Profiling of impurities in illicit methamphetamine by high-performance liquid chromatography and capillary electrochromatography

High performance liquid chromatography (HPLC) with photodiode array (PDA) UV and fluorescence (FL) detection, and capillary electrochromatography (CEC) with laser-induced fluorescence (LIF) detection were investigated for the analysis of acidic extracts derived from illicit methamphetamine. These compounds include major impurities from the hydriodic acid/red phosphorous reduction method, i.e., 1,3-dimethyl-2-phenylnaphthalene and 1-benzyl-3-methylnaphthalene, and other trace-level, structurally related impurities. For certain of these solutes, HPLC with conventional FL detection gave at least a 60× increase in sensitivity over UV detection. In addition, other highly fluorescent impurities were detected in methamphetamine produced via four other synthetic routes. The use of a rapid scanning FL detector (with acquisition of “on the fly” excitation or emission) provided structural information and gave “optimum” excitation and emission detection wavelengths. CEC with LIF detection using UV laser excitation provided greatly improved chromatography over HPLC, with good detection limits in the low ng/ml range. Both methodologies provide good run-to-run repeatability, and have the capability to distinguish between samples.     

Nitrofuran antibiotic residues in pork: The FoodBRAND retail survey

Use of nitrofuran drugs in food-producing animals has been prohibited within the EU because they may represent a public health risk. Monitoring compliance with the ban has focused on the detection of protein-bound nitrofuran metabolites which, in contrast to the parent compounds, are stable and persist in animal tissues. As part of the “FoodBRAND” project, an extensive survey of pork was undertaken across 15 European countries. Samples (n = 1500) purchased at retail outlets were analysed for the nitrofuran metabolites AOZ, AMOZ, AHD and SEM using LC–MS/MS determination of nitrobenzaldehyde derivatives. Limits of quantification for the method were 0.1 μg/kg (AOZ, AMOZ), 0.2 μg/kg (SEM) and 0.5 μg/kg (AHD). Of the 1500 samples tested, measurable residues of nitrofuran metabolites were confirmed in 12 samples (0.8% incidence overall) of which 10 samples were purchased in Portugal (AOZ, 0.3 μg/kg; AMOZ, 0.2–0.6 μg/kg) and one sample each in Italy (AMOZ, 1.0 μg/kg) and Greece (AOZ, 3.0 μg/kg).     

Evaluation of triclosan and biphenylol in marine sediments and urban wastewaters by pressurized liquid extraction and solid phase extraction followed by gas chromatography mass spectrometry and liquid chromatography mass spectrometry

Analytical methods, based on GC–MS and LC–MS, for the determination of traces of 2,4,4′-trichloro-2′-hydroxydiphenyl ether (triclosan) and biphenylol in urban wastewater and marine sediments were developed. These methods involve the use of diverse analytical techniques, such as solid phase extraction (SPE) and pressurized liquid extraction for sample preparation, and GC–negative chemical ionization MS and LC–electrospray ionization (ESI) MS–MS for identification and quantification. The recoveries of triclosan and biphenylol were 84 and 80% in wastewater and 100 and 73% in sediments, respectively. Detection limits obtained were in the range of ppb and ppt. To prove their applicability to real samples and as part of a more extensive monitoring program, the developed methods were applied to the analysis of wastewater samples, coming from an urban wastewater treatment plant (UWWTP), and of marine sediment samples collected at the outflow of two UWWTPs to the sea. Results obtained reveal the presence of triclosan in all the samples at concentrations that ranged from 0.8 to 37.8 μg/l in wastewater and from 0.27 to 130.7 μg/kg in sediments. These preliminary data reinforce the interest for further research on this topic.     

New extraction procedure to improve the determination of quinolones in poultry muscle by liquid chromatography with ultraviolet and mass spectrometric detection

The present article aims to develop a new extraction procedure to improve the determination of quinolones in chicken muscle. This new determination method was validated using liquid chromatography–ultraviolet detection (LC–UV) and liquid chromatography–mass spectrometry detection (LC–MS), which has special bearing on stability studies. The results obtained by using the method were compared with the results obtained with a previous methodology. The new extraction procedure presents a sensitivity low enough to determine concentration of these drugs below the permissible maximum residue limits (MRL) in chicken muscle and is less time consuming than the previous methodology.     

