Determination of pharmacologically active compounds in root extracts of Cassia alata L. by use of high performance liquid chromatography

A simple high performance liquid chromatography (HPLC) method was developed and validated for the determination of six phenolic compounds, five anthraquinones (rhein, aloe-emodin, emodin, chrysophanol and physcion) and a flavonoid (kaempferol), in root extracts from Cassia alata L. Solid-phase extraction, using C(18) cartridges, was used to remove interfering substances from the root extracts. The extracts were analyzed on a C(18) column using an isocratic mobile phase which consisted of acetonitrile, methanol, and 10mM aqueous ammonium acetate (25:55:20, v/v). Identification of the analytes was performed by use of standards and on-line mass spectrometric detection using atmospheric pressure chemical ionization. The concentration of the phenolic compounds in the root extracts was determined using HPLC with ultraviolet detection at 260nm. The limits of detection obtained for the anlytes were in the range of 0.23-4.61ppm. The overall R.S.D. precision values (intra- and inter-day) for the retention times and peak-areas were lower than 0.16 and 2.10%, respectively. In addition, the recovery of the developed method for the analysis of these phenolic compounds was determined, and ranged from 81.2+/-4.3 to 106+/-2%.     

Drug detection in hair by chromatographic procedures.

This article reviews the analysis of 31 drugs and drug metabolites in human hair by thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gas chromatography-mass spectrometry and mass spectrometry. The most important detection method after chromatographic separation of the components is the mass spectrometry because of its sensitivity and specificity. Washing steps to exclude external contamination, extraction, derivatization, stationary phases, detection modes and detection limits of the mass spectrometric and gas chromatographic-mass spectrometric procedures are presented in five tables. Additionally, a method for a gas chromatographic-mass spectrometric screening procedure is presented.     

Analysis of low-molecular-mass inorganic and organic anions by ion chromatography-atmospheric pressure ionization mass spectrometry.

Different nonvolatile mobile phases have been tested for the combination of ion-exchange chromatography combined with mass spectrometric detection of anions and organic acids. Buffer systems based on carbonate, sulfate, oxalate and citrate as the eluting species have been used. Among these, citrate proved to be the most versatile eluent allowing the separation of anions with absolute detection limits between 0.4 and 0.7 ng and of organic acids with detection limits between 0.4 and 4 ng in the non-suppressed mode. In the suppressed eluent mode iodate, bromate and chlorate could be separated using sodium carbonate as the mobile phase resulting in detection limits of 50 pg. The method was applied to the analysis of water samples containing oxyhalides originating from ozonization. Additionally, organic acids were separated by chromatographic separation techniques like reversed-phase, ion-pair or ion-exclusion chromatography and the compatibility with mass spectrometry was investigated with special respect to sensitivity of this detection mode.     

Identification of organic sulfur compounds in coal bitumen obtained by different extraction techniques using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometric detection

The determination of organic sulfur compounds (OSC) in coal is of great interest. Technically and operationally these compounds are not easily removed and promote corrosion of equipment. Environmentally, the burning of sulfur compounds leads to the emission of SO _x gases, which are major contributors to acid rain. Health-wise, it is well known that these compounds have mutagenic and carcinogenic properties. Bitumen can be extracted from coal by different techniques, and use of gas chromatography coupled to mass spectrometric detection enables identification of compounds present in coal extracts. The OSC from three different bitumens were tentatively identified by use of three different extraction techniques: accelerated solvent extraction (ASE), ultrasonic extraction (UE), and supercritical-fluid extraction (SFE). Results obtained from one-dimensional gas chromatography (1D GC) coupled to quadrupole mass spectrometric detection (GC–qMS) and from two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC × GC–TOFMS) were compared. By use of 2D GC, a greater number of OSC were found in ASE bitumen than in SFE and UE bitumens. No OSC were identified with 1D GC–qMS, although some benzothiophenes and dibenzothiophenes were detected by use of EIM and SIM modes. GC × GC–TOFMS applied to investigation of OSC in bitumens resulted in analytical improvement, as more OSC classes and compounds were identified (thiols, sulfides, thiophenes, naphthothiophenes, benzothiophenes, and benzonaphthothiophenes). The roof-tile effect was observed for OSC and PAH in all bitumens. Several co-elutions among analytes and with matrix interferents were solved by use of GC×GC.     

Identification of derivatives of bisphenol A diglycidyl ether and novolac glycidyl ether in can coatings by liquid chromatography/ion trap mass spectrometry

A reversed-phase liquid chromatographic method combined with fluorescence and multiple mass spectrometric detection in series is presented for the separation and structure elucidation of bisphenol A diglycidyl ether (BADGE) and novolac glycidyl ether (NOGE) derivatives. Atmospheric pressure chemical ionization in the positive ion mode and collision induced fragmentation in the ion trap allowed identification of BADGE- and NOGE-related compounds originating from reactions of the glycidyl ethers with bisphenols, solvents, and chain stoppers. Two extracts from food-can coatings were investigated in detail. It was possible to elucidate the structures of many substances and consequently to draw conclusions about the production of the lacquers.     

