Characterization and determination of genipin-1-β-gentiobioside in gardenia fruit by high-performance liquid chromatography with ultraviolet detection following electrospray ionization mass spectrometry

A practical method for the analysis of genipin-1-β-gentiobioside in gardenia fruit was developed by high-performance liquid chromatography with ultraviolet monitoring and electrospray ionization mass spectrometry characterization (HPLC/UV/ESI-MS). Efficiency of ultrasonic extraction (UE), accelerated solvent extraction (ASE) and high-speed homogenizer extraction (HHE) on genipin-1-β-gentiobioside in gardenia fruit was compared. UE gave higher extraction efficiency and lower matrix interference, and it was employed. Analysis of genipin-1-β-gentiobioside was performed using a Supelco C₁₈ column with methanol–20 mM NH₄Ac (30:70, v/v) as the eluent. Flow rate was at 0.8 ml/min, and the detection was at 240 nm. The limit of detection (LOD), using HPLC/UV, was about 0.25 μg/ml, whereas the overall recovery was better than 96.0%. Both full scan MS and MS² of genipin-1-β-gentiobioside with positive polarity were obtained and elucidated. The specific ions were chosen to characterize genipin-1-β-gentiobioside in gardenia fruit sample. The proposed method has been successfully applied to measure the real samples and the content profiles of genipin-1-β-gentiobioside in gardenia fruit samples were obtained and evaluated.     

Separation and identification of ceramides in the human stratum corneum by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and electrospray multiple-stage mass spectrometry profiling

Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum coracum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramdies than that achievable by thin-layer chromatography. Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum corneum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramides than that achievable by thin-layer chromatography.     

Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono‐hexsosyl ceramide groups with good peak area repeatability (RSD% < 10) and linearity (R² > 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co‐existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.     

Simultaneous analysis of ten phytohormones in Sargassum horneri by high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry

Phytohormones have attracted wide attention due to their important biological functions. However, their detection is still a challenge because of their complex composition, low abundance and diverse sources. In this study, a novel method of high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry was developed and validated for the simultaneous determination of ten phytohormones including indole‐3‐acetic acid, isopentenyladenine, isopentenyl adenosine, trans‐zeatin riboside, zeatin, strigolactones, abscisic acid, salicylic acid, gibberellin A3, and jasmonic acid in Sargassum horneri (S. horneri). The phytohormones were extracted from freeze‐dried S. horneri with methanol/water/methanoic acid (15:4:1, v/v/v) analyzed on a Hypersil Gold C₁₈ column and detected by electrospray ionization tandem triple quadrupole mass spectrometry in the multiple reaction monitoring mode. The experimental conditions for the extraction and analysis of phytohormones were optimized and validated in terms of reproducibility, linearity, sensitivity, recovery, accuracy, and stability. Distributions of the phytohormones in the stems, blades, and gas bladder of the S. horneri in drift, fixed, and semi‐fixed growing states were investigated for the first time. The observed contents of the phytohormones in S. horneri range from not detected to 5066.67 ng/g (fresh weight). Most phytohormones are distributed mainly in the stems of S. horneri in drift and semi‐fixed states.     

啤酒中单糖的衍生化HPLC-ESI-MS测定方法研究

单糖类样品在溶液中非常稳定,难于离子化,不适合于进行ESI-MS检测。采用1-苯基-3-甲基-5-吡唑啉酮(PMP)将糖类物质衍生化,HPLC-ESI-MS在线联用,选择性离子扫描方式对几种啤酒样品中的5种单糖进行了分离检测。检出限可达到80pg。     

Determination of microcystin-LR in surface water using high-performance liquid chromatography/tandem electrospray ionization mass detector

The need for a rapid, sensitive, and reliable analytical method for microcystin-LR has been emphasized by the awareness of toxic cyanobacteria as a human-health risk through drinking water. Microcystin-LR is the most commonly reported microcystin which is produced by cyanobacteria. The WHO guideline for microcystin-LR in drinking water is 1 μg/l. In this paper, an effective method has been developed by application of high-performance liquid chromatography/tandem electrospray ionization mass detector in the determination of microcystin-LR in surface water sample. At the LC-MS-MS CID-full scan mode, different relative collision energies have been tested with 30% being used for further microcystin-LR analysis. The possible mass dissociation path has been proposed. Based on 30% relative collision energy, present method has an excellent method detection limit (MDL), which is as low as 2.6 ng/l. To the best of our knowledge, this represents one of the most sensitive methods in existence for the microcystin-LR analysis. This method has also been validated by evaluation of the calibration linearity, precision, accuracy, recovery, and mass ratio stability.     