Surface and wastewater quality monitoring: combination of liquid chromatography with (geno)toxicity detection,diode array detection and tandem mass spectrometry for identification of pollutants

Identification of unknown water pollutants with liquid chromatography and tandem mass spectrometry (LC–MS–MS) is often more complex and time consuming than identification with gas chromatography and mass spectrometry (GC–MS). In order to focus the identification effort on relevant compounds, unknown peaks need to be selected carefully. Based on its frequency of occurrence in the LC–Diode Array Detection (LC–DAD) chromatograms of surface and infiltrated waters, an unknown peak was selected for identification with LC–MS–MS. This compound was identified as hexamethoxymethylmelamine (HMMM), a chemical often used in the coating industry. This is the first time the presence of this chemical in surface waters has been reported. In addition to HMMM, two other structurally related compounds were found to be present in the investigated surface water. A standard mixture of HMMM and its by-products did not exhibit (geno)toxicity under the test conditions applied in this study. In another example, a genotoxic fraction of an industrial wastewater was isolated and examined by LC–MS–MS using a modern quadrupole–orthogonal acceleration-time-of-flight mass spectrometer (Q-TOF). Four compounds were detected. The structures of two compounds present are proposed to be 9-amino-2-hydroxy-acridine and 9-hydroxy-acridine-N-oxide or its structural isomer dihydroxy-acridine. Confirmation with standards could not be carried out, as pure compounds are not available. The other two compounds (structural isomers) could not be identified based on the data available within this study.     

Determination of theophylline and its metabolites in biological samples by liquid chromatography-mass spectrometry

Liquid chromatography–mass spectrometry (LC–MS) is a powerful tool for analysis of drugs and their metabolites. We used a column-switching system in combination with atmospheric pressure chemical ionization LC–MS (LC–APCI–MS) for the determination of theophylline and its metabolites in biological samples. The separation was carried out on a reversed-phase column using methanol–20 mM ammonium acetate as a mobile phase at a flow-rate of 1 ml/min in 30 min. In the mass spectrum, the molecular ions of these drugs and metabolites were clearly observed as base peaks. This method is sufficiently sensitive and accurate for the pharmacokinetic studies of these drugs.     

Analysis of acidic drugs in the effluents of sewage treatment plants using liquid chromatography-electrospray ionization tandem mass spectrometry

Azaspiracid poisoning (AZP) is a new human toxic syndrome that is caused by the consumption of shellfish that have been feeding on harmful marine microalgae. A liquid chromatography–mass spectrometry (LC–MS) method has been developed for the determination of the three most prevalent toxins, azaspiracid (AZA1), 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3) as well as the isomeric hydroxylated analogues, AZA4 and AZA5. Separation of five azaspiracids was achieved on a C₁₈ column (Luna-2, 150×2 mm, 5 μm) with isocratic elution using acetonitrile–water containing trifluoroacetic acid and ammonium acetate as eluent modifiers. Using an electrospray ionisation (ESI) source with an ion-trap mass spectrometer, the spectra showed the protonated molecules, [M+H]⁺, with most major product ions due to the sequential loss of two water molecules. A characteristic fragmentation pathway that was observed in each azaspiracid was due to the cleavage of the A-ring at C₉–C₁₀ for each toxin. It was possible to select unique ion combinations to distinguish between the isomeric azaspiracids, AZA4 and AZA5. Highly sensitive LC–MS³ analytical methods were compared and the detection limits were 5–40 pg on-column. Linear calibrations were obtained for AZA1 in shellfish in the range 0.05–1.00 μg/ml (r²=0.9974) and good reproducibility was observed with a relative standard deviation (%RSD) of 1.8 for 0.9 μg AZA1/ml (n=5). The %RSD values for the minor toxins, AZA4 and AZA5, using LC–MS³ (A-ring fragmentation) were 12.3 and 8.1 (0.02 μg/ml; n=7), respectively. The selectivity of toxin determination was enhanced using LC–MS–MS with high energy WideBand activation.     

