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.     

Comparison between liquid chromatography-UV detection and liquid chromatography-mass spectrometry for the characterization of impurities and/or degradants present in trimethoprim tablets

The characterization of impurities and/or degradants present in pharmaceutical compounds is an important part of the drug development process. Although LC–UV is commonly employed for impurities and degradant compound determination, LC–MS techniques are proposed in this work to be a viable modern alternative for the characterization of these compounds. LC–UV and LC–MS were compared for the detection of impurities present in different brands of trimethoprim tablets by using an in-line LC–UV–MS system with atmospheric pressure chemical ionization source (APCI) coupled with a reversed-phase gradient HPLC system. It was shown that, although chemical noise was higher when using full-scan LC–MS compared to LC–UV, low level impurities were better detected by mass spectrometry (MS) when modern software algorithms are employed. These included the “Contour” chromatogram algorithm and/or the “component detection algorithm” (CODA). In addition, MS allowed for the simultaneous determination of the molecular masses and some structural information of the impurities and/or degradants. The results also showed a large difference in the purity of trimethoprim among different manufacturers. LC–MS and tandem MS techniques were employed to acquire fragmentation patterns for trimethoprim and its degradants to gain insight into their structures.     

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.     

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.     

Progress in liquid chromatography-mass spectrometry instrumentation and its impact on high-throughput screening

In the past 10 years, liquid chromatography–mass spectrometry (LC–MS) has rapidly matured to become a very powerful and useful analytical tool that is widely applied in many areas of chemistry, pharmaceutical sciences and biochemistry. In this paper, recent instrumental developments in LC–MS-related interfacing, ionization and mass analysis are reviewed from the perspective of the application of LC–MS in high-throughput screening of combinatorial libraries and the related high-throughput quantitative bioanalysis in early drug-discovery studies, such as early adsorption, distribution, metabolism and excretion studies.     

Quantitative analysis of cytokinins in plants by liquid chromatography–single-quadrupole mass spectrometry

A sensitive method for the quantitative analysis of all natural isoprenoid cytokinins in plant material by electrospray single-quadrupole mass spectrometry is presented. A baseline chromatographic separation of 20 non-derivatised naturally occurring cytokinins has been developed. Precise analyses of O-glucoside and ribonucleotide fractions were also performed by the high-performance liquid chromatography–mass spectrometry (HPLC–MS) but run separately from the basic cytokinin metabolites. Using post-column splitting, the flux from narrow-bore (2.1 mm i.d.) reversed-phase liquid chromatography column was simultaneously introduced into the diode array and mass detector. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. When low cone voltage (20 V) was applied, all studied cytokinins were determined in aqueous methanol as dominant quasi-molecular ions of [M+H]⁺ with limits of detection ranging between 10 and 50 fmol. For routine analysis a linearity range between 25 (75) fmol and 100 pmol was obtained. Developed liquid chromatography–mass spectrometry (LC–MS) method in selective ion monitoring mode was employed to quantify cytokinin species in tobacco BY-2 suspension culture and poplar leaves (Populus×canadensis Moench, cv Robusta).

Purified plant cell (BY-2) and plant tissue (poplar leaves) extracts were obtained by using two different ion-exchange chromatography steps, in combination with immunoaffinity purification using a broad-spectrum monoclonal anti-cytokinin antibody. The antibody strongly recognises the presence of N⁶-substituent on purine skeleton and thus does not bind adenine and related compounds. The presence of authentic cytokinins in the extracts quantified by LC–MS was further verified by enzyme-linked immunosorbent assays (ELISAs) with prior LC preparation. The combination of liquid chromatography–single-quadrupole mass spectrometry with immunoaffinity chromatography offers an efficient and elegant method for detection and quantification of cytokinin metabolites.     

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.     

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.     

