensitivity of the standard ion detectors decreases with increasing ion mass. Here we perform a protein profiling study of the snake venom Sistrurus miliarius barbouri, comparing 2D gel electrophoresis and reversed-phase high-performance liquid chromatography (HPLC) with a high mass cryodetector MALDI-TOF instrument (Macromizer), whose detector displays an uniform sensitivity with mass. Our results show that such MS approach can render superior analysis of protein complexity compared with that obtained with the electrophoretic and chromatographic approaches. The summation of ion impacts allows relative quantification of different proteins, and the number of ion counts correlates with the peak areas in the reversed-phase HPLC. Furthermore, the sensitivity reached with the high mass cryodetection MS technology clearly exceeds the detection limit of standard high-sensitivity staining methods.     

GC/MS characterization of urinary metabolites of doxylamine succinate: Identification of the aglycones formed from intestinal microflora metabolism of the polar glucuronide metabolites

This study describes the use of high performance liquid chromatography (HPLC) and capillary gas chromatography/mass spectrometry (GC/MS) in the characterization of polar glucuronide conjugates of doxylamine and their subsequent aglycones following deconjugation. Rat urinary extracts which contained doxylamine and both nonconjugated and conjugated doxylamine metabolites, were examined by HPLC before and after incubation with rat intestinal microflora. The subsequent deconjugated urinary metabolites and the nonconjugated products remaining in the urinary extracts were then isolated, acetylated, and assayed by GC/MS. Incubation with the intestinal microflora indicated that anaerobic bacteria were capable of effecting hydrolytic cleavage of these polar O-glucuronide metabolites of doxylamine and its demethylated products to their subsequent aglycones. GC/MS analysis was performed using a fused silica DB-5 GC column and was utilized for the identification of these deconjugated metabolites.     

Optimized liquid chromatography-mass spectrometry approach for the isolation of minor stress biomarkers in plant extracts and their identification by capillary nuclear magnetic resonance

A LC-MS approach is presented for the isolation of minor key plant biomarkers, in view of their characterization by NMR at the microgram scale. Due to the complexity of plant extracts, the purification of metabolites present in low concentrations is critical. The strategy used relies on the optimization of the chromatographic analysis using ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOF-MS), thanks to modelling software. The optimized method is then transferred to semi-preparative LC conditions with MS detection. The approach is illustrated by the isolation of wound-induced jasmonate derivatives revealed by a metabolomic study in Arabidopsis thaliana leaves and their subsequent characterization by capillary NMR (CapNMR).     

Two-dimensional liquid chromatography/mass spectrometry set-up for structural elucidation of metabolites in complex biological matrices

For absorption, distribution, metabolism and excretion (ADME) studies of drug candidates, mass spectrometry (MS) has become an indispensable tool for the characterization of biotransformation pathways. Samples from in vivo animal studies such as plasma, tissue extracts or excreta contain vast amounts of endogenous compounds. Therefore, the generation of metabolite patterns requires dedicated sample pre-treatment and sophisticated separation methods. Methodologies used for metabolite separation are often inappropriate for structure elucidation. Therefore, a two-dimensional liquid chromatography (LC) approach in combination with MS was developed. Study samples were analyzed using high-performance liquid chromatography (HPLC) for the generation of a qualitative and quantitative metabolite pattern (first dimension) with high reproducibility and recovery without extensive sample pre-treatment. Selected radioactive metabolite fractions were then applied to micro-HPLC with off-line radioactivity monitoring and subsequent MS detection (second dimension). Applying the two-dimensional HPLC/MS approach not only major metabolites could be identified, even minor and trace metabolites were characterized. The usage of sampled metabolite fractions allowed also the re-analysis of specific metabolites for additional investigations (e.g. H/D exchange experiments or product ion scanning experiments). It could be clearly shown that the two-dimensional HPLC/MS approach showed mass spectra with higher sensitivity and selectivity significantly improving the characterization of minor and trace metabolites in in vivo ADME studies.     

Determination of theophylline in serum by high performance liquid chromatography/mass spectrometry with atmospheric pressure chemical-ionization (APCI HPLC/MS)

 A method for the determination of theophylline (TH), without derivatization, in serum by isotope dilution mass spectrometry using labelled [1, 3-¹⁵N₂-2-¹³C]theophylline (LTH) as internal standard is described. After deproteinization, the analyte is directly injected into a high performance liquid chromatography – mass spectrometer operating with atmospheric-pressure chemical-ionization (APCI HPLC/MS). The concentrations of TH in sera measured by APCI HPLC/MS are compared with results from gas chromatography – isotope dilution mass spectrometry (GC-ID/MS), high performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA). The accuracy, precision and recovery of the APCI HPLC/MS and GC-ID/MS methods are discussed. The coefficient of variation (CV) determined from duplicate samples was less than 2%. The detection limit was 10 ng/ml at a signal-to-noise ratio of 3:1. Received: 17 January 1996/Revised: 26 March 1996/Accepted: 5 April 1996     

Application of liquid chromatography-tandem mass spectrometry to the analysis of benzodiazepines in blood

A sensitive and selective high-performance liquid chromatography/atmospheric pressure chemical ionisation tandem mass spectrometry (HPLC/APCI-MS/MS) method for the simultaneous detection of 18 benzodiazepines and metabolites in human blood is described. The procedure utilises butyl chloride extraction at alkaline pH followed by reversed-phase liquid chromatography. The technique is suitable for screening analyses and confirmation of identity of the benzodiazepines at their lowest reported therapeutic concentrations using 500 microL of blood. The method has been successfully applied in forensic cases involving low concentrations of benzodiazepines.     

Mycosporine-glutamicol-glucoside: a natural UV-absorbing secondary metabolite of rock-inhabiting microcolonial fungi

Microcolonial ascomycetes are known to inhabit bare rock surfaces in cold and hot deserts and thus are habitually exposed to high levels of solar radiation. Several of these stress-tolerant fungal isolates, cultivated in the laboratory under daylight illumination, were studied for the presence of effective UV-radiation protection substances. Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses allowed for efficient separation and structure clarification of two mycosporines. It was demonstrated that both mycosporine-glutamicol-glucoside and mycosporine-glutaminol-glucoside are natural and constitutive secondary metabolites of microcolonial fungi. The function and relation of these substances in the fungal cell are discussed.     

Determination of chloroacetanilides, triazines and phenylureas and some of their metabolites in soils by pressurised liquid extraction, GC-MS/MS, LC-MS and LC-MS/MS

Pressurised liquid extraction (PLE) technique was used for the simultaneous extraction of phenylureas, triazines and chloroacetanilides and some of their metabolites from soils. Extractions were performed by mixing 15 g of dried soil with 30 mL of acetone under 100 atm at 50 degrees C, during 3 min and with three PLE cycles. Prior to the analysis of naturally contaminated soils, each of the five representative soil matrices used as blanks (of different depths) was spiked in triplicate with standards of each parent and degradation compound at about 10, 30 and 120 microg/kg. For each experiment, isoproturon-D6 and atrazine-D5 were used as surrogates. Analysis of phenylureas and metabolites of triazines and phenylureas was carried out by reversed phase liquid chromatography/mass spectrometry (LC-MS) and LC-MS/MS in the positive mode. Gas chromatography (GC)/ion trap mass spectrometry was used in the MS/MS mode for the parent triazines and chloroacetanilides. The average extraction recoveries were above 85%, except for didesmethyl-isoproturon, and quantification limits were between 0.5 and 5 microg/kg. The optimised multi-residue method was applied to soils and solids below the root zone, sampled from agricultural plots of a small French hydrogeological basin.