Quantitative determination of sulfonamide in meat by liquid chromatography-electrospray-mass spectrometry

This paper describes a newly developed liquid chromatography–electrospray-mass spectrometry (LC–ES-MS) method for the quantitative determination of nine commonly used sulfonamides (sulfadiazine, sulfapyridine, sulfamerazine, sulfamethazine, sulfamonomethoxine, sulfisoxazole, sulfadimethoxine, sulfaquinoaline and sulfaphenazole) in meat. [M+H]⁺ and [M+Na]⁺ were the two major ions detected in positive ion mode. Selective ion monitoring was employed for quantitative determination. Satisfactory linearity, 0.1–10 μg ml⁻¹, of each compound was obtained. Blank meat samples were fortified at levels between 50 and 500 μg kg⁻¹. [Phenyl-¹³C6]sulfamethazine was used as internal standard. Sulfonamides were isolated from meat with a solvent extraction procedure and then determined by LC–ES-MS. The limits of detection were below 10 μg kg⁻¹. The application of this newly developed method was demonstrated by analyzing various beef, pork and chicken samples from local markets.     

Energetic heterogeneity of the surface of a molecularly imprinted polymer studied by high-performance liquid chromatography

A sequential combination of reversed-phase liquid chromatography–mass spectrometry (LC–MS) and capillary electrophoresis (CE) has been explored in order to perform separation and characterization of a multicomponent peptide mixture from the synthesis of leuprolide. The mixture was first analyzed and fractionated by LC–MS, and the collected fractions were subsequently separated by CE. Unambiguous identification of the electrophoretic peaks was achieved by injecting the collected fractions separately and spiking the leuprolide crude mixture. Furthermore, structural information about the components of the mixture provided by several semi-empirical migration models has been used to check the accuracy of the structures previously proposed by LC–MS. Combination of the two orthogonal techniques results in an enhancement of their individual selectivity characteristics.     

Analysis on residues of estrogens,gestagens and androgens in kidney fat and meat with gas chromatography-tandem mass spectrometry

The use of estrogens, gestagens and androgens (EGAs) in animal fattening is prohibited in the European Community. Based on the general detection capabilities of Belgian laboratories, National Minimum Required Performance Limits (National MRPLs) for a number of EGAs have been imposed by the inspection services. Selective hyphenated techniques, e.g. GC–MS and GC–MS², with high detection capability are needed. β-Trenbolone, which is meant to be a “problem” molecule for GC–MS, can be detected at the 2 μg/kg level using GC–MS². Based on the National MRPLs in different matrices, our laboratory has divided the EGAs into a class system. In this set-up, analysis of EGAs in kidney fat and meat is discussed.     

Liquid chromatography-inductively coupled plasma mass spectrometry

It is known that while many elements are considered essential to human health, many others can be toxic. However, because the intake, accumulation, transport, storage and interaction of these different metals and metalloids in nature is strongly influenced by their specific elemental form, complete characterization of the element is essential when assessing its benefits and/or risk. Consequently, interest has grown rapidly in determining oxidation state, chemical ligand association, and complex forms of a many different elements. Elemental speciation, or the analyses that lead to determining the distribution of an element’s particular chemical species in a sample, typically involves the coupling of a separation technique and an element specific detector. A large number of methods have been developed which utilize a multitude of different separation mechanisms and detection instruments. Yet, because of its versatility, robustness, sensitivity and multi-elemental capabilities, the coupling of liquid chromatography to inductively coupled plasma mass spectrometry (LC–ICP–MS) has become one of the most popular techniques for elemental speciation studies. This review focuses on the basic principles of LC–ICP–MS, its historical development and the many ways in which this technique can be applied. Different liquid chromatography separations are discussed as well as the factors that must be considered when coupling each to ICP–MS. Recent applications of LC–ICP–MS to the speciation of environmental, biological and clinical samples are also presented.     

