Strategy of glycerolipid separation and quantitation by complementary analytical techniques : Plenary lecture

Although improved systems for chromatographic resolution continue to be developed there is good reason to believe that no single method will be capable of complete separation of all lipid mixtures including the geometric, positional and stereochemical isomers in each molecular species. Furthermore, the chromatographic systems giving the highest resolution usually yield the least complete recoveries of components and require separate procedures of quantitation. It is therefore necessary to develop appropriate strategies that yield the required resolution as a result of consecutive application of complementary analytical techniques. At the present time, the original combination of thin-layer and gas— liquid chromatography has been joined by the combination of thin-layer and liquid, and liquid and gas—liquid chromatography with both liquid and gas—liquid chromatography being frequently coupled to mass spectrometry with computerized data processing. Internal standardization with hydrogen flame ionization provides a simple quantitative detection for gas chromatography, while mass spectrometry serves a similar purpose in liquid chromatography, although a much more extensive calibration may be required for quantitation. Special advantages for both separation and quantitation of most neutral lipid mixtures are derived from enzymic and chemical modification of the samples prior to chromatography. With imaginative work-up of samples, superior qualitative and quantitative results can frequently be obtained by appropriate combination of chromatographic techniques of limited resolving power.     

The determination of (-)-(S)- and (+)-(R)-ifosfamide in plasma using enantioselective gas chromatography: a validated assay for pharmacokinetic and clinical studies

An enantioselective gas chromatographic method has been developed and validated for the determination of the plasma concentration of the enantiomers of the anticancer drug ifosfamide (IFF). In this approach, the IFF enantiomers are separated from the plasma matrix by solid phase extraction, chromatographically resolved by gas chromatography on a chiral stationary phase, and detected by mass selective detection using selective ion monitoring. The assay has been validated for routine clinical and pharmacokinetic use and has a limit of detection in plasma of 250 ng/ml of each isomer.     

Detection of the illegal use of ethinylestradiol in cattle urine by gas chromatography—mass spectrometry

A method for the detection of ethinylestradiol in cattle urine is described, based on enzymic hydrolysis of the sample, clean-up by means of disposable octadecyl and amino solid-phase extraction columns, fractionation by reversed-phase high-performance liquid chromatography, and detection by gas chromatography—mass spectrometry (selected-ion monitoring). Identification is based on both gas chromatographic and mass spectrometric data. The method has been tested on urine samples for a collaborative study and all the results found were correct.     

Hippophae rhamnoides L.: chromatographic methods to determine chemical composition, use in traditional medicine and pharmacological effects

There is an increasing interest in the usage of chromatographic methods on the analysis of chemical compounds present in Hippophae rhamnoides L. In this paper, the chromatographic techniques applied for the determination, separation and identification of chemical compounds of H. rhamnoides L. are reviewed. We examined the existing chromatographic methods based on separations by paper and thin-layer chromatography, gas chromatography, high-performance liquid chromatography and capillary electrophoresis and also methods of detection by ultraviolet absorption, fluorescence, refractive index, electrochemical and mass spectrometry. Biological properties of the plant and its pharmacological effects and use in traditional medicine have also been reviewed.     

Chromatographic separation of 2,3-benzodiazepines

A review of chromatographic methods for the determination of 2,3-benzodiazepines (2,3-BZs) is presented. The determinations are performed to investigate the presence of potential impurities in drug substances and to study their pharmacokinetic profile in biological samples, either in animals or in humans. Several methods dealt with a pretreatment of samples, i.e., liquid–liquid extraction by using a variety of solvents, solid-phase extraction, direct injection of specimens into the chromatographic apparatus. Different chromatographic techniques have been used. High-performance liquid chromatography allows optimal sensitivity and specificity by using ultraviolet or diode array detection methods. Gas chromatography-mass spectrometry and gas chromatography with nitrogen-phosphorous or electron-capture detectors have been also reported. Suitable methods for the separation of enantiomers of 2,3-BZs have been described. Thin-layer chromatography has been shown to be capable to isolate analytes from biological samples as urine or faeces. The reported chromatographic techniques are currently applied to define the metabolic pathways of 2,3-BZs in experimental and clinical studies.     

