Analytical investigation of secondary metabolites extracted from Camellia sinensis L. leaves using a HPLC‐DAD‐ESI/MS data fusion strategy and chemometric methods

Considerable attention has been paid to the study of green tea leaves because of their high consume and beneficial effects on human health. In this work, an appropriate strategy is proposed to investigate and resolve the major metabolites extracted from Camellia sinensis tea leaves. Statistical design mixtures of ethanol, ethyl acetate, dichloromethane, and chloroform were used to study the effects of different solvents and their mixtures on the extraction of the secondary metabolites of C. sinensis tea leaves from two different harvest seasons. Extracted samples were analyzed by high performance liquid chromatography‐diode array detection‐electrospray ionization mass spectrometer allowing the resolution of a large amount of tea metabolites with high relative abundances, especially when their extraction was performed in pure ethanol and with solvent mixtures with ethanol. Resolution of the more relevant metabolites was achieved by the simultaneous analysis of the fused diode array detection and mass spectrometer detectors data from the same samples using the multivariate curve resolution‐alternating least squares chemometric method. Peak areas finally resolved were further analyzed by orthogonal signal correction and partial least squares‐discrimination analysis to discriminate among C. sinensis tea samples. Using the Variable Importance in Projection variable selection method, epigallocatechin and caffeine were finally selected as the two more important chemical constituents of tea leaves that were discriminating more between the tea samples from two different harvest seasons. Copyright © 2016 John Wiley & Sons, Ltd.     

Analysis of global components in Ganoderma using liquid chromatography system with multiple columns and detectors

In present study, a multiple columns and detectors liquid chromatography system for analysis of global components in traditional Chinese medicines was developed. The liquid chromatography system was consist of three columns, including size exclusion chromatography column, hydrophilic interaction chromatography column, and reversed phase chromato‐graphy column, and three detectors, such as diode array detector, evaporative light scattering detector, and mass spectrometry detector, based on column switching technique. The developed multiple columns and detectors liquid chromatography system was successfully applied to the analysis of global components, including macromolecular (polysaccharides), high (nucleosides and sugars)‐, and low (triterpenes)‐polarity small molecular compounds in Ganoderma, a well‐known Chinese medicinal mushroom. As a result, one macromolecular chromatographic peak was found in two Ganoderma species, 19 components were identified in Ganoderma lucidum (two sugars, three nucleosides, and 14 triterpenes), and four components (two sugars and two nucleosides) were identified in Ganoderma sinense. The developed multiple columns and detectors liquid chromatography system was helpful to understand comprehensive chemical characters in TCMs.     

Tests for liquid chromatographs

Summary The paper describes tests for liquid chromatographs to assess the autosampler and pump flow rate precision; suitability of the instrument for normal phase chromatography; UV detector performance; and external band broadening characteristics. Tests for mixing and proportioning characteristics of solvent pumps and performance of diode array detectors are also considered. These tests are designed to evaluate the performance of both newly released liquid chromatography equipment and equipment already in operation. Data from evaluations carried out by instrument manufacturer’s and suppliers is reported and discussed. This data offers specifications to a common format for initial selection of instruments prior to purchase.     

Fast ultra‐high‐performance liquid chromatography with diode array and mass spectrometry method for determination of tadalafil drug substance and its impurities

Tadalafil is used for the treatment of erectile dysfunction. Its related patents expired in 2016, and so related generic drug production is predicted to be increased. This work is focused on developing a fast ultra‐high‐performance liquid chromatography with diode array detection and/or mass spectrometry detection for the separation and determination of tadalafil and its impurities in pharmaceutical samples. A modern reversed‐phase stationary phase with sub‐2 μm particle size, Zorbax StableBond Rapid Resolution High Definition with octylsilane chemically bonded phase to totally porous silica particles, was used for the solving this problem. Column temperature was set at 40 ± 0.1°C. A mobile phase consisting of acetonitrile and aqueous solution of 0.1% (v /v) trifluoroacetic acid for diode array detection detection and 0.05% (v /v) formic acid, both running at a flow rate of 0.62 mL/min, were used to achieve the required separation of all components within a 5 min run. The limit of detection was 3.5 μg/L and the limit of quantification was 10.0 μg/L for the method for both UV and MS detectors. Accurate mass spectra of tadalafil's related impurities are shown for advanced confirmation. The method is directly transferable to routine analysis of tadalafil in pharmaceutical and control laboratories.     