Preparation and evaluation of polyacrylate-coated fused-silica capillaries for reversed-phase open-tubular liquid chromatography

By in situ photopolymerization of acrylates, thick polyacrylate films can be immobilized in 10-μm fused-silica capillaries. Up to 1.9-μm thick films can be produced in 0.3–1.2 m long capillaries, resulting in phase ratios> 1.0 and leading to columns with high mass loadability. Owing to the high mass loadability, application of “on-column” UV detection is possible. The effect of incorporating alkyl acrylates in the film on retention behaviour and efficiency was extensively investigated. The success rate of the preparation of silicone—ethylhexyl acrylate films is almost 100%. The polyacrylate films are stable towards basic solutions up to pH 12. The column efficiency is demonstrated with separations of anthracene derivatives. A fast separation of methyl-substituted benzenes with on-column UV detection is shown.     

Developing strategies for isolation of minor impurities with mass spectrometry-directed fractionation

Efficient and automated purification of new chemical entities/potential drug substances and isolation of minor impurities are important aspects of early drug discovery and development strategies, especially when combinatorial synthesis is applied. LC–MS controlled preparative LC and automated fraction collection have been developed for this purpose. The success of such an approach is greatly determined by the quality of the software controlling the application, the coordination between software and hardware, and the reliability of the hardware. The performance of a commercially-available LC–MS controlled autopurification system was evaluated by fractionating four impurities of buspirone as a model compound, eluting closely to the major component under both acidic and basic mobile-phase conditions. A purification strategy for these four components is proposed.     

Packed capillary liquid chromatography–electrospray mass spectrometry analysis of organophosphorus chemical warfare agents

Packed capillary column liquid chromatography (LC)–electrospray mass spectrometry (ESI-MS) was used for the first time to detect and identify four common organophosphorus chemical warfare agents in aqueous samples. Aqueous samples containing the organophosphorus chemical warfare agents in the 0.01 to 0.1 mg/ml range were analyzed directly by packed capillary LC–ESI-MS with the chemical warfare agents and several minor related impurities being well resolved under acetonitrile–water gradient elution conditions. The ESI-MS data for isopropyl methylphosphonofluoridate (sarin or GB), O-ethyl N,N-dimethylphosphoramidocyanidate (tabun or GA), cyclohexyl methylphosphonofluoridate (GF) and pinacolyl methylphosphonofluoridate (soman or GD) were acquired with a sampling cone voltage setting that promoted collisionally activated dissociation, and resulted in the acquisition of informative mass spectra containing both molecular and product ion information. The developed method appears to be an attractive alternative to GC–MS for the analysis of aqueous samples containing organophosphorus chemical warfare agents and their hydrolysis products, since they may be analyzed directly without the need for additional sample handling.     

Liquid chromatography-(tandem) mass spectrometry of selected emerging pollutants (steroid sex hormones,drugs and alkylphenolic surfactants) in the aquatic environment

Among the various compounds considered as emerging pollutants, alkylphenolic surfactants, steroid sex hormones, and pharmaceuticals are of particular concern, both because of the volume of these substances used and because of their activity as endocrine disruptors or as causative agents of bacterial resistance, as is the case of antibiotics. Today, the technique of choice for analysis of these groups of substances is liquid-chromatography coupled to mass spectrometry (LC–MS) and tandem mass spectrometry (LC–MS–MS). In the last decades, this technique has experienced an impressive progress that has made possible the analysis of many environmental pollutants in a faster, more convenient, and more sensitive way, and, in some cases, the analysis of compounds that could not be determined before. This article reviews the LC–MS and LC–MS–MS methods published so far for the determination of alkylphenolic surfactants, steroid sex hormones and drugs in the aquatic environment. Practical considerations with regards to the analysis of these groups of substances by using different mass spectrometers (single quadrupole, ion trap and triple quadrupole instruments, etc.), interfaces and ionization and monitoring modes, are presented. Sample preparation aspects, with special focus on the application of advanced techniques, such as immunosorbents, restricted access materials and molecular imprinted materials, for extraction/purification of aquatic environmental samples and extracts are also discussed.     