Analyses of steroids in saliva using highly selective mass spectrometric techniques

Highly selective techniques of gas chromatography mass spectrometry have been used in the unequivocal identification of salivary steroids at concentrations ranging from 20 pg ml-1 to 20 ng ml-1. Oestradiol-17 beta, for example, has been identified in pregnancy saliva by gas chromatography high resolution mass spectrometry selected ion monitoring of the bis-TMS ether and by gas chromatography mass spectrometry metastable peak monitoring of the bis-tert-butyldimethylsilyl ether. Dehydroepiandrosterone sulphate has been identified in saliva, following enzymic hydrolysis, by gas chromatography high resolution mass spectrometry selected ion monitoring of the tert-butyldimethylsilyl ether and methyloxime tert-butyldimethylsilyl ether. These initial analyses have been designed to guide the development of routine immunoassay procedures which may subsequently be validated by comparison with reference gas chromatographic mass spectrometric methods.     

Characterization of mixtures of dipeptides by gas chromatography/mass spectrometry

The gas chromatographic and mass spectrometric behavior of over 120 different dipeptides has been investigated. The dipeptides were analyzed as their N,O-perfluoropropionyl methyl ester derivatives by combined gas chromatography/mass spectrometry. Mass spectra of the dipeptides were obtained using both electron impact and chemical ionization. Gas chromatographic retention times were obtained for each of the dipeptides studied and utilized for the prediction of the retention times for most of the 400 common dipeptides. These techniques enable the unambiguous identification of dipeptides in mixtures.     

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 specifity. 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.     

A comparison of thermospray and direct liquid introduction high-performance liquid chromatography/mass spectrometry for the analysis of candidate antimalarials

The analysis of antimalarials by high-performance liquid chromatography (HPLC)/mass spectrometry demonstrates a new dimension in specificity along with increased sensitivity compared to conventional HPLC detection methods. Both direct liquid introduction and thermospray HPLC/mass spectrometry interfaces provided molecular weight information as well as characteristic fragment ions for antimalarials not normally amenable to direct probe or gas chromatographic/mass spectrometric techniques. The direct liquid introduction interface, which incorporated a 1/100 split, showed a detection limit of 30 ng using selected ion monitoring. The thermospray technique showed less than 1 ng detection limits using selected ion monitoring.     

Screening of β-2 agonists and confirmation of fenoterol,orciprenaline, reproterol and terbutaline with gas chromatography–mass spectrometry as tetrahydroisoquinoline derivatives

A new method for a comprehensive screening and confirmation of β-2 agonists in human urine is presented based on gas chromatography–low-resolution mass spectrometry (GC–MS) using electron impact ionisation (EI). After hydrolysis of the conjugates with β-glucuronidase/arylsulfatase a derivatisation step with formaldehyde converts fenoterol, orciprenaline, reproterol and terbutaline to one derivative, a tetrahydroisoquinoline, while the other β-2 agonists remain unchanged. Liquid–liquid extraction and trimethylsilylation follow. The tetrahydroisoquinoline derivatives show good gas chromatographic and mass spectrometric behaviour. The detection limit of these four β-2 agonists in the screening using low-resolution mass spectrometry is 10 ng/ml of urine. The other β-2 agonists are detected as parent compounds with the same recovery after sample preparation with and without formaldehyde. The EI mass spectra of the tetrahydroisoquinoline derivatives are presented.     

Quantification of plasma phenylethylamine by combined gas chromatography/electron capture negative ion mass spectrometry of the N-acetyl-N-pentafluorobenzoyl derivative

An ultrasensitive method capable of detection and quantification of beta-phenylethylamine in 1 ml of human plasma has been developed using gas chromatography/electron capture negative ion mass spectrometry. Phenylethylamine and tetra-deutero phenylethylamine internal standard in plasma were acetylated, extracted into organic solvent and then further acylated with pentafluorobenzoyl chloride. The N-acetyl-N-pentafluorobenzoyl-phenylethylamines were detected by high-resolution single ion monitoring of the molecular ions. Normal plasma levels were found to be 41.5 +/- 10.7 pg ml-1, in accordance with results of a previous high-performance liquid chromatographic method.     