Determination of clindamycin in animal plasma by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

A method for the quantification of clindamycin in animal plasma using high-performance liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS/MS) is presented. Lincomycin is used as the internal standard. The sample preparation includes a simple deproteinization step with trichloroacetic acid. Chromatographic separation is achieved on an RP-18 Hypersil column using gradient elution with 0.01 M ammonium acetate and acetonitrile as mobile phase. Good linearity was observed in the range 0-10 microg ml(-1). The limit of quantification of the method is 50 ng ml(-1) and the limit of detection is 1.3 ng ml(-1). The method was shown out to be of use for pharmacokinetic studies of clindamycin formulations in dogs.     

高效液相色谱-电喷雾串联质谱法测定饲料中的N-氨基甲酰-L-谷氨酸

建立了高效液相色谱-电喷雾串联质谱(HPLC-ESI-MS/MS)测定饲料中N-氨基甲酰-L-谷氨酸(NCG)含量的方法。饲料样品经甲醇提取、混合型强阴离子交换反相固相萃取(PXA)柱净化、HPLC分离后,采用ESI-MS/MS在正离子多反应监测(MRM)模式下进行检测,以碎片离子m/z 148.0和m/z 84.0进行定性,以碎片离子m/z 130.0进行定量。NCG的检出限(S/N >3)为24 μg/kg,定量限(S/N >10)为80 μg/kg,在20~1000 μg/L的质量浓度范围内峰面积与含量的线性关系良好,相关系数为0.9999。对饲料中NCG在80、200、500 mg/kg等3个添加水平下的回收率进行了测定,分别为104.0%、103.5%、95.3%,相对标准偏差分别为7.5%、6.3%、5.8%。结果表明,该方法操作简单,净化效果好,快速,灵敏度和准确度高,符合对饲料样品中NCG检测分析的要求。     

Structural characterization and identification of dibenzocyclooctadiene lignans in Fructus Schisandrae using electrospray ionization ion trap multiple-stage tandem mass spectrometry and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry

The electrospray ionization ion trap multiple-stage tandem mass spectrometry (ESI-MSⁿ) and electrospray ionization Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-ICR-MSⁿ) have been applied successfully to the direct investigation of a number of dibenzocyclooctadiene lignan constituents from the methanol extracts of the Fructus Schisandrae in the positive ion mode. The detailed structural characterization of the same skeleton and different peripheral substituents had been studied and the precise elemental compositions of ions at high mass resolution had been obtained. So the fragmentation mechanisms could be clarified. And the lignan components in Schisandra chinensis (Turcz.) Baill. fruits (SCF) and Schisandra sphenanthera Rehd. et Wils. fruits (SSF) were identified by comparing the structural information and fragmentation mechanisms. Then a pair of isobaric compounds was differentiated. Meanwhile these two similar fruits were distinguished. The research results demonstrated that ESI-MSⁿ technique is a sensitive, selective and effective tool for the direct analysis and rapid determination of constituents in complex mixtures from nature products. And these should be useful for the identification of similar compounds and differentiation of similar species from Chinese herbs.     

Nano-flow multidimensional liquid chromatography with electrospray ionization time-of-flight mass spectrometry for proteome analysis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the top five cancers with the highest incident of a disease worldwide. To understand the mechanisms of hepatocarcinogenesis, proteomics analysis provides a powerful tool to identify proteins that associate with HCC. We developed a two-step procedure for mapping of HCC proteomics. In the first step, in order to simplify the complexity of proteomics of HCC, the subfractionation of complex protein mixtures in HCC into “subproteomes” is presented based on the solubility of protein. While in the second step an automate comprehensive two-dimensional (2D) separation system, coupling strong cation-exchange (SCX) in the first dimension with capillary reversed-phase chromatography (cRPLC) in the second dimension is developed further to separate and analyze proteins associated with HCC. By using this system, complex sample can be injected, desalted, separated and analyzed in complete automatization. The procedure for proteomics analysis was found to be applied for proteins with great molecular mass (>100 000), small molecular mass (<20 000), highly basic (pI > 9.5) and hydrophobicity, which are not well resolved in 2D-gel electrophoresis. In total 229 proteins were identified by using the described proteomics platform. Among them, several proteins related to the process of carcinogenesis were investigated further.     