Applications of solid-phase microextraction in food analysis

Food analysis is important for the evaluation of the nutritional value and quality of fresh and processed products, and for monitoring food additives and other toxic contaminants. Sample preparation, such as extraction, concentration and isolation of analytes, greatly influences the reliable and accurate analysis of food. Solid-phase microextraction (SPME) is a new sample preparation technique using a fused-silica fiber that is coated on the outside with an appropriate stationary phase. Analyte in the sample is directly extracted to the fiber coating. The SPME technique can be used routinely in combination with gas chromatography (GC), GC–mass spectrometry (GC–MS), high-performance liquid chromatography (HPLC) or LC–MS. Furthermore, another SPME technique known as in-tube SPME has also been developed for combination with LC or LC–MS using an open tubular fused-silica capillary column as an SPME device instead of SPME fiber. These methods using SPME techniques save preparation time, solvent purchase and disposal costs, and can improve the detection limits. This review summarizes the SPME techniques for coupling with various analytical instruments and the applications of these techniques to food analysis.     

Simple ion chromatographic method for the determination of chlormequat residues in pears

Current methods for quantitative determination of chlormequat residues in food crops are characterized by rather low recoveries and the need for derivatization (in case of gas chromatography, GC), or by high capital investment (in case of liquid chromatography–mass spectrometry, LC–MS). We propose a cation-exchange chromatography method for the analysis of chlormequat in pears. The method is based on extraction of the target compound with 40 mM HCl, followed by centrifugation and filtration. The filtrate is directly injected into an ion chromatograph equipped with a commercially available cation-exchange column and a suppressed conductivity detection system. While the limit of detection (LOD) (0.5 mg/kg) may not be small enough to allow dietary analysis, the method meets all validation requirements and is an alternative for the existing GC and LC–MS methods in quality control.     

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.     

Quantitation of anabolic hormones and their metabolites in bovine serum and urine by liquid chromatography-tandem mass spectrometry

A specific and sensitive method based on tandem mass spectrometry with on-line high-performance liquid chromatography using atmospheric pressure chemical ionisation (LC–APCI-MS–MS) for the quantitation of anabolic hormone residues (17β-19-nortestosterone, 17β-testosterone and progesterone) and their major metabolites (17-19-nortestosterone and 17-testosterone) in bovine serum and urine is reported. [²H₂]17β-Testosterone was used as internal standard. The analytes were extracted from urine (following enzymatic hydrolysis) and serum samples by liquid–liquid extraction and purified by C₁₈ solid-phase extraction. Ionisation was performed in a heated nebulizer interface operating in the positive ion mode, where only the protonated molecule, [M+H]⁺, was generated for each analyte. This served as precursor ion for collision-induced dissociation and two diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring LC–MS–MS. The overall inter-day precision (relative standard deviation) ranged from 6.37 to 2.10% and from 6.25 to 2.01%, for the bovine serum and urine samples, respectively, while the inter-day accuracy (relative error) ranged from −5.90 to −3.18% and from −6.40 to −2.97%, for the bovine serum and urine samples, respectively. The limit of quantitation of the method was 0.1 ng/ml for all the hormones in bovine serum and urine. On account of its high sensitivity and specificity the method has been successfully used to confirm illegal hormone administration for regulatory purposes.     

Mass spectrometric determination of ergosterol in a prairie natural wetland

Packed capillary liquid chromatography–electrospray mass spectrometry (LC–ESI-MS) was used for the analysis of a snow sample that was accidentally contaminated with an organophosphorus chemical warfare agent during the destruction of a chemical munition. Sarin, its hydrolysis products and a number of related compounds were identified on the basis of acquired LC–ESI-MS data. Full mass spectra were acquired for 14 compounds, with all exhibiting MH⁺, [MH+ACN]⁺ ions and/or protonated dimers that could be used to confirm molecular mass. Sampling cone voltages from 20 to 70 V were utilized with the higher sampling voltages enhancing formation of structurally important product ions in the ESI interface. All data were acquired with a time-of-flight mass spectrometer with a resolution of 5000 (50% valley definition), a resolution that aided in the assignment of elemental composition of the observed ions. The application of LC–ESI-MS to snow analysis appears to be an attractive alternative to the GC–MS methods, since both chemical warfare agents and their hydrolysis products may be analysed directly, eliminating the need for additional sample handling and derivatization steps.     

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).     