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.     

Thin-layer chromatography/electrospray ionization triple-quadrupole linear ion trap mass spectrometry system: analysis of rhodamine dyes separated on reversed-phase C8 plates

The direct analysis of separated rhodamine dyes on reversed-phase C(8) thin-layer chromatography plates using a surface sampling/electrospray emitter probe coupled with a triple-quadrupole linear ion trap mass spectrometer is presented. This report represents continuing work to advance the performance metrics and utility of this basic surface sampling electrospray mass spectrometry system for the analysis of thin-layer chromatography plates. Experimental results examining the role of sampling probe spray end configuration on liquid aspiration rate and gas-phase ion signal generated are discussed. The detection figures-of-merit afforded by full-scan, automated product ion and selected reaction monitoring modes of operation were examined. The effect of different eluting solvents on mass spectrum signal levels with the reversed-phase C(8) plate was investigated. The combined effect of eluting solvent flow-rate and development lane surface scan rate on preservation of chromatographic resolution was also studied. Analysis of chromatographically separated red pen ink extracts from eight different pens using selected reaction monitoring demonstrated the potential of this surface sampling electrospray mass spectrometry system for targeted compound analysis with real samples.     

Increasing the sensitivity of an LC-MS method for screening material extracts for organic extractables via mobile phase optimization

Organic extractables (substances extracted from materials used in pharmaceutical packaging) are discovered, identified, and quantified via screening of extracts with analytical methods including liquid chromatography with mass spectrometric detection (LC-MS). Because extractables include a large number of diverse compounds that are typically present in plastic extracts at low levels, the LC-MS methods must be broad scope and sensitive. To accomplish these objectives, screening studies typically couple gradient reversed-phase separations with electrospray MS detection (both positive and negative ion modes). While such methods are generally applicable for a number of extractables, they are not optimal for some commonly encountered extractables due to either poor chromatographic performance (e.g., peak tailing) or poor MS response. Modifications to mobile phase composition (e.g., pH adjustment) were examined to improve the performance of an LC-MS screening method. The use of 0.1% acetic acid with 1 mM ammonium acetate (pH 3.6) as the aqueous portion of the mobile phase provided favorable sensitivities for a number of extractables both in positive and negative ion modes. In positive ion mode, the acidic mobile phase improved responses for moderately weak basic compounds by increasing their degree of protonation. For very weak basic compounds such as amides, ammonium ions in the mobile phase promoted proton adduct responses. In negative ion mode, an acidic mobile phase containing acetate anion improved ESI responses for acidic compounds, primarily due to gas phase effects.     

Analysis of intact bacteriohopanepolyols from methanotrophic bacteria by reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry

Direct detection of most intact biohopanoids is not possible using conventional GC-MS techniques due to their highly functionalised and amphiphilic nature. Here we report the application of a new reversed-phase high-performance liquid chromatography method for the direct analysis of acetylated, intact bacteriohopanepolyols in solvent extracts of methanotrophic bacteria. Atmospheric pressure chemical ionisation mass spectrometric detection provides structural information relating to the number and types of functional groups present in the four biohopanoids detected: bacteriohopanetetrol, aminobacteriohopanetriol, -tetrol and -pentol. The method should facilitate the assessment of hopanoid composition of both bacteria and environmental samples.     

Chemical fingerprint analysis of phenolics of Albizia chinensis based on ultra-performance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry and antioxidant activity

Albizia species have been shown to have anti-inflammatory and anti-allergic properties. However, efficient analytical methods for identification of their active constituents are still lacking. Ultra-performance liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) was used to study the phenolic composition of the ethanolic extracts of different parts (flowers, leaves, pods and bark) of A. chinensis. In addition, the antioxidant activity of the ethanolic extracts was evaluated by the 1,1-diphenyl-2-picryl hydrazyl (DPPH) free-radical and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical cation scavenging methods. Four compounds were isolated from the ethanolic extract of the flowers and characterized by 1H and 13C NMR spectroscopy as quercetin-3-O-rhamnoside, quercetin, quercetin-3-O-arabinofuranoside, and myricetin-3-O-rhamnoside. Separation and quantification of the phenolics was accomplished using a reversed-phase BEH C18 column with the mobile phase of methanol-water (0.05% formic acid), and detection wavelengths of 360 and 254 nm.     