Fesoterodine stress degradation behavior by liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry

In the present study, a rapid validated stability-indicating LC method was established and comprehensive stress testing of fesoterodine was carried out according to ICH guidelines. Fesoterodine was subjected to stress conditions of acid and basic hydrolysis, oxidation, photolysis and thermal decomposition. The degradation products formed under stress conditions were investigated by LC-UV and LC-ESI-MS. Successful separation of the drug from its degradation products was achieved on a monolithic C₁₈ column (100 mm × 4.6 mm i.d.) maintained at 45 °C using acetonitrile-methanol-0.03 mol L⁻¹ ammonium acetate (pH 3.8) (30:15:55, v/v/v) as the mobile phase. The flow rate was 2.4 mL min⁻¹ and the detection wavelength was 208 nm. Validation parameters such as specificity, linearity, precision, accuracy, and robustness were evaluated. Chromatographic separation was obtained within 2.5 min and it was suitable for high-throughput analysis. Fragmentation patterns of degradation products formed under different stress conditions were studied and characterized through LC-ESI-MS fragmentation. Based on the results, a drug degradation pathway was proposed, and the validated LC method was successfully applied to the quantitative analysis of fesoterodine in tablet dosage forms, helping to improve quality control and to assure therapeutic efficacy.     

Authentication and quantitative analysis on the chemical profile of Xanthium fruit (Cang-Er-Zi) by high-performance liquid chromatography-diode-array detection tandem mass spectrometry method

A high-performance liquid chromatographic method using diode-array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) was developed for the qualitative and quantitative analysis on Xanthium fruit, a commonly used traditional Chinese medicine. In this study, 7 characteristic components, 1-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, chlorogenic acid, 1,5-O-di-caffeoylquinic acid, 1,3-O-di-caffeoylquinic acid, 4,5-O-di-caffeoylquinic acid and 1,3,5-O-tri-caffeoylquinic acid were identified and quantified by a validated HPLC-DAD method, and a fingerprint comprised of 12 markers was established under the same operating conditions. Furthermore, HPLC-ESI-MS/MS method was successfully used to deduce the structure of three main constituents. On the basis of the established chromatographic profiles, 30 populations of cocklebur samples including 3 related species and 1 unknown species were divided into 3 chemotypes, indicated that place of origin significantly influences the kinds and content of components in cocklebur, and hence affects their quality. The simultaneous determination of 7 caffeoylquinic acids in the 30 samples showed a great variety in the amounts of caffeoylquinic acids present. The study indicated that some species such as Xanthium mongolicum of the genus Xanthium might be suitable for development as new alternative sources of caffeoylquinic acids to supplement the officially listed Xanthium species, and the abundant constituents such as chlorogenic acid perhaps should be recorded in some authorized publications and applied to the quality control or quality evaluation for Xanthium in China. The entire analytical procedure is reproducible and suitable for the authentication and quantification of Xanthium fruits.     

Determination of cefquinome in pig plasma and bronchoalveolar lavage fluid by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

The aim of this study was to develop a rapid and sensitive method for the quantification of cefquinome in animal plasma and bronchoalveolar lavage (BAL) fluid using high-performance liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESI-MS/MS). Cefadroxil is used as internal standard. For plasma, the sample preparation includes a simple deproteinization step with a Microcon filter. This allows detecting the unbound cefquinome concentration, which is correlated with the concentration in other body fluids, such as BAL fluid. To be able to detect the total plasma concentration, deproteinization with acetonitrile, followed by a back-extraction of actonitrile with dichloromethane was performed. The BAL fluid is centrifuged to precipitate floating particles. Chromatographic separation is achieved on a PLRP-S column using 0.005% formic acid and methanol as mobile phase. For plasma, good linearity was observed in the range of 5-2500 ng ml(-1) for both the unbound and total concentration. The response in BAL fluid was linear in the range of 4-1000 ng ml(-1). The limit of quantification (LOQ) was set at 5.00 ng ml(-1) for plasma and at 4.00 ng ml(-1) for BAL fluid. The limit of detection (LOD) was 3.12 ng ml(-1) and 0.41 ng ml(-1) for the unbound and total concentration in plasma, respectively, and was 1.43 ng ml(-1) for BAL fluid. The method was shown to be of use in a pharmacokinetic study in pigs, where the correlation between cefquinome concentrations in plasma and BAL fluid of pigs was studied.     