Chemometric strategies for enhancing the chromatographic methodologies with second-order data analysis of compounds when peaks are overlapped

This overview covers the different chemometric strategies linked to chromatographic methodologies that have been used and presented in the recent literature to cope with problems related to incomplete separation, the presence of unexpected components in the sample, matrix effect and changes in the analytical signal due to pre-treatment of sample.Among the different chemometric strategies it focuses on pre-treatment of data to correct background and time shift of chromatographic peaks and the use of second-order algorithms to cope with overlapping peaks from analytes or from analytes and interferences in liquid chromatography coupled to diode array, fast-scanning fluorescence spectroscopy and mass spectrometry detectors. Finally the review presents the strategies used to deal with changes in the analytical response as result of matrix effect in liquid and gas chromatography, as well as the use of standardization strategies to correct modifications in the analytical signal as a consequence of sample pre-treatment in liquid chromatography.     

Assessing stability‐indicating methods for coelution by two‐dimensional liquid chromatography with mass spectrometric detection

Chromatographic analysis of trace organic impurities/degradants coeluting in the midst of active pharmaceutical ingredient can be challenging given similarities in their structures and differences in their relative levels/intensities. Conventional detection techniques such as diode array detection and mass spectrometry are often inadequate to detect/identify these residual coeluting impurities and could result in a false negative. Application of two‐dimensional chromatography to address/evaluate coelution in conventional chromatography is presented. Areas of interest, usually corresponding to the main component, are transferred to secondary column/s for further separation termed as pseudocomprehensive two‐dimensional liquid chromatography. Coelution, if any, in the rest of the chromatogram is monitored using conventional detectors. In this work, the use of similar and complementary phases in both dimensions is presented. The use of the same phase in both dimensions to resolve coeluting impurities (especially in the front and tail of the main component differing by orders of magnitude) is an easier alternative to finding complementary column/s, as hydrophobicity dominates reversed‐phase separation. The same phase separation is practical as relative levels of impurities and main component in some transferred fractions are comparable enabling their separation. The results were confirmed using mass spectrometry. This work has significant bearing as a method assessment tool in pharmaceutical and other industries.     

A Comparison of the Properties of Contactless Conductivity and Diode‐Array Photometric Detectors in Analyses of Low‐Molecular,Biologically Active Substances by Capillary Electrophoresis in Acetic Acid Solutions

The performance of the contactless conductivity (C⁴D) and diode array photometric (DAD) detectors has been compared for CE separations of creatinine, arginine and 3‐methylhistidine in acetic acid background electrolytes. The contactless conductivity detector response has also been modeled. It has been found that the two detectors provide similar responses and can readily be used for dual CE detection. Changes in the acetic acid concentration affect the C⁴D noise less than the DAD noise, but their effect on the C⁴D response to the analytes is greater than with DAD. In general, C⁴D provides better detection results at higher acetic acid concentrations, while DAD is more sensitive and reliable at very low ones. Capillaries with greater internal diameters are preferable for both detectors, provided that the separation efficiency is not adversely affected. Acetic acid is a suitable background electrolyte for CE separations of small, basic organic molecules.     

Simultaneous extraction and analysis by high performance liquid chromatography coupled to diode array and mass spectrometric detectors of bixin and phenolic compounds from annatto seeds

This study was designed to identify and quantify the carotenoids and phenolic compounds from annatto seeds using high performance liquid chromatography coupled to diode array and mass spectrometer detectors (HPLC-DAD-MS/MS). Furthermore, using response surface methodology, an optimized procedure for simultaneous extraction of these compounds was established. In addition to bixin, known to be the main carotenoid in annatto seeds, hypolaetin and a caffeoyl acid derivative were identified as the main phenolic compounds. The optimized procedure involved 15 extractions using acetone:methanol:water (50:40:10, v/v/v) as solvent, a solid-liquid ratio of 1:9 (m/v) and an extraction time of 5 min. Validation data indicated that the HPLC method proposed provided good linearity, sensitivity, procedure accuracy, system precision and suggested its suitability for the simultaneous analysis of phenolic compounds and carotenoids in annatto seeds.     