Identification of multiple components in Guanxinning injection using hydrophilic interaction liquid chromatography/time-of-flight mass spectrometry and reversed-phase liquid chromatography/time-of-flight mass spectrometry

An approach for the identification of multiple components in traditional Chinese medicine injections (TCMIs) using a combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) coupled with time-of-flight mass spectrometry (TOFMS) was developed for the quality control of Guanxinning injection (GXNI), a widely used TCMI, composed of Salvia miltiorrhiza and Ligusticum Chuanxiong. A total of 50 compounds from five compound classes, including saccharides, amino acids, organic acids, phenolic acids and phthalides, were identified or tentatively characterized on the basis of accurate mass measurements and subsequent TOFMS product ions. Six groups of isomers of phenolic acids and saccharides were tentatively distinguished. It was observed that the ESI-TOFMS fragmentation behavior of phthalides was different in negative and positive ion mode, and the fragmentation pathways were tentatively elucidated using structurally-relevant product ions. Several highly polar constituents were characterized for the first time from GXNI by HILIC/TOFMS. In addition, all the constituents identified from GXNI were further assigned in the two individual crude drugs. The integrated strategy has provided a powerful approach for the separation and identification of the multiple components in GXNI, and it has also assisted in the establishment of methods for the comprehensive safety and quality evaluation of TCMIs.     

Sensitive analysis of 1alpha,25-dihydroxyvitamin D3 in biological fluids by liquid chromatography-tandem mass spectrometry.

A liquid chromatographic-tandem mass spectrometric assay using 5% bovine serum albumin as the calibration matrix has been developed for the quantitative analysis of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] in biological fluids. The analyte was extracted from the matrix after protein precipitation using an automated solid-phase extraction procedure involving both a reversed-phase and normal-phase procedure on a single C18 cartridge. The analytical chromatography was performed using a Symmetry C8 50 x 2.1 mm, 3.5 microm column. The mobile phase was a linear gradient from 75 to 99% methanol with a constant concentration of 2 mM ammonium acetate. 1alpha,25(OH)2D3 and the internal standard [2H6]1alpha,25(OH)2D3 were detected by using MS-MS. The ion source was operated in the positive electrospray ionisation mode. The assay is specific, sensitive, and has a capacity of more than 100 samples per day, with a limit of quantitation of 20 pg ml(-1) for a 1.0-ml sample aliquot. The assay has been used for the analysis of 1alpha,25(OH)2D3 in serum from rats and pigs simultaneously with the analysis of the vitamin D analog seocalcitol.     

Reversed-phase ion-pairing liquid chromatography/ion trap mass spectrometry for the analysis of negatively charged,derivatized glycans

The significant complexity, similar polarity and lack of ionizable sites make the analysis of glycans an analytical challenge. These compounds are often derivatized and separated by normal-phase high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) followed by UV or fluorescence detection. Due to widespread use of reversed-phase chromatography coupled to electrospray mass spectrometry as an analytical tool, our laboratory has developed this methodology for the analysis of glycans derivatized with a negatively charged tag, 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). It is possible to exploit the ability of this negatively charged tag to interact with a mobile phase ion-pairing reagent, allowing retention on a reversed-phase C(18) column for subsequent on-line UV or MS analysis. ANTS-derivatized samples, including a maltooligosaccharide ladder and glycans released from bovine ribonuclease B, bovine fetuin, and chicken ovalbumin, were analyzed using this method. In addition to reversed-phase retention, ribonuclease B and ovalbumin derivatives displayed highly desirable isomeric separation. With the use of mass spectrometric detection for glycan identity, this allowed relative quantitation of individual components.     

Collection of analytical data for benzodiazepines and benzophenones

On April 1st, 1986, amendments were made to Schedule 2 to the Misuse of Drugs Act 1971 such that 33 benzodiazepines became controlled as Class C drugs in the U.K. An analytical database has been prepared to aid the detection and identification of controlled benzodiazepines. Chromatographic properties have been measured including gas chromatography (GC) retention index values, high-performance liquid chromatography (HPLC) capacity factors and thin-layer chromatography (TLC) RF x 100 values. UV spectroscopic and mass spectrometric (MS) data have also been recorded. Analytical data (GC, TLC and MS) are also presented for benzophenones which were produced by acid hydrolysis of the parent benzodiazepines.     

Neuropeptide analysis with liquid chromatography-capillary electrophoresis-mass spectrometric imaging

Herein we report the first attempt of coupling multidimensional separations to matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging detection. Complex neuropeptide mixtures extracted from crustaceans were first fractionated by reversed-phase liquid chromatography (RPLC), and then subjected to a capillary electrophoresis-mass spectrometric imaging platform. With a specific focus on orcokinin family neuropeptides, we demonstrated that these trace-level analytes from complex neural tissue samples can be fully separated from chemical noise and interfering components and visualized as mass spectrometric imaging signals. A total of 19 putative orcokinins were detected, with highly efficient separations within the family being achieved for the first time. The results indicate that two-dimensional separation coupling to mass spectrometric imaging can serve as a novel and powerful tool in proteomics and peptidomics studies.     