Drug analysis by direct liquid introduction micro liquid chromatography mass spectrometry

The analytical capabilities of a micro high performance liquid chromatograph interfaced to an unchanged quadrupole mass spectrometer are presented. Continuous monitoring of the total micro liquid chromatographic effluent allows full scan chemical ionization mass spectra of from one to five nanograms of drugs and their metabolites to be recorded. The interface is a simple, inexpensive device which can be assembled from commercially available components. An eight microliter per minute flow rate of the micro liquid chromatographic eluant allows separation and identification of biologically important substances not amenable to gas chromatography mass spectrometry techniques. The sensitivity of micro liquid chromatography mass spectrometry performed as described is comparable with gas chromatography mass spectrometry and is achieved by introducing the total micro liquid chromatographic effluent into the chemical ionization ion source of the mass spectrometer. Selected ion monitoring provides 20 pg detection limits of phenothiazine tranquilizers injected on column.     

Detection of metabolites of toxic alkylmethylphosphonates in biological samples.

The major metabolites and breakdown products of some toxic organophosphonates are their respective alkymethylphosphonic acids. These acids ionize at physiological pH and are not amenable to gas chromatographic analysis in their underivatized forms. Their detection in biological samples has been difficult because of their presence at only trace levels. Existing analytical methods were developed mainly for measuring these phosphonic acids in environmental samples and at higher concentrations. In this study, we devised a gas chromatographic/mass spectrometric method to provide confirmation and quantification of the organophosphonic acids of soman (GD), sarin (GB) and GF in blood and urine. This report describes the various derivatization conditions that we have studied and demonstrates the characteristic mass spectra by different ionization techniques.     

LC/CE-MS tools for the analysis of complex arabino-oligosaccharides

Recently, various branched arabino-oligosaccharides as present in a sugar beet arabinan digest were characterized using NMR. Although HPAEC often has been the method of choice to monitor the enzymatic degradation reactions of polysaccharides, it was shown that HPAEC was incapable to separate all known linear and branched arabino-oligosaccharides present. As this lack of resolution might result in an incorrect interpretation of the results, other separation techniques were explored for the separation of linear and branched arabino-oligosaccharides. The use of porous-graphitized carbon liquid chromatography with evaporative light scattering and mass detection as well as capillary electrophoresis with laser-induced fluorescence and mass detection demonstrated the superiority of both the techniques toward HPAEC by enabling the separation and unambiguous identification of almost all the linear and branched arabino-oligosaccharides available. The elution behavior of all arabino-oligosaccharides for the three tested separation techniques was correlated with their chemical structures and conclusions were drawn for the retention mechanisms of the arabino-oligosaccharides on the different chromatographic and electrophoretic systems. The combination of the elution/migration behavior on LC/CE and the MS fragmentation patterns of the arabino-oligosaccharides led to the prediction of structures for new DP6 arabino-oligosaccharides in complex enzyme digests.     

Determination of saccharide content in pneumococcal polysaccharides and conjugate vaccines by GC-MSD

A simple and sensitive gas chromatographic method was designed for quantitative analysis of Streptococcus pneumoniae capsular polysaccharides, activated polysaccharides, and polysaccharide conjugates. Pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F polysaccharide or conjugate were subjected to methanolysis in 3N hydrochloric acid in methanol followed by re-N-acetylation and trimethylsilylation. Derivatized samples were chromatographed and detected using gas chromatography with mass selective detector. Gas chromatographic results were compared with colorimetric values with agreement of 92 to 123% over the range of all samples tested. Monosaccharides released during methanolysis included hexoses, uronic acids, 6-deoxy-hexoses, amino sugars, and alditols. Quantitative recovery of monosaccharides was achieved for all serotypes by the use of a single methanolysis, derivatization, and chromatography procedure. Response factors generated from authentic monosaccharide standards were used for quantitation of pneumococcal polysaccharides and conjugates with confirmation of peak assignments by retention time and mass spectral analysis. This method allows saccharide quantitation in multivalent pneumococcal vaccine intermediates and final drug products with low-level detection (10 pg) and peak purity.     

Determination of MK-287, a new platelet-activating factor antagonist, in plasma and serum by gas chromatography chemical ionization mass spectrometry

MK-287 is a novel platelet-activating factor antagonist. A sensitive and specific gas chromatographic/mass spectrometric assay has been developed for the determination of the drug in serum and plasma. The assay utilizes an extraction with methyl-t-butyl ether and subsequent trimethylsilylation of the hydroxyl function. The gas chromatographic/mass spectrometric determinations are carried out with temperature-programmed capillary gas chromatography and ammonia negative chemical ionization mass spectrometry. The method has sufficient sensitivity, precision, accuracy and selectivity for the analysis of drug concentrations in clinical samples.     