Metabolic profile of glyburide in human liver microsomes using LC‐DAD‐Q‐TRAP‐MS/MS

The sulfonylurea urea drug glyburide (glibenclamide) is widely used for the treatment of diabetes milletus and gestational diabetes. In previous studies monohydroxylated metabolites were identified and characterized for glyburide in different species, but the metabolite owing to the loss of cyclohexyl ring was identified only in mouse. Glyburide upon incubation with hepatic microsomes resulted in 10 metabolites for human. The current study identifies new metabolites of glyburide along with the hydroxylated metabolites that were reported earlier. The newly identified drug metabolites are dihydroxylated metabolites, a metabolite owing to the loss of cyclohexyl ring and one owing to hydroxylation with dehydrogenation. Among the 10 identified metabolites, there were six monohydroxylated metabolites, one dihydroxylated metabolite, two metabolites owing to hydroxylation and dehydrogenation, and one metabolite owing to the loss of cyclohexyl ring. New metabolites of glyburide were identified and characterized using liquid chromatography–diode array detector–quadruple‐ion trap–mass spectrometry/mass spectrometry (LC‐DAD‐Q‐TRAP‐MS/MS). An enhanced mass scan–enhanced product ion scan with information‐dependent acquisition mode in a Q‐TRAP‐MS/MS system was used to characterize the metabolites. Liquid chromatography with diode array detection was used as a complimentary technique to confirm and identify the metabolites. Metabolites formed in higher amounts were detected in both diode array detection and mass spectrometry detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison of High Performance Liquid Chromatography Methods with Different Detectors for Determination of Steroid Hormones in Aqueous Matrices

This paper presents the development and comparison of procedures for the qualitative and quantitative determination of five estrogenic compounds (17-α-ethynylestradiol, estrone, estradiol, estriol, and progesterone) in drinking water and wastewater samples. Five extraction columns and two disks were tested for their efficiency. The C₁₈ columns were superior on the basis of cost for solid phase extraction of drinking water or sewage. However, the best recoveries were achieved using extraction disks. High-performance liquid chromatography with diode array, fluorescence, and tandem mass spectrometry detectors were compared for selectivity, repeatability, and linearity of response. Solid phase extraction with high-performance liquid chromatography and tandem mass spectrometry was the most sensitive, efficient, and precise for the determination of hormones in drinking water and wastewater. This method satisfied analytical validation criteria and was characterized by a low limit of detection at the pg-ng/L level. The study also considered challenges that emerged for steroidal hormone determination in aqueous samples.     

Determination of benzoylurea insecticide residues in tomatoes by high-performance liquid chromatography with ultraviolet-diode array and atmospheric pressure chemical ionization-mass spectrometry detection

A simple and sensitive method using high-performance liquid chromatography/ mass spectrometry (LC/MS) was developed and validated for simultaneous determination of 5 benzoylurea insecticides-diflubenzuron, triflumuron, teflubenzuron, lufenuron, and flufenoxuron-in tomatoes. Residues were successfully separated on a C18 column by methanol-water isocratic elution. Detection was carried out by an ultraviolet diode array detector (UV-DAD) coupled with a quadrupole mass spectrometer, using atmospheric pressure chemical ionization (APCI) in negative-ion mode. The main ions were the deprotonated molecules [M-H]- for triflumuron, and the anions formed by elimination of hydrofluoric acid [M-H-HF]- for diflubenzuron and flufenoxuron, and [M-2H-HF] for lufenuron and teflubenzuron. The calibration plots were linear for both detectors over the range 0.05 to 10 microg/mL, and the method presented good quality parameters. The limits of detection for standard solutions were 0.008-0.01 mg/L (equivalent to 0.08-0.1 ng injected) for both detectors, and the limits of quantification (LOQs) were approximately 10 times lower than national maximum residue levels (MRLs). Depending on the compound and the detector, the LOQ values ranged from 0.2 to 0.4 ng injected. The optimum LC-UV-DAD/APCI-MS conditions were applied to the analysis of benzoylureas in tomatoes. The obtained recoveries from fortified tomato samples (50 g), extracted with ethyl acetate and purified by solid-phase extraction on silica sorbent, were 88-100 and 92.9-105% for the UV-DAD and MS detectors, respectively, with precision values (relative standard deviations) of 2.9-11 and 3.7-14%, respectively. The method was applied to 12 tomato samples from local markets, and diflubenzuron and lufenuron were detected in only one sample at concentrations lower than the MRLs. The results indicate that the developed LC/MS method is accurate, precise, and sensitive for quantitative and qualitative analysis at low levels of benzoylureas required by legislation.     