Comparison of different extraction procedures for the comprehensive characterization of bioactive phenolic compounds in Rosmarinus officinalis by reversed-phase high-performance liquid chromatography with diode array detection coupled to electrospray time-of-flight mass spectrometry

In the present work, a comparative study between two environmentally friendly and selective extraction techniques, such as supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) have been carried out focusing in the bioactive phenolic compounds present in Rosmarinus officinalis. For the analysis of the SFE and PLE extracts, a new methodology for qualitative characterization has been developed, based on the use of reversed-phase high-performance liquid chromatography (RP-HPLC), equipped with two different detection systems coupled in series: diode array detector (DAD) and time of flight mass spectrometry (TOF-MS) detector connected via an electrospray ionization interface (ESI). The use of a small particle size C(18) column (1.8 μm) provided a great resolution and made possible the separation of several isomers. Moreover, UV-visible spectrophotometry is a valuable tool for identifying the class of phenolic compounds, whereas MS data enabled to structurally characterize the compounds present in the extracts. The applied methodology was useful for the determination of many well-known phenolic compounds present in R. officinalis, such as carnosol, carnosic acid, rosmadial, rosmanol, genkwanin, homoplantaginin, scutellarein, cirsimaritin and rosmarinic acid, as well as other phenolic compounds present in other species belonging to Lamiaceae family.     

Characterization of peptides by liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a post-column complexant

Two model peptides, des-Arg1-bradykinin (DAB) and bradykinin (B), were cationized by Ag+ after their separation by reversed-phase liquid chromatography (RPLC) prior to mass spectrometry (MS). Silver nitrate solution was used as a post-column reagent. The RPLC and MS experimental conditions were optimized using flow injection in order to obtain sufficiently abundant silver adducts to permit MS/MS experiments. The use of water-methanol with 0.1% formic acid as mobile phase allowed a good chromatographic separation of the two peptides with a polymeric stationary phase and sufficiently abundant silver-containing adducts, [M + Ag + H]2+ and [M + 2Ag]2+. The gas-phase dissociation of [DAB + Ag + H]2+ and [DAB + 2Ag]2+ led to interpretable mass spectra during the on-line cationization experiment. Most of the ions obtained by dissociating [DAB + Ag + H]2+ and [DAB + 2Ag]2+ species are silver-containing ions but the ions produced depend on the parent. The ions coming from the dissociation of the doubly charged silver adducts [DAB + Ag + H]2+ or [DAB + 2Ag]2+ are of interest compared with those coming from the singly charged silver species or doubly charged protonated species. The fragmentation of the doubly charged silver adducts provides ions over the entire mass range. Although the presence of several prolines in des-Arg1-bradykinin prevents the formation of some expected ions, the observation of triplets [an-H + Ag]+, [bn-H + Ag]+ and [bn + OH + Ag]+ produced by the dissociation of on-line Ag(+)-cationized peptides could contribute to greater success of automatic sequencing of peptides.     

Investigations on the degradation of aspartame using high-performance liquid chromatography/tandem mass spectrometry

Aspartame is a widely used sweetener,the long-term safety of which has been controversial ever since it was accepted for human consumption.It is unstable and can produce some harmful degradation products under certain storage conditions.A high-performance liquid chromatography/tandem mass spectrometry method was developed for the simultaneous analysis of aspartame and its four degradation products,including aspartic acid,phenylalanine,aspartyl-phenylalanine and 5-benzyl-3,6-dioxo-2-piperazieacetic acid in water and in diet soft drinks.Aspartame and its four degradation products were quantified by a matrix matched external standard calibration curve with excellent correlation coefficients.The limits of detection were 0.16–5.8 mg/L,which exhibited higher sensitivity than common methods.This method was rapid,sensitive,specific and capable of eliminating matrix interferences.It was also applied to the study of the degradation of aspartame at various pH and temperatures.The results indicated that aspartame was partly degraded under strong acidic or basic conditions and the extent of degradation increased with increasing temperature.     