Solid-phase extraction of polar hydrophilic aromatic sulfonates followed by capillary zone electrophoresis-UV absorbance detection and ion-pair liquid chromatography-diode array UV detection and electrospray mass spectrometry

A comprehensive comparison of four different polymeric solid-phase extraction (SPE) materials for the extraction of 22 different aromatic sulfonates of environmental concern was performed. The investigated adsorbents were the polystyrene–divinylbenzene materials LiChrolut EN from Merck, Isolute ENV+ from International Sorbent Technology, HR-P from Macherey–Nagel and the new Oasis HLB poly(divinylbenzene-co-N-vinylpyrrolidone) copolymer from Waters. Different SPE parameters like the elution solvent and the drying step of the cartridges were optimized. Analyses were performed by capillary zone electrophoresis–UV absorbance detection (CZE–UV) and ion-pair liquid chromatography–diode array UV detection coupled in series with electrospray mass spectrometry (IP-LC–DAD-ESI-MS) in the negative ionization mode. LC–MS offers a higher separation efficiency than CZE. The best adsorbents were LiChrolut EN and HR-P followed by Isolute ENV+ and Oasis HLB. The recoveries for most of the onefold negatively charged aromatic sulfonates were >50% for the extraction from spiked ground water at 50 μg/l. Recoveries for LiChrolut EN and HR-P were approximately 20% higher than for Isolute ENV+. Very hydrophilic sulfonates containing more than one negative sulfonate group could not be extracted by any of the tested adsorbents.     

Capillary electrophoresis—matrix-assisted laser-desorption ionization mass spectrometry of proteins

An “off-line” combination of capillary electrophoresis (CE) with matrix-assisted laser-desorption mass spectrometry (MALDI-MS) has been developed for the structural characterization of CE-separated peptides and proteins. Using a sheath flow interface, similar to that developed for “on-line” CE—fast atom bombardment MS and CE—electrospray MS, an efficient sample isolation procedure has been developed which is applicable to bioorganic compounds in aqueous buffer solutions. This isolation procedure, with subsequent transfer to the MALDI-MS sample target, has been successfully used for the direct analysis of CE-separated proteins of M _r up to 67 000, and a mixture of apolipoprotein AII monomer and homodimer, using sample amounts of less than 1 pmol.     

Determination of nitro aromatic, nitramine, and nitrate ester explosive compounds in explosive mixtures and gunshot residue by liquid chromatography and reductive electrochemical detection

Reductive and oxidative electrochemical detection with liquid chromatography is applied to the determination of nitro aromatics, nitrate esters, nitramines, and diphenylamines in military explosives and single and double base smokeless gunpowders. A sensitive and highly selective method is presented for the detection of organic “gunshot residue” on the hand of individuals who have discharged a weapon. The detection limits at S/N = 3 are of the order of 0.5, 1, 2, and 0.3 picomol for nitro aromatic, nitramine and nitrate ester explosive compounds, and diphenylamines, respectively.     

Evaluation of synthetic liquid chromatography—diode array detection—mass spectrometry data for the determination of enzyme kinetics

In this paper, we investigate the accuracy and precision of the results from diode array detector (DAD) data and mass spectrometry (MS) data as obtained subsequent to chromatographic separations using computer simulations. Special attention was given to simulations of multiple injections from a developing enzymatic reaction. These simulations result in three-way LC–DAD–MS kinetic data; LC–DAD and LC–MS data were also evaluated independently in this investigation. The noise characteristics of the MS detector prevent accurate determination of the individual reaction rate constants by the analysis method. Using the data from the DAD in combination with the MS detector results in improved estimation of the rate constants. The results also indicate that the higher resolving power of the MS information compensates for the lower signal-to-noise ratio in these data, compared to DAD data.     

Simultaneous determination of paracetamol and chlorpheniramine in human plasma by liquid chromatography-tandem mass spectrometry

An analytical method for the determination of paracetamol and chlorpheniramine in human plasma has been developed, validated and applied to the analysis of samples from a phase I clinical trial. The analytical method consists in the extraction of paracetamol and chlorpheniramine with diethyl ether, followed by the determination of both drugs by an LC–MS–MS method, using 2-acetamidophenol as internal standard. The intra-assay and inter-assay precision and accuracy of this technique were good and the limit of quantitation was 0.5 μg/ml of plasma for paracetamol and 0.2 ng/ml for chlorpheniramine. The concentration working range was established between 0.5 μg/ml and 25 μg/ml for paracetamol and between 0.2 ng/ml and 50 ng/ml for chlorpheniramine. This method has been used for analyzing more than 1200 human plasma samples from a clinical study with 24 volunteers.