Verapamil: new insight into the molecular mechanism of drug oxidation in the human heart

Verapamil is a commonly prescribed cardiovascular drug, but surprisingly its metabolism in the target tissue of pharmacotherapy is basically unknown. We therefore investigated its biotransformation in human heart tissue and correlate the production of metabolites with the gene expression of major drug metabolising enzymes. Using electrospray LC–MS–MS and LC–MS³ experiments, a total of nine metabolites were observed in incubation experiments with verapamil and microsomes isolated from the human heart tissue, and this included a carbinolamine-, N-formyl-, ahemiacetale-, and formate-intermediate of N-demethyl- and O-demethylverapamil. We also observed a hydroxylation product at the benzylic position of atom C-7 (M9). Metabolites M5–M9 are novel and were not observed in previous studies with liver or other human tissues. A fine example of the considerable metabolic competence of human heart is the formation of M1–M4, e.g. dealkylverapamil, norverapamil and isomers of O-demethylverapamil, which were believed to be exclusively produced by the liver.     

Analysis of scopolamine and its eighteen metabolites in rat urine by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method is described for the determination of scopolamine and its metabolites in rat urine by combining liquid chromatography and tandem mass spectrometry (LC–MS/MS). Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of scopolamine. After extraction procedure, the pretreated samples were injected into a reversed-phase C18 column with mobile phase of methanol/ ammonium acetate (2 mM, adjusted to pH 3.5 with formic acid) (70:30, v/v) and detected by an on-line MS/MS system. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (ΔM), retention-times and full scan MSⁿ spectra with those of the parent drug. The results revealed that at least 18 metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, hydroxyscopolamine, hydroxyscopolamine N-oxide, p-hydroxy-m-methoxyscopolamine, trihydroxyscopolamine, dihydroxy-methoxyscopolamine, hydroxyl-dimethoxyscopolamine, glucuronide conjugates and sulfate conjugates of norscopolamine, hydroxyscopolamine and the parent drug) and the parent drug existed in urine after ingesting 55 mg/kg scopolamine to healthy rats. Hydroxyscopolamine, p-hydroxy-m-methoxyscopolamine and the parent drug were detected in rat urine for up 106 h after ingestion of scopolamine.     

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.     

Analysis of glycolytic intermediates in Saccharomyces cerevisiae using anion exchange chromatography and electrospray ionization with tandem mass spectrometric detection

The purpose of this study is to selectively and quantitatively analyze several glycolytic intermediates in cells of Saccharomyces cerevisiae using high-performance anion exchange chromatography (HPAEC) coupled to electrospray ionization tandem mass spectrometry for the analysis. A sodium hydroxide gradient is used to separate the glycolytic compounds and after the column sodium hydroxide is reduced by proton exchange with a membrane device prior to introduction to the mass spectrometer. The detection limits for 10 μl samples are down to the 0.4–5 pmol range. This corresponds for the intracellular metabolites to a range of 2–20 nmol per gram biomass dry weight (DW). Standard addition did reveal some influence of the sample matrix on the measured concentrations. Separation and analysis is hardly affected by the high sulfate and phosphate concentrations (1 mM) in the fermentation medium and by the intracellular matrix. Validation of the glucose-6-phosphosphate LC–MS–MS analysis results with enzymatic analysis showed an excellent agreement between the two methods. The suitability of the method was clearly shown by analyzing a series of steady state S. cerevisiae samples from a carbon limited aerobic chemostat culture.     

Analysis of pesticides in fruit, vegetables and cereals using methanolic extraction and detection by liquid chromatography–tandem mass spectrometry

A method for analysing carbamates and other relatively polar pesticides by LC–MS–MS with electrospray ionisation has been developed. The method is based on extraction by ultrasonication using a methanolic ammonium acetate–acetic acid buffer. After centrifugation the samples are filtered in Miniprep filter HPLC vials and detected by LC–MS–MS. To compensate for variations in the MS response [¹³C₆]-carbaryl was used as internal standard and matrix-matched pesticide solutions were used as external standards for the quantification. The method has been validated for the matrices apple, avocado, carrot, lettuce, orange, potato and wheat at the spiking levels—0.02; 0.04 and 0.20 mg kg⁻¹. Recoveries were generally in the range 70–120%. Results from participation in three intercomparisons proved the accuracy of the method. As the analytical procedure does not include any concentration or cleanup steps, it is easy and fast to perform, making it applicable for routine analysis in large pesticide monitoring programmes.