Separation and identification of compounds in Rhizoma chuanxiong by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry

A comprehensive two-dimensional liquid chromatographic separation system based on the combination of a CN column and an ODS column is developed for the separation of components in a traditional Chinese medicine (TCM) Rhizoma chuanxiong. Two columns are coupled by a two-position, eight-port valve equipped with two storage loops and controlled by a computer. The effluent is detected by both the diode array detector and atmospheric pressure chemical ionization (APCI) mass spectrometer. More than 52 components in the methanol extract of R. chuanxiong were resolved and 11 of them were preliminary identified according to their UV and mass spectra.     

Deconvolution of mass spectra measured with a non-uniform detector array to give accurate ion abundances

Array detectors are under active development since they offer large improvements in the efficiency of mass spectrum measurements. High quality is always a requirement whether array detectors are used for ions, electrons, UV, x-rays, etc., but in mass spectrometry the very high accuracy frequently needed in ion abundance measurements for isotope ratios is unique. These demands necessitate modelling the measurement process and careful deconvolution of the measured data. A linear model in terms of matrix algebra is presented in which the incident spectrum (unknown) and the measured spectrum are represented by column matrices and the action of the detector array on the incident spectrum is represented by an experimentally measurable square 'A' matrix. Residual noise in this matrix can be minimized following a singular value decomposition procedure and its use in 'forward deconvolution' of measured spectra is discussed. The random error in the incident ion counts is accounted for after the deconvolution since this is not an error from the perspective of the detector. The microchannel plate electron multiplier gain distribution is an important feature of the deconvolution.     

On-Line Screening and Identification of Radical Scavenging Compounds Extracted from Flos Lonicerae by LC-DAD–TOF-MS

Flos Lonicerae is commonly used in traditional Chinese medicine. The ethanolic extracts prepared from Flos Lonicerae were used for the rapid evaluation of free radical scavenging activity by using the extracts to clear on-line 2,2′-diphenyl-1-picrylhydrazyl (DPPH·) free radical with LC serving as the separating tool of bioactive compounds, two diode array detectors (DAD) and a time-of-flight mass spectrometer (TOF-MS) as qualitation tools. The LC-DPPH on-line method of Flos Lonicerae extracts revealed the presence of four radical scavenging compounds, which were simultaneously identified as chlorogenic acid, rutin, and two isomers of isochlorogenic acid by high-resolution TOF-MS. To the best of our knowledge, it is original to demonstrate the rapid and successful use of LC-DPPH-DAD–TOF-MS to directly and simultaneously screen and identify the anti-free radical compounds in the extracts of Flos Lonicerae without any purification. The present method provides a useful improvement on the reducing of workload     

Simultaneous determination of antidepressants by non-aqueous capillary electrophoresis-time of flight mass spectrometry

Simultaneous determination of 20 antidepressants in plasma samples was carried out by non-aqueous capillary electrophoresis with time of flight mass spectrometry via electrospray ionization, where a mixture of 60 mM ammonium acetate and 1M acetic acid in acetonitrile, and water, as well as methanol (100:1:0.5, v/v/v) was selected as the background electrolyte. By using time of flight mass spectrometry, accurate mass information was obtained and the background noise was dramatically decreased, thus causing a great improvement in qualitative ability. As for the plasma sample, solid phase extraction with Oasis HLB was used. The limits of detection and quantification were in the range of 0.5-1 and 1-5 ng/ml, respectively. The sensitivity of the present method was found better, i.e. approximately 10-60 folds compared to that using photo diode array detectors because the analyte peak could be clearly distinguished from the background derived from the plasma. The present method was found very useful and practical as regards to routine analysis of plasma samples.     

High performance liquid chromatography/electrospray mass spectrometry of Hypericum perforatum extracts

Hypericum perforatum L. (St. John's Wort) is a widely distributed herbaceous perennial plant which has been well known as a medicinal plant since antiquity. In recent years, H. perforatum has received increasing attention for the treatment of depression and other neuralgic disorders. The main constituents of H. perforatum extract include flavonoids, naphthodianthrones, phloroglucinols, essential oils and xanthones. The present work reports the analysis of naphthodianthrones and phloroglucinols in H. perforatum extracts by means of high performance liquid chromatography (HPLC) coupled simultaneously to a diode array detector (DAD) and electrospray mass spectrometry (ESI-MS). Hypericin, pseudohypericin, hyperforin and adhyperforin were separated and identified on the base of their on-line UV and mass spectra. Quantitative analysis of hypericin derivatives in different extracts of H. perforatum using DAD and MS detectors was performed. In addition, direct infusion ESI-MS of H. perforatum extracts was applied to obtain rapid mass fingerprints of constituents present in the sample.     

Detection systems for mass spectrometry imaging: A perspective on novel developments with a focus on active pixel detectors

Instrumental developments for imaging and individual particle detection for biomolecular mass spectrometry (imaging) and fundamental atomic and molecular physics studies are reviewed. Ion‐counting detectors, array detection systems and high mass detectors for mass spectrometry (imaging) are treated. State‐of‐the‐art detection systems for multi‐dimensional ion, electron and photon detection are highlighted. Their application and performance in three different imaging modes – integrated, selected and spectral image detection – are described. Electro‐optical and microchannel‐plate‐based systems are contrasted. The analytical capabilities of solid‐state pixel detectors – both charge coupled device (CCD) and complementary metal oxide semiconductor (CMOS) chips – are introduced. The Medipix/Timepix detector family is described as an example of a CMOS hybrid active pixel sensor. Alternative imaging methods for particle detection and their potential for future applications are investigated. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a system to evaluate compound identity, purity, and concentration in a single experiment and its application in quality assessment of combinatorial libraries and screening hits

The development and use of a new assay system for the simultaneous determination of identity, purity, and concentration of sample components from combinatorial libraries produced by parallel synthesis are described. The system makes use of high-performance liquid chromatography with UV/vis photodiode array (PDA), evaporative light scattering (ELSD), chemiluminescent nitrogen (CLND), and time-of-flight mass spectrometer (TOFMS) detectors (HPLC-PDA-ELSD-CLND-TOFMS). Although these detectors have previously been utilized separately for the analysis of combinatorial chemistry libraries, the use of TOFMS along with CLND provides a synergistic combination enabling target and side-product structures to be identified and their concentrations and purities determined in a single experiment from a solution containing microgram levels of material. The CLND was found to give a linear response based on the number of moles of nitrogen present. Therefore, if the number of nitrogens per molecule is known, the concentration of each nitrogen-containing sample component may be determined utilizing an unrelated co-injected standard. A molecular formula for an impurity may often be calculated from the exact mass determined by the TOFMS and knowledge of the chemistry involved. Thus, if the sample components contain nitrogen, the concentration of every identified HPLC peak may be determined even in the absence of primary standards. This combination of detectors enabled the characterization of both target compounds and byproducts in combinatorial libraries, allowing the optimization of library synthetic procedures. This system was also used to survey the quality of libraries, enabling the selection of the best libraries for screening. This method also facilitated the characterization of samples from combinatorial libraries found as hits in high-throughput screening to establish the potency of the leads based on their actual concentration. In addition, concentrations and potencies of impurities were determined after identification of their structures, utilizing exact mass data, determination of charge states, and knowledge of the synthetic chemistry.     

Detection, characterization and identification of crucifer phytoalexins using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry

We have analyzed 23 crucifer phytoalexins (e.g. brassinin, dioxibrassinin, cyclobrassinin, brassicanals A and C) by HPLC with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) using both negative and positive ion modes. Positive ion mode ESI-MS appeared more sensitive than negative ion mode ESI-MS in detecting this group of compounds. A new HPLC separation method, new LC-MS and LC-MS(2) data and proposed fragmentation pathways, LC retention times, and UV spectra for selected compounds are reported.