Gas chromatographic–mass spectrometric assay for rocuronium with potential for quantifying its metabolite, 17-desacetylrocuronium, in human plasma

A rapid, sensitive and selective method has been developed for the quantification of plasma concentrations of neuromuscular blocking drug, rocuronium, using gas chromatography with mass spectrometric detection. 3-Desacetylvecuronium served as the internal standard. The method involved iodide ion pair formation and a single-step liquid–liquid extraction with dicholoromethane. This method also permits simultaneous determination of its putative metabolite, 17-desacetylrocuronium, although the high detection limit for the metabolite limits the practical application of this method in pharmacokinetic study of the metabolite. The extraction efficiency was 75% for rocuronium and 50% for 17-desacetylrocuronium. The limit of quantification was 26 ng/ml for rocuronium and 870 ng/ml for its metabolite. The assay was used successfully in a patient undergoing liver transplantation and receiving rocuronium as a constant rate infusion and in a patient undergoing general elective surgery receiving the drug as an intravenous bolus. This assay is a time-saving alternative to published gas or liquid chromatographic methods for assaying rocuronium.     

Gas chromatographic/mass spectrometric analysis of specific isomers of polychlorodibenzofurans

The gas chromatographic/mass spectrometric characteristics of 26 congeners of polychlorinated dibenzofurans, previously characterized by specific synthetic routes and by standard spectroscopic techniques, have been evaluated. The electron impact mass spectra are not particularly isomer-specific, though 2,3,7,8-tetrachlorodibenzofuran is distinguishable on this basis from the three other tetrachloro isomers investigated in this work. Positive ion methane chemical ionization mass spectra do show a greater degree of isomer distinction, and are reasonably reproducible. Electron attachment negative ion spectral characteristics are also presented. Preliminary results on negative ion chemical ionization mass spectra, obtained using methane plus small amounts of oxygen as reagent gas, are reported.     

Determination of aminoethylcysteine ketimine decarboxylated dimer in human plasma and cultured cells by high-performance liquid chromatography with electrochemical detection

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural compound with antioxidant properties of a new family of sulfur-containing amino acids. It has been detected in human urine and plasma, in mammalian cerebellum and, more recently, in dietary vegetables. In the present study, a simple, highly sensitive method using a high-performance liquid chromatography system with electrochemical detection (ECD) has been developed. The method showed excellent precision and accuracy. It has been found to be about 100-fold more sensitive than gas chromatographic method and 2000-fold more sensitive in respect to the liquid chromatography method with UV detection. The method showed the required features of specificity and sensitivity to detect aminoethylcysteine ketimine decarboxylated dimer in human plasma and in cultured cells after in vitro supplementation.     

Sample preparation and high-resolution separation of mycotoxins possessing carboxyl groups

The chromatographic analysis of carboxyl-containing mycotoxins, such as fumonisin B₁, ochratoxin A, and citrinin, presents a continual challenge. Toxins must first be extracted from foods or tissues and then cleaned up before chromatographic separation and detection. Liquid–liquid extraction efficiencies for some carboxylic mycotoxins are marginal for spiked samples and uncertain for incurred residues. Immunoaffinity columns may be useful for concentrating mycotoxins from samples before chromatography. In almost every case, more than one analytical method must be used to confirm the identification of the mycotoxin. The fumonisins are especially troublesome to analyze because they are relatively insoluble in organic solvents, they are not separated easily by gas chromatography, and they do not respond to the usual absorbance or fluorescence detectors used in liquid chromatography. Fluorescence derivatization and electrospray liquid chromatography–mass spectrometry have now made it possible to detect trace levels of mycotoxins. The purity of mycotoxin standards for toxicological studies can be determined by liquid chromatography with either an evaporative light scattering detector or electrospray mass spectrometer. New developments in capillary electrophoresis, nonporous microsphere liquid chromatography, and detection methods for low-volatility compounds show promise for improving the analysis of mycotoxins in the future.