Quantitative determination of domperidone in human plasma by ultraperformance liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for determining domperidone in human plasma. The analyte and internal standard (IS; mosapride) were isolated from plasma samples by protein precipitation with methanol (containing 0.1% formic acid). The chromatographic separation was performed on an Xterra MS C(18) Column (2.1 x 150 mm, 5.0 microm) with a gradient programme mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.30 mL/min. The total run time was 4.0 min. The analyses were carried out by multiple reaction monitoring using the parent-to-daughter combinations m/z 426 --> 175 and m/z 422 --> 198 (IS). The areas of peaks from the analyte and IS were used for quantification of domperidone. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification was 0.2 ng/mL, and the assay exhibited a linear range of 0.2-60.0 ng/mL and gave a correlation coefficient (r(2)) of 0.999 or better. Quality control samples (0.4, 0.8, 15 and 50 ng/mL) in six replicates from three different analytical runs demonstrated an intra-assay precision (RSD) 4.43-6.26%, an inter-assay precision 5.25-7.45% and an overall accuracy (relative error) of <6.92%. The method can be applied to pharmacokinetic and bioequivalence studies of domperidone.     

Simultaneous quantification of three isoflavonoid glycosides in rabbit plasma after oral administration of Astragalus mongholicus extract by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A selective and sensitive liquid chromatography coupled with triple stage quadruple tandem mass spectrometry (HPLC/TSQ-MS/MS) was developed and validated for simultaneous quantification of calycosin-7-O-β-d-glycoside (CCSG), formononetin-7-O-β-d-glycoside (Ononin) and (6R,10R)-9,10-dimethoxypterocarpan-3-O-β-d-glycoside (DPG) in rabbit plasma. Plasma samples were extracted with solid-phase extraction (SPE), separated on an Inertsil ODS-3 column and detected by tandem mass spectrometry with electrospray ionization (ESI) interface in positive selective reaction monitoring (SRM) mode. 3,7,8-Trimethoxy-xanthone-1-O-primaverose was used as internal standard (IS) for quantitative measurement. For each analyte, one major product ion was chosen and used for screening of it. Calibration curves were generated over the range of 2-1000 ng mL⁻¹ with the correlation coefficients greater than 0.99 by using a weighted (1/χ) least squares linear regression. The method had the lower limit quantification of 0.15, 0.21 and 0.19 for CCSG, Ononin and DPG, respectively, with precision less than 20%. The intra- and inter-day precisions ranged from 2.48 to 6.38% and 4.81 to 11.78% (R.S.D.%), respectively. This assay is suitable for determining the above three trace glycosides in rabbit plasma simultaneously and thus investigating the pharmacokinetics of glycosides from Astragalus mongholicus extract in rabbits.     

Identification of indigoid dyes in natural organic pigments used in historical art objects by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry

Pigments are among the most important components of historical paintings and textiles and their nature provides the unique character of color. They can be divided into two main groups: inorganic and organic, extracted from plants or animals. Their identification is a necessary stage in the conservation of art objects. Reversed-phase liquid chromatography with electrospray ionization mass spectrometry (ESI-MS) and UV/visible spectrophotometric methods were elaborated for the identification of indigoid (indigo, indirubin, isoindigo, isoindirubin) color components of natural dyestuffs and their natural or synthetic precursors (indican, isatin, indoxyl, 2-indolinone). ES-MS offers detection limits in the range 0.03-5.00 microg ml(-1) for the color compounds examined. The method developed made it possible to identify indigo and its isomers in genuine Indian indigo, indigo from woad and Tyrian Purple. It was applied to the identification of natural dyes on fiber from a 19th century Japanese tapestry, 'Cranes in the landscape'. A procedure based on freezing and grinding of a sample before the extraction of dyes from the textile was developed. The components of the extract obtained were identified after acidic hydrolysis as indigotin and methylene blue.