High-performance liquid chromatographic-mass spectrometric assay of busulfan in serum and cerebrospinal fluid.

A liquid chromatographic-mass spectrometric method has been developed to determine busulfan concentrations in the cerebrospinal fluid and serum of some children undergoing bone marrow autotransplantation. After two liquid-liquid extraction steps with dichloromethane on a biological matrix, the separation of busulfan was carried out by isocratic reversed-phase chromatography. The mass spectrometric system was operated in electron-impact mode. Principal ions at m/z 175, 111 and 79 were observed for busulfan, but only m/z 175 was chosen for the quantification of the analyte. The retention time of busulfan was 2.5 min. The detection limit of 100 ng/ml allowed the determination of cerebrospinal fluid and serum busulfan concentrations during the four days of high-dose (1 mg/kg) treatment prior to autotransplantation in five child patients.     

Identification of phenolic constituents in Radix Salvia miltiorrhizae by liquid chromatography/electrospray ionization mass spectrometry

The phenolic components from Radix Salvia miltiorrhizae Bunge, a well-known herbal medicine (Dan-Shen in Chinese), have been investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). HPLC analyses were performed on a reversed-phase C18 column using gradient elution. In the ESI mass spectra a predominant [M-H]- ion was observed in negative mode and provided molecular mass information. ESI-MS/MS spectra of the [M-H]- ions were used for structural analysis, based on the spectra of standards. It was found that caffeic acid and its monomeric analogs containing a carboxyl group readily lost CO2, while dimers, trimers and tetramers of caffeic acid expelled successively danshensu or caffeic acid or their esters. Twenty-eight phenolic compounds in S. miltiorrhizae were characterized, of which eight compounds were positively identified by comparison with standards. The remaining twenty phenolics for which standards were not available were tentatively identified based on their UV spectra and MS/MS fragmentation characteristics.     

Determination of endotoxins by gas chromatography: evaluation of electron-capture and negative-ion chemical-ionization mass spectrometric detection of halogenated derivatives of beta-hydroxymyristic acid

The sensitivity and selectivity of gas chromatography for analysing several halogenated ester derivatives of beta-hydroxymyristic acid were studied using both selected-ion monitoring detection with negative-ion chemical-ionization mass spectrometry and electron-capture detection. Six different derivatization methods were compared with respect to yield, chemical stability and formation of by-products. Procedures for removal of excess reagents using disposable silica columns and thin-layer chromatography were elaborated. The 3-O-pentafluorobenzoyl-methyl ester was the preferred derivative since it provided high sensitivity and had the molecular ion as the base peak in the mass spectrum. The detection limit was 0.5 pg with electron-capture detection and 0.3 pg with the mass spectrometric system. Using beta-hydroxymyristic acid as a chemical marker it was possible to detect Escherichia coli endotoxin in aqueous solution at a level of 1 ng/ml.     

Combined high-performance liquid chromatographic-continuous-flow fast atom bombardment mass spectrometric analysis of acylcoenzyme A compounds

A high-performance liquid chromatographic method for the analysis of coenzyme A thioesters which employs continuous-flow fast atom bombardment mass spectrometric detection is presented. The chromatographic system utilizes gradient elution with reversed-phase conditions using ammonium acetate-acetonitrile from both standard analytical (3.9 mm I.D.) and microbore (1 mm I.D.) columns. Applications to coenzyme A thioesters of various acyl group chain length (C2-C18) and functionality (-COOH, -OH, -C = C-) are described. The system is also applied to an in vitro enzyme reaction (crotonase) to directly follow the disappearance of substrate and appearance of product. The mass spectrometry of coenzyme A thioesters, their chromatographic behavior, system stability, and sensitivity of detection are discussed.     

Simple extraction method and gas chromatography-mass spectrometry in the selective ion monitoring mode for the determination of phenols in wine

The concentrations of 22 components of wine, including most of those that have been shown to possess significant biological properties, have been determined by a fast and simple analytical method based on gas chromatography with mass spectrometric detection in the selective ion monitoring mode (GC/MS-SIM). The procedure involves an easy liquid-liquid extraction and derivatization methods of flavanols, phenols and carboxylic acid, using a very small wine volume. The average recovery (Rcv) ranged from 73 to 107% and linear regression coefficients (r(2)) were in a range of 0.981or=0.999. The GC/MS-SIM technique gives good specificity and sensitivity, and can therefore be suitable for routine monitoring of the concentration of individual phenolic antioxidants during winemaking